BACK
JEFF BROWN
Public Defender
MICHAEL BURT
Deputy Public Defender
555 Seventh Street, Second Floor
San Francisco, California 94103
(415) 553-9650

Attorneys for Defendant
ROBERT NAWI

SUPERIOR COURT OF CALIFORNIA

COUNTY OF SAN FRANCISCO


THE PEOPLE OF THE STATE OF ) CALIFORNIA, )
)
Plaintiff, ) )
vs. )
)
ROBERT NAWI )
)
Defendant. )
___________________________________ ) SCN: 176527
MCN: 1812436

Date: January__, 2000
Time:
Department:

OPPOSITION TO PEOPLE'S MOTION TO ADMIT DNA TEST RESULTS AND REQUEST FOR KELLY-FRYE HEARING

TO TERENCE HALLINAN, DISTRICT ATTORNEY FOR THE CITY AND COUNTY OF SAN FRANCISCO, STATE OF CALIFORNIA, TO ASSISTANT DISTRICT ATTORNEY JOHN FARRELL AND TO THE ABOVE-ENTITLED COURT:

PLEASE TAKE NOTICE that on ,________ 2000 at a.m. in Department __ of the above-entitled court, the defendant herein will oppose the People's anticipated Motion to Admit DNA Test Results and will and hereby does request a Kelly-Frye hearing on the grounds that (1) the People's DNA evidence is inadmissible under Kelly-Frye and under Evidence Code Section 352; (2) this opposition raises issues pursuant to Kelly-Frye that have not been decided by any California appellate court; and, (3) the defendant is entitled in any event to a hearing to determine whether correct scientific DNA testing procedures were used in this particular murder case.
This Motion is based on this Notice, the attached memorandum of points and authorities, the testimony and exhibits in the Kelly-Frye hearing in People v. Bokin et al, S.C. No. 168461, judicial notice of which is hereby requested pursuant to Evidence Code Section 452(d), and any oral and documentary evidence and argument as may be produced at the hearing on said motion.
DATED: December 20, 1999
Respectfully Submitted,

____________________________
MICHAEL N. BURT
Attorneys for Defendant
ROBERT NAWI

 

 I. INTRODUCTION
When science is imported from the laboratory into the courtroom the leap from one context to another must be carefully analyzed. Because DNA evidence based on seemingly astronomical statistical probabilities by its very nature is particularly compelling to lay jurors, and because DNA typing is used in the criminal context for purposes of identifying perpetrators of particularly heinous crimes, extreme circumspection in evaluating the techniques and protocols of DNA typing is mandated. See People v. Venegas (1998) 18 Cal.4th 47, 81-84 (noting "DNA evidence is different", and that "[t]his is an instance in which the method of scientific proof is so impenetrable that it would ‘...assume a posture of mystic infallibility in the eyes of a jury...'"); People v. Kelly (1976) 17 Cal.3d 24, 31-32 ("When identification is chiefly founded upon an opinion which is derived from utilization of an unproven process or technique, the court must be particularly careful to scrutinize the general acceptance of the technique.") In addition, because DNA typing methodologies for use in the forensic arena has been the subject of several appellate decisions, keen attention to the issues that have been decided, and those that are still unresolved is required.

A. FUNDAMENTALS OF DNA
DNA is the carrier of genetic information in every plant, animal, and mircroorganism including bacteria. DNA consists of two strands that are held together at regular intervals to form a double helix. A useful, and common, analogy is a ladder: a DNA molecule looks like a twisted ladder or a spiral staircase. The rungs are made up of two bases that are bound together, and are attached to strands of sugar molecules. These strands are the sides of the ladder. There are only four different bases: adenine (A), guanine (G), cytosine (C), and thymine (T). The base A can only bind with the base T and C only with G. The binding qualities of the bases result in two complementary strands (picture the ladder split from top to bottom) that are not identical. For example, if the sequence of bases on one strand (one half of the ladder) is ATTGCT, the bases on the other half of the ladder will have the sequence TAACGA.
When a human being or any other living organism grows, it must duplicate its DNA so that each new cell will have a complete copy of that organism's DNA. The first step of the duplication, or reproduction, process is that the DNA molecule is first "unzipped": the strand splits lengthwise. The bonds between the two bases are broken by enzymes and the DNA becomes single-stranded. Enzymes present in the cell then make a new strand of DNA using the two existing strands as templates, resulting in two complete double-stranded pieces of DNA.
Genes, often called markers, are the genetic units responsible for inherited traits such as eye and hair color and genetic diseases such as sickle cell anemia. Genes consist of specific sequences of bases. In other words, a gene refers to a particular part of the DNA ladder, identified by the rungs at that part of the ladder. The base sequence of a gene is generally unique to that gene. However, a gene can have variations, usually small changes in the base sequence resulting in a different type of allele of the same gene or marker. Each double-stranded segment of DNA contains only one variant or allele.
Genes are organized in the human cell on structures called chromosomes, which are made up of DNA molecules and found in the nucleus of the cell. Humans have twenty-three chromosomes and each cell has two copies of each chromosome, resulting in a total of forty-six chromosomes. In humans, a person inherits twenty-three chromosomes from their mother and twenty-three chromosomes from their father. A gene exists at only one location on a single chromosome. Thus, an individual has two copies or alleles of each gene, one on either chromosome. Even if the two copies are the same variant, they are called alleles of the same gene.
For example, consider a fictional gene, Gene Z. This gene is found on human chromosome six. There are two different variants or alleles of Z found in the human population, Z-1 and Z-2. If Individual A is homozygous for Z-1, that means that Individual A has two copies of Z-1, one on each copy of chromosome six. If Individual B is heterozygous for Z that means Individual B has allele Z-1 on one copy of chromosome six and has allele Z-2 on the other copy of chromosome six.

B. DNA TYPING TECHNOLOGY
"DNA typing" is a catch-all term for a wide range of methods for studying genetic variations. Advances in DNA technology paved the way for detection of variation (polymorphism) in specific DNA sequences. DNA typing is the analysis of a DNA sample to identify either variations in the length of specific pieces of DNA (known as RFLP typing) or whether or not a specific piece of DNA is present (known as PCR typing). DNA can be extracted from various bodily fluids, such as semen and blood, and the amount of DNA obtained is proportional to the number of nucleated cells present in the sample. RFLP requires a relatively large sample, and is commonly known as "DNA fingerprinting" as it can result in a positive exclusion of an individual. PCR requires a relatively small sample, and also can be used to positively exclude a donor.
RFLP examines loci characterized by a variable number of long tandem repeats (VNTRs). At some DNA loci, a core sequence of base pairs, typically 15 to 35 base pairs in length, is repeated a number of times consecutively along the chromosome. Alleles differ in number of repeats and are differentiated on the basis of size. Steps in the RFLP analysis include restriction enzyme digestion of the DNA; separation of DNA fragments by electrophoresis, Southern blotting, and probing with locus-specific radioactive or chemiluminescent probes. The result is a gel with bands; the bands show the lengths of loci from the samples taken from known and unknown sources. In other words, the gel allows the analyst to compare the lengths of the VNTRs of samples taken from the crime scene, including from the victim's body, with samples taken from the victim and the defendant. Once the analyst determines that the length of the VNTRs "match" the analyst must then determine the significance of that match. Specifically, the analyst determines, using databases of known samples, what is the likelihood that the reported match is a true match. The results often are extremely high numbers, and represent the chance that there is a single randomly selected person would match the trace evidence. Koehler, supra, at 30.
Forensic DNA typing evolved from medical diagnostic techniques. Medical diagnostic DNA typing involves adequate, clean samples from known sources, a comparison of discrete alternatives, and it requires no knowledge of the distribution of patterns in the general population.
In contrast, forensic DNA typing involves samples that are often degraded, contaminated, and from multiple, unknown sources. It also involves matching of couples from a wide range of alternatives present in the population and thus lacks built-in consistency checks. And, except in cases where the DNA evidence excludes a suspected donor, assessing the significance of an apparent match requires statistical analyses of population frequencies. (NRC 1 at 27-63.)

C. THE BIOLOGICAL BASIS FOR PCR-BASED TESTING
The natural process of DNA replication has been duplicated in PCR technology. PCR is not a genetic test, "but merely a tool to increase the amount of genetic material to be tested." Thompson, supra, at 215. The National Research Council describes PCR as "a laboratory process for copying a chosen short segment of DNA millions of times." National Research Council, The Evaluation of Forensic DNA Evidence 69 (1996) [hereinafter "NRC 2"]. Scientists in the laboratory isolate DNA, and, using heat instead of enzymes, unzip the strands of DNA. The scientist then adds a primer, a short piece of DNA with a sequence known to the scientist, to the unzipped DNA. The primer "marks" the section on the DNA molecule that will be amplified. An enzyme called polymerase is added to the primer-DNA mixture. As in the living cell, the polymerase creates a new complementary strand from each of the original strands present. This simple process is repeated multiple times, each time separating and duplicating the existing DNA. In this way , large quantities of a specific DNA sequence can be made from a very small amount of sample DNA.
The quality of the copy created by PCR is only as good as the quality of the original sample. Contamination of samples by bacteria or other substances containing DNA affect the reliability of the amplification. NRC 1 at 64-65; NRC 2 at 82-83. In other words, any contamination in a sample will be amplified along with the DNA from the sample. "[A]mplification of contaminant could lead to spurious typing results." NRC 1 at 65.
Again, PCR is used to replicate small segments of the DNA strand. Scientists have identified several small segments, or loci, where the DNA strand varies among groups of people. Where a locus is highly variant, it is called polymorphic.
Simply because a locus is polymorphic does not mean that that locus is appropriate for use in forensic DNA testing. See NRC 1 at 63 (noting that "some loci can cause systematically unfaithful amplification"). Several criteria should be met before a locus is deemed appropriate for forensic DNA testing. These criteria include:
1. The marker should be highly polymorphic and have a high level of genetic heterozygosity;
2. The target sequence should be easily and specifically amplified;
3. Methods for detecting allelic variation should be uncomplicated and thoroughly reliable;
4. Population data on genotype frequencies must be available in order to assign estimates of the marker's power of discrimination and the probability of false inclusion;
5. The marker systems should be inherited independently so that frequencies derived from on marker system can be multiplied with those from others, thereby increasing the power of discrimination. Independent inheritance occurs when the markers are on separate chromosomes or are in linkage inequilibrium when present on the same chromosome.
Kamrin T. MacKnight, Comment, The Polymerase Chain Reaction (PCR): The Second Generation of DNA Analysis Methods Take the Stand, 9 Santa Clara Computer and High Tech. L.J. 287, 307 (1993) citing Cecilia H. von Beroldingen et al., Applications of PCR to the Analysis of Biological Evidence, in PCR Technology: Principles and Applications for DNA Amplification 209, 201 (Henry A. Erlich ed. 1989).
Loci used in forensic testing are of two types: those loci that vary by sequence and those that vary by length. Sequence variants are differences in the actual sequence of bases at a particular location on the DNA molecule. Length variants look at the number of times a sequence of bases repeats. NRC 2 at 74. The San Francisco Crime Lab conducts forensic DNA typing on both sequence variants and length variants.
Tests used to identify sequence variants in a DNA sample employ a reverse-dot blot method. Probes (single stranded pieces of DNA that are complementary to the genes or markers amplified) are fixed to a membrane in dots along a strip. The probes are "synthetic" copies of the alleles of each gene or marker used. The amplified DNA which has been denatured is applied to the strips in a buffered solution and the amplified DNA is allowed to bind to its complementary probe on the strip. When amplified DNA binds to a particular dot, the dot appears blue because of a chemical tag that is added to the DNA during the amplification process. The intensity of the blue color is important in determining the genotype of the sample. Dot blots appear with varying intensities, and judging whether the appearance of a faint dot constitutes the presence of an allele, or simply contamination is an extremely subjective determination. On the strip, each of the dots is labeled to correspond to an allele of the marker, and a pattern or combination of these dots gives the genotype of the person.
HLADQ [hereinafter "DQ Alpha" or DQ ] is a sequence variant locus. This locus codes for production of a protein. NRC 2 at 71. DQ Alpha has eight alleles, although the tests used only detect six alleles. Ibid. at 74. With six alleles, there are 21 possible genotypes: six homozygous and 15 heterozygous. Ibid. at 71. At the time of the first NRC report in 1992, only the DQ Alpha locus was used for forensic testing. NRC 1 at 70. Test kits were purchased from the Cetus Corporation. These kits contained the primers, polymerase and typing strips necessary to conduct DQ Alpha typing of a sample. The San Francisco Police Crime Lab ("Crime Lab") does not use this kit, but uses a newer kit manufactured by Perkin-Elmer that simultaneously types the DQ Alpha locus with five other loci, known together as the Polymarkers.
Instead of amplifying only one gene, like the DQ Alpha test, the polymarker ("PM") test amplifies five loci simultaneously: LDLR (low-density lipoprotein receptor), GYPA (glycophorin A), HBGG (hemoglobin gamma globin), D7S8 (a marker on chromosome 7) and GC (group-specific component). Each of the loci are bi- or tri- allelic, totaling 972 genotypic combinations. NRC 2 at 74. The first forensic polymarker tests were conducted with kits designed only to amplify the polymarker loci. As mentioned above, the Crime Lab uses a newer kit which amplifies and types not only the polymarker loci, but also the DQ Alpha locus.
In addition to typing DQ Alpha and polymarker loci, the Crime Lab also types the D1S80 locus. D1S80 is a length-variant locus classified as a VNTR. The base sequence of D1S80 is 16 bases long. There are more than 30 alleles. Because D1S80 varies by length, the alleles are differences in the number of times the base sequence repeats. NRC 2 at 72. D1S80 does not confirm the sequence of the repeating bases, and "some ambiguous alleles are caused by insertion or deletion of a single base …." Id.
The D1S80 locus is typed using electrophoretic separation in a technique called PCR-amplified fragment length polymorphism technique (AMP-FLP.). This technique has characteristics of both PCR and RFLP typing. After the fragments are amplified using PCR, their lengths are determined using a technique called electrophoresis. Electrophoresis is a process where the amplified sample is placed on one end of a gel. An electric current is applied to the gel. The DNA fragments move across the gel; the shorter fragments move faster than the longer fragments. At the end of the process the fragments, which appear as bands, are lined up along the gel according to size. The length of the fragments are compared to a control sample, known as an allelic ladder. The determination of the actual length of the unknown samples is a subjective determination made by the technician conducting the analysis.The San Francisco Crime Lab began PCR-based DQ Alpha + Polymarker and D1S80 typing in February, 1997, but did not issue its first formal report using these tests until April, 1997. The DQ Alpha+Polymarker kit was used in this case in June,1998. Although the D1S80 kit has not yet been used in this case, it is anticipated that such results are forthcoming.
Yet another type of length-variant loci used for forensic typing by the Crime Lab are Short-Tandem Repeat ("STR") loci. These loci are 2 to 7 base pairs long. Bernd Brinkmann, Overview of PCR-Based Systems in Identity Testing in Forensic DNA Profiling Protocols (Patrick J. Lincoln and Jim Thomson, eds. 1998) 106. Like D1S80, the STR loci vary by length, so typing the loci do not reveal the base sequence of the locus. However, unlike D1S80,and unlike some early versions of STR testing, in which one locus is typed by means of gel electrophoresis and visualized by staining, the STR loci were typed in this case by a true "black box" technology in which several loci are run simultaneously through a automated capillary electrophoresis system and then typed not by a human, but by complicated computer analysis which uses a flourescent-based laser detection system. In March, 1998, The Crime Lab began typing three STR loci ( THO1, TPOX, and CSF), and a sex determination of the amelogenin gene, using a "Green I" typing kit, a 310 Genetic Analyzer, and a fully automated capillary electrophoresis system. It was this typing system that Superior Court Judge Robert Dondero recently ruled in People v. Bokin et al., SC. No.168461 did not meet Kelly- Frye standards. A copy of Judge Dondero's written seventeen page opinion, issued after an extensive five month hearing, is attached to this pleading as exhibit A.
Finally, as part of their flourescent-based STR testing, the Crime Lab in February 1999 began to use a new STR typing kit called "Profiler Plus", which types nine additional STR loci
( D3S1358, vWa, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317, D7S820). The present case is apparently the first time the Crime Lab has used this kit in actual casework. Significantly, in December, 1998 a federally funded Audit Team conducted a long delayed audit of the Crime Lab, and on April 7, 1999 the Audit team issued a highly critical report detailing deficiencies in many areas. Importantly, the report concludes that " (t)he deficiencies observed should be addressed before the program purses casework DNA typing involving the Profiler-Plus and Cofiler systems." A copy of the Audit Report, as well as a letter from the Crime Lab responding to the Report, is attached hereto as Exhibit B.
Once an evidentiary sample collected from a crime scene, and blood from the victim and suspect(s) has been extracted and the genotypes of each determined, the technician will calculate a frequency for the occurrence of a given genotype in either the general population or within a given racial or ethnic population. In other words, the frequency of a given allele in a random collection of samples is used to calculate the probability of the occurrence of a particular genotype or collection of alleles. One expert describes this number as the "random match probability … a theoretical likelihood that a randomly selected person from the general population (of from the population of certain large ethnic or racial groups) would genetically match the trace evidence as well as the defendant." In this case, the Crime Lab claims in its report that the random match probabilities are as follows: 1 out of 19 thousand trillons for Caucasians, 1 out of 280 thousand trillions for Blacks, and 1 out of 340 thousand trillions for Mexican- Americans.
Based on the genotypes determined, the technician will use a database for the particular marker or groups of markers, to determine the frequency of the genotype in the population. The databases contain frequencies of genotypes. To illustrate, if the DQ test of the suspect's blood gives a type of 1.1, 2, the technician will chose the frequencies of 0.036 for Caucasians, 0.036 for African Americans, and 0.022 for Hispanics. The inverse of each number is taken to determine the occurrence of the genotype in the population, in this example, 1 in 28 for Caucasians and African Americans, and 1 in 45 for Hispanics.
When more than one marker is tested such as in the PM test or when the PM test is combined with the DQ test, the initial frequency calculation is done for the alleles at each gene or marker. The frequencies for each gene are then multiplied together using the Product Rule, and an overall frequency is determined. The inverse of this number is then taken and the overall frequency is found. Again, to illustrate, a suspect has the genotype of DQ 1.1, 2 and the LDLR of A, A. The calculations are as follows:
Caucasian African American Hispanic
DQ 0.036 0.036 0.022
LDLR 0.23 0.08 0.23
LDLR x DQ 0.008 0.003 0.005
Inverse 1 in 125 1 in 333 1 in 200
As shown above, the addition of each marker will increasingly narrow the number of individuals within the population that will have the given genotype. However, these tests do not and cannot show whether the suspects blood is the blood present in the unknown sample, even if the suspect's genotype matches that of the unknown sample. The only conclusive result from any of the tests conducted in this case is to exclude an individual.
The validity of using the Product Rule rests on a number of assumptions. First, the population studied engages in random mating--that mates are not chosen based on their genotype at the markers used in DNA testing. NRC 2 at 90. If the alleles of a gene are subject to random mating, then the alleles should be in what is called Hardy-Weinberg (HW) equilibrium. A population is in HW equilibrium if the actual observations of each genotype are consistent with that expected under the HW test. Id. at 91-92. A population will not be in equilibrium if the occurrence of a particular genotype in a population is significantly different than expected after statistical analysis. The lack of equilibrium indicates that there is some substructure to the population so that the alleles of a given gene are not assorting independently from generation to generation.
When multiple genes are used, as they are in this case, an additional assumption must be made, that is that the loci are in linkage equilibrium. NRC 2 at 106. Linkage equilibrium is the point at which the frequency of a multi-locus genotype is equivalent to the product of the genotype at each loci in a randomly mating population. Id. In other words, the frequency of a particular genotype involving more than one gene or marker, is equal to the frequency of one marker multiplied by the frequency of the second marker.
To summarize, to conduct bloodtyping analysis using one of the above methods, the following steps must be followed. First, a sample containing human DNA must be extracted from a forensic sample. The DNA must be amplified using PCR. The amplified sample is then typed for alleles at specific loci. Once the sample has been typed, the results are compared to a known sample of DNA, which has also been typed at the same loci, using the same techniques. If there is a reported match, then, using a database of genotype frequencies, the analyst determines the random match probability that the known sample and unknown sample are from the same source.

D. THE SCOPE OF KELLY-FRYE
The prosecution seeks to introduce DNA evidence derived from pooling of fingernail scrapings which were collected over 12 years ago, which allegedly contain DNA profiles of several people, and which was derived from four commercially developed test kits: the DQ Alpha + Polymarker kit, the D1S80 kit, the Green I kit, and the Profiler-Plus kit. The particular kits and methodologies used in this case are novel: they have never been reviewed for admissibility by an appellate court in California . Even the best established methods (those based on RFLP analysis) are controversial and have recently been held inadmissible by the Supreme Court of California. People v. Venegas (1998) 18 Cal.4th 47(reversal of rape conviction is required for lack of compliance by the FBI with procedures recommended in 1992 by the National Research Council (NRC) for determining the statistical probability of a random match).
The newer PCR-based systems have produced conflicting opinions in the trial courts. In the Riverside county case of People v. Scott, S. C. No. CR-48638, Judge Bailey conducted an extensive evidentiary hearing before concluding in February, 1997 that the D1S80 typing kit did not meet prong one of the three part Kelly-Frye standard. A copy of his ruling is attached as exhibit C. In People v. Bokin, Judge Dondero found that the faults with the D1S80 kit identified in Scott could be addressed under prong three of the three prong test of Kelly, but he also ruled that the Green I STR typing kit did not meet Kelly -Frye standards under prong one because it was not shown that it was generally accepted in the broader scientific community, which includes not only forensic scientists, but also molecular biologists. In a third case, People v. McClanahan, S. C. No. 162412, Judge Kramer considered the Green I kit but not D1S80, and he ruled that the Green I kit met prong one because it was generally accepted by forensic scientists. Needless to say, the evidentiary use of PCR- based testing systems is controversial.Cf., People v. Soto (1999) 21 Cal. 4th 512, 535-539 (reviewing the ebb and flow of scientific and judicial opinion regarding RFLP testing).
According to critics of DNA testing procedures, "the major lesson of the controversy is that forensic laboratories are lacking in scientific rigor and that new forensic techniques receive inadequate scientific scrutiny before they are presented in court. By this account, an atmosphere of hostility surrounds the issue because promoters of forensic DNA tests have responded to legitimate criticisms by attacking the critics rather than heeding their message."Thompson, Evaluating the Admissibility of New Genetic Identification Tests: Lessons From the 'DNA War', 84 J. of Crim. Law & Criminology 22
Nowhere is this hostility better illustrated than in the recent intemperate and unfair remarks of DNA expert Ed Blake, who in 1996 was awarded a lucrative city contract to supervise the San Francisco Crime Lab's DNA Program. Lashing out at the recent ruling of Superior Court Judge Robert Dondero in People v. Bokin, which was highly critical of the SFPD's DNA Program, Blake has called for the removal of Judge Dondero from office on the outrageous ground that the judge is "an arrogant, ignorant, stupid judge who is unable to get through the smoke thrown at him by a very skilled lawyer.""Judge Rejects DNA Test Evidence", San Francisco Examiner, May 7, 1999, p1; see also, Comments of Professor William Thompson, People v. Bokin: An Internet Discussion, http://CA_v_Bokin.pdf@www.scientific.org ("We will get a lot farther through respectful exchanges...than we will by publically declaring that those who disagree with us are ignorant, stupid or callous.") Ironically, Blake is the so-called "impartial" expert that gave crucial, but misleading (see infra at pp. 38-40) testimony in People v. Morganti (1996) 43 Cal. App. 4th 643, a case the People ask this Court to follow here.
Prosecutors and commercial vendors of DNA test kits typically portray forensic DNA testing as a science that admits of no error in the identification of individuals. See e.g., "Judge Rejects DNA Test Evidence", San Francisco Examiner, May 7, 1999, p1 ( " Frank Gannon, Foster City public relations manager for the manufacturer, said his firm was confident the ruling [of Judge Dondero] would be overturned. ‘Our tests show this test 100 percent accurate,'he said. ‘We have never had a mistake. We've never put someone at the scene of a crime who was not there. It's impossible.'") ; Rockne P. Harmon, "‘Barney' the Dinosaur: While Court Dawdles, DNA Admissibility Standard Fossilizes, San Francisco Daily Journal, June 8, 1998, p.4 (Alameda County prosecutor claims that all DNA testing methods are infallible and that the decision in Venegas and the Court's inaction in other pending DNA cases means that "clarification of four years of positive Court of Appeal DNA opinions is unlikely until long after the November election in which three of the Supreme Court justices are up for confirmation.")
Prosecutors and manufacturers views, however, do not generally reflect an understanding of the several sciences that comprise the foundations of forensic DNA analysis. The techniques that are employed in forensic DNA laboratories represent a blend of the old and the new in molecular biology, chemistry, population genetics, and statistical science. The fact that various elements of the new technology have been used successfuly in other applications for some years does not mean that those elements can be combined in a manner that will result in a fully matured science at the outset. Yet that is how DNA testing has been portrayed since its initial appearence at criminal trials in the late 1980's. Fortunately, our Supreme Court, unlike some others, has not been so easily misled.
1. The Admissibility of DNA Evidence Must Be Determined Under The Standard of People v. Kelly.

The admissibility of each item of DNA evidence depends on whether it was derived from a method that is generally accepted to be reliable. To make this determination, the court must apply the standard set forth in People v. Kelly (1976) 17 Cal.3d 24, 130 Cal.Rptr 144. The Kelly standard has three "prongs":
(1) it must be established, usually by expert testimony, that the scientific methods utilized are generally accepted as reliable by the relevant scientific community,
(2) the witness furnishing such testimony must be properly qualified as an expert to give an opinion on the subject, and
(3) the proponent of the evidence must demonstrate that correct scientific procedures were used in the particular case.

Kelly, 17 Cal.3d at p. 30 [emphasis in original].
Importantly for the present case, Venegas recently clarified that "the Kelly test's third prong does not apply the Frye requirement of general acceptance -it assumes the methodology and technique in question has already met that requirement. Instead, it inquires into the matter of whether the procedures actually utilized in the case were in compliance with that methodology and technique, as generally accepted by the scientific community. ...The third prong inquiry is thus case specific; ‘it cannot be satisfied by relying on a published appellate decesion.'" 18 Cal 4th at 78 (emphasis added).
In People v. Farmer (1989) 47 Cal. 3d 888, 913 the Court had stated that " careless testing affects the weight of the evidence and not its admissibility..." However, in Venegas the Court clearly retreated: " Our reference to ‘careless testing affecting the weight of the evidence and not its admissibility' in Farmer ...was intended to characterize short-comings other than the failure to use correct, scientifically accepted procedures such as would preclude admissibility under the third prong of the Kelly test." 18 Cal. 4 th at p.80 (emphasis in original). See also, People v. Soto (1999) 21 cal. 4th 512, 519 ("The proponent of the evidence must also demonstrate that correct scientific procedures were used.")
As Venegas teaches, the goal of the Kelly standard as applied to DNA evidence is to "forestall the jury's uncritical acceptance of scientific evidence or technology that is so foreign to everyday experience as to be unusually difficult for laypersons to evalute." 18 Cal. 4th at p.80. "DNA evidence is different", and " is an instance in which the method of scientific proof is so impenetrable that it would ‘...assume a posture of mystic infallibility in the eyes of a jury...'"18 Cal.4th 47, 81-84. There is also concern that efforts to challenge controversial scientific evidence through cross-examination and expert testimony will consume inordinant amounts of time and raise issues "beyond the scope of critical analysis by the average lay person." In re Amber B. (1987) 191 Cal.App.3d 682, 690-91.; Accord People v. McDonald 37 Cal.3d 351, 372 (1984). The greatest danger arises when a technique carrying an "aura of infallibility" is, in fact, quite fallible, and when an analysis of the reliability of the technique involves technical issues that are difficult for lay individuals to understand. Again, DNA evidence " is an instance in which the method of scientific proof is so impenetrable that it would ... assume a posture of mystic infallibility in the eyes of a jury." 18 Cal. 4th at 83-84
The role of the Court, when applying the general acceptance test to DNA evidence, is to determine "the existence, degree, (and) nature of a scientific consensus or dispute (with) the interpretative assistance of qualified live witnesses subject to a focused examination in the courtroom."People v. Soto (1999) 21 Cal. 4th 512, 540-541 n. 31 The court must " conduct a 'fair overview' of the subject, sufficient to disclose whether 'scientists significant either in number or expertise publicly oppose [a technique] as unreliable.People v. Reilly (1987) Cal.App.3d 1127, 1148, quoting from People v. Brown (1985) 40 Cal.3d 512, 533.
As Venegas teaches, "(i)n determining the question of general acceptance, courts must consider the quality, as well as quantity, of the evidence supporting or opposing a new scientific technique." 18 Cal. 4th at 85(emphasis added) And as Venegas also makes clear, the court's role in the inquiry is not one of abdication to the scientists. Thus, in Venegas, the Court adopted the modified ceiling approach advocated in the 1992 NRC report, notwithstanding that the 1996 NRC report, as well as numerous other scientific articles authored by well-credentialed experts, and testimony from such experts at the Kelly hearing, all assailed the approach as scientifically unsound. 18 Cal 4th at 86-89. More recently, in People v. Soto, supra, the Court emphasized that "in a context of rapidly changing technology, every effort should be made to base (decision) on the very latest scientific opinions..." 21 Cal. 4th at 540-541 n. 31 The point here is that the court's role in the inquiry is not one of a potted-plant.

2. Kelly Requires That Each Major Step of a DNA Typing Procedure Be Generally Accepted as Reliable by The Scientific Community

In reviewing the admissibility of DNA evidence under Kelly, this court does not write on a blank slate. Several California appellate rulings on DNA evidence have helped establish the nature and scope of the issues reviewable under Kelly. See, People v. Soto, supra; People v. Venegas, supra; People v. Allen (1999) 72 Cal. App. 4th 1093; People v. Wright (1998) 62 Cal. App.4th 31; People v. Morganti (1996) 43 Cal. App. 4th 643; People v. Axell (1991) 235 Cal.App.3d 836 ; People v. Barney (1992)8 Cal.App.4th 798; People v. Wallace (1993)14 Cal.App.4th 651. Because of the importance in this case of Allen,Wright and Morganti, these decisions are discussed in section IIB infra. One key point established by the other cases is that forensic DNA testing requires a series of distinct steps, and that the methods employed at each major step are independently reviewable under Kelly; each step must be generally accepted. For example, Soto, Venegas, Axell and Barney identified three major steps in DNA tests that employ RFLP analysis and made it clear that failure to use a generally accepted method for any of these steps would preclude admissibility of the resulting evidence. For newer technologies, this court will need to identify the steps in the testing procedure and ascertain that a generally accepted method is being used at each step, particularly at critical steps (i.e., points where serious errors might occur).
Statistical computation will obviously be a critical step for every DNA testing procedure that purports to find a "match" between samples with respect to their genetic characteristics. See People v. Venegas 18 Cal 4th at 82 ( " A determination that the DNA profile of an evidentiary sample matches the profile of a suspect establishes that the two profiles are consistent, but the determination would be of little significance if the evidentiary profile also matched that of many or most other human beings."); People v. Barney, 8 Cal. App. 4th at 803 ("[t]he statistical calculation step is the pivotal element of DNA analysis, for the evidence means nothing without a determination of the statistical significance of a match of DNA patterns."); Axell, supra, 235 Cal.App.3d 836, 866 ("We find that...a match between two DNA samples means little without data on probability..."); People v. Wallace (1993) 14 Cal. App. 4th 651 n. 3 (1993) (without valid statistics DNA evidence is "meaningless").
Recent scientific commentary has clarified the necessary elements of the statistical estimation procedure. In particular, it is now clear that no statistical procedure is acceptable unless it addresses the probability of two events that could cause a "match" to be reported between samples from different people: (1) a coincidental match between different individuals who happen to have the same genetic characteristics, and (2) a false positive (false match) due to laboratory error. The probability of a coincidental match is typically estimated by determining the frequency of matching genetic characteristics (genotypes) in a suitable reference population (or populations). The probability of a false positive is estimated by determining the laboratory's rate of errors in proficiency tests. See generally, L. Mueller, The DNA Controversy And NRC II, in Statistical Methods in the Health Sciences: Genetics, ME Halloran & S. Geisser (eds., 1999); D.H. Kaye, DNA, NAS, NRC,DAB, RFLP, PCR, and More: An Introduction to the Symposium on the 1996 NRC Report on Forensic DNA Evidence, (1997) 37 Jurimetrics: The Journal of Law, Science, and Technology 395.
Another critical step for every DNA testing procedure is the exact methodology used to determine the various DNA types within each testing system. In People v. Venegas, (1998) 18 Cal. 4th 47, the Supreme Court's discussion of prong one did not focus on a particular locus or even on a generic methodology such as RFLP, but rather on the "general scientific acceptance of the FBI's RFLP methodology used in this case...." See also, People v. Brown (1985) 40 Cal. 3d 512, 528, 534 ("[T]he prosecution failed to satisfy the Kelly/Frye rule by demonstrating at trial the scientific acceptance of the tests performed." Moreover, the issue could not be resolved on appeal because " the electrophoretic method itself is apparently performed under substantial chemical and electrical variations, and considerable training and experience are necessary to interpret the visual results."); People v. Wright , (1998) 62 Cal. App. 3d 31, 38 ( PCR methods used to analyze DQ alpha and polymarker loci were admissible in part because witnesses testified that " the PCR technique, and the DQ Alpha and polymarker methods used to conduct the PCR analysis in this case, are both generally accepted as reliable and valid in the scientific community."); People v. Barney (1992) 8 Cal. App. 4th 798, 824 ( " Moreover, the survival of the third-prong inquiry is a matter of common sense. If it is not established that correct scientific procedures were used in the particular case, it cannot be known whether the test actually conducted was the one that has achieved general scientific acceptance."); People v. Axell (1991) 235 Cal. App. 3d 836, 849 (" After reviewing the protocols used by Cellmark labs and laboratory notes in this case, Doctor Roberts [and six other scientists were] of the opinion that the procedures set forth in the protocol were recognized as reliable in the scientific community, and, as performed in this case, conformed to the protocols."); People v. Taylor (1995) 33 Cal App. 4th 262, 266 ("Kelly's test of general acceptance in the scientific community applies not only to the techniques for laboratory analysis of the DNA material but also to the statistical calculations which indicate the likelihood that the crime scene sample of DNA would match with some individual other than the defendant.... At the time of the Kelly hearing conducted in this case in February l993 there was authority for the proposition that the laboratory processes and protocols used in DNA analysis by Cellmark Diagnostic-the testing company which performed the test conducted in this case-met with general scientific acceptance.")
The focus on the specific methods and procedures used in the particular case is universal in Kelly- Frye litigation. As stated in State v. Jackson (Neb. 1998) 255 Neb. 68, 582 N. W. 2d 317, 325 a case in which the Nebraska Supreme Court recently ruled that the results of an unspecified STR procedure should not have been admitted absent a foundation that the lab had followed its own testing protocols, the admissibility of PCR- based testing methods is generally held to turn on the following issues:" (1) whether the witnesses on the DNA issue are experts in the relevant scientific fields, (2) whether the type of DNA testing used in the case under consideration is generally accepted by the relevant scientific communities, including the accompanying statistical analysis; (3) whether the testing protocol used in the case under consideration is generally accepted as reliable if performed properly, (4) whether the test conducted properly followed the protocol; (5) whether DNA analysis evidence is more probative than prejudicial ... and (6) whether statistical probability evidence interpreting the DNA analysis results is more probative than prejudicial."See also, United States v. Beasley (1996 8th Cir.) 102 F. 3d 1440, 1445 ("[T]he PCR method of DNA typing using the DQ alpha Amplitype test kit and the Polymarker test kit has achieved general acceptance within the forensic science community."); United States v. Lowe (D. Mass 1997) 954 F. Supp. 401, 411(Changes in the FBI's RFLP methodology are significant enough to require Daubert analysis even though general RFLP methodology has already been ruled admissible);United States v. Gaines (S.D. Fla 1997) 979 F. Supp. 1429,1437 (" For DQA1 and PM, the process used for determining a match involves the use of the reverse dot blot test. For D1S80 and Amelogenin, the process used is gel electrophoresis. Since the processes are different, they will be addressed separately.")Commonwealth v. Blasioli, (1998) 552 Pa. 149, 1998 WL 313388 ("This Court has generally required that both the theory and technique underlying novel scientific evidence must be generally accepted.") Commonwealth v. Sok, (Mass. 1997) 425 Mass. 787,683 N.E. 2d 671,798 ("Here we are primarily concerned with whether the particular test kits were reliable when utilized by CBR."); State v. Harvey, (N.J. 1997) 151 N.J. 117, 699 A. 2d 596,622-623
( " Proving general acceptance ‘ entails the strict application of the scientific method, which requires an extraordinarily high level of proof based on prolonged , controlled, consistent, and validated experience.'...Here, the State's burden is to prove that the polymarker test and the interpretation of its results are non-experimental, demonstratable techniques that the relevant scientific community widely, but perhaps not unanimously, accepts as reliable.")( acceptance of PM generally did not preclude challenge to dot -intensity analysis, as " a court must examine each step of a scientific process or technique."); People v. Dalcollo (1996) 282 Ill. App.3d 944,669 N.E.2d 378( " [W]e hold that the Frye test requires that both the theory and the techniques or procedures implementing the theory must be generally accepted in the relevant scientific community.")(collecting numerous out of state cases in support) Franson v. Micelli (1994) 269 Ill. App. 3d 20, 645 N.E. 2d 404, vacated on other grounds, 666 N.W. 2d1188 ( "The first reason we reject the Lipscomb/Miles cases is because these courts improperly applied the Frye test. The Lipscomb court reasoned that Frye applies to new scientific principles and the principle involved is the general DNA forensic analysis and not the procedures used within that framework. Thus, the court asserted that the fact that different companies may use different procedures in arriving at their results only goes to the weight of the evidence. The first flaw in this reasoning is that, although ‘general DNA forensic analysis' may be the new scientific principle, Frye requires that ‘the thing from which the deduction is made' must be generally accepted. It cannot be disputed that the theory behind DNA testing is well accepted. This does not mean, however, that simply because the concept is recognized the procedures used are not subject to a Frye analysis and any challenge to their validity only goes to the weight of the evidence. Under Frye, if the procedure or ‘thing' upon which the DNA result was determined is not generally accepted, then the result is inadmissible.") Along with these courts and the first NRC Report, the defense urges the Court to hold that " (i)t is meaningless to speak of the reliability of DNA typing in general-without specifying a particular method." NRC I at p. 8.(emphasis added)
In the recently conducted Kelly-Frye hearings before Judge Dondero in People v. Bokin, the People's formulation of the prong one issue was improperly focused on " STR's and D1S80", which are not methods at all, but rather a "class of repeated units"(STR's)(NRC II, p. 70) and a locus(D1S80) (NRC II, p. 72). The method used in this case to detect alleles at the DQ Alpha and polymarker is a reverse-dot blot method utilizing the DQ Alpha +Polymarker test kit. The method used in this case to detect alleles at the D1S80 locus is gel electrophoresis utilizing the Perkin Elmer D1S80 kit. The method used in this case to detect alleles at the thirteen STR loci is automated capillary electrophoresis utilizing the 310 Genetic Analyzer and the Green I and Profiler-Plus kits. The method used to make frequency calculations in this case is the unmodified product rule derived from the particular databases used by the San Francisco Police Department. These testing methods, not PCR generally (see, United States v. Hicks (9th Cir. 1996) 103 F. 3d 837,844 ["The PCR method itself is not a genetic test; it is a mere amplification technique."], and certainly not the loci themselves, should be the proper focus of prong one.
The failure to grasp this distinction led the People astray from the outset in Bokin and in People v. McClanahan, before Judge Kramer. For example, in Bokin, instead of having their sole expert Dr. Holt review the protocols used and render an opinion about the general acceptance of the specific procedures outlined in the protocols, as was done in Axell and many other cases, the People contented themselves with leaving the witness in the dark about the specific procedures used by the San Francisco Crime Lab (see, R.T. 394) and asking such vague and meaningless questions as " Are the STR loci THO1, TPOX,...and CSF1PO...generally accepted in the scientific community as a reliable means of human identification?" (R.T. 45) At this level of generality, and without specific definition of either the relevant scientific community or the specific methods being utilized, the answer is predictable, but irrelevant, much as it would be in a hypothetical case in which a group of psychics, thinking they are scientists and believing they can make valid fingerprint comparisons by holding the known and questioned prints to their turbaned heads, are asked the question, "Is fingerprinting generally accepted in the scientific community as a reliable means of human identification?" The People's presentation in both Bokin and McClanahan was that meaningless.
3. The Court Must Look Beyond Forensic Science For Evidence of General Acceptance.

The general acceptance test of Kelly cannot be met by showing that promoters and practitioners of the method accept it to be reliable. The test is not whether a method is accepted by those who have a personal or professional stake in its acceptance, but rather, whether it is "accepted as reliable by the larger scientific community in which it originated." People v. John W. 185 Cal.App. 3d 801, 805 (1986); People v. Shirley 31 Cal.3d 18, 54 (1982).
Courts have also recognized that promoters and practitioners of a particular method "may be too closely identified with the endorsement of [the technique] to assess fairly and impartially the nature and extent of any opposing scientific views." Kelly, supra, 17 Cal.3d at 38. Thus, when applying the Kelly standard, the court must look to experts who are "'impartial,' that is, not so personally invested in establishing the technique's acceptance that he might not be objective about disagreements within the relevant scientific community." People v. Brown, supra, 40 Cal.3d 512, 530; accord People v. Venegas, 18 Cal. 4th at 77 (" FBI Agent Lynch, though vigorously defending the merits of the FBI's RFLP analytical procedures she had followed in this case, did not purport to be qualified, as a molecular biologist or otherwise, to testify on questions of general scientific acceptance of the validity of those prosedures.") In this regard, the court should bear in mind that employees of forensic labs "have a clear pecuniary interest in the acceptance of DNA evidence by the courts. The success of their employers and the stability of their own employment depends upon continued use of DNA testing." Dan L. Burk, DNA Identification: Possibilities and Pitfalls Revisited, 31 Jurimetrics J. 53, 79-80. Obviously, employees of laboratories that hold patent rights to a new forensic technique have a similar pecuniary interest in the acceptance of the technique in court.
As Judge Dondero recently held in Bokin, the relevant scientific community for purposes of the prong one inquiry is the broader community of molecular biologists and population geneticists and not simply the community of forensic scientists using DNA technology, as was held by Judge Kramer in McClanahan. This principle is dictated by relevant case law. See, People v. Soto, 21 Ca. 4th at 515 (Issue is whether the RFLP statistical methodology " is generally accepted in the relevant scientific community of population geneticists."); People v. Venegas 18 Cal. 4th at 77 ("FBI Agent Lynch...did not purport to be qualified, as a molecular biologist or otherwise, to testify on questions of general scientific acceptance of the validity of...procedures.");People v. Axell (1991) 235 Cal. App. 3d 836, 857("The search for scientific consensus must be from within ‘the particular field in which it belongs'. Since DNA profiling is an amalgamation of primarily two disciplines, molecular biology and population genetics, it appears logical to consider its acceptance by those communities for forensic use."); People v. Reilly (1987) 196 Cal. App. 3d 1127, 1138-1139(same).
The failure of the People to recognize this principle during the course of two hearings resulted in inadequate evidentiary showings which myopically focused solely on the views of the forensic community. In McClanahan, Dr. Holt's entire testimony was strictly limited to whether she felt that "STRS" were generally accepted by "the forensic DNA community."(R.T. 118; 127,133,159,260). D1S80 was not an issue in that case. In Bokin, the People again relied exclusively on Dr. Holt and had her testify that "STR and D1S80 typing" are generally accepted in the "relevant scientific community".(R.T. 44) Although that community was never explicitly defined, it is clear from the overall context of her answers and her emphasis on the annual gathering of forensic DNA analysts at the Promega Proceedings, and on the widespread use of STR methods by law enforcement personnel, that she was once again talking about the forensic science community. Indeed, at one point in cross examination she acknowledged this narrow focus (R. T.405), she admitted that " I read mostly forensic journals", and she conceded she was not current on the literature of the criticisms within the medical community, outside of forensics, about the use of STR's and PCR. (R.T. 295-296). The same narrow focus was evident in McClanahan, where Judge Kramer evidently and erroneously accepted the view that the relevant scientific community could be restricted to the forensic community. By contrast, Judge Dondero's mid-hearing ruling that the relevant scientific community extended beyond forensic scientists gave the People fair warning that they had to broaden the scope of their prong one presentation. They never did, with the result that the Bokin record contained a glaring failure of proof on prong one issues, a failure made most conspicuous by a comparison with other cases in which prosecutors have successfully established the general acceptability of other DNA testing methods. See e.g., People v. Soto, 21 Cal. 4th at 527 (" During the 11 days of Kelly hearing testimony... the parties elicited extensive DNA testimony from 3 prosecution experts: the criminalist, Robert Keister, and two human population geneticists, Ranajit Chakraborty and Bruce Kovacs."); People v. Axell (1991) 235 Cal. App. 3d 836, 849,856 (" At the hearing, the People presented four expert witnesses in their case- in- chief and two in rebuttal.")
The history of litigation on forensic DNA tests shows the wisdom of looking beyond forensic science to the "larger scientific community" to determine what is and is not accepted. When RFLP-based DNA evidence was first introduced in U.S. courts in 1987, it faced almost no opposition. Few scientists, other than those who were employees or consultants of the forensic laboratories knew the details of forensic testing procedures, and few had occasion to think about the difficulties posed by the transfer of DNA technology to the forensic arena.
Forensic DNA tests were ... developed by commercial laboratories whose procedures were proprietary, whose work product is generally not available for examination by outsiders (except when used in a court hearing), and whose laboratory protocols were initially available only by court order. The forensic scientists who developed and validated the techniques in both commercial and government laboratories were well aware that they would be called upon to defend the new tests when their admissibility was challenged in litigation, and they had a professional stake in the outcome of that litigation. In that atmosphere, they had little incentive to air and discuss problems, and strong incentive to suppress doubt and uncertainty for fear that candid statements would be used against them in court.

Thompson, Evaluating the Admissibility of New Genetic Identification Tests: Lessons From the 'DNA War', 84 J. of Crim. Law & Criminology 22, at 95.
During the first few years that these tests were used, there was little hint of the "raging controversy" that would later arise, leading to their exclusion in Venegas, Barney and Wallace. Indeed, nearly two years passed before the first scholarly critique of forensic DNA test procedures appeared in the scientific literature. Eric Lander, DNA Fingerprinting on Trial, 339 Nature 501 (1989).
The absence of scholarly criticism did not mean that the methods used by forensic DNA laboratories were "generally accepted." Many laboratories were, in fact, using methods that have since been rejected by the scientific community.
Without the benefit of open scientific scrutiny, some testing laboratories initially used methods (for such fundamental steps as identifying patterns, declaring matches, making comparison with a databank, and correcting for band shifting) that they later agreed were not experimentally supported.
NRC 1 at 56.
Before the methods could become controversial, they had to become known and understood. The absence of criticism when the tests were first introduced was not proof that the tests would be generally accepted in the scientific community; silence does not imply assent. Thus, to conclude that a technique is "generally accepted" when only a few scientists (other than its promoters) have evaluated it, is perilous indeed.

4. Neither Acceptance of PCR For Nonforensic Scientific Purposes Nor Widespread Law Enforcement Use of PCR Testing Kits For Databasing and Other Nonevidentiary Purposes Establishes That Forensic Tests Employing PCR Are Reliable or Generally Accepted For Courtroom Use

The People in McClanahan and Bokin made the argument that forensic DNA tests must necessarily be accepted in the scientific community because they employ procedures (e.g., PCR) that are used and accepted elsewhere in science for other purposes. Their argument is syllogistic, viz.: PCR analysis is accepted; SFPD's Crime Lab uses PCR; therefore the Lab's testing in all its variations is accepted. The problem with this argument is that it fails to recognize the difficulties that may arise from the transfer of technology from one application to another. It is widely recognized that forensic DNA testing is more technically demanding than other applications of RFLP and PCR technology, and that it involves additional critical steps that do not arise in other applications (such as the matching and statistical estimation steps). What must be considered under Kelly is whether the method that produced the incriminating evidence is accepted to be reliable as it was applied, not whether a similar method is accepted for another purpose.
The simpleminded notion that any forensic DNA test employing RFLP or PCR is, of necessity, reliable and accepted was flatly rejected by the National Research Council:
Before a method can be accepted as valid for forensic use, it must be rigorously characterized in both research and forensic settings to determine the circumstances under which it will and will not yield reliable results. It is meaningless to speak of the reliability of DNA typing in general--i.e., without specifying a particular method.
NRC 1 at 51-52.
Moreover, even as to forensic use of a particular method, our Supreme Court has explicitly rejected "widespread use" by law enforcement as a surrogate for a searching inquiry into whether impartial scientists accept a particular technique. In People v. Leahy (1994) 8 Cal. 4th 587, 605-606, the Court stated unambiguously :
The People observe that HGN testing has been used by law enforcement agencies for more than 30 years....
In determining whether a scientific technique is "new" for Kelly purposes, long-standing use by police officers seems less significant a factor than repeated use, study, testing and confirmation by scientists or trained technicians...To hold that a scientific technique could become immune from Kelly scrutiny merely by reason of long- standing and persistent use by law enforcement outside the laboratory or the courtroom, seems unjustified.

The United States Supreme Court agrees: "Respondent argues that because the Government--and in particular the Department of Defense--routinely uses polygraph testing, the Government must consider polygraphs reliable. Governmental use of polygraph tests, however, is primarily in the field of personnel screening, and to a lesser extent as a tool in criminal and intelligence investigations, but not as evidence at trials.... Such limited, out of court uses of polygraph techniques obviously differ in character from, and carry less severe consequences than, the use of polygraphs as evidence in a criminal trial. They do not establish the reliability of polygraphs as trial evidence, and they do not invalidate reliability as a valid concern supporting Rule 707's categorical ban." United States v. Scheffer (1998) 523 U.S. 303, 118 S.Ct 1261, 1266 n. 8 In other words, all Dr Holt's and the People's talk about the "worldwide acceptance" of STR and D1S80 "loci" for CODIS or other law enforcement or investigative purposes is simply beside the point.

5. A Particular DNA Typing Method Is Not Considered Generally Accepted Unless Certain Essentials Of Correct Scientific Procedure Are Met: Developmental and Internal Validation, Publication and Peer Review, Accreditation, Proficiency Testing, Contamination Controls, and Regular Audits

How do particular methods become generally accepted by the relevant scientific community? One overriding principle put forth by the community itself is that "(f)orensic DNA anlysis should be governed by the highest standards of scientific rigor in analysis and interpretation." NRC 1 at 52; see also, NRC 2 at75 ("It is important that forensic laboratories use strict quality-control standards to minimize the risk of error.") These strict standards were first widely publicized in the 1992 NRC Report so heavily relied upon by the Supreme Court in Venegas. The 1992 NRC Report takes no position on the validity or scientific acceptability of any particular forensic DNA test, but sets forth a set of requirements that it regards as "essential" for assuring the reliability of any forensic DNA test. The NRC II follows the same approach and specifically recommends that "(l)aboratories should adhere to high quality standards (such as those defined by TWGDAM and the DNA Advisory Board)..." Id at 88 Hence, one way to judge whether or not a new technique would be generally accepted in the scientific community (if better known) is to determine whether it meets the "essential" requirements established by the NRC Reports, the TWGDAM Guidelines, and the DAB Advisory Board Standards.
The NRC Reports, TWGDAM Guidelines, and DAB Standards establish what is scientifically acceptable procedure for conducting PCR-Based or other DNA testing. Compliance with these and other guidelines, including the lab's own testing protocols, is accordingly a prerequisite to admissibility of DNA evidence in this case.
The court in People v. Barney (1992) 8 Cal. App. 4th 798, 812-813 specifically addresses this question:
The NRC Report concludes there is indeed a need for standardization of laboratory procedures and proficiency testing (as well as appropriate accreditation of laboratories) to ensure the quality of DNA laboratory analysis. But the absence of such safeguards does not mean that DNA analysis is not generally accepted....Rather, the absence of these safeguards goes to the question whether a laboratory has complied with generally accepted standards in a given case, or, as stated in terms, whether the prosecutor has shown that "correct scientific procedures were utilized in the particular case. (Emphasis added)

See also, People v. Venegas (1998) 18 Cal. 4th at 53,( "lack of compliance by the FBI with procedures recommended in 1992 by the National Research Council (NRC) for determining the statistical probability of a random match" renders DNA evidence inadmissible); United States v. Beasley (8th Cir. 1996) 102 F.3d 1440,1448( " In every case, of course, the reliability of the proffered test results may be challenged by showing that a scientifically sound methodology has been undercut by sloppy handling of the samples, failure to properly train those performing the testing, failure to follow the appropriate protocols, and the like."); State v. Jackson (Neb. 1998) 255 Neb. 68, 582 N. W. 2d 317, 325(the results of an unspecified STR procedure should not have been admitted absent a foundation that the lab had followed its own testing protocols). The California Legislature also recognizes the importence of these guidelines. See, Penal Code Section 277(d)("Nothing in this section precludes laboratories meeting Technical Working Group On DNA Analysis Methods (TWGDAM) guidelines or standards promulgated by the DNA Advisory Board...from performing forensic identification analysis, including DNA profiling..."
One crucial requirement established by all of these guidelines and standards is that forensic DNA tests must be developmentally validated through extensive empirical studies. See,DAB Standard 8.1( "The laboratory shall use validated methods and procedures for forensic casework analysis....Novel forensic DNA methodologies shall undergo developmental validation to ensure the accuracy, precision and reproducability of the procedure."); TWGDAM Guidelines 4.1.2
(" Validation studies must have been conducted by the DNA laboratory or scientific community prior to the adoption of a procedure by the DNA laboratory.") According to NRC I, developmental validation is necessary to establish an objective and quantitative procedure for identifying the pattern of a sample and for declaring a match, to identify possible artifacts that could lead to incorrect interpretation if not recognized,to understand the limits of each DNA typing method for sensitivity to quantity, mixture,and contamination, to establish the statistical frequency of the genetic markers and the error rate of the procedure.NRC I at 52-55
TWGDAM Guideline 4.1.5.1 sets forth the minimal requirements which must be meet in order to satisfy the requirement of developmental validation, including studies in the following areas: standard specimen studies, consistency studies, population studies, reproducability studies, mixed specimen studies, environmental studies, matrix studies, nonprobative evidence studies, nonhuman studies, minimum sample studies, and on-site evaluation studies. DAB Standard 8.1 contains a similar listing.
The 1992 NRC Report also emphasizes that the key validating studies must not only be done but published and peer reviewed before a laboratory can claim that its methods are generally accepted.
If a new DNA typing method (or a substantial variation on an existing one) is to be used in court, publication and scientific scrutiny are very important. Extensive empirical characterization must be undertaken. Results must be published in appropriate scientific journals. Publication is the mechanism that initiates the process of scientific confirmation and eventual acceptance or rejection of a method.

NRC 1 at 56; see also, TWGDAM Guideline 4.1.5.12 ("It is essential that the results of the developmental validation studies be shared as soon as possible with the scientific communiy through presentations at scientific/professional meetings. It is imperative that details of these studies be available for peer review through timely publications in scientific journals."). The existence of an "extensive literature in peer-reviewed journals" was key to the Supreme Court's recent ruling in People v. Soto, 21 Cal. 4th at 540, that population substructure does not impact significantly upon DNA RFLP population frequency estimates.
A second fundamental requirement is internal validation, which requires that the laboratory gain "a solid base of experience in forensic application" before it uses a new DNA typing method.NRC I at 55; DAB Standard 8.1.3("Internal validation shall be performed and documented by the laboratory.");TWGDAM Guideline 4.5("Prior to implementing a new DNA analysis procedure or an existing DNA procedure...the forensic laboratory must first demonstrate the reliability of the procedure in-house.")
According to NRC I, internal validation involves five separate steps:
1. familiarity with the system using fresh samples (i.e., fresh blood);
2. test marker "survival" in dried stains (i.e., blood);
3. test system on simulated samples that have been exposed to various environmental conditions;
4. establish "basic competence" in the use of the system by blind trials;
5. test the system on nonprobative evidence samples in which the origin is known, to check reliability.

The NRC I recommends that when a DNA technique is initially developed, all five steps should be carefully followed. Id p. 55 It further recommends that as laboratories adopt a particular technique, "it will not always be necessary for them to repeat all the steps, but they must demonstrate familiarity and competence by following steps 1, 4, and 5." Id.
The 1995 TWGDAM Guidelines are even more rigorous. Guideline 4.5 provides that internal validation "must" include the following:
4.5.1 The method must be tested using known samples.
4.5.2 If a modification which materially effects the results of an analysis has been made to an analytical procedure, the modified procedure must be compared to the original using identical samples.
4.5.3 Precision (eg. Measurement of fragmented lengths) must be determined by repetitive analysis to establish criteria for matching.
4.5.4 The laboratory must demonstrate that its procedures do not introduce contamination which would lead to errors in typing.
4.5.5 The method must be tested using proficiency test samples. The proficiency test may be administered internally, externally, or collaboratively.

DAB Standard 8.1.3 also requires internal validation, and adds requirements not found in TWGDAM. For instance, Standard 8.1.3.1 requires that the " procedure shall be tested using known and nonprobative evidence samples", and that "(t)he laboratory shall moniter and document the reproducibility and precision of the procedure using human DNA controls." Standard 8.1.3.3 adds the requirement that "(b)efore the introduction of a procedure into forensic casework, the analyst or examination team shall successfully complete a qualifying test."
In addition to requiring both developmental and internal validation, the NRC Reports, the TWGDAM Guidelines, and the DAB Standards recommend other measures to ensure a mininum level of quality assurance. NRC I states that " courts should require that a proponent of DNA typing evidence have appropriate accreditation--including documentation of external, blind proficiency testing (as well as other accreditation that might be mandated by government or come to be generally accepted in the profession)--for its evidence to be admissible." NRC I at 106-107 The Report explains that
There is strong legal foundation for such a position. As a number of courts have correctly recognized, the admissibility of scientific evidence depends not just on a technology's being sound in principle, but on the testing laboratory's having applied it in the case at hand according to generally accepted standards. Courts should view the absence of appropriate accreditation as constituting a prima facia case that the laboratory has not complied with generlly accepted standards.
Id. at 107


See also, NRC II at 88 ("Laboratories should...make every effort to be accredited for DNA work (by such organizations as ASCLD-LAB)."; DAB Standards, Preface("Throughout its deliberation concerning these quality standards, the DNA Advisory Board recognized the need for a mechanism to ensure compliance with these standards. An underlying premise for these discussions was that accreditation would be required to demonstrate compliance with the standards and therefore assure quality control and a quality program. Accordingly, the Board recommends that forensic laboratories performing DNA analysis seek such accreditation with all deliberate speed.").
The California Legislature has embraced the accreditation requirement wholeheartedly in the DNA and Forensic Identification Data Base And Data Bank Act of 1998. Penal Code Section 297(a) provides that "(t)he laboratories of the Department of Justice that are accredited by the American Society of Crime Lab Directors Laboratory Accreditation Board (ASCLD/LAB), and any crime designated by the Department of Justice that is accredited by the ASCLD/LAB, are authorized to analyze crime scene samples..." Penal Code Section 297(b) provides that "(a) biological sample obtained from a suspect in a criminal investigation for the commission of any crime may be analyzed for forensic identification profiles, including DNA profiles, by the DNA Laboratory of the Department of Justice, which is accredited by the ASCLD/LAB, or any crime laboratory designated by the Department of Justice that is accredited by the ASCLD/LAB. Penal Code Section 297(c) states explicitly that "(a)ll laboratories, including the Department of Justice DNA laboratories, contributing DNA profiles for inclusion in California's DNA databank shall be accredited by the ASCLD/LAB.Additionally, each laboratory shall submit to the Department of Justice for review the annual report required by the ASCLAD/LAB which document's the laboratory's adherence to ASCLAD/LAB standards."
As indicated, the NRC I also recommends the requirement of "rigorous external proficiency testing via blind trials" as a prerequisite to admissibility of DNA evidence. In fact, NRC I emphasizes this requirement above all others:
Most important, there is no substitute for rigorous proficiency testing via blind trials. Such proficiency testing constitutes scientific confirmation that a laboratory's implementation of a method is valid not only in theory, but also in practice. No laboratory should let its results with a new DNA typing method be used in court, unless it has undergone such proficiency testing via blind trials.
NRC 1 at 55.

The NRC II and TWGDAM Guidelines also indicate the importance of regular proficiency testing, including blind proficiency testing. See, NRC II at 88 (" Regular proficiency tests, both within a laboratory and by external examiners, are one of the best ways of ensuring high standards. To the extent that it is feasible, some of the tests should be blind."); TWGDAM Guideline 9.2("It is highly desirable that the DNA laboratory participate in a blind proficiency test program, and every effort should be made to implement such a program.") The DAB Standards are somewhat less emphatic on the requirement of blind trials, but still it is clear that rigorous external proficiency testing is a prerequisite to reliable DNA testing under the Standards. See,Standard 13.1(Examiners...who are actively engaged in DNA analysis shall undergo, at regular intervals of not to exceed 180 days, external proficiency testing in accordance with these standards."
Another requirement established in all the reports, guidelines, and standards is the absolute necessity of controlling for the problem of contamination in PCR testing. Forensic DNA testing based on the PCR technique is very different than RFLP testing in this regard. There is universal recognition in the scientific community that special and peculiarly difficult problems must be resolved if a reliable transfer of the extremely sensitive PCR technology from its present use in research laboratories and clinical (medical) diagnostic laboratories to the forensic arena is to be accomplished.
One point that emerges immediately in considering the general acceptance of PCR based forensic testing is that the scientific community has unequivocally warned about special dangers of sample contamination. These dangers embrace both the methods used for the initial collection, preservation, and handling of samples by crime scene technicians as well as the methods used for processing the samples in the laboratory. The NRC 1 Report expressed its most "serious concern" about the problem of "contamination of evidence samples with other human DNA," and warns that " (e)ven the simple act of flipping the top of a plastic tube might aerosolize enough DNA to pose a problem." NRC 1 at 65 The Report goes on to underscore that reliable methods must be developed to prevent and control contamination problems unique to forensic PCR based testing in both the sample gathering and laboratory testing processes. NRC 1 at 66. The NRC II likewise warns that "any procedure that uses PCR methodology is susceptable to error by contamination" and and that "(i)f the contaminating DNA is present at a level comparable to the target DNA, its amplification can confound the interpretation of typing results, possibly leading to an erroneous conclusion." NRC II at 23. The NRC II is also emphatic that " the integrity of the chain of custody is of paramount importance" , and that "(t)his means meticulous care, attention to detail, and through documentation of every step of the process, from collecting the evidence material to the final laboratory report." Id. at 25;See also, TWGDAM Guideline 4.5.4("The laboratory must demonstrate that its procedures do not introduce contamination which would lead to errors in typing."); DAB Standard 6.1("The laboratory shall have a facility that is deigned to provide adequate security and minimize contamintion."), Standard 7.1("The laboratory shall have and follow a documented evidence control system to ensure the integrity of physical evidence. This system shall ensure that...the laboratory follows documented procedures that minimize loss, contamination, and/or deleterious change of evidence.")
Therefore, it must be emphasized that a Kelly inquiry concerning PCR based testing must embrace the methods used by the crime scene investigtors and laboratories for identifying, correcting, and preventing contamination in the collection, preservation, and handling of crime scene samples. In addition, for reasons already stated, a Kelly inquiry must separately review the general acceptance of each PCR-based technique, and its application by the Crime Lab in this case, whether one chooses to characterize the inquiry as a Prong 1 or Prong 3 question.
Finally, the NRC Reports, the TWGDAM Guidelines, and the DAB Standards stress the need for regular external audits and the need to take corrective action in response to any deficiencies uncovered in the audit process.See NRC I at 106(recommending periodic on-site inspections of accredited labs); NRC II at 78-80(" Proficiency-testing and audits are key assessment mechanisms in any program for critical self-evaluation of laboratory performance....Regular audits of laboratory operations complement proficiency-testing i the monitoring of general laboratory performance."); TWGDAM Guideline 10(" Audits are an important aspect of th QA program....Audits or inspections should be conducted at least once every 2 years by individuals separate from and independent of the DNA testing laboratory...Records of each inspection should be maintained and should include...remedial action taken to resolve existing problems..."); DAB Standard 14 (The laboratory shall establish and follow procedures for corrective action), Standard 15(The laboratory shall conduct audits annually and once every two years, a second agency shall participate in the annual audit)
These emphatic pronouncements, from distinguished scientific panels, following many years of study of the issues, hold an important lesson. The court should not take seriously the claim that a new DNA typing method is "generally accepted" within the meaning of Kelly if the method has not been developmentally and internally validated by extensive publication and peer review, and if the particular lab does not meet the accreditation, proficiency testing, contamination control, and audit requirements set forth in nationally recognized standards for the quality assurance of forensic DNA testing laboratories.




II.
DEFENDANT ROBERT NAWI IS ENTITLED TO CHALLENGE THE BLOODTYPING RESULTS IN THIS CASE UNDER KELLY-FRYE BECAUSE ISSUES RAISED IN THIS OPPOSITION HAVE NOT BEEN RESOLVED BY ANY APPELLATE COURT IN CALIFORNIA, NOR IN ANY CASE PENDING REVIEW BEFORE THE CALIFORNIA SUPREME COURT

Citable case law does not answer all of the questions that arise when deciding the admissibility of DNA typing evidence. Some issues regarding the admissibility of a small number of DNA typing tests have been addressed by the California Supreme Court and by California Courts of Appeal. However, as will be discussed in detail below, neither citable California case law nor the cases that for many years were pending review before the California Supreme Court contain issues that definitively decide the issue of admissibility of PCR-based DNA testing.
A. THE ISSUES RAISED IN THIS MOTION ARE NOT INCLUDED IN ANY CASE CURRENTLY PENDING BEFORE THE CALIFORNIA SUPREME COURT

Criminal lawyers in California, defense and prosecution alike, have been anxiously awaiting decisions by the California Supreme Court in nine cases, all of which raise Kelly-Frye issues regarding the admissibility of either RFLP or PCR-based DNA testing. On May 11, 1998 the California Supreme Court issued a much-anticipated decision in People v. Venegas, supra. On August 23, 1999, the Court decided People v. Soto, supra. Although Venegas and Soto involved a Kelly-Frye challenge of RFLP technology, the holding in these cases provides the California Supreme Court's most recent interpretation of the parameters of Kelly-Frye. Seven other cases were for many years on hold at the Supreme Court, pending action in light of the decisions in Venegas and Soto . Very recently, on October 27, 1999, the Court dismissed review in all seven cases , thus leaving the law as it relates to PCR-based testing in some doubt. The defense recognizes that these seven cases are not citable authority. However, the following discussion is presented for the purpose of distinguishing the issues raised by defendant Robert Nawi in this motion from those issues for which resolution by the Supreme Court was anticipated.
On March 16, 1995 the California Supreme Court granted review of the Fifth District appellate decision in People v. Venegas (1995) 31 Cal.App.4th 234. Sergio Venegas was charged with rape and other offenses. The FBI conducted RFLP testing on samples taken from the defendant, semen stains on the bedspread on which the rape occurred and vaginal swabs taken from the victim. Venegas, supra, at 234. The FBI concluded that for three of the four tests conducted, the defendant's DNA matched the DNA in the crime scene samples. Id. A fourth test was inconclusive. Applying statistical methods, the FBI concluded that the likelihood of another individual being the source of the DNA found at the scene was one in 65,000 of the general population and one in 30,000 of the southwestern Hispanic population. Id.
In the court of appeal, the defendant prevailed on a "prong one" Kelly claim that the trial court had erred in finding general acceptance in the scientific community for the RFLP method and the statistical methods used in conjunction with RFLP. In addition, the defendant prevailed on a "prong three" that the FBI failed to use correct scientific procedures in this case in violation of Kelly, supra, 17 Cal.3d at 30.
The Supreme Court only partially agreed with the Court of Appeals. Regarding Mr. Venegas's prong one challenge to the general acceptance of RFLP, the Supreme Court followed the rulings in People v. Barney and People v. Axell and held that the RFLP methodologies used by the FBI were generally accepted in the scientific community.
The Supreme Court also ruled on Mr. Venegas's prong three challenge. After a discussion of the nature of the third prong of Kelly, the Supreme Court found that the FBI failed to follow the recommendations of the 1992 NRC Report in the statistical calculation step of their analysis. Thus, the Supreme Court upheld the Court of Appeal's reversal of Mr. Venegas's conviction, and remanded the case. In two footnotes, the court expressly left open any Kelly issues regarding PCR testing 18 Cal.4th at 58, n.5 and the issue of whether the statistical calculations must account for laboratory error, 18 Cal.4th at 95, n.42.
On the same day that the Supreme Court granted review in Venegas, that Court granted review to People v. Soto (1995) 30 Cal.App.4th 340. Frank Soto was convicted of one count of attempted rape. Using the RFLP method, the Orange County Sheriff's Department laboratory compared DNA from defendant's blood with DNA taken from semen found at the crime scene and concluded that, using the "fixed bin"statistical method the likelihood that someone other than the defendant was the source of the semen stain was one in 189 million Hispanics and one in 38 million Caucasians.
On appeal the defendant challenged the use of the product rule, a step in the statistical analysis where the probability of each locus of specific lengths appearing in the general population are multiplied together. The product results in extremely high probabilities that the defendant is the source of the sample in question. The Court of Appeal found that although there were scientists who did not agree that the product rule was not appropriately used in RFLP statistical calculations, the product rule is generally accepted in the relevant scientific community.
In People v. Soto (1999) 21 Cal. 4th 512, the Supreme Court upheld the court of appeals decision, finding that " (i)t is clear from the evidence in the record, the clear weight of judicial authority, and the published scientific commentary, that the unmodified product rule, as used in the DNA forensic analysis of this case, has gained general acceptance in the relevant scientific community and therefore meets the Kelly standard for admississibility." Id at 541 However, the Court's holding was founded in the facts adduced at the hearing that an "extensive literature in peer-reviewed journals" has accumulated in support of the conclusion that population substructuring does not impact significantly upon RFLP population frequency estimates. Id. at 540-541. The Court cited a worldwide study of RFLP frequency data by the FBI; the publication of the NRC 2 in 1996 which concluded that if the tests are properly conducted the unmodified product rule can be used; and the proliferation of peer reviewed literature supporting the conclusion that for RFLP tests, the use of the product rule is appropriate. The court concluded: "It is clear . . . that the unmodified product rule, as used in the DNA forensic analysis in this case, has gained general acceptance." Id. at 541 [emphasis added]. The Court went on to limit its holding to RFLP cases in which the probabilities of a random match are very rare--one in the multimillions or billions: " We have no occassion in this case to consider whether substructuring could be a cause of material variation in much higher frequencies, e.g., one in several hundred." Id. at 541.
Thus, nothing in the Soto decision impacts on the issues in this case. First, it was an RFLP case in which the Court attached the "greatest significance" to NRC II. 21 Cal. 4th at 539. The NRC II stresses the tentative nature of our current state of knowledge with respest to PCR-based systems, as well as a fundamental distinction between RFLP (VNTR) loci and PCR loci:
PCR-based systems have several advantages, the most important being that they can be used when source material is sparce or degraded and a second being that there need not be uncertainties of measurement. But there are also disadvantages. VNTRs have many allelles, none of which is at a high frequency. Presumably, the high mutation rate accounts for that and for the small differences in frequencies among subgroups. The VNTR's used for forensics also occur at loci that have no function and therefore are probably not affected by natural selection. Some of the loci used in PCR-based systems have only a small number of alleles, and the loci are at functional genes, which means that there is less assurance for HW (Hardy Weinberg equilibrium) and LE (linkage equilibrium). Many more loci are required to produce the same probability levels than are required for VNTRs.
Yet, the statistical uncertainties with VNTRs...make it desirable to bring new loci into the system. The extensive activity in mapping genes is leading to the rapid discovery of many more possible markers, some of which are expected to have the kinds of properties that are desirable for forensic use: high mutation rate, multiple alleles, lack of function (which increases the possibility of neutrality), speed of analysis, low cost, and unambigious identification of alleles. We encourage the development and validation of such systems.
NRC II at 120-121

NRC II also explicitly recognizes that "(t)here has not been the extensive sampling of subpopulations and geographic areas for PCR-based systems that has been done with VNTR's." Id. at 35. Based on this lack of research and based further on emerging data which indicates both that PCR loci are at functional genes, and that population substructuring does impact significantly upon PCR population frequency estimates, prominent population geneticists testified in Bokin that the unmodified product rule is inappropriate for PCR-based tests. Moreover, NRC II itself clearly proposes modifications of the product rule for RFLP and PCR-based systems, such as the "2p rule", the use of a (theta) factor, and the use of confidence intervals. Id. at 119-120, 146. Nothing in Soto abrogates the use of these modifications of the frequency calculations using the product rule.
Second, Soto solely involves statistics and the use of the unmodified versus the modified product rule to calculate RFLP match frequencies in an unmixed sample, and not questions of methodology. See, People v. Soto, 21 Cal. 4th at 518 (" He focused his challenge, not on the threshold RFLP analysis or on the comparison of his DNA with that found on the bedspread, but on the second stage of the analysis: the population frequency determination.") Here, Mr. Nawi's challenge is focused on all aspects of PCR analysis: the threshold DQ alpha, Polymarker, Green I, and Profiler Plus analysis, the comparison analysis for each of these systems, and the population frequency determination on a mixed sample. Soto does not address these questions.

Also on March 15, 1995, the Supreme Court agreed to review the Fourth Appellate District's decision in People v. Wilds (1995) 31 Cal.App.4th 636. Defendant Wilds was convicted of robbery, two counts of forcible oral copulation and rape of one victim; robbery, three counts of forcible oral copulation and rape of another victim; and burglary of a third victim. From DNA testing conducted by the RFLP method, the police concluded that the sperm cells found in the victims were "identical" to the DNA taken from the defendant's blood. Experts testified that the probability of someone other than the defendant being the source of the sperm as one in 4.5 million.
On appeal Wilds argued that both the RFLP methodology and the statistical calculations were not generally accepted in the scientific community, and that the trial court erred in so finding. The First Appellate District did not agree. Citing People v. Axell (1991) 235 Cal.App.3d 836; People v. Barney (1992) 8 Cal.App.4th 798; People v. Leahy (1992) 8 Cal. 4th 587, 612; and People v. Soto (1994) 30 Cal.App.4th 340, the court concluded that RFLP typing and the related statistical methods are admissible under Kelly. As indicated above, on October 27, 1999, the Court dismissed the petition for review under Cal. Rules of Court 29.4(c). That Rule provides that the Supreme Court may dismiss review of a cause as "improvidently granted", and that upon the dismissal, the opinion of the Court of Appeal shall become final but shall remain unpublished unless the Supreme Court expressly orders otherwise. No such publication order was entered in Wilds.
One month after Venegas, Soto, and Wilds the Supreme Court granted review to another DNA bloodtyping case. In People v. Marlow (1995) 48 Cal.App.4th 1440, review granted, April 25, 1995 (No. S046996) defendant Gustavo appealed his convictions of, inter alia, two counts of murder, rape, and kidnaping. Marlow argued that not only was RFLP not generally accepted in the scientific community, but also the lab that conducted the RFLP in his particular case, failed to follow proper procedures in conducting their analysis. The Sixth District Court of Appeal found that, although there was some disagreement, RFLP is generally accepted in the scientific community, and that the lab used proper procedures. On October 27, 1999, the Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
Venegas, Soto, Wilds and Marlow each raised essentially the same issues: whether there is general acceptance in the relevant scientific community of RFLP and the statistical methods used in conjunction with RFLP, and, in the case of Venegas and Marlow, whether proper procedures were used in their particular cases. The next case granted review by the Supreme Court raised a new issue. In People v. Amundson, (1995) 34 Cal.App.4th 1151, review granted August 10, 1995 (No. S047242) Wayne Amundson challenged, inter alia, his conviction of murder with a special circumstance allegation that the murder was committed during a rape. Cellmark diagnostics conducted RFLP analysis. The Serological Research Institute ("SERI"), using PCR to amplify the DNA samples, compared the alleles of DQ alpha locus in samples taken from the defendant and samples taken from the crime scene. In addition to challenging the statistical calculation step of RFLP, the defendant argued that amplifying the DQ alpha locus using the PCR technique for the purposes of identifying an individual as the source of DNA was not generally accepted in the scientific community.
The Court of Appeals for the Fourth Appellate District did not agree. Basing its conclusion on the trial record, case law from other jurisdictions, and scientific literature, the court found that "PCR testing" is generally accepted in the scientific community. Amundson, supra, at 803. The Court also held that the statistical methods used in RFLP protocols were also admissible under Kelly-Frye. On October 27, 1999, the Supreme Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
Next in the line of cases on hold for many years was People v. Burks (1995) 41 Cal.App.4th 652, review granted Nov. 16, 1995 (No. S048916). Melvin Burks was convicted of, inter alia, forcible rape, and two counts of sexual battery by restraint. Using RFLP methods, a lab hired by the prosecution compared semen found at the scene of both attacks with a DNA sample taken from the defendant, and declared a match. Id.
On appeal, the defendant argued that the DNA evidence was inadmissible because the use of the modified ceiling principle in statistical calculations is not generally accepted in the scientific community. The court found that because the " ‘modified ceiling' method employed here in the statistical calculation step of RFLP analysis of DNA is more favorable to Burks than the ‘unmodified product rule' rejected in Barney and is generally accepted withing the scientific community … RFLP analysis using the more conservative ‘modified ceiling' approach satisfies Kelly evidentiary requirements." On August 12, 1998, the Supreme Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
On April 25, 1996 the Supreme Court granted review to People v. Smith (1996) 42 Cal.App.4th 204, review granted April 25, 1996 (No. S052233). In this case defendant Frank Smith appealed his conviction of, inter alia, two counts of forcible rape. Smith argued to the Fourth Appellate District that the trial court erred in admitting bloodtyping evidence. Specifically, Smith challenged the statistical methods, specifically the product rule, used as part of the RFLP analysis by the Orange County Crime Lab.
In rejecting Smith's argument, the Court of Appeal noted that the trial court had found that the Orange County Crime Lab had followed correct procedures in that case, and that the question regarding the use of the product rule in RFLP cases had been decided in People v. Axell, supra. The court concluded that the use of the product rule was generally accepted in the scientific community. On October 27, 1999, the Supreme Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
The Supreme Court also granted review in People v. Givens (1997) 53 Cal.App.4th 554 review granted May 28, 1997 (No. S060563). Harold Givens was convicted of rape, forcible oral copulation, and burglary. The convictions arose out of attacks on four women. For each victim, PCR and RFLP testing was conducted on samples taken from vaginal swabs. PCR testing was conducted by Alan Keel, the criminalist in the case at bar; RFLP analysis was conducted by Cellmark, a private laboratory.
The focus of Givens's claims to the Court of Appeal and of that Court's decision was the use of the unmodified product rule calculations. Givens argued that the trial court improperly admitted the results of the DNA tests because the unmodified product rule was not generally accepted in the scientific community. Although the general acceptance of PCR was at issue in the Kelly-Frye hearing held by the trial court, this claim was not addressed by the Court of Appeal.
The First District disagreed with Givens, "[b]ased on the quality and the quantity of evidence produced in the instant case" Givens, supra, 53 Cal.App.4th at 668, and held that the DNA typing evidence was properly admitted. As with the other cases, on October 27, 1999, the Supreme Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
Finally, and most recently, the Court granted review in People v. Daniels (1998) 62 Cal. App. 4th 1529, review granted June 17, 1998.Mr. Daniels was convicted of rape based on a blood sample that was analyzed by RFLP, DQ alpha, and D1S80 testing. The PCR testing was done by the Santa Clara County Crime Lab. At trial, the defendant did not object to the PCR test results, but did object to the RFLP statistical calculations performed by the F.B.I. On appeal, the defendant again raised the objection to the use of the product rule for RFLP testing, and also claimed that his trial counsel was ineffective for failing to object to the use of the product rule for the PCR testing. In a brief, two paragraph discussion the court rejected both arguments. With respevt to PCR and the possible applicability of Barney, the court stated that "there is no basis for extrapolating its conclusions about RFLP match frequencies to PCR procedures, a completly different method of DNA analysis." Id. As with the other cases, on October 27, 1999, the Supreme Court dismissed the petition for review under Cal. Rules of Court 29.4(c), with no order that the Court of Appeal opinion be published.
Equally important as determining which issues have been in front of the Supreme Court is identifying which have not been in front of that Court. First, Amundson and, to a limited degree, Givens raised only one issue relating to PCR-based tests. In Amundson SERI amplified only the DQ Alpha locus. In addition, as mentioned above, the general acceptance of PCR-based testing was not raised on appeal in Givens. Thus, the only issue that could have been resolved if the Supreme Court had not dismissed review in Amundson and Givens was whether using PCR-based testing to amplify the DQ Alpha locus is generally accepted in the scientific community. In any event, the Court's action in these cases has left even this narrow question unresolved by the Supreme Court. This opposition includes challenges to the DQA1+PM, D1S80, Green I and Profiler-Plus tests employed by the Crime Lab in Mr. Nawi's case.Moreover, none of the cases before the Court involve the use of twelve year old fingernail samples allegedly containing complicated mixtures of several people, which raises issues unique to this case.
The cases recently before the Court also do not consider the issue of whether the databases used in determining the significance of similarities between PCR samples are reliable and generally accepted, or whether an error rate or other statistical modifications are necessary for PCR-based systems.. Although other states have considered some of these issues, see, e.g., State v. Carter (Neb. 1994) 524 N.W.2d 763, overruled on other grounds, 571 N.W.2d 276, 293, this issue is not presently before the Supreme Court nor, as will be detailed below, has it been decided by a California Court of Appeal.
Finally, neither Venegas, Soto, nor any of the other cases raised the issue of whether the San Francisco Police Crime lab uses proper procedures in conducting PCR-based testing, although Venegas does make clear that a hearing must be conducted on this issue in this case.

B. ISSUES RAISED IN THIS MOTION HAVE NOT BEEN RESOLVED BY ANY CALIFORNIA COURT OF APPEAL.
In 1998, the First District issued a written opinion on the admissibility of DNA testing using an earlier version of Roche Molecular Systems' DQ Alpha kit and an earlier version of the Polymarker kit. People v. Wright (1998) 62 Cal.App.4th 31. The Wright court relied solely on the holding in People v. Morganti, infra, in holding that both the DQAlpha and PM kits were admissible under Kelly-Frye, stating "[t]he PCR matching technique, at the very least, has certainly acquired general acceptance in the scientific community." Id. at 1832. For a number of reasons, Wright cannot be considered precedent or controlling on the issue of whether the Crime Lab's PCR based DNA testing methods meet Kelly-Frye standards.
First, the Wright court misread the holding of Morganti. Wright states that "(f)ollowing Morganti, we agree with the trial court's ruling here. The PCR method has obviously acquired general acceptance in the scientific community." Id. at 1834. Morganti did not hold that the PCR method was generally accepted; Morganti only considered the acceptance of an early version of Cetus's original DQAlpha kit, 43 Cal. App. 4th 643, 662 n. 9, which as laid out above, and which will be developed further infra, differs from the DQ Alpha +PM kit, the Green I kit, and the Profiler Plus kit employed in this case. In addition, when DQ Alpha is combined into a single kit with the five polymarker loci and then used in conjunction with two STR kits to come up with a combined probability, issues not relevant to DQ arise that could not have been addressed in Morganti, specifically the use of population genetics and statistical analysis of multiple genes, and the effects on reliability of combining a number of loci into a single DQ Alpha + Polymarker kit. As the NRC II recognized following the decision in Morganti,
the Polymarker system "was only beginning to be widely used" in 1996. NRC II at 72. As use of this system has developed, more recent scientific literature calls into serious question whether the community of molecular biologists accept as reliable the DQ alpha + Polymarker kit used in this case. For example, one recent article regarding the combined DQ Alpha +PM kit found that attempts at typing samples using this multiplex kit resulted in failed hybridization and allelic dropout. Donald E. Riley, Compromised Optimal Hybridization Temperatures May Explain PM Plus DAQ1 Anomalies, 44 Biochemistry and Molecular Biology International 59, 63 (1998). Dr. Riley testified in the Bokin hearing and explained extensively his study and the basis for his expert opinion that the DQ alpha + PM kit was not generally accepted as reliable by the community of molecular biologists. Dr. Riley's article was not and could not have been considered in either Wright or Morganti, because it was not published until after the decisions in those cases. As Soto emphasizes, "(i)n the context of rapidly changing technology, every effort should be made to base (decision) on the very latest scientific opinions, including those published during the appellate phase of the case." 21 Cal. 4th at 540 n. 31
What the Wright court failed to consider is that the while PCR is a method used in DNA testing, it is only one step of many used to determine a genotype and to calculate population frequencies.See, United States v. Hicks (9th Cir. 1996) 103 F. 3d 837,844 [ "The PCR method itself is not a genetic test; it is a mere amplification technique."]. An analogy to the Wright court's myopic focus on PCR would be a court holding that using ink to take fingerprints was generally accepted but failed to consider the further steps taken to analyze the print. It was not until our Supreme Court issued its ruling in Venegas, decided three months after Wright, that it became clear that California courts were required to scrutinize each step of DNA analysis under Kelly. Venegas, and not Wright, sets forth the requirement of addressing DNA evidence at each stage of the process.
The NRC in its first report recognized the distinction between different tests using the common step of PCR, recommending that in "PCR-based typing, the committee makes a number of technical recommendations, including recommendations for thorough characterization of each PCR assay for definition of the range of conditions under which it will perform reliably." NRC 1, at 72-73 [emphasis added]. If all tests that used PCR as a basis could be lumped together under the general title "PCR" then the recommendations of the NRC to characterize the different assays used and the validation studies done on each new marker or locus, see infra, would be meaningless.
Therefore, the holding of Wright is not controlling in this case because it incorrectly lumped together all tests using PCR technology in its interpretation of Morganti. As explained above, the DQ Alpha + PM kit differs from DQ Alpha alone in that it analyzes different genes or makers and therefore use different primers in the amplification step and different reverse dot blot strips in the hybridization step, and the use of multiple markers also complicates the statistical calculations. Also, as demonstrated below, the D1S80 kit, the Green I kit, and the Profiler Plus kit raise unique reliability problems which were not and could not have been considered in either Wright or Morganti.
Second, Wright and Morganti are not controlling precedent because new evidence of a controversy is now available and must be considered under the reasoning of Kelly. Although Kelly states that once a trial court's decision to admit new scientific evidence is affirmed on appeal and the "precedent so established may control subsequent trials," the court also recognized that the decision will only be precedent "until new evidence is presented reflecting a change in the attitude of the scientific community." 17 Cal.3d at 32 [emphasis added].
The holding in Morganti, supra, was limited to the admissibility of DQ PCR testing under Kelly-Frye. Id. at 663. The PCR analysis conducted and reviewed by the Morganti court involved only the DQ gene. Id. at 662. The court described the procedure in the following manner:
In the forensic setting, PCR analysis of DQ alpha involves three general steps. First, DNA is extracted from the nucleus of cells present in an unknown bloodstain. Second, the DQ alpha is replicated or amplified by a process which involves combining the DNA with a commercially available solution or ‘cocktail' and then subjecting the solution to a series of controlled temperature cycles. Finally, the amplified gene is typed in order to identify the alleles present in the amplified DNA.
Id. [emphasis added].
At the Kelly-Frye hearing, the People called two witnesses: Edward Blake and Gary Harmor, identified by the trial court as "forensic serologists." Id. at 664. Harmor testified that "he had not read any publications which criticize PCR analysis of DQ alpha as flawed or unreliable. And he was not aware of any criticism of the statistical data he used." Id. Blake testified that the technique was accepted as reliable in the scientific community and that he was "not aware of any scientist who work in this field or of any publications expressing the view that PCR analysis is unreliable." Id. at 644 n.11. Blake further testified that the NRC 1 report says that the PCR is an "acceptable reliable process." People v. Morganti, No. 19691, Superior Court of Sonoma County, April 5, 1993 (R.T. 2935).
The only witness who challenged the assertions of Blake and Harmor was Dr. Grunbaum, who stated that while PCR was generally accepted among scientists, its application in the forensic arena was not generally accepted. Id. at 664. The court rejected Dr. Grunbaum's opinion as having little weight because Dr. Grunbaum appeared to be biased against Harmor, he had little forensic experience and his opinions were "unsupported by reason or lacked foundation." Id. The fatal flaw of Morganti is that neither the trial court nor the court of appeal was provided with a fair overview of the current underlying controversy regarding PCR, as is done in section III of this brief. Most of this controversy developed after the Kelly hearing in Morganti thus bringing it within the ambit of Kelly's rule that new evidence can be considered if there is a change in the attitude of the scientific community.
At the time of the Kelly-Frye hearing in Morganti, the Polymarker kit was not available. The Kelly-Frye hearing was held in April 1993, a few months prior to the introduction of the PM test kit by Perkin-Elmer. Therefore, at the time of the hearing, the court could not have, nor did it, consider the use of the DQ Alpha in conjunction with the use of the PM kit. Finally, since the time of Morganti, a new kit was developed that allows the amplification and typing of the DQ and PM markers together. This combination required extensive new testing and changes in the protocols used. See Ann Marie Gross & Richard Guerrieri, HLA DQA1 and Polymarker Validations for Forensic Casework: Standard Specimens, Reproducibility, and Mixed Specimens, Journal of Forensic Sciences (1996) 41(6):1022. As indicated above, one recent article regarding the combined DQ Alpha +PM kit found that attempts at typing samples using this multiplex kit resulted in failed hybridization and allelic dropout. Donald E. Riley, Compromised Optimal Hybridization Temperatures May Explain PM Plus DAQ1 Anomalies, 44 Biochemistry and Molecular Biology International 59, 63 (1998). Surveying prior studies, Dr. Riley states that "three independent studies reported false typings with either DQ Alpha and Polymarker. In two of these studies, the false typings were confirmed by other techniques such as DNA sequence data or by comparisions with nonmultiplex systems." Id. p. 64 Other recent scientific commentary regarding the general unreliability of PCR-based testing is surveyed in section III infra.
In addition, with the use of multiple markers, new issues regarding the use of population genetics and statistical analysis in calculating genotype frequencies have arisen based solely on the use of more than one gene as a marker. See, infra.
Moreover, Wright itself recognizes a variation of the "change in scientific attitude rule" of Kelly when it observed that "appellant also does not provide any arguments specifically directed against the result reached in the Morganti decision." 62 Cal. App. 4th at 38. Here, however, such arguments are being raised. In addition to the issues raised in Point III infra, it is important to stress that both the trial court and the court of appeal in Morganti relied on Blake's representation of the opinion of the National Research Council and the scientific community. Review of the NRC 1 shows that Blake provided misleading testimony to the trial court.
NRC 1, which was published in 1992, does not state that PCR or the DQ Alpha system is reliable or generally accepted. Instead, NRC 1 provides only technical descriptions and passing comments about the potential uses of PCR- based testing of forensic samples. Indeed, NRC 1 identifies areas of research that need to be pursued. "PCR provides excellent starting material for direct DNA sequencing, and sequence analysis might ultimately be the approach used for personal identification. But it will require improvements in automated sequencing technology and the generation of larger databases on sequence variability." NRC 1 at 44. The NRC went on to note that " further experience should be gained with respect to PCR in identity testing." NRC 1 at 70. Finally, the NRC concluded, "PCR analysis is extremely powerful in medical technology, but it has not yet achieved full acceptance in the forensic setting." NRC 1 at 70.
Recently, misrepresentation of the views of the National Research Council by an expert to the court resulted in a reversal of a defendant's convictions of first degree murder, burglary, and sexual battery. Murray v. Florida (1997) 692 So.2d 157, 163. In Murray the Supreme Court of Florida found that the prosecution had failed to meet its burden to show that PCR methodologies were generally accepted because the prosecution's witness avoided questions regarding his testing procedures, gave "unenlightening" testimony regarding his probability calculations and, most importantly, "affirmatively misled the trial court as to the NRC's acceptance of PCR DNA methodology at the time of the hearing." 62 So.2d at 163. The court explained that
[Prosecution expert] Nippes told the court that the report of the [NRC 1] ‘was an endorsement of the forensic DNA application to forensic science whether it was RFLP or PCR." In fact, the NRC's 1992 report expressly withheld endorsement of PCR methodology. The committee has explained that although the PCR method has "enourmous promise" "it has not yet achieved full acceptance in the forensic setting." NRC Report at 70. Specifically, in relation to the use of PCR testing with commercial kits, one of which was used in this case, the NRC report cautioned: "The Committee sees a potential for introduction of unreliable kits and the musiuse of kits. The existence of kits suggests ease of use and low chance of technical error. The committee believes that nonexpert laboratories will run a significant chance of error in using kits" NRC Report at 69.
692 So.2d at 160, n.5
The court went on to hold that "the paucity of information in this record concerning the application of the PCR methodology to the DNA evidence at issue here leads us to the conclusion that even if the trial court had attempted to determine whether this evidence met the Frye standard, there is no way the court could have found it admissible." Id. at 63. This court should follow Murray and conclude, as suggested in Wright itself, that because of the misrepresentation of Dr. Blake, there are valid arguments specifically directed against the result reached in Morganti.
Moreover, it should go without saying that whatever his status when the Kelly hearing was held in Morganti in April 1993, Dr. Blake is currently " too closely identified with the endorsement of [the technique] to assess fairly and impartially the nature and extent of any opposing scientific views." Kelly, supra, 17 Cal.3d at 38. Thus, when applying the Kelly standard, the court must look to experts who are "'impartial,' that is, not so personally invested in establishing the technique's acceptance that he might not be objective about disagreements within the relevant scientific community." People v. Brown, supra, 40 Cal.3d 512, 530; accord People v. Venegas, 18 Cal. 4th at 77 (" FBI Agent Lynch, though vigorously defending the merits of the FBI's RFLP analytical procedures she had followed in this case, did not purport to be qualified, as a molecular biologist or otherwise, to testify on questions of general scientific acceptance of the validity of those prosedures.")
Nowhere is Dr. Blake's lack of impartiality better illustrated than in his recent intemperate and unfair personal attacks against Judge Robert Dondero. Blake, who has admitted in several Kelly hearings that he makes his living promoting and conducting PCR-based forensic testing, was in 1996 awarded a lucrative city contract to supervise the San Francisco Crime Lab's DNA Program. Lashing out at the recent ruling of Superior Court Judge Robert Dondero in People v. Bokin, which was highly critical of the SFPD's DNA Program, Blake has called for the removal of Judge Dondero from office on the outrageous ground that the judge is "an arrogant, ignorant, stupid judge who is unable to get through the smoke thrown at him by a very skilled lawyer.""Judge Rejects DNA Test Evidence", San Francisco Examiner, May 7, 1999, p1. His intemperence has not escaped the criticisms of his scientific collegues. See, Comments of Professor William Thompson, People v. Bokin: An Internet Discussion, http://CA_v_Bokin.pdf@www.scientific.org ("We will get a lot farther through respectful exchanges...than we will by publically declaring that those who disagree with us are ignorant, stupid or callous."). To give credence to the views of Blake in these circumstances would make a mockery of Kelly's prong two requirement of impartiality. The same most certainly must be said for the Roche Molecular Systems and Cetus employees who authored the alleged "validation" studies upon which the Morganti court relied. 43 Cal. App. 4th at 664 665. Scientists who are affiliated with companies that deliberately lie and distort scientific data to the Unites States government in order to obtain lucrative patent rights cannot be considered impartial, and their so-called scientific "studies" cannot be allowed to pollute the waters of justice.
Finally, Wright should not be considered controlling due the manner in which the Kelly-Frye hearing was conducted in the trial court. The defense did not call any expert witnesses to testify at the Kelly-Frye hearing and chose instead to call an expert to attack the DNA evidence at trial. The court of appeal refused to apply the defense experts testimony to the issue of Kelly-Frye, holding the decision to not call the expert at the hearing a tactical decision by the trial attorney. 62 Cal. App. 4th at 41 Because of this tactical decision, the trial court and the court of appeal were presented with a Kelly-Frye hearing in which three prosecution expert witnesses testified with no opposition from the defense. The Wright court's resolution of this issue, and its refusal to consider the defense expert's testimony, are disapproved in Soto, which emphasizes that, "(i)n the context of rapidly changing technology, every effort should be made to base (decision) on the very latest scientific opinions, including those published during the appellate phase of the case." 21 Cal. 4th at 540 n. 31
The hearing in Wright leaves this Court in a position to consider new evidence of a controversy that was not presented in Wright and that is presented here. Therefore, this Court cannot find that Wright precludes a Kelly-Frye hearing in this case. In this case, evidence that was not presented in either Wright or Morganti regarding the use of both the Dq + PM, the D1S80 kit, the Green I kit , and the Profiler Plus kit, alone and in combination, has been presented to this Court, thus requiring a Kelly-Frye hearing.
The People have argued in McClanahan and Bokin that California case law holds that all PCR-based tests pass Kelly-Frye, and that there is no need for additional Kelly-Frye hearings when all that is being changed is the gene which is being amplified and visualized. The National Research Council and the numerous cases cited in section D. 2. Supra do not agree. As the NRC I summarises the issue, "[T]he use of various detection technologies for PCR products might require a pretrial hearing about the characteristics of the detection method and its sensitivity to artifacts. In each case, the court can properly limit inquiry to the substantially novel aspects of the technology, focusing on whether the method is accepted for scientific applications and whether it has been validated for forensic identification." NRC 1 at 144-45.
The People have further argued that the NRC 2 makes quite clear that it finds PCR technology, including the methodologies and genetic loci typed in this case to be reliable. The People's representation of the NRC 2 is misleading. The People's have relied on quotes from the NRC 2 involving only general statements about "DNA typing." These quotes, coming from a small group of primarily statisticians and lawyers, reveal nothing about the general acceptance or reliability of the typing methodologies used by the Crime Lab. The People have also in the past quoted the NRC 2's description of the advantages of PCR-based tests over RFLP testing. Again, this statement provides no insight regarding the reliability and general acceptance of the typing methodologies used in this case, and more importantly, the People tellingly omit the remainder of the NRC's discussion which focuses on the disadvantages of PCR-based testing, including that
" any procedure that uses PCR methodology is susceptable to error by contamination." NRC II at 23 Next, the People have referred to the reaffirmation by the NRC 2 that "the molecular technology is thoroughly sound and that the results are highly reproducible" , quoting NRC 2 at 23. The NRC 1 found that the technology of PCR amplification in general to be sound, but did not endorse genotyping of any particular locus. See, e.g., NRC 1 at 44.
The People have cited the NRC 2 for the proposition that the polymarker, D1S80, and STR loci have been validated for forensic use. However, as will be discussed in more detail below, the tests used in this case have not been validated in the manner required by NRC 1, the TWGDAM Guidelines, or the DAB Standards. Also as will be discussed below, there exists controversy regarding the reliability of typing these loci. The broad statements in the NCR 2 cannot be interpreted as general acceptance of the DQA1+PM D1S80, and STR kits used in this case, especially since the STR kits did not even become available until after the publication of NRC II in 1996.
Finally, the last two quotes provided by the People in other cases are taken out of context. The first, "[w]e conclude that PCR-based systems should be used." NRC 2 at 119, comes at the end of a chapter on population genetics. This chapter discusses the population biology of the loci that are studied in PCR-based systems. Specifically, the chapter discusses issues such as independence of alleles. This chapter does not discuss at all the reliability of amplifying particular loci or the genotyping of particular loci. The paragraph containing this quote ends with admonishment to use population data from PCR-based systems conservatively. The People also have quoted another broad statement by the NRC regarding "profiling technology", quoting NRC 2 at 73. This vague statement comes at the end of a chapter containing descriptions of all available DNA profiling techniques, including RFLP, PCR-based tests, and newer tests known as mitochondrial DNA testing. If the NRC intended to state that all PCR-based genotyping systems are reliable and generally accepted, surely they would have simply made that statement instead of painting with such broad strokes. In any event, as Venegas makes clear in its adoption of the 1992 NRC Report over the 1996 Report, and as Soto reconfirmed by focusing on the scientific literature that had developed on RFLP testing after the publication of the NRC II, the NRC II is not the final word on California Kelly-Frye DNA issues.
Nor are the issues raised in this case addressed by the recent case of People v. Allen (1999) 72 Cal. App. 4th 1093. The same misperception of the science of PCR-based testing evidenced in Wright and Morganti with respect to DQ alpha and polymarker testing is also evident in Allen with respect to Short Tandem Repeat testing kits. Without citation to the Venegas holding that " FBI Agent Lynch, though vigorously defending the merits of the FBI's RFLP analytical procedures she had followed in this case, did not purport to be qualified, as a molecular biologist or otherwise, to testify on questions of general scientific acceptance of the validity of those prosedures" (18 Cal. 4th at 77), the court in Allen stated that it failed to see why the testimony of a Cellmark employee could not alone show general acceptance of "STR's (which test three [undisclosed] genetic markers.)" Id. at 1097, 1099.
Contrary to all of the cases discussed in section D. 2. , supra, the court focused not on the specific STR kits employed in the case, but on the vague concept of "STR testing" in general. In finding that the People had established through the testimony of a single interested witness that "STR testing" was generally accepted by an undefined "scientific community", the court relied heavily on a Massachusetts case, Commenwealth v. Rosier (1997) 425 Mass. 80 in which the Massachusetts Supreme Court quoted the NRC II report out of context for the proposition that
" STR testing is similar in principle to the RFLP (or VNTR) method, which has been found to be reliable." Id at 1100. In fact, the portions of NRC II referred to in Rosier and Allen state that STR methods "are usually capable of unique allelic identification" (NRC II at 34), that "(a)s more STR's are developed and validated, this system is coming into wide use" (Id. at 71), and that such methods " will soon be on the same solid footing as VNTRs" (NRC II at 117). Such statements are a far cry from the Allen court's apparent understanding that somehow NRC II had validated all STR testing platforms for all time. The court cited no California cases standing for the proposition that one STR based test is the same as any other and cited no California cases that stated that general acceptance of PCR as a method to amplify DNA means that all applications of PCR-based testing is generally accepted as reliable. The court's loose reading of Kelly, and its failure to even acknowledge Venegas, was apparently prompted by the court's understanding that "the results generated by a scientific test once considered valid can be challenged by evidence the test has since been invalidated", and that "defendant is not foreclosed from showing new information which may question the continuing reliability of the test in question or to show a change in the consensus within the scientific community concerning the scientific technique." Id. at 1100-1101.
As Venegas clearly holds, an appellate decision upholding one type of DNA methodology is only binding on another DNA methodology " (i)n the absence of proof of any material scientific distinction between the two methodologies." 18 Cal. 4th at 54. Here, the Court has before it the declaration of Dr. Christie Davis attached hereto as Exhibit D. Dr. Davis has a Ph.D. in Microbiology and Immunology and is familiar with the hearing in Allen and with the technology at issue in this case. Dr. Davis in no uncertain terms lays out the facts that make the tests before this Court scientifically distinct from those in Allen, thus allowing this Court to hold a prong one hearing on the Green I and Profiler Plus kits as well as the ABI Prism 310 capillary electrophoresis system.
In ¶ 7, Dr. Davis states that the PCR process is not a test itself, but is a means to increase the number of copies of genetic markers and that the process itself is complex and varies depending on the genetic marker of interest. In ¶ 8, Dr. Davis states that the test at issue in Allen, is different than either of the two STR kits challenged here. It was a kit produced by a different manufacturer, Promega, and while amplifying the three of the four markers from Green I, the test used a silver stained gel rather than capillary electrophoresis. As Dr. Davis notes, the primers are different than those in the Green I kit, the protocol used is different than the one for the Green I kit, including the thermocycler program as well as the number of cycles of amplification. Id. at ¶ 10.
Perhaps most important is that the results that are obtained from the Promega kit used in Allen can vary from the results obtained using the Perkin-Elmer Green I kit. Id. at ¶ 11. Thus one piece of evidence analyzed with the Green I kit will have a different genetic profile than one analyzed with the Promega CTT kit. If the Court follows the reasoning of the People and of Allen, then both results and both kits would be considered reliable. This is logically inconsistent and would lead to absurd results. For example, if one lab tested the evidence using a Promega kit and the results were inculpatory, and then another lab retested the same evidence with Green I and the results were exculpatory, then one expert would have to testify that both kits were reliable and generally accepted. This example illustrates the fallacy of the argument and the reasoning that STR based tests are interchangeable because they involve STR markers as a class.
The same reasoning applies to the Profiler Plus kit. As a test, it is completely different than Green I and the Promega CTT kit. Declaration of Dr. Davis, at ¶ 12-15. The nine loci tested are different than those found in Green I and Promega CTT, and include one locus, vWA, that has produced several papers that "address serious problems that can ultimately change the genetic profile obtained."Declaration of Dr. Davis, at ¶ 14. See also, C. Alves, et. al., vWA STR Genotyping: Inconsistency Between Perkin Elmer's Profiler Plus Kit And Promega's Geneprint, International Society for Forensic Haemogenitics, Eighteenth International Congress Abstracts, August 17-21, 1999, San Francisco, Ca., p. 30 (simultaneous study of vWA locus by the Perkin Elmer's Profiler Plus Kit and the Promega Geneprint Kit produced an inconsistency between the genotyping in each kit: using Profiler it was found to be 18 and with Geneprint 16-18. " Since primer sequences were not available from the manufacturers we could not sequence the corresponding regions. However, it is tempting to interpret the inconsistency as a result of a Perkin Elmer primer annealing failure...The finding now reported evidences the need for caution when comparing genotypes or gene frequencies made in amplicons and produced by different primers."); M.C. Kline, et. al., Nonamplification of a vWA Allele, J. Forensic Sci. 1998 Jan., 43(1):250(National Institute of Standards and Technology researcher documents same inconsistency and indicates that Perkin Elmer " is aware of the problem and they are actively pursuing an explanation for this allelic dropout by sequencing the sample"); S. Walsh, Commentary on Kline, MC, Non-Amplification of a vWA Allele, J. Forensic Sci. 1998 Sept., 43(5) 1103( Perkin Elmer admits the problem exists, and claims it is caused by a flanking sequence mutation. "Our laboratory has observed flanking sequence mutations in several STR loci, including the vWA loci reported here, D16S539, TPOX. Other laboratories have reported flanking sequence mutations at D13S317 and DS7820." Perkin Elmer admits that the problem will continue, but claims that it can be avoided by using Perkin Elmer products exclusively.) As these articles demonstrate, the primers in the three kits are different. The reaction conditions are different. In no way are the three kits interchangeable. The Green I kit could not be used to test for CTT nor could either the Green I and CTT kits components be used to test for the Profiler Plus markers. There is no question that each is an independent and wholly different testing system that must be evaluated prior to its acceptance.
The logic of the defense position in this case, that acceptance of Promega CTT does not allow untested acceptance of other STR based kits, is supported by the People's own witnesses in the Bokin hearing. Dr. John Tonkyn of the California Department of Justice, testified that "[t]he kit requires 4.1, developmental validation, before it can be utilized. In addition, each laboratory that uses this must comply with Section 4.5, internal validation." (R.T. 5456). Significantly, Dr. Tonkyn conceded on cross examination that "a validation study which dealt, for instance, with the Promega CTT system, using the Promega primers in a silver staining system, would not validate developmentally or internally the Green I Kit, which uses an entirely different set of primers, an entirely different software, and entirely different parameters." (R.T. 5477).
Therefore, the Court has before it through Dr. Davis' declaration and Dr. Tonkyn's testimony, that general acceptance of one kit does lead to the general acceptance of another kit. The Green I and Profiler Plus kits differ in a materially scientific manner from the Promega kit at issue in Allen. The Court must hold a hearing to inquire into whether or not the Profiler Plus and Green I kits are generally accepted as reliable.
Moreover, the method used to visualize the amplified DNA is materially different than that used in Allen. There, the tests were conducted using silver stain gels. Declaration of Dr. Davis, at ¶ 16-19. That method is simple as compared to the capillary gel electrophoresis method. Capillary electrophoresis is an entirely different technology that is automated; uses a different polymer system; different chemistry; and uses two computer programs to analyze the raw data. Id. at ¶16-17. The systems is known to produce artifacts and no validation studies have been published. Id.
This Court must take seriously the gatekeeper function delineated in Kelly and require that this new technology that carries so much weight with lay jurors be subject to the most stringent examination. This Court should not be taken in by the illogical and simplistic arguments of the People that STRs are STRs.

C. THE RULING IN PEOPLE V. MCCLANAHAN IS NOT BINDING ON THIS COURT, AND THE RULING IN PEOPLE V. BOKIN IS MORE PERSUASIVE AND SHOULD BE FOLLOWED

The oral ruling by the Honorable Judge Richard Kramer in McClanahan regarding the Green I kit is not binding on this Court. The extensive five month hearing in Bokin came after that in McClanahan and is supported by detailed findings of fact and conclusions of law set forth in Judge Dondero's sixteen page ruling . (See, Exhibit A). Judge Dondero ruled that the Green I kit was not generally accepted as reliable within the relevant scientific community. See, People v. Bokin, SCN 168461, dated May 5, 1999. As the decision in Bokin makes clear, the standards not only of the scientific community of molecular biologists and geneticist, but of the forensic community as delineated in the TWGDAM and DAB guidelines were not minimally satisfied as regards the validation and testing of the Green I kit. Therefore, the defendant respectfully requests that this Court take judicial notice of the testimony and evidence in Bokin and hold that the Green I kit and the tests performed in this case using the Green I kit, are inadmissible under Kelly.
New evidence was presented in Bokin that was not presented in McClanahan.First, the studies of the California Department of Justice on the Green I kit were not before Judge Kramer. Those studies indicated that the Green I kit did not perform as Dr. Cynde Holt testified that they did. High levels of stutter were observed as well as "reverse profiles." Additionally, Dr. Holt did not bring to Judge Kramer's attention the fact that the phenomenon of allelic dropout had been observed. In particular, Dr. Holt did not inform Judge Kramer that different results were obtained using Perkin-Elmer's kits, as compared to the same set of markers manufactured by Promega. This omission was made despite Dr. Holt's admission in Bokin that she was aware of the controversy when she testified in McClanahan, and despite the fact that she stridently urged the court in McClanahan that there were absolutely no reliability problems with any Perkin Elmer product. Evidence of this allelic dropout problem was brought out in the Bokin case and is documented in the articles cited above, one of which (the Walsh article) is authored by a Perkin Elmer employee.
Additionally, Dr. Holt mislead Judge Kramer in her testimony regarding the validation studies done by Perkin-Elmer and the forensic community. In particular, Dr. Holt testified that the California Department of Justice had validated Green I and that she had personally attended the talk given by Dr. Tonkyn in which he presented his results. This was directly contradicted by Dr. Tonkyn and by others at the Department of Justice. Dr. Lance Gima filed a declaration with the court in Bokin, stating that the studies conducted by his lab were internal studies and did not amount to validation of the Green I kit, in part because they had abandoned the study. Dr. Tonkyn testified that he did not present any results of his Green I study and that he announced at the talk that Dr. Holt stated that she attended, that he had not finished the study.
Finally, Dr. Holt testified that certain tests described in the Green I kit User's Manual, were conducted by California Department of Justice. In Bokin, Dr. Tonkyn testified that some of the tests described in the Manual as being conducted by his lab, had not in fact been conducted.
This Court should cast a wary eye upon the order issued by Judge Kramer. Dr. Holt, the only expert called by the People in McClanahan, misrepresented evidence and withheld critical information from the court. Based on her misrepresentation, her credibility is suspect and should not in a case such as this, prevent this Court from taking a close and critical look at the Profiler Plus kit, another product of Perkin-Elmer
III.
DEFENDANT ROBERT NAWI RAISES UNRESOLVED ISSUES PURSUANT TO KELLY-FRYE

As discussed above, several controversies remain that are unresolved by case law that binds this court. Also discussed above, Venegas clearly mandates a hearing on the third prong of Kelly to determine whether the Crime Lab's testing procedures meet scientifically acceptable standards. Keeping in mind the parameters of case law that binds this court, the defense submits that the court should hold a hearing to determine several other issues raised below.
A. THE METHODS USED IN THIS CASE FOR THE COLLECTION, PRESERVATION, HANDLING, AND PROCESSING OF CRIME SCENE SAMPLES FOR FORENSIC PCR BASED DNA TESTING --THE DQ ALPHA + POLYMARKER, D1S80, GREEN I, AND PROFILER-PLUS KIT TECHNIQUES -- ARE NOT GENERALLY ACCEPTED AS RELIABLE AMONG THE RELEVANT SCIENTIFIC COMMUNITY

1. The Failure of The SFPD Crime Lab to Follow Correct Scientific Procedures As Outlined In TWGDAM and DAB Guidelines, The NRC Reports, The Manufacturer's Protocols, And the Lab's Own Protocols Renders Any DNA Test Results Inadmissible

The prosecution's burden under Venegas is a very heavy one indeed. There are no California appellate cases approving the methods of the San Francisco Crime Lab, which uses commercially distributed amplification and typing kits. The use of a commercially distributed standardized "kit," however, does not mean a laboratory employs adequate methods and controls to do reliable testing. Individual laboratory validation is required, and many scientists, and the NRC itself, believe that additional scientific controls are necessary to make PCR based forensic testing reliable. The NRC makes this point clear in its comments on Roche's DQ-alpha "kit":
One commercial kit for forensic PCR analysis has been marketed. Other such kits will probably be ready for commercial distribution soon. The committee sees a potential for introduction of unreliable kits and the misuse of kits. The existence of a kit suggests ease of use and low chance of technical error. The committee believes that nonexpert laboratories will run a significant chance of error using kits. We therefore recommend that a standing committee (discussed later in this chapter) consider the issue of regulatory approval of kits for commercial use in forensic DNA analysis. Even though no precedent exists for the regulation of tests in forensic DNA applications, we believe that it might be necessary for a government agency to test and approve kits for DNA analysis before their actual forensic use.
NRC Report, at 69.
Moreover, the use of PCR based DQ Alpha +Polymarker, D1S80, and STR test kits, which commenced in San Francisco in March, 1997 in the case of DQ Alpha + Polymarker and D1S80, in March, 1998 in the case of Green I, and in February, 1999 in the case of Profiler-PlusFe, is clearly, for the Crime Lab, at the earliest experimental stages. The forensic use of these techniques by this lab is barely known to the scientific community, much less generally accepted.
Furthermore, the prosecution will be unable to demonstrate at the Kelly hearing that the SFPD has, or employs, a generally accepted method for the collection, preservation, and handling of crime scene samples for purposes of PCR-based testing. As indicated above, the NRC II is emphatic that such controls are necessary becauser "any procedure that uses PCR methodology is susceptable to error by contamination" and thus "the integrity of the chain of custody is of paramount importance." NRC II at 23,25. Moreover, the risk of sample contamination created by unreliable preservation and handling practices is seriously compounded by the fact that the SFPD DNA laboratory does not employ generally accepted methods for identifying, preventing, and controlling contamination in its processing of samples for PCR testing. Put simply, the methods employed by SFPD are not generally accepted as reliable because they create scientifically unacceptable risks of sample contamination for purposes of PCR testing on the crime samples in this case.
As recently revealed in Bokin, the failure of the SFPD Crime Lab to follow correct scientific procedures is substantial.The major deficiencies can be quickly summarized. The crime lab is not accredited (see, Penal Code Section 297(c) ["All laboratories, including the Department of Justice DNA laboratories, contributing DNA profiles for inclusion in California's DNA databank shall be accredited by the ASCLD/LAB."]), it lacks critical resources and managerial structure, and until his recent resignation it is operated by a "technical manager" (Allen Keel) who was unqualified and operated without adequate supervision and oversight. The lab has not adopted and enforced sound and conservative interpretative guidelines and its proficiency and internal validation testing progam is sporadic and insufficient. The lab lacks adequate security and contamination control protocols and it failed to follow industry standards after contamination was found in its water supply, which required that the lab shut down and run anew all casework.The lab improperly makes DQA and PM calls in the absence of C or S dots and fails to use appropriate positive and controls in its testing.
The Lab also does not have " a managerial staff with the authority and resources needed to discharge their duties and meet the requirements of the standards..." DAB Guideline 4.1; TWGDAM Guideline 1.3 According to a letter Mr. Keel himself recently wrote to state auditors, "the lab has no senior or supervising criminalist overseeing anything other than Ms. Blake who manages the overall laboratory operation in a provisional capacity as senior criminalist...The failure of the audit report to even comment on the disarray of the SFPD laboratory formal organizational and hierarchical structure is glaring absent." (Bokin Exhibit 8K).
The evidence in Bokin also convincingly demonstrates that DNA forensic analysis is a technically demanding and subjective enterprise that demands specialized training, good judgment, the ability to learn from one's mistakes, constant continuing education efforts in a rapidly changing field, impartiality and a proper scientific attitude. Even well trained PhDs such as Department of Justice employee Dr. John Tonkyn admitted in Bokin that he had made critical mistakes in interpretation because of his carelessness and failure to follow sound conservative guidelines.
The DAB guidelines specifically require that a technical manager such as Mr. Keel, "shall have at a minimum a Master's degree in biology, chemistry, or forensic related area and successfully complete a minimum of 12 semester or equivalent credit hours of a combination of undergraduate and graduate course work covering the subject areas of biochemistry, genetics, and molecular biology or other subjects which provide a basic understanding of the foundation of foresnic DNA analysis as well as statistics and or population genetics as it applies to forensic DNA analysis." DAB 5.2.1. The TWGDAM guidelines are similar but slightly less demanding. See guideline 2.2.3.1
Although Mr. Keel represented to Judge Dondero on direct examination that he was qualified "pursuant to TWGDAM and DAB guidelines" (R.T. 2026) it is clear from his cross examination that he does not have a Master's degree and that he has not yet obtained a waiver of the above requirement pursuant to DAB guideline 5.2.1.1. The evidence in Bokin further establishes that Mr. Keel has a B.S. in zoology and that he has taken an unspecified number of different zoological courses including immunology, human physiology, and the struction and function of muscle. He has also taken some early UC extension classes in nucleic acid biochemistry. That is the extent of his formal training (R.T. 2133, 2135). His CV and his testimony further indicate that his formal educational efforts stopped arround 1996. His case notes for his proficiency tests and for the cases before the Court in Bokin indicate multiple instances of mistakes, shoddy record-keeping procedures, and failure to follow proper protocol. The cross examination on these issue revealed many irregularities, including crossed out and changed results, self grading, and failure to submit the tests on time. The cross examination also revealed that while employed at the Oakland Crime Lab, he failed serology proficiency tests because of interpretational and sample mix-up errors, and committed the same kinds of mistakes in his DNA casework.
Reviewing all of this evidence, Judge Dondero specifically found that " Alan Keel's credentials are not in compliance with DAB guidelines. He lacks the appropriate academic background mandated by DAB 5.2.1 in that he has no Master's degree in the particular sciences identified in the guideline, nor the semester hours in particular courses listed. More importantly, he has not complied with the waiver option pursuant to 5.2.1.1. Familarity with the record involving past efforts at proficiency testing by Keel suggests lapses in meeting deadlines. On the record beore the Court, Keel would not satisfy DAB requirements for any contemporary analysis. This Court cannot be nore critical of this particular circumstance."Exhibit A at 14
Mr. Keel's overall attitude about the work he does is best summed up by his response to criticism in a 1995 ASCLD-LAB audit report indicating that the Crime Lab was not in compliance with TWGDAM Guideline 5.3, which requires that "a PCR laboratory will require special laboratory configuration and sample handling" to avoid the ever present danger of contamination. Keel asserted, contrary to every other expert who testified in the hearing, that , "THESE SEPARATIONS ARE RECOMMENDED TO ENSURE PCR SAMPLES WILL NOT BE CONTAMINATED. THESE ARE RECOMMENDATIONS. THIS WORK CAN BE DONE IN THE SAME ROOM IN A BARN, AS LONG AS THE -- THE PROPER CONSIDERATIONS AND GUIDELINES ARE IN PLACE TO CONTROL FOR THINGS LIKE CONTAMINATION. THERE IS NO ABSOLUTE REQUIREMENT IN REGARD TO DEALING WITH THESE FACTORS." (RT 3275-3276) Despite the ludicrousness of this statement, Keel stuck with his position upon further cross examination:
Q. SO YOUR PERSONAL OPINION WOULD BE THAT PCR SETUP, AMPLIFICATION, AND THE OTHER STEPS IN THE PROCEDURE COULD ALL BE CONDUCTED IN A BARN IN ONE ROOM WITHOUT ANY PROBLEM WITH CONTAMINATION?

A. I THINK THAT'S POSSIBLE, YES.

Q. AND DO YOU KNOW ANYBODY ELSE IN YOUR PROFESSION, OR IN THE WORLD THAT SHARES THAT VIEW?

A. I BELIEVE THAT ANYBODY WITH A DEGREE OF COMMON SENSE WOULD SHARE THAT OPINION.
RT at 3276

Judge Dondero's ruling specifically called this remark "perhaps the most memorable yet disturbing declaration in the entire record.". Exhibit A, p. 15 n. 13 As Judge Dondero correctly concluded, "(a)ny relevant documentation on this topic and certainly the case law which controls the admissibility of Keel's work product mandates that DNA analysis is technical and rigorous analysis....It certainly should not be something so basal that it can be performed in a barn." Exhibit A at 15.
Keel's carelessness is further reflected in his numerous self-graded proficiency test record "cross-outs" in which he apparently changed analytical results after being provided the target values. Denying such a practice, he admitted that in at least three self-graded tests there was evidence of cross-outs, and most remarkably he testified as follows:
MR. BURT: Q. WHAT DO YOU THINK THE CHANCES ARE, MR. KEEL, OF YOU TAKING THREE PROFICIENCY TESTS AND IN EACH AND EVERY ONE OF THOSE TESTS COMMITTING TRANSCRIPTIONAL ERRORS, ALL OF WHICH HAPPEN TO CORRESPOND WITH CHANGING VALUES TO REFLECT THE TARGET VALUES IN TESTS THAT YOU YOURSELF GRADED?

A. NONE OF THE TARGET VALUES WERE CHANGED, MR. BURT. AND AS FAR AS MY ERROR RATE IN MAKING TRANSCRIPTIONAL ERRORS, I MAKE TRANSCRIPTIONAL ERRORS VIRTUALLY EVERY TIME I PICK UP A PEN.

RT at 3969

His past work performance includes an evaluation by his supervisor that Mr. Keel is "not always as aware of the fundamental theoretical bases of some of the techniques (he) use(s) as might be desirable." (R.T. 4603, 4604) The supervisor also observed that Mr. Keel "occassionally seems to resent technical comments which may question (his) analytical approach or may contain constructive criticism from a peer." (R.T. 4606) His attitude toward counsel during Bokin indicates that this disability is still with Mr. Keel. That attitude, as reflected in a declaration filed in Bokin, was, as found by Judge Dondero, " beyond advocacy--it indicated a critical attitude toward the defense function in a criminal case." Exhibit A at 15 As also found by Judge Dondero, "(w)hile on the stand, Keel regularly indicated he chose to ignore particular protocols suggested in the literature, including that of Perkin-Elmer and peer reviewed journals." Id at 15 By his own admission, he currently sees no need to follow suggested remedial advice given to him by federally funded DNA Consortium auditors who visited his lab in late 1998 who were highly critical of his practice of failing to sign and date his numerous "cross-outs."(RT 5087) Clearly, Mr. Keel is not competent to be the technical manager of the San Francisco DNA Lab or even to be a competent analyst.
Futhermore, from the moment Bokin began and Mr. Keel submitted an incredible April 13, 1998 declaration (exhibit 4L) complaining about his discovery obligations, accusing defense witnesses and counsel of improper and criminal motives, and misleading the Court about the results of a December 1997 proficiency test survey, this witness exhibited a siege mentality that posits that a small "clique" of misguided scientists and defense counsel are out to get him. As indicated, Judge Dondero found his bias to be "beyond advocacy." Exhibit A at 14-15
Mr. Keel's hostile attitude has apparently prompted him to take on the role of an overzealous advocate. During the course of Bokin, he sat at the prosecution's table day in and day out, passing notes to the prosecutors and to witnesses, and he did this despite his complaints to Judge Dondero and to the media that he is overwhelmed with case work. His hostile attitude while testifying was evident throughout the hearing and his hostility extends even to his own supervisor, Ms. Blake, who was forced to apologize for his conduct to state auditors in a letter in which she wrote that she, "must apologize a bit for his tone. He is totally overwhelmed with work and angry about our circumstances." (exhibit 8K) The comment by Keel that prompted this apology was his assertion that, "the lab has no senior or supervising criminalist overseeing anything other than Ms. Blake who manages the overall laboratory operation in a provisional capacity as senior criminalist...the failure of the audit report to even comment on the disarray of the SFPD laboratory formal organizational and heirearchical structure is glaring absent." Id.
The bias he exhibits in his casework is of a different and more dangerous sort. The evidence before the Court in Bokin indicated that Mr. Keel has not established or adhered to the "sound conservative guidelines" touted by Dr. Holt, but rather approaches each case on an ad hoc basis and manipulates the analytical procedures until a desired result is obtained. His analysis in the Franklin and Long case in which he reran tests over and over again until he got the desired result is the best example of this bias. (R.T. 3210-3215) As indicated in NRC II, "bias in forensic science usually leads to sins of ommission rather than commission possibly exculpating evidence might be ignored or rejected. Contradictory test results or evidence of sample mixture may be discounted. Such bias is relatively easy to detect if test results are reviewed critically." NRC II pg. 84-85. Test results were reviewed critically in Bokin and the hearing in that case demonstrates that Mr. Keel's bias renders him incapable of fairly conducting DNA analysis.
Moreover, although Judge Dondero ultimately ruled that " the prosecution only barely satisfied prong three" (Exhibit A at 16)in the cases before the Court, he also concluded that his critical comments on the lab should " serve as a forewarning", and that "the prosecution should see that problems identified in Tab 32, as well as the Audit Reports and Grand Jury study are finally resolved." Exhibit A at 15-16 Tab 32, an annotated summary of Keel's testimony showing his numerous departures from the TWGDAM Guidelines, DAB Standards, the manufacturer's protocols, and Keel's own protocols is attached hereto as Exhibit E. The Audit reports before the Court were the 1995 ASCLD-Lab audit and a 1998 audit report by the State Board of Auditors, which Mr. Keel himself described as superficial. The Grand Jury study before the court was a 1995 civil grand jury report which was highly critical of the Lab.
Significantly, however, what was not before the Court were the results of a December, 1998 audit conducted by the federally-funded DNA Consortium, which was designed to be a full audit to assess compliance with TWGDAM Guidelines and DAB Standards.This audit report, which was not issued until April 7,1999 and did not become available to the defense until May 25,1999 was not available in time for Judge Dondero's May 6, 1999 ruling. The report, and the lab's response to it, are attached as exhibit B. The report removes any doubt as to whether the Crime Lab can satisfy prong three in Mr. Nawi's case. Although the report must be read in its entirety to appreciate all of the deficiencies identified by the auditors, it is important to note that the auditors found numerous deficiencies, including the following substantive problem areas:
1.Insufficient space
2.Inadequate number of personnel for caseload
3. A policy allowing casework with materials not previously QC-checked
4. A DQA1/PM interpretation policy unsupported by external validation
5. Lack of adjudicated-type samples for internal validation of Green STR I
6.Initiation of casework prior to authorization of methods
7. Inadequate documentation of technical and administrative reviews,and
8. Inadequate audit trail for equipment and materials used in testing
In regard to item 6, initiation of casework prior to authorization of methods, the auditors specifically rejected the rationale put forth by Keel in Bokin to the effect that he did not have to undergo any internal validation in San Francisco because he had already done so in Oakland. The auditors explained that this reasoning was "problematic":
The purpose of internal validation(also known as in-house) validation is to demonstrate acceptable performance of the total system: analysts, facilities,equipment, and materials. PCR systems can be sensitive to differances between particular instruments, water quality, and ambient temperature, among other things. One member of the audit team (John Hartman, also a member of the TWGDAM committee that drafted the Guidelines) states that such a transfer of validation was not invisioned by TWGDAM, hence the adjectives internal and in-house. In defense of SFPD's position, it should be pointed out that even if its internal validation had taken place within its doors and using its own equipment,over time all of the personnel, equipment, materials and even facilities could be changed without its formally needing to revalidate following each change.
Exhibit D at 8

In any event, the auditors specifically concluded that "(t)hese deficiencies and those of the documentation system should be addressed before the program implements Profiler-Plus and CoFiler testing. A follow-up external audit at that time is highly recommended." Id at 16. To the defense's knowledge, after the auditor's visit, Mr. Keel resigned and no such follow-up audit has been conducted. Moreover, the Lab's response to the audit indicates, per the pattern established in Bokin, that Mr. Keel refuses to even acknowledge that any defiencies exist, much less that he plans to take any action to remedy them. On this record, the "bare" preponderance found present in Bokin is totally lacking here as to prong three of the Kelly test.

2. The Reason That Forensic PCR Testing Has Not Achieved General Acceptance In The Scientific Community Of Molecular Biologists Is Because Serious Problems Have Been Encountered. There Are At Least Six Flaws In The Application Of Current Forensic PCR Testing Methods: Contamination, Degradation, Misincorporation, Differences In Qualitative And Quantitative Fidelity, Differential Amplification, and False Positives.

The PCR technique was first developed by Dr. Kary Mullis while working for the Cetus Corporation in 1985. Dr. Mullis won a Nobel prize for this work.See generally, Kary Mullis, Dancing Naked in the Mind Field (1998); Paul Rabinow, Making PCR: A Story of Biotechnology (1996).
Cetus owned a patent on the PCR technique which survived a lawsuit by DuPont, which claimed the technique was not patentable. Then, Cetus, and the PCR patent, were purchased by Hoffman-LaRoche (Roche). As indicated in n. 15 supra, on December 7, 1999, in Hoffman-La Roche, Inc. and Roche Molecular Systems, Inc v. Promega Corp __F. Supp. __ (N.D. Cal. Dec. 7, 1999), a patent infringement case, Federal District Court Judge Vaughn Walker recently issued a sweeping ruling in which he concluded, based on extensive trial testimony, that Roche's patent on the AmpliTaq® DNA polymerase enzyme must be invalidated because of Cetus's numerous instances of fraudulent conduct in securing the patent. Significantly for this case, the instances of misrepresention involved Cetus and Roche scientists lying about in- house scientific studies.
The PCR technique is used widely in research laboratories and many clinical laboratories, most notably immunological laboratories doing clinical work on organ transplants. Nonetheless, the scientific community has consistently expressed caution about the transfer of PCR technology to forensic applications. For example, the Office of Technology Assessment of the United States Congress reported in July, 1990:
At present, however, the enthusiasm of some for PCR applied to forensic casework is tempered. Cautionary voices warn that, compared to RFLP analysis, all the possible artifacts and steps necessary to avoid them have not been fully identified. Some believe that additional studies of PCR on simulated or real samples is necessary to ensure that problems often encountered with real samples, including DNA and non-DNA contaminants, do not interfere with accurate PCR use in forensic applications.

More recently, the NRC I began its discussion of forensic PCR testing with this observation:
PCR is a relatively new technique in molecular biology, having come into common use in research laboratories only in the last four years. Although the basic exponential amplification procedure is well understood, many technical details are not, including why some primer pairs amplify much better than others, why some loci cause systematically unfaithful amplification, and why some assays are much more sensitive to variations in conditions. Nonetheless, it is an extremely powerful technique that holds great promise for forensic applications because of its great sensitivity and the potential of its use on degraded DNA.

And the NRC ended its discussion of PCR by concluding:
PCR analysis is extremely powerful in medical technology, but it has not yet achieved full acceptance in the forensic setting. The theory of PCR analysis, even though it is the analysis of synthetic DNA as opposed to the natural sample, is scientifically accepted and has been accepted by a number of courts. However, most forensic laboratories have invested their energy in the development of RFLP technology and have left the development of forensic PCR technology to a few other laboratories. Thus, there is no broad base of experience in the use of the technique in identity testing. [emphasis added]

The reason that forensic PCR testing has not achieved general acceptance in the scientific community of molecular biologists is because serious problems have been encountered. There are six basic difficulties with the application of current forensic PCR testing methods: contamination, degradation, misincorporation, differences in qualitative and quantitative fidelity, differential amplification, and false positives.
a. Contamination
Contamination is the single greatest problem in the transfer of PCR technology to forensic testing. This is because the extraordinary ability of PCR to reproduce a single copy of DNA is also its greatest disadvantage. "PCR is not discriminating as to the source of the DNA it amplifies, and it can be exceedingly sensitive." (NRC I at 65). " Any procedure that uses PCR is susceptable to error caused by contamination leading to amplification of the wrong DNA." (NRC II at 71)
Contamination can arise in a myriad of ways. The general categories of contamination outlined by the two NRC reports are:
a) Contamination from handling in the field during collection, either by cross-contaminating samples with each other directly, cross-contaminating samples through DNA carry-over on evidence gathering instruments or the gatherer's clothing, mixing up samples, or inadvertent contribution from the biological products from the evidence gatherer him or herself (sweat, sneezing, dandruff, etc.). "The important consequences of those sorts of contamination are that samples might appear to be mixtures from several persons and, in the worst case, that only the contaminating type might be detected. The concern is greater with PCR-based typing methods than with VNTR analysis because PCR can amplify very small amounts of DNA. A false match could occur if the genetic type of the contaminating materials by chance matched the genetic type of a principle (such as a suspect) in a case or worse, if the contaminant itself came from a suspect in the case." (NRC II at 83). In this case, Allen Keel testified in Bokin that he does not get involved in monitoring the procedures for evidence collection because such procedures are implemented by crime scene investigators not subject to his control( See Exhibit E, summary of Keel testimony at 16-17);
b) In the laboratory, cross-contamination of samples to each other, or contamination emanating from the evidence handlers and their instruments, while samples are being manipulated, sorted, labeled, dried, cut, before, during, and after the DNA extraction process. "For example, in the loading of an electrophoresis gel, a sample loaded in one lane might leak into an adjacent lane, which might then appear to contain a mixed sample. Confusion resulting from lane-leakage problems is typically avoided by leaving alternate lanes empty or by placing critical samples in nonadajacent lanes, and this should always be done." (NRC II at 82) In this case, the evidence in Bokin established that Mr. Keel has had numerous lane-leakage and broken test tube problems and that "as a matter of convenience" he does not leave alternative lanes empty. (Exhibit E at 14, 24) The evidence also showed that he does not follow the recommendation of the NRC II to seperately test evidence and reference samples.(Id. at 8) It also established that Keel does not believe in, and therefore does not follow, the recomendations of the F.B.I. and others that DNA lab technicians should wear protective hair nets and face masks.(Id. at 6). It further established that he uses scissors to cut evidence and then cleans the scissors with ethanol or water, a procedure all other experts opinioned was totally inadequate to control for examination. (Id. At 5-6) As indicated above, Keel incredibly believes and acts on the belief that PCR contamination is not a concern and that PCR testing can be carried out reliably in a barn. (RT 2378);
c) Contamination from contaminants in solutions, reagents, aerosols, water, and even in Taq DNA polymerase. As the NRC I warns, "(e)ven the simple act of flipping the top of a plastic tube might aerosolize enough DNA to pose a problem." (NRC I at 66). The NRC II recommends that " adherence to a standard QC program provides safeguards against these kinds of laboratory error." (NRC II at 82). In this case, two of the specific criticisims of the recent December 1998 federally sponsored audit were that the SFPD DNA lab had " (a) policy of allowing casework with materials not previously QC-checked", and that the lab had "(i)nadequate audit trail for instrumentation and material used in testing."( Exhibit B at p. 2). The evidence in Bokin also established that the lab routinely ignored indications of contamination in its positive and negative controls, that it keeps more than one test tube open at a time contrary to the kit manufacturer's protocol, and that at one point, prior to the testing in this case, the lab discovered contamination in its water supply and yet, contrary to standard practice, the lab continued to operate instead of shutting down and redoing all casework conducted to date. (Exhibit E at 6-9, 17-19).One article produced at the hearing (See, section III A. 2.c.iii, # 5, infra)indicated that contamination has been documented even in Taq DNA polymerase, which is the essential ingredient in all the labs PCR reactions. Mr. Keel testified he was unaware of the article or the problem. (RT 3096);
d) PCR product carryover contamination - the contamination of evidence samples or reaction solutions with PCR products from prior amplifications. "The most serious problem is contamination of evidence samples and reaction solutions with PCR products from prior amplifications." (NRC I at 66). " A false match can occur if the genetic type of a contaminant matches by chance the genetic type of a principle in the case; in the worst case, the contaminant originates from another party in the case." (NRC II at 84). The NRC II recommends among other precautions the maintenance of a one-way flow of material and the use of chemical sterilization precautions. In this case, the SFPD follows neither of these recommendations and the federal auditors specifically found that although " post-PCR manipulations are restricted to a single room...photographs of PCR product gels and dot-blot strips are returned to the DNA extraction room for storage in three-ring binders with other analytical records." (Exhibit B at 13); and
e) Mixed samples -- contamination from the fact that a crime scene sample, such as blood stain, could be a mixture of bloods or other biological fluids or substances. NRC I at 65-67; NRC II at 84. The NRC I states that "(a)s a rule, mixed samples should be interpreted with great caution....Interpretations based on quantity can be particularly problematic--eg., if one saw two allelles of strong intensity and two of weak intensity, it would be improper to assign the first pair to one contributor and the second pair to a second contributor unless it had been firmly established that the system was quantiatively faithful under the conditions set." (NRC I at 66). In this case, as is set forth more fully below, Keel can include mr. nawi as a possible contributor to a twelve year old fingernail sampling only by "inferring" the DQ alpha type of the sample based on a dot blot intensity analysis that is not even endorsed by Keel's then reviewer and now employer Dr. Blake. In sum, no matter where one turns in this case, there are major methodological flaws in the PCR testing performed by Allen Keel and his assistants.
Recognized leaders in the scientific community, unaffiliated with laboratories who have a vested interest in the immediate implementation of current forensic PCR methods, have urged against the use of such testing until more stringent controls can be employed to guard against sample contamination, including independent blind proficiency testing of laboratories. The views of these scientists alone preclude a finding of general acceptance.
First, consider the observations of Dr. Richard Lewontin, a pre-eminent geneticist who has been profoundly influential in the scientific debate over forensic DNA testing:

The usual scenario in a criminal trial is that a small amount of dried blood, semen or tissue scraping is recovered from the scene of a crime. A suspect is then identified, and a sample of his or her blood is taken on cubic-centimeter amounts. DNA from the crime scene scraping and from the suspect's blood sample are then compared side by side in the same laboratory. (Sometimes the tissue or dried blood is found on property of the suspect and it is the victim's DNA that is to be matched with it.) While there is more than enough DNA recoverable from the suspect's large blood sample to carry out the needed procedures, the very small, and often degraded, sample from the crime scene does not contain sufficient DNA for the comparison. To obtain sufficient material, the DNA from the crime scene is "amplified," that is, copied thousands or millions of times in a procedure known as polymerase chain reaction (PCR). As the name of the procedure suggests, the original small number of DNA molecules are copies once, then these copies plus the original are copied a second time and so on for a number of cycles, increasing the total population of molecules exponentially until a sufficient amount has been produced for the matching procedure. The problem with the PCR technique is that because of its chain nature, contaminant molecules in the original sample may also be amplified and, since the original crime scene sample contained so few molecules, contaminants may overwhelm the original in the amplification. In addition, small differences in DNA sequence can have very large effects on the relative amplification of the components in an original mixture.
Now consider the actual practice in a forensic DNA laboratory. A technician is handling two samples. One is the very large DNA sample from the suspect's blood, the other is the minuscule DNA sample from the crime scene, which is then amplified by PCR. The situation is ideal for PCR contamination, with the result that the suspect's DNA will not really be compared with that from the crime scene, but with his or her own DNA that has just been replicated in the PCR reaction. The result will be a perfect match.
All of us who use the PCR technique regularly are acutely conscious of the contamination problem, and the best laboratories have suffered occasionally from it. The perspiration and "oils" on fingertips have provided enough DNA contamination in PCR experiments to give completely artifactual results. Only careful replication catches these errors, and some errors have not been caught until much later when another laboratory found conflicting results. In the forensic context, where the liberty and even life of the suspect is in question, it is essential that courts be assured that laboratories are taking careful precautions against these contamination errors, not to speak of grosser errors of recording, etc. Representatives of commercial laboratories that have previously been found to provide erroneous results have told interviewers that they have "cleaned up their act." Perhaps they have, but we cannot know without independent checks, and anyway what about the people convicted before they "cleaned up their act"? The FBI has consistently refused to allow independent quality control checks, relying on their own internal procedures.
In a forensic context, where the liberty and even life of a suspect is at stake, there must be frequent, independent and unannounced inspections and tests of DNA laboratories, on the model of the inspections carried out by radiation safety officers and the Department of Energy of laboratories using radioactive materials. The issue is certainly too important to be dismissed by the unsubstantiated opinion of someone not familiar with the technical procedures at first hand. If the data themselves are unreliable, questions of probabilities of alternative suspects are irrelevant.

R. C. Lewontin, Comment: The Use of DNA Profiles in Forensic Contexts, in "DNA Fingerprinting: A Review of the Controversy," 9 Statistical Science, 259-260 (1994).
Dr. Richard J. Roberts won the Nobel prize in genetics for his work in the area of restriction enzymes. He has testified numerous times as a prosecution expert in cases involving RFLP analysis and traditional Southern blotting methods. Dr. Roberts has submitted a number of affidavits opposing the use of forensic PCR analysis. In an affidavit submitted for consideration at a Frye hearing in Washington v. Gentry, he had this to say:

First, I will make some general comments about PCR technology. The technique is an extremely powerful one. In principle, and occasionally in practice, a small region of a single DNA molecule can be amplified sufficiently to allow its detection. This means that DNA from a single cell in principle can be detected by the method. It is this exquisite sensitivity that makes it desirable for use in a forensic setting where often only limited amounts of material may be available. Unfortunately, it is this very sensitivity that makes it also prone to the possibility of contamination. This can result in the amplification, not of the small amount of sample that was intended, but rather of a small amount of an unintentional contaminant. Indeed, problems of contamination are a major source of error in the laboratory and extraordinary precautions need to be taken when performing multiple PCR experiments to avoid amplifying samples that have become contaminated through aerosols or sloppy laboratory procedures. Of course, knowing that such contamination can take place means that when working in a laboratory one takes the necessary precautions to avoid it. Unfortunately, the samples that are collected from crime scenes are, by their very nature, usually contaminated to begin with! It thus becomes extremely important that proper checks and controls are carried out to be certain that the product amplified from a particular sample is from the sample and not from the contaminant. How can this be done? Unfortunately, to the best of my knowledge there is no generally accepted method by which this can be guaranteed at the present time. The reasons are described below.
PCR is a technique that is still relatively new. It has only been in routine use in academic laboratories during the last two years and so our experience with the method is itself fairly limited. This experience for the most part has involved the pristine academic laboratory setting. Much less experience is available for forensic samples. In a laboratory setting it is usually possible to take the great precautions that are necessary to avoid dealing with mixed samples. Nevertheless, it occurs and occasionally amplification results in bands other than the ones expected. In the laboratory one can usually go back and repeat the experiment, perhaps changing reagents or merely being more careful about sample handling. That such artifactual amplification will happen from time to time is to be expected.

Bokin Exhibit 27,Roberts Aff., February 22, 1991, Washington v. Gentry, at 2-3 [emphasis added].
Finally, and perhaps most significantly, there is the position of Dr. Kary Mullis himself, the man who developed the PCR technique and won a Nobel prize for doing so.
Dr. Mullis believes that the PCR DQ-alpha test should be used for exclusions but not inclusions. He has publicly taken this position, and criticized the inadequacy of controls being used to protect against contamination, in two Kelly hearings in the past two years. See, Bokin Exhibit 28, Mullis testimony, People v. McIntosh & Schlaepfer and People v. Moffet.
In the course of his testimony in People v. McIntosh & Schlaepfer, Case No. 33026, Tulare County Superior Court, on August 23, 1994, Dr. Mullis touched upon the major issues the defense is raising here.
First, Dr. Mullis specifically addresses why special care must be taken in the collection, handling, and preservation of crime scene samples when PCR testing is going to be done, and why such procedures must be considered a part of the forensic PCR testing method:
Q. Okay. What I'm pointing at, for the record, is the DNA technology in Forensic Science by the National Research Council that was published in '92. I forgot what exhibit it is.
But, in any event, you are familiar with some of the findings and the information that's contained in that report they submitted in '92?
A. Yes.
Q. Looking at page 52 where they distinguish between the DNA diagnostics and DNA forensic where they give descriptions, I'm just curious whether this accurately represents what you're describing here when you're talking about diagnostics. You're usually involving clean tissue samples, known sources, repeated -- usually can be repeated comparison of discrete alternatives. Is this the kind of stuff that you're acquainted with?
A. Right. You have control over everything. Not over everything, but over many more things when you're in a diagnostic situation than if you're just selecting samples that have been laying around.
Q. So where they describe the forensic differences that are involved in forensic typing, they're speaking of degraded and contaminated possibilities, multiple unknown sources, inability to repeat always, matching of samples that can be from a wide range of alternatives. These are the kinds of things that you're aware of?
A. Yes, it's much more difficult to know that your results are really what they seem to be in that situation. I think -- criminalists, I think, are the people that go around and collect that stuff. They probably will get better at it. They'll eventually know what you can and can't do. But it's a vast problem compared to what you have in a clinic or in a hospital.
McIntosh, Mullis at 2340, line 17 to 2342 line 3.
Q. I didn't ask the question very well, I'm sure. But I think you answered it. Just to make sure though, what I'm talking about is that let's say you have samples that are picked up at a crime scene and not handled carefully and contamination could occur, a cross-mixing of various samples of DNA. Because of PCR's inherent sensitivity, if you ran PCR procedure on that DNA and then attempted to type it using the dot blot method, for example, would it be-- tend to be more sensitive to that error because of the evidence that was handled in PCR versus some other type of typing?
A. Yeah, I think I answered saying in certain cases when you wouldn't always know when that was, it certainly would be. In other cases, it introduces an uncertainty there that you don't have it you don't amplify. Because if you don't amplify and you get a thousandth part contamination, you know it will still be a thousandth part when you get to the analysis. It will be a wispy little thing somewhere and you'll say that's probably an artifact.
If you're going to amplify it and you pick up a contaminant that happens to be a lot easier to amplify than the sample that you really want to amplify, then that could become a real problem, because ten cycles gives you a thousand fold amplification, 20 cycles gives you a million, 30 cycles, which these people are doing, 30 or 32, gives you a billion.
Q. Thank you. Now, you said something this morning when you were talking about the application of PCR to the research and diagnostics area and then you were asked about the application in the forensics area. I think you mentioned that forensics is not the same as research and diagnostics. And then you said the certainty level is a lot lower, and I'm not sure what you meant by that.
In other words, when you're using PCR in the forensic area, is the certainty level lower than you would expect it to be?
A. I don't think the certainty level of the PCR reaction inherently would be any lower. It doesn't matter what it's being used for. The certainty level of the whole analysis involves the collection, the maintaining, the processing of the samples.
McIntosh, Mullis at 2387 line 2 - 2388 line 24.
Similarly, Dr. Mullis decisively rebuts a common prosecution contention that PCR-based tests must be assumed to be free of contamination if the negative amplification control (the "blank control") that comes with the Roche kit does not light up. Echoing the NRC's warning that a blank control will not necessarily reveal contamination ("[E]ven in a laboratory contaminated with PCR carryover, blank controls do not necessarily become contaminated on every occasion." NRC 1 at 67), Dr. Mullis describes the dimensions of the problem:
Q. Now, you said earlier something, I won't go into the details, but it is listed here on page 67, that blank controls do not necessarily become contaminated on every occasion. This is a problem?
A. That's a tough problem, too. It is. How many blank controls are you going-- like I said, you cannot ever show that there wasn't one contaminating molecule in a bottle without taking the whole bottle and putting it into the PCR reaction. Once you've done that, you got to make another bottle. Just like you can't ever know if in fact there's just one, you know, virus in your blood stream if you're looking for a dangerous virus. You can't sample the whole thing. You don't sample the whole bottle of a particular reagent when you take a sample of it.
If it's highly contaminated, the statistics say you stick your pipette in there and take it out and you would get at least one of the contaminants. If there's only ten or fifteen in there, it will only be ten or fifteen times when you're using that bottle that you end up pulling one of them out. You never know when that would be. So you have to have five controls and they come out blank and then the unlucky time you might stick it down in there and put it into one of your experimental tubes and it will show up there, but not in any of your controls.
McIntosh, Mullis at 2375 line 21 to 2376 line 23.
On cross examination Dr. Mullis remained firm in his contention that forensic PCR tests were not as reliable as other diagnostic clinical applications, particularly when analysis was being performed on degraded samples.
Q. As far as the actual basic science of the diagnostic laboratories using PCR, that is no different than a forensic lab using PCR, correct?
A. The basic science is basic science. I suppose the problems in the forensic lab, we have tried to list some of the ones that are in a forensic lab where it's very difficult or impossible to even make a good shot at solving them. Where in a clinical setting it is possible to take precautions that make your results much more reliable. That's what I was talking about just before the break which had to do with being able to know exactly how much DNA you're adding, how many copies of what it is you're looking for to expect and to set up the conditions of the amplification in accord with that.
If you have a sample that's degraded, you don't know how much DNA's in it, you just have to take a shot at it. And you might go too many cycles and, therefore, bring a contaminant up to a level that it will look like if it wasn't a contaminant.
McIntosh, Mullis at 2420 line 1 to line 24.
Finally, Dr. Mullis rejected the idea that combining, as the prosecution has done in this case, several different DNA systems would make the PCR testing any more reliable:
Q. In a case where we use several different systems, one including DQ-alpha as a site, we use RFLP markers as other sites, conventional serological evidence as other sites, would the combination of all those sites be useful?
A. If there weren't so much trouble with reliability on the DQ-alpha thing, that would certainly be data that you could add up together and say well, the probability based on DQ-alpha is that he's in a 17 percent group. The probability based on blood typing is that he's on a 30 percent group. The probability that you do with some RFLP or a couple of those puts him in an even smaller group. But you still would come up with maybe a number of, say, two out of a hundred people might or two out of a thousand or one out of a million maybe, if you're lucky. Depends on the data that you collect, whether it's rare data or whether he's got common things at those places.
But the reliability of the RFLP data, it says why add that to this whole mishmash of stuff that you've got? Why take some piece of evidence that you can find plenty of scientists will come in and say I wouldn't trust it on myself? I would repeat it over and over. I've got this problem with contamination. I've got this one with differential amplification.
This method relies on too many things that are easy to get wrong to make it really worth pursuing. That's all I'm saying. I'm saying why pick a method, why use a method that is so subject to human error and to just things that are way beyond our control? Why pick a method like that to use in a Court of Law? You wouldn't use that on your taxes.
McIntosh, Mullis at 2450 line 12 to 2451 line 21.
Other molecular biologists from the academic community and the clinical community have expressed similar objections to the concerns voiced by Drs. Lewontin, Roberts, and Mullis. For instance, Dr. Ashok Bhagwat, a professor in the Chemistry Department at Wayne State University, had extensive experience studying molecular biology at Cold Spring Harbor where he received his post doctoral training working under Dr. Richard Roberts. In Washington v. Gentry, Dr. Bhagwat offered these succinct views:
Q: Dr. Bhagwat, is PCR technology generally accepted in the scientific community for use on crime scene evidence?
A: No, it's not.
Q: And could you describe to the Court some of the general reasons why you have that opinion?
A: There are several problems with the material that you are likely to obtain at a crime scene. It is most likely to be contaminated. And let me just slightly digress and say, PCR is so powerful that a single cell contamination could be devastating to the result, and hence, contamination is a big potential problem and it is unlikely that it is easily solvable for evidence arising from crime scenes.
Bokin Exhibit 29, Washington v Gentry testimony, 28 RP 2238-2239.
Dr. John Gerdes is clinical director at Immunological Associates of Denver, a reference laboratory that uses the same PCR technology involved in this case in conjunction with transplants (matching organ donors and recipients) and screening for AIDS and other infectious diseases. He has testified over twelve times expressing, from the hands-on perspective of one who runs a clinical laboratory, the same objections raised by Drs. Lewontin, Roberts and Mullis. Dr. Gerdes testified most recently in the McIntosh case, in tandem with Dr. Mullis, and the defense submits this transcript for the Court's edification.(See, Bokin Exhibit 30).
Most of the discussion so far has focused on cross-contamination of forensic samples in their collection, preservation, handling, and processing. Before leaving the subject of contamination, however, special mention must be made of the phenomenon of PCR product contamination: contamination of samples by PCR product from previously run reactions in a laboratory. This is sometimes called "carryover contamination." One of the developers of the Cetus/Roche kit, Dr. Russell Higuchi, describes the phenomenon:
More unique to PCR is the possibility of carryover contamination from a completed PCR to another sample yet to be amplified. Because by the nature of PCR, PCR product will seed production of more PCR product, the sheer number of copies of PCR product after amplification can make the consequences of such contamination more dramatic. A typical PCR could have 1012 copies of an amplified gene. If a preparer inadvertently transfers, as before, 0.1 l of PCR sample A into sample B, even though sample B has a relatively high concentration of human DNA, the number of copies of the target, single-locus gene that derive from sample A far outnumber the copies that actually stem from sample B. Thus, the DNA type obtained will be that of A and not B, and the relative amount of the B type is so small that it would not even show up in the test, eliminating the possibility that the presence of more than two alleles would flag the occurrence of the contamination.

The NRC highlighted the problem of PCR carryover contamination as a key area of vulnerability, declaring "it has become clear that carryover products from one PCR reaction to another must also be eliminated." NRC 1 at 67. Accordingly, the NRC commented that "[m]ethods of detecting and preventing contamination from one PCR reaction to another in forensic laboratories are generally still in their early stages, and additional development should be encouraged."
It is plain, however, that forensic PCR laboratories have not responded appropriately. For example, the NRC pointedly suggested that it would be a good idea to treat all evidence samples with uracil N-glycose (UNG) before amplification to destroy any PCR carryover from previous PCR reactions. Id. Clinical laboratories have followed up on this idea. Dr. Gerdes reports that his and other clinical laboratories "don't do anything without UNG anymore," McIntosh, Gerdes, 1915 line 6, and that the UNG control was featured in a PCR chlamydia kit which won recent FDA approval. Id., 1914 line 19. Forensic PCR laboratories, including the one which did testing in this case, do not use UNG or any other chemical treatment to protect against carryover contamination.(RT 3012-3013)
Most importantly, since PCR carryover contamination can be a systemic, cumulative problem in a laboratory, the NRC demanded that controls must be in place to monitor "general contamination" in forensic PCR laboratories:
In view of the problem of contamination due to handling and carryover, laboratories must incorporate contamination control into their standard operating procedures. And outbreaks of contamination and the steps taken to correct the problem should be documented.

NRC 1 at 67.
In this case, as indicated above, in December 1998, two federally sponsored auditors concluded after careful inspection that the SFPD DNA lab had " (a) policy of allowing casework with materials not previously QC-checked", and that the lab had "(i)nadequate audit trail for instrumentation and material used in testing." ( Exhibit B at p. 2).The evidence in Bokin also established that the lab routinely ignored indications of contamination in its positive and negative controls, that it keeps more than one test tube open at a time contrary to the kit manufacturer's protocol, and that at one point, prior to the testing in this case, the lab discovered contamination in its water supply and yet, contrary to standard practice, the lab continued to operate instead of shutting down and redoing all casework conducted to date. (Exhibit E at 6-9, 17-19).

b. Degradation
One of the reasons both TWGDAM and the DAB Standards require extensive developmental and internal validation of each PCR testing platform is to ensure that each platform can reliably type specific kinds of samples under a variety of environmental circumstances encountered in crime scene samples. TWGDAM Section 4.1.5.6 specifically requires that the required developmental validation must include environmental studies "so that the effects of factors such as matrix, age, and degradative environment (temperature, humidity,UV) on a sample are considered." See also, DAB Standard 8.1.2.2 (The developmental validation shall include... species specificity, sensitivity, stability, and mimixture studies...").
In this case, the Crime Lab purports to be able to accurately type thirteen year old fingernail and victim reference samples allegedly containing DNA profiles of at least three persons. As indicated above, before such testing is admitted into evidence, it is generally accepted that developmental and internal validation must be conducted to demonstrate that typing of aged fingernails is possible. Although the scientific community has begun to examine this issue, the existing data is fragmentary and raises more questions than it answers. It is far to early to conclude that typing of aged mixed fingernail scrapings is generally accepted as reliable.
Even as to the typing of fresh fingernails, problems have been encountered because of the unique nature of fingernails. So far, it has been possible on fresh samples to obtain only highly degraded DNA. One recent article discussed below proposes the following possible explanation:

Although DNA profiles are currently reproduced from different biological sources( hair roots, semen, faeces), keratin cutaneous appendages have been traditionally regarded as refractory to DNA extraction and genomic analysis....Nails, as other cutaneous structures, derive from hyperkeratinization of epidermal cells... A tentative explanation for the highly degraded status of harvest DNA...could be in the apoptosis ( cellular programmed death, associated to nuclear DNA fragmentation) to which keritinized tissues undergo. To follow their psysiological fate, nail cells are likely to activitate specific biochemical pathways, leading to nuclear membrane disintegation and keritin content increase. In this process, the DNA is set free in the cytoplasm, it is attacked and psysiologically degraded by nucleases.
C. Capelli, et. al., Fingerprints From Fingernails?, Advances in Forensic Haemogenetics 7 (1997) p. 100-102.

To date, other studies considering the problem of degradation in general or the typing of fingernails specifically have been equally tentative, or, if not, have indicated definite problems in typing degraded material. A fair overview of this literature is as follows:

1. L. Potsch et al, HLA DQ alpha Typing of Human Fingernails,Advances in Forensic Haemogenetics 4 (1991) 99-101: "In contrast to the extensive knowledge about protein biochemestry of keratinization, little is known about the fate of nuleic acids during these processes. It has been suggested that both DNA and RNA completly degenerate in the initial phase of keratinization. However, from electron microscopial studies it is known that nuclear remnants are present. From these findings and our own observations one might expect that nucceic acids should still be available...Although our study describes preliminary experiments and must be confirmed by further investigations the persistence of high-molecular-mass DNA within (fresh fingernail) cells which die in their normal differentiation may give new aspects to the process of keratinization."

2. R. Reynolds, et al., The Development and Evaluation of New Genetic Markers for the Application of PCR to Forensic Casework,Advances in Forensic Haemogenetics 4 (1991) 29-31: "Frequently forensic DNA samples are degraded to some extent. It has been demonstrated previously that amplification of severely degraded DNA samples yields either the correct HLA DQA type or no type. More recent studies indicate it is possible to obtain ambigious and even incorrect D1S80 types from moderately degraded DNA samples."

3.P.T. Bilous, et. Al. DNA Analysis of human Blood Recovered from Explosion Debris,Advances in Forensic Haemogenetics 5 (1993) 265-267: "The majority of bloodstains (11 of 15 ) yielded DNA typing profiles ( single locus RFLP analysis) consistent with the donor control samples. Partial digestion, altered migration, and weak intensity bands, were encountered in the remaining four samples. Three of these samples were found exposed to the soil."

4.G.A. Drago, et. Al., The Degradation of a VNTR Polymorphic Site In Various Biological Specimens When Subjected To Different Temperature Conditions,Advances in Forensic Haemogenetics 5 (1993) 271-274: "These results indicate that DNA may be more labile than previously observed....DNA from liquid semen shows some degradation even within 1 week. Material extracted from liquid blood and semen stored at 37 degrees C shows weak band patterns after one week and no bands at all were detected after 4 weeks. The observation in some instances in material from liquid semen and semen stains, both stored at room temperature, of bands additional to those expected gives cause for concern and warrants further investigation. In this limited study although such bands were seen in association with the bands which were expected to be present in the sample they could appear stronger than the expected bands." (RFLP)(Department of Haematology, The london Hospital Medical College)

5.M. Prinz, et. al., Effect of Degradation on PCR Based DNA Testing,Advances in Forensic Haemogenetics 5 (1993) 375-378: " Using DNA polymorphisms with short alleles and the PCR approach it is possible to acheive results from minimal of DNA and degraded samples. But, as has been described e.g. by Reynolds et al. (1993) for D1S80, the degradation can lead to dubious or even wrong typing results through causing preferential or sole amplification of the smaller allele. In cases where the degree of degradation cannot be tested beforehand, degradation artefacts will not always be expected and recognized.

6. P. Wiegand et al., DNA Typing if Debris From Fingernails, Int. J. Leg. Med. (1993) 106:81-83:" especially important is the method of preparation. A careful but not extensive cleaning procedure is the most successful because more extensive procedures lead to an excess of DNA from the fingernails of the scratcher. This can give rise to allelic drop-out or to a quantitative suppression of the other contributor."

7. P. Fattorini et al., False Results in the HLA-DQ alpha Typing: Two Cases Reported, Advances in Forensic Haemogenetics 6 (1995) 272-274: " two cases are reported where ambigious results by HLA-DQ alpha typing...were obtained. The case one refers to the DNA typing of 13 -year -old bloodstains. The reasons of these results are unknown and several mechanisms could be involved: contamination by iexogenous DNA, inhibitors, etc."

8. C. Augustin et al., Identification Of Human Remains:Short Tandem Repeat Analysis Versus Mitochondrial DNA,Advances in Forensic Haemogenetics 7 (1997) 83-84: Parts of a severely decomposed body were analyzed at 8 STR loci. "PCR-analysis... led to contradictory results between the bone and muscle tissue of the corpse. ..(I)t remained unclear why the bone did not fir to muscle tissue and hair of the corpse"

9. C. Capelli, et. Al., Fingerprints From Fingernails?,Advances in Forensic Haemogenetics 7 (1997) 100-102: " although DNA profiles are currently reproduced from different biological sources ( hair roots, semen, faeces), keratin cutaneous appendages have been traditionally regarded as refractory to DNA extraction and genomic analysis....Nails, as other cutaneous structures, derive from hyperkeratinization of epidermal cells...The aim of this study is to evaluate nails as a candidate to forensic analysis and to test several inherent extraction procedures to optimize the harvest of DNA from this tissue...Short incubation and pK concentrations ... returned highly degraded DNA from fragments down to 100 bp. Unexpectedly, these samples were not amplifiable (at STR loci), by the presumable coextraction of inhibitors (keratin itself could be one). In this case, inhibitors are possibly not destroyed, as is with prolonged enzymatic digestion. On the other hand, incubation with very low pK concentration should let both DNA and inhibitors be released and the resulting samples still amplifiable. A tentative explanation for the highly degraded status of DNA harvest DNA...could be in the apoptosis ( cellular programmed death, associated to nuclear DNA fragmentation) to which keritinized tissues undergo. To follow their psysiological fate, nail cells are likely to activitate specific biochemical pathways, leading to nuclear membrane disintegation and keritin content increase. In this process, the DNA is set free in the cytoplasm, it is attacked and psysiologically degraded by nucleases.)

10. R. Roby, The Extraction of DNA From Human Nail Material, Proceedings From the Seventh International Symposium on Human identification (Promega Corp. 1996) p. 133: "In order to process nails as evidence, a validation study was performed to demonstrate that DNA could be susuccessfully extracted from human nail material. Organic extraction of aged (8 year old) human nail material yielded a sufficient quantity of DNA for successful mtDNA sequencing; however, STR analysis was unsuccessful." (Study by The Department of Defense DNA Registry).

This review of the literature indicates persuasively that although some initial validation work has been done on the typing of human fingernails, the state of scientific knowledge is not such that typing of thirteen year old fingernails scrapings is accepted as reliuable even by forensic scientists, let alone by the broader community of molecleur biologists. In fact , if the last quoted study by the United States Department of Defense is to be credited, reliable typing of such aged stains is not possible.

c.Misincorporation, Differences in Qualitative and Quantitative Fidelity, and Differential Amplification.

PCR amplification products do not always faithfully represent the starting material in the sample, either qualitatively or quantitatively.
During PCR amplification nucleotides are known to be "misincorporated" at the rate of one per 10,000 nucleotides per cycle. If amplification is performed on a sample that has a large number of molecules, and if the misincorporation is random ("stochastic fluctuation"), then the low frequency of random errors will not skew typing results. The NRC I warns, however, that "for systems in which misincorporation is not random," difficulties will arise. NRC I, at 64. In particular, in DNA systems that contain tandem repeat sequences, "the DNA polymerase can slip during amplification, introduce or delete copies of the repeat, and produce a heterogeneous collection of fragments, often making interpretation difficult." Id. Recent literature shows that the misincorporation problem has been the most serious for STRs and D1S80 (See, section III A. 2.c.iii, # 1-4, 6, 16, 18 infra).
Differential amplification is also a potential problem:
In some cases, PCR can be qualitatively faithful but quantitatively unfaithful, because some alleles amplify more efficiently than others. A sample might contain a 50:50 mixture of two alleles and yield an amplified product with a 90:10 ratio. Differential amplification can arise through several mechanisms. It has been observed in the amplification of allelic products of different sizes (larger products tend to amplify less efficiently than shorter products) and in the amplification of sequences that differ significantly in GC content (because of differing denaturation efficiencies). In some cases, faithful amplification occurs at some temperatures and differential amplification at other temperatures. The possibility of differential amplification needs to be addressed in the design and development of amplification protocols for each genetic marker system. The safeguards to ensure that differential amplification does not occur should be defined and documented.

NRC 1 at 64-65.
When dealing with mixed forensic samples, especially small degraded samples of the kind at issue in this case, quantitative analysis with current forensic PCR methods can be, in the NRC's words, "problematic." Id. at 65. The example selected by the NRC I as representative of this problem is the exact dot blot intensity analysis used by Mr. Keel in this case. The NRC I also notes that " (i)deally, primers should amplify only the desired target locus. However, nonspecific amplification can be seen, if one amplifies for extended cycle numbers. Limits on the cycle number might be required as a safeguard against nonspecific products" Id. at 65. The evidence in Bokin indicates clearly that Mr. Keel does not monitor his cycling parameters. (See, Exhibit E at 5)
It should be noted that Dr. Mullis believes that PCR based sequencing methods, such as those being developed now on mitochondrial DNA, can avoid the problems of quantitative and qualitative amplification:
But the sequencing -- the reason I was so strong on sequencing is that it doesn't rely -- sequencing doesn't rely on the quantitative accuracy in the amplification of different bands. The quantitative aspect is not as important. Just as long as you've got enough to sequence, it won't matter if you were sort of inefficient in your amplification if you have enough to sequence. If you don't, you can just amplify some more until you do...
If you're sequencing -- if you're sequencing with PCR amplification, [inefficient amplifications] won't change the results. It will say well, you didn't get enough to even get a sequence, so go back and amplify some more. It won't change the sequence. The data that you come out with will be the same, regardless of whether any of those things are wrong.
McIntosh, Mullis, 2377 line 13 to 2378 line 13.

d. False Positives
i. Introduction In their original points and authorities in support of admission of DNA evidence in both Bokin and McClanahan, the People provided the court with an impressive looking Appendix containing 95 citations to articles in forensic science trade journals.This list of titles was presented for the apparent purpose of demonstrating an alleged universal acceptance of all forms of PCR-based testing in every aspect of science and medicine. The court was also informed that "(t)he People are not aware of any publications questioning the scientific reliabilty of PCR-based DNA analysis and population databases." ( Bokin Points and Authorities at p. 76 )
The People's claimed ignorance is reminiscent of the prosecution's two Kelly-Frye experts in People v. Morganti (1996) 43 Cal. App. 3rd 656, both of whom testified in April, 1993 that they were "not aware of any scientists who work in this field or of any publications expressing the view that PCR analysis is unreliable." Id. P. 664; See also, People v. Wright (1998) 62 Cal. App. 4th 31 (one of the same experts, Ed Blake, provided the same testimony in August, 1994 )
A close look at the articles cited by the People reveals that many of them do not consider the specific methods and procedures used by the San Francisco Crime Lab in this case. Moreover, most, if not all, of the articles have been authored by law enforcement personnal whose livelihood is dependent on court acceptance of PCR or by employees of companies who stand to make huge profits from such acceptance. As noted in the National Research Council's first report on DNA Technology in Forensic Science (NRC I ): " There is reason to be skeptical of entrusting any important regulatory matters to a self-regulating organization.... There has been a notable dearth of published research in forensic DNA testing by scientists unconnected to the companies that market the tests." (p.162)
More fundamentally, it was not true when Morganti and Wright were litigated, and it is certainly not true today, that there are no peer reviewed publications, by disinterested scientists, questioning the scientific reliabilty of PCR-based DNA analysis and population databases. The People's pleading in Bokin made the claim that the defense had not cited any scientific literature which post-dates the Wright appellate decision in 1998. But since Wright, contrary to Soto, explicitly refused to consider any testimony by defense experts that was elicited after the Kelly hearing in that case ( 62 Cal. App. 4th at 41-42 ), and Morganti expressly but erroneously holds that " (s)ince... a court should ordinarily confine itself to the record on matters of a highly complex nature, we have not consulted outside literature in this case " ( 43 Cal. App. 4th at 663 n.10) , the defense urges that the dates of the Kelly hearings in those cases should be the proper focus of the inquiry as to whether " the prevailing scientific opinion has materially changed". People v. Venegas (1998) 18 Cal. 4th 47 In what follows, the defendant will attempt to provide a summary of only some of the most recent general scientific and forensic literature which calls into serious question the reliabilty of PCR-based DNA testing in medical science in general and forensic science in particular. Responsive to the People's pleadings in Bokin, the focus here is on the more recent literature in order to demonstrate that to the extent they are relevent at all, Morganti and Wright should not be controlling in this case because the prevailing scientific opinion has materially changed since those cases were litigated in April 1993 and August 1994.

ii.Recent Scientific Literature Demonstrates That Prevailing Scientific Opinion Has Materially Changed With Respect To The Reliability Of PCR-Based DNA Testing In Medical Science

A recent MEDLINE search of scientific articles on "false positive PCR tests" revealed a total of 315 general nonforensic medical science articles in the period 1993 to the present, as compared to only 82 such articles for the period 1989 -1992. Abstracts of only some of the most recent articles are as follows:
1. 7. Borrow R; Guiver M; Sadler F; Kaczmarski EB; Fox AJ False positive diagnosis of meningococcal infection by the IS1106 PCR ELISA FEMS Microbiol Lett, 162(2):215-8 1998 May 15 : At a time when optimal case ascertainment for meningococcal infection is a high priority, the need for non-culture case confirmation, in particular by DNA amplification, is seen as being of vital importance to assist contact management and cluster recognition. A solution hybridisation assay with colorimetric microtitre plate detection (polymerase chain reaction-enzyme-linked immunosorbent assay (PCR ELISA) has been developed using the multicopy insertion sequence IS1106 which had reportedly achieved a specificity of 100% and was described as being meningococcal specific. This PCR ELISA assay was evaluated on specimens from over 5000 patients at the national Meningococcal Reference Unit (MRU) between late 1995 and early 1997 and was found to be highly sensitive. Insertion sequences, however, are genetically mobile with the ability to spread between species and even genera. During the evaluation period of the IS1106 PCR ELISA a number of false positives proved to be caused by organisms other than N. meningitidis were recorded resulting in the withdrawal of this assay as a front line screening assay for routine confirmation of meningococcal infection.
2. Sakugawa H; Nakasone H; Nakayoshi T; Kinjo F; Saito A; Yakabi S; Zukeran H; Miyagi Y; Taira R; Koja K; et al High proportion of false positive reactions among donors with anti-HCV antibodies in a low prevalence area J Med Virol, 46(4):334-8 1995 Aug :Among 39,656 voluntary blood donors in Okinawa Prefecture, Japan, 115 (0.29%) were repeatedly reactive for antibody to hepatitis C virus (anti-HCV) by second generation (2nd-gen) passive hemagglutination assay (PHA). Positive serum samples were tested for anti-HCV using three different enzyme immunosorbent assays (ELISAs; Abbott 2nd EIA, UBI-HCV-EIA, JCC-2) and for HCV-RNA by the polymerase chain reaction (PCR). The 115 2nd-gen PHA-positive sera were divided into three groups according to the agglutination titers; > 2(10) (high titer group), 2(7)-2(9) (median), 2(5)-2(6) (low). All but one serum (44/45) in the high PHA titer group reacted in each of the three second screening ELISAs. Furthermore, 43 (97.7%) of the 44 sera contained HCV-RNA by PCR. In the median titer group, 11 of the 13 samples tested were positive by each of the three ELISAs, and 4 (36.4%) of the 11 showed reaction by PCR. On the other hand, all of the 38 sera tested in the low titer group were negative for HCV-RNA by PCR, and 24 of the 38 were also negative by each of the three ELISAs. Most of the low titer positive reactions in the 2nd-gen agglutination assay seemed to be false positive.
3. Kaul K; Luke S; McGurn C; Snowden N; Monti C; Fry WA Amplification of residual DNA sequences in sterile bronchoscopes leading to false-positivePCR results J Clin Microbiol, 34(8):1949-51 1996 Aug : PCR has been used successfully for the direct detection of Mycobacterium tuberculosis in uncultured patient samples. Its potential is hindered by the risk of false-positive results as a result of either amplicon carryover of cross-contamination between patient samples. In the present study, we investigated whether residual amplifiable human or M. tuberculosis DNA could remain in sterile bronchoscopes and potentially be a cause of false-positive PCR results in subsequent patient samples. Sterilized bronchoscopes were flushed with sterile saline, and the collected eluate was submitted for PCR amplification of IS6110 sequences and exon 8 of the human p53 gene. Of a total of 55 washes of sterile bronchoscopes from two institutions, 2 (3.6%) contained amplifiable M. tuberculosis DNA and 11 (20%) contained residual human DNA. These findings indicate that residual DNA can remain in sterilized bronchoscopes and can be a source of false-positive PCR results.
4. Keum WK; Park CE; Lee JH; Khil LY; Kang I; Kim SS; Jung JC; Oh SM; Woo HJ; Lee JH; Kim YC; Yoon Y; Choi JW; Ha J Primers determine the sensitivity of PCR-mediated hepatitis B virus DNA detection and pretreatment of PCR mixture with 8-methoxypsoralen eliminates false-positive results Mol Cells, 7(2):244-50 1997 Apr 30: Most methods for the diagnosis of hepatitis B virus (HBV) infection largely depend on viral DNA detection by polymerase chain reaction (PCR) or radioimmunological assay of viral antigens or antibodies. The quality assurance program recently established in Europe reported that PCR-mediated HBV DNA detection methods used in many laboratories produced a high rate of false-positive and false-negative results. Thus, we attempted to improve the conditions of current PCR methods for detection of HBV DNA. In the present study, we applied a recently developed method of releasing HBV DNA from virion by NaOH treatment of patient serum. Using four different primer sets specific to the HBV core region, we found that the sensitivity of first-round PCR can be improved by more than two orders of magnitude depending on the primers. The second round of PCR using nested primers was sensitive enough to detect up to 10(-6) pg of the HBV DNA, which is equivalent to approximately 3 copies of the HBV genome. Among the approximately 800 HBV-infected patient sera investigated in our laboratory, more than 60% of the tested samples gave positive results in the first-round PCR. The rate of positive results obtained using our experimental conditions is very high in comparison with other reports. The reamplification of the first-round PCR reaction mixture with the nested primers produced practically 100% positive results. For diagnosis of HBV infection, we routinely used 1 microliter of patient serum, which was found to be optimum in our laboratory. Surprisingly, from 20% of our positive results, even serum diluted to 1/100 (0.01 microliter) produced a stronger signal than 1 microliter. This observation suggests that direct PCR amplification of HBV DNA released from serum by NaOH treatment has to be compensated by other DNA detection methods for correct quantitation. In order to eliminate the false positive signal resulting from the carry-over due to massive screening of a large number of samples, PCR reaction mixture containing 8-methoxypsoralen was exposed to ultraviolet light prior to thermal cycle amplification. This exercise did not decrease the sensitivity of the detection method, but almost completely removed the false positive results caused by contaminated templates. We are in the process of improving PCR-mediated HBV DNA detection methods to attain more reliable and easily applicable methods.
5. Teo IA; Shaunak S Polymerase chain reaction in situ: an appraisal of an emerging technique Histochem J, 27(9):647-59 1995 Sep : Polymerase chain reaction (PCR) in situ is a new technique which promises to enhance considerably our ability to detect a few copies of target nucleic acid sequences in fixed tissues and cells. It has an enormous potential for application in diagnostic histopathology of viral diseases and in the study of gene expression. PCR in situ is, however, technically difficult, and amplification of the target DNA is only 30-300 fold. In this article we present an overview of PCR in situ techniques used to amplify both DNA and RNA targets (RT-PCR in situ). We also identify problems which can reduce the efficiency of the technique or which can give rise to false-positive results. They include (1) the inhibitory effects of cross-linking of histones to DNA or PCR amplification, (2) abstraction of PCR reagents by tissue-bonding agents which are used to coat glass slides, (3) poor denaturation of target DNA and subsequent DNA renaturation due to extensive cross-linking of histones to DNA, or because of incorrect temperature regulation of thermal cyclers, (4) false-positive results which arise from end-labelling of DNA strand breaks by Taq polymerase, and (5) diffusion of PCR products into and out of cells leading to false-positive results. We present some of the approaches that have been used to overcome some of these difficulties and suggest new avenues for investigation to improve this technique further.
6. Teo IA; Shaunak S PCR in situ: aspects which reduce amplification and generate false-positive results Histochem J, 27(9):660-9 1995 Sep : PCR in situ promises the ability to amplify and detect very low levels of target nucleic acid in tissues. Despite considerable effort, the technique is still technically difficult and has not yet proved to be reliable or reproducible. We have now identified a number of factors which can contribute to the poor amplification of the target DNA and to the generation of false-positive signals. These factors include the effects of fixation, reagent abstraction, DNA degradation, DNA end-labelling and product diffusion. We present evidence to show that formaldehyde fixation cross-links histones to DNA and thus restricts the subsequent amplification of target sequences by PCR. End-labelling of DNA occurs when direct incorporation is used to detect amplified products and this gives rise to false-positive signals. Amplified products can also diffuse out of cells and into neighbouring cells which do not contain target sequences. They can undergo re-amplification within these cells giving rise to false-positive signals. We believe considerable caution should be exercised in the interpretation of results generated using PCR in situ.
7. Boyd AS; Annarella M; Rapini RP; Adler-Storthz K; Duvic M. False-positive polymerase chain reaction results for human papillomavirus in lichen planus. Potential laboratory pitfalls of this procedure J Am Acad Dermatol, 35(1):42-6 1996 Jul :BACKGROUND: The polymerase chain reaction (PCR) is a common tool in laboratory evaluation of tissue samples. It has been particularly useful in testing for nucleic acids of infectious organisms implicated in the pathogenesis of cutaneous diseases. OBJECTIVE: Our purpose was to evaluate archival biopsy material (paraffin-embedded) of lichen planus for human papillomavirus (HPV) DNA with PCR and in situ hybridization (ISH). METHODS: Tissue sections were obtained from 13 specimens of lichen planus and 10 control tissues. Standard methods for PCR and ISH were used to evaluate the presence of HPV DNA. RESULTS. Initial PCR results demonstrated HPV DNA in 11 of 13 specimens and in none of the control tissue. However, testing for specific HPV types revealed nucleic acid contamination. ISH was negative in all specimens. CONCLUSION: Accurate evaluation of tissue with PCR is difficult because of the procedure's profound sensitivity. Positive results reported in the literature should be viewed with caution. Potential causes for false-positive and false-negative results should be considered.
8. Wiedbrauk DL; Werner JC; Drevon AM Inhibition of PCR by aqueous and vitreous fluids.J Clin Microbiol, 33(10):2643-6 1995 Oct : The detection of viral nucleic acids in intraocular fluids and tissues by PCR has become increasingly important in clinical ophthalmology. While much attention has been directed toward minimizing false-positive reactions resulting from specimen contamination or amplicor carryover, relatively little attention has been given to the causes of false-negative PCRs. This report describes a PCR inhibitor in normal aqueous and vitreous fluids that can produce false-negative PCR results. As little as 0.5 microliter of vitreous fluid and 20 microliters of aqueous fluid can completely inhibit DNA amplification in a 100-microliters PCR mixture. This inhibition was not primer specific, nor was it due to chelation of Mg2+ ions or DNase activity in the ocular fluid. The inhibitor was completely resistant to boiling for 15 min. However, the inhibitory effects were completely removed by a single chloroform-isoamyl alcohol (24:1) extraction. The extent of PCR inhibition depended upon the type of thermostable DNA polymerase used in the reaction. Taq DNA polymerase was very sensitive to the inhibitor, while thermostable DNA polymerases from Thermus thermophilus HB-8 (Tth) and Thermus flavus (Tfl) were completely resistant. Thus, the inhibitory effects of intraocular fluids on PCRs can be removed by diluting the specimen, by chloroform extraction, or by using Tth or Tfl DNA polymerases
9. Smith KC; Starke JR; Eisenach K; Ong LT; Denby M Detection of Mycobacterium tuberculosis in clinical specimens from children using a polymerase chain reaction .Pediatrics, 97(2):155-60 1996 Feb : OBJECTIVE: We evaluated the usefulness of the polymerase chain reaction (PCR) using the insertion sequence IS6110 as the target for DNA to detect Mycobacterium tuberculosis in clinical specimens from children. STUDY DESIGN: This was a prospective, controlled, blinded study comparing PCR on clinical specimens, mycobacterial culture, and clinical diagnosis. PATIENTS: Sixty-five hospitalized children were evaluated, 35 with tuberculosis disease and 30 controls. Cases were defined by culture and/or specific clinical criteria. Controls included patients with tuberculosis infection but no detectable disease as well as patients free of tuberculosis infection and disease. RESULTS: Polymerase chain reaction had a sensitivity of 40% and a specificity of 80% compared with clinical diagnosis. Mycobacterial culture had a sensitivity of 37%. The combination of culture and PCR identified 19 of 35 children (54%) with clinically diagnosed tuberculosis. There were six children with false-positive PCR results: One had tuberculosis infection without disease, two had Mycobacterium avium lymphadenitis, and three had diagnoses unrelated to tuberculosis. CONCLUSIONS: The sensitivity of PCR is comparable to that of culture for detecting M tuberculosis in children, and may strengthen and hasten the clinical diagnosis in culture-negative patients. However, because of the limitations in specificity, the results of PCR alone are insufficient to diagnose tuberculosis in children. Although ongoing refinements in PCR techniques should improve the specificity of this test, epidemiologic and clinical information continue to be the most important consideration in the diagnosis of tuberculosis in culture-negative children.
10.Bahrmand AR; Bakayev VV; Babaei MH Use of polymerase chain reaction for primary diagnosis of pulmonary tuberculosis in the clinical laboratory. Scand J Infect Dis, 1996, 28:5, 469-72: A nested polymerase chain reaction (PCR) has been used for the rapid detection of tubercule bacilli in respiratory specimens from 287 patients suspected of tuberculosis. The results of PCR testing were compared with isolation methods (conventional culture and Bactec system) in 110 smear-positive and 177 smear-negative patients. There were only 4 false negative results by PCR in the 171 specimens that were M. tuberculosis complex culture-positive. Of 92 PCR-positive samples prepared from the smear-positive specimens 90 (97.8%) were confirmed by culture. However, a poor correlation was obtained between initial 122 PCR-positive results and combined 81 culture recovered organisms in smear-negative patients. After verification of the efficacy of isolation method, retesting PCR-positive culture-negative samples, and studies of patients' clinical histories, only 18 of the cases were found to be associated with the disease. The other 29 results out of the original 47 discrepants were considered PCR false positives, possibly due to contamination. In conclusion, the PCR assay described is suitable for implementation in daily routine work with respiratory specimens, however it should be validated with culture, especially for the smear-negative patients.
11.Schwartz DH, Laeyendecker OB, Arango-Jaramillo S, Castillo RC, Reynolds MJ Extensive evaluation of a seronegative participant in an HIV-1 vaccine trial as a result of false-positive PCR. Lancet 1997 Jul 26;350(9073):256-259: BACKGROUND: In the USA, more than 2000 volunteers have received one or more experimental HIV-1 vaccines in phase I and II clinical trials, and there have been breakthrough HIV-1 infections among participants receiving vaccine and placebo. Serological diagnosis of new HIV-1 infections in vaccine-trial participants will become increasingly complicated as more viral components are used in vaccines. Recognition of this problem has led to a reliance on viral-genome measurement to distinguish vaccine-induced immunity from HIV-1 infection. Currently, quantitative RNA measurement is expensive, prone to contamination, and reliable only in laboratories certified by manufacturers or that have quality-control programmes. METHODS: A high-risk vaccinee presented after an acute febrile episode and was tested for HIV-1 infection by reverse transcriptase (RT) PCR of viral RNA. Further extensive tests were required to clarify the HIV-1 infection and immune status of the vaccinee, including repeat RT-PCR, nested DNA PCR, western blot, lymphoproliferation assay, cytotoxic T-cell lysis, CD8-depleted co-culture, and HIV-1 challenge culture. FINDINGS: Initial testing of plasma by RNA RT-PCR was reported as positive. This result was not confirmed by viral cultures, nested DNA PCR, western blot, or EIA. Additional RNA RT-PCR assays gave positive results from earlier occasions in the vaccine trial. Eventually, testing of all previously reactive samples by RNA RT-PCR was repeated in a quality-controlled laboratory, and confirmed the negative HIV-1 status of the individual. INTERPRETATION: This case report exemplifies the difficulties with use of viral-genome measurement as a screening test to diagnose HIV-1 infection, particularly in individuals who have ever participated in HIV-1 vaccine trials. Monitoring of large numbers of phase III vaccinees by RNA RT-PCR will not be feasible. The design of efficacy trials for new vaccines should be in parallel with development of antibody-based diagnostic tests that are capable of differentiating between immunisation and true HIV-1 infection.
12.Busch MP, Henrard DR, Hewlett IK, Mehaffey WF, Epstein JS, Allain JP, Lee TH, Mosley JW Poor sensitivity, specificity, and reproducibility of detection of HIV-1 DNA in serum by polymerase chain reaction. The Transfusion Safety Study Group. J Acquir Immune Defic Syndr 1992;5(9):872-877 A series of recent studies have reported detection by the polymerase chain reaction (PCR) of cell-free human immunodeficiency virus type 1 (HIV-1) DNA (as opposed to virion RNA) in serum from both seropositive and seronegative persons. To evaluate the sensitivity, specificity, and reproducibility of PCR detection of cell-free HIV-1 DNA, we distributed coded panels containing 98 serum specimens obtained from well-characterized, infected individuals and control blood donors to the two laboratories with reported experience with this technique. Positive results were reported with HIV-1 gag primers (SK38/39) for 48 of 188 separate PCR determinations on DNA extracts from 44 serum samples from seropositive patients (25.5% sensitivity). HIV-1 gag signal was also reported for 28 of 151 PCR determinations on 34 samples from noninfected blood donors (18.5% false-positive rate). PCR for HIV-1 env DNA performed in one laboratory was negative on all specimens from seropositive and seronegative patients. Results for cell-free HIV-1 gag and human genomic (beta-globin or HLA DQ-alpha) DNA were inconsistent on replicate and serial specimens evaluated within each laboratory and between laboratories. These results indicate that current techniques for detecting cell-free HIV-1 DNA in serum lack adequate sensitivity, specificity, and reproducibility for widespread clinical applications.
13.McHugh TD, Ramsay AR, James EA, Mognie R, Gillespie SH Pitfalls of PCR: misdiagnosis of cerebral nocardia infection. Lancet 1995 Nov 25;346(8987):1436: "Histology...did not show features suggestive of T gondii infection. Pcr resulted in a PCR product similar to that from the T gonii control.....Our study identifies an unrelated but clinically significant organism as a source of crossreactivity and confirms that before P(CR)30 assay can be applied to patient diagnosis, DNA from organisms likeky to be found in clinical specimens must be examined for similar false-positive results."
14.Holliman RE, Patel B, Johnson JD, Mangan J, Savva D Pitfalls of PCR: misdiagnosis of cerebral nocardia infection. Lancet 1996 Feb 3;347(8997):335-336 : " Our results confirm the generation of a PCR product when applying the P30 based assay to some nocardia stains. We also found cross-reactivity with Mycobacterium tuberculosis.... Established good practice dictates that PCR should not be the sole diagnostic test for toxoplasmosis in any clinical setting."
15.Trinker M, Hofler G, Sill H False-positive diagnosis of tuberculosis with PCR. Lancet 1996 Nov 16;348(9038):1388 :" This case demonstrates that the results of pcr assays-although highly specific for the detection of mycobacterial DNA-should be interpreted only in conjunction with clinical information in order to avoid inappropriate treatment. PCP assays are not standardized;false positive results may be due to individual laboratory procedures, contamination, or might reflect amplification of dna from nonviable myco bacteria."
16.Chen G, Chen Z Effects of reagents used in polymerase chain reaction on sensitivity and specificity of detection of Mycobacterium tuberculosis. Chung Hua Chieh Ho Ho Hu Hsi Tsa Chih 1996 Oct;19(5):294-296: OBJECTIVE: To explore the effects of reagents used in polymerase chain reaction on sensitivity and specificity of detection of mycobacterium tuberculosis. METHODS: 205 tuberculosis and 105 nontuberculosis cases' sputum specimens were examined simultaneously by smear, polymerase chain reaction using three different reagents and culture. RESULTS: The positive rates of Mycobacterium tuberculosis by polymerase chain reaction using three different reagents were 82.4%, 71.7% and 61.0% respectively in 205 tuberculosis cases, while 18.0%, 11.4% and 2.8% respectively in 105 nontuberculosis cases. The differences between the above three different groups was statistically significant (P < 0.005). CONCLUSIONS: The sequences of primers and the length of amplified products of different reagents may be associated with the positive and the false positive rates of Mycobacterium tuberculosis in polymearase chain reaction.
17.Doucet-Populaire F, Lalande V, Carpentier E, Bourgoin A, Dailloux M, Bollet C, Vachee A, Moinard D, Texier-Maugein J, Carbonnelle B, Grosset J A blind study of the polymerase chain reaction for the detection of Mycobacterium tuberculosis DNA. Azay Mycobacteria Study Group. Tuber Lung Dis 1996 Aug; 77(4):358-362:SETTING: Nine French laboratories routinely involved in mycobacterial work. OBJECTIVE: To assess the detection of Mycobacterium tuberculosis in experimental samples by polymerase chain reaction (PCR) using the insertion sequence IS6110 as a target for deoxyribonucleic acid (DNA) amplification. DESIGN: Nine laboratories participated in a blind study of the detection of M. tuberculosis by PCR in 20 coded samples containing either a definite number of M. tuberculosis complex (positive samples) or environmental mycobacteria (four samples) or no mycobacteria (five samples). RESULTS: Five laboratories reported false-positive PCR results, with an average rate of 7%. All laboratories except one reported positive PCR results for samples containing 10(5) cfu/ml or more. M. tuberculosis DNA was detected in two thirds of samples containing 10(4) and 10(3) cfu/ml, and in one third of the samples containing 10(2) cfu/ml. CONCLUSION: The results of the study suggest that PCR using IS6110 as a target for DNA amplication is neither very sensitive nor really specific for the detection of M. tuberculosis.
18.Noordhoek GT, Kolk AH, Bjune G, Catty D, Dale JW, Fine PE, Godfrey-Faussett P, Cho SN, Shinnick T, Svenson SB, et al Sensitivity and specificity of PCR for detection of Mycobacterium tuberculosis: a blind comparison study among seven laboratories. Clin Microbiol 1994 Feb;32(2):277-284 :PCR is, in principle, a simple and rapid test for use in the detection of Mycobacterium tuberculosis. However, virtually no data are available on the reliability and reproducibility of the method. In order to assess the validity of PCR for the detection of mycobacteria in clinical samples, seven laboratories participated in a blinded study of 200 sputum, saliva, and water samples containing either known numbers of Mycobacterium bovis BCG cells or no added organisms. Each laboratory used its own protocol for pretreatment, DNA extraction, and detection of the amplification product. Insertion sequence IS6110 was the target for DNA amplification. Several participating laboratories reported high levels of false-positive PCR results, with rates ranging from 3 to 20% and with one extreme value of 77%. The levels of sensitivity also ranged widely among the different participants. A positive PCR result was reported for 2 to 90% of the samples with 10(3) mycobacteria. Although most participants did include control tests to check the sensitivity and specificity of the PCR, the sequence of operations from sample pretreatment to purification of DNA from bacteria was not always monitored adequately. During these procedures cross-contaminating DNA was introduced and/or bacterial DNA was lost. The results of the study show that the implementation of an effective system for monitoring sensitivity and specificity is required before the PCR can be used reliably in the diagnosis of tuberculosis.
19.du Toit R, Victor TC, van Helden PD Empirical evaluation of conditions influencing the polymerase chain reaction: enterotoxigenic Escherichia coli as a test case. Eur J Clin Chem Clin Biochem 1993 Apr31(4):225-231: False polymerase chain reaction (PCR) results may be obtained under unfavourable reaction conditions. Therefore optimal conditions for the different factors influencing a specific PCR method should be determined before introduction to a clinical diagnostic laboratory. This study has concentrated on the detection of heat-labile enterotoxin-producing E. coli by PCR, with empirical evaluation of various factors. Template was prepared by heat-lysis of E. coli, and this was shown to be adequate for PCR detection. The results showed that deviation from the optimal conditions of any of the following conditions may lead to false results: lysis of bacterial cells, denaturation temperature during cycling, annealing temperature, primer concentration, enzyme concentration, magnesium concentration and ion concentration. Three different detection methods for PCR product were also evaluated. As little as one bacterium can be detected after 35 cycles of PCR amplification with 32P-labelled oligonucleotide probe. An alkaline phosphatase-labelled probe was 10-fold less sensitive, whereas 100 bacteria in 10 microliters of the original sample suspension were necessary to give a positive signal after gel electrophoresis. The information in this study may be useful to those who wish to introduce PCR tests to diagnostic laboratories
20.Wood GS, Salvekar A, Schaffer J, Crooks CF, Henghold W, Fivenson DP, Kim YH, Smoller BR Evidence against a role for human T-cell lymphotrophic virus type I (HTLV-I) in the pathogenesis of American cutaneous T-cell lymphoma. J Invest Dermatol 1996 Sep;107(3):301-307 :We used a standard polymerase chain reaction (PCR)/Southern blot assay (sensitivity > 10(-5)) to detect human T-cell lymphotrophic virus type I (HTLV-I) proviral pX, pol, and env genes in the lesional skin of 42 American patients with cutaneous T-cell lymphoma (CTCL). As in some prior reports using similar methods, a variable proportion of PCR tests were positive (seven of 42 for pX, three of 42 for pol, and two of 37 for env), resulting in an overall positive test rate of 12 of 121 (10%). To determine the significance of these positive test results, we performed several additional studies. D1S80 polymorphism analysis of CTCL cases and HTLV-I PCR analysis of non-CTCL dermatosis controls showed no evidence that positive PCR tests resulted from sample mislabeling, gross HTLV-I contamination, or human endogenous retroviruses. We then modified the standard PCR assay to incorporate ultraviolet (UV) light to destroy low-level PCR contamination. With this modified assay (sensitivity > 10(-5)), only three of 12 previously positive cases were still positive, suggesting that the earlier positives were due to trace contamination of PCR reagents or trace contamination of sample DNA. This interpretation was also supported by: (i) a match between pX and pol sequences cloned from one PCR-positive specimen and the MT4-positive control, (ii) our inability to confirm HTLV-I in any PCR-positive case using genomic dot blotting (sensitivity > 10(-2)), and (iii) negative PCR results when new samples from two of the remaining positive cases were analyzed. Finally, we used our modified UV/ PCR/Southern blot assay to test an additional 28 cases of American CTCL for pX. All of them were negative. Although these studies of 70 cases of American CTCL do not exclude the possibility that another virus is involved in the pathogenesis of this disease, they provide strong evidence against a role for HTLV-I. Furthermore, they emphasize the need for special strategies to control for false-positive PCR tests that can result from even trace levels of contamination with viral DNA. As a consequence, associations between diseases and viruses should be viewed skeptically if they are based primarily on conventional PCR data. 21.Wilke WW, Jones RN, Sutton LD Department of Pathology, University of Iowa College of Medicine USA. Automation of polymerase chain reaction tests. Reduction of human errors leading to contamination. Diagn Microbiol Infect Dis 1995 Apr;21(4):181-185: We compared the performance over 21 months of manually performed polymerase chain reaction (PCR)-based DNA analysis experiments with 25 months of automated PCR performed by a Zymark robotic system. Automation of the PCR technique resulted in a sixfold reduction in the number of experiments reporting carryover contamination and decreased the overall rate of contamination among total reactions 68-fold. Whereas contamination occurrences among manual experiments were evenly dispersed over the study interval and correlated with the lack of experience of laboratory personnel, the contamination that occurred with the robotic system was confined to the first 10 months of operation. In manual experiments, many of the 81 no-target false positives were sufficiently strong to result in the invalidation of 151 samples and positive controls. The seven no-target control false positives in the automated system were weak bands that were easily subtracted as background. Because none of the negative samples had DNA bands, no sample on the automated system has ever been invalidated as a result of contamination. Automation of PCR tests appears to offer great promise in reducing contamination to acceptable levels
22.Kelley PK, McClain KL Department of Pediatrics, Baylor College of Medicine, Respiratory contamination of polymerase chain reactions by human herpesvirus 6. Am J Hematol 1994 Dec;47(4):325-327: Human herpesvirus type 6 DNA derived from human breath was discovered to contaminate PCR reactions during routine reaction preparation. Parallel PCR experiments were conducted in which expiratory secretions were blocked by a surgical mask, while others were performed without any attempt to circumvent respiratory contamination. The experimenter was previously determined to harbor HHV-6 DNA in the saliva. All reactions in which expiration was obstructed were negative for HHV-6 DNA via PCR. Reactions in which there was no attempt to obstruct respiratory secretions were positive for HHV-6 DNA. These data suggest that PCR assays investigating the presence of HHV-6 may be highly susceptible to contamination from the experimenter leading to false positive results.

iii. Recent Scientific Literature Demonstrates That Prevailing Scientific Opinion Has Materially Changed With Respect To The Reliabilty Of PCR-Based DNA Testing In Forensic Science

Among the more recent of the scientific articles on the forensic application of PCR-based DNA testing are the following:
1.Smith JR, Carpten JD, Brownstein MJ, Ghosh S, Magnuson VL, Gilbert DA, Trent JM, Collins FS National Center for Human Genome Research, National Institutes of Health, Approach to genotyping errors caused by nontemplated nucleotide addition by Taq DNA polymerase. Genome Res 1995 Oct;5(3):312-317: Thermostable DNA polymerases can catalyze nontemplated addition of a nucleotide to the 3' end of amplification products. This presents a potential source of error in genotyping studies employing Taq DNA polymerase to amplify microsatellite loci. Although the activity is marker specific, experimental variation is often seen in the degree of modification. Consequently, for a given microsatellite marker, an allele may be inconsistently identified as either the unmodified or modified amplification product. Full automation of high-throughput genotyping has been hampered by the need for manual editing of data because of this source of allele misidentification. In this study we estimate a 1% to 3% error rate attributable to nontemplated nucleotide addition in the ABI PRISM genotyping system. We present a PCR-based strategy to minimize this source of error.
2.. Brownstein MJ, Carpten JD, Smith JR National Institute of Mental Health, National Institutes of Health Biotechniques 1996 Jun;20(6):1004-1006 Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Taq DNA polymerase can catalyze non-templated addition of a nucleotide (principally adenosine) to the 3' end of PCR-amplified products. Recently, we showed that this activity, which is primer-specific, presents a potential source of error in genotyping studies based on the use of short tandem repeat (STR) markers. Furthermore, in reviewing our data, we found that non-templated nucleotide addition adjacent to a 3' terminal C is favored and that addition adjacent to a 3' terminal A is not. It was clear, however, that features of the template in addition to the 3' terminal base also affect the fraction of product adenylated. To define consensus sequences that promote or inhibit product adenylation, we transplanted sequences between the 5' ends of the reverse primers of markers that are adenylated and those of markers that are not adenylated. It proved difficult to identify a single sequence capable of protecting the products of all markers from non-templated addition of nucleotide. On the other hand, placing the sequence GTTTCTT on the 5' end of reverse primers resulted in nearly 100% adenylation of the 3' end of the forward strand. This modification or related ones (called "PIG-tailing") should facilitate accurate genotyping and efficient T/A cloning.
3. Magnuson VL, Ally DS, Nylund SJ, Karanjawala ZE, Rayman JB, Knapp JI, Lowe AL, Ghosh S, Collins FS National Center for Human Genome Research, National Institutes of Health Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques 1996 Oct;21(4):700-709: The Applied Biosystems PRISM fluorescence-based genotyping system as well as the Invitrogen TA Cloning vector system are influenced by the tendency of Taq DNA polymerase to add an adenine nucleotide to the 3' end of PCR products after extension. Incomplete addition of adenine to a majority of PCR product strands creates problems in allele-calling during genotyping and potentially diminishes the cloning efficiency of such products. Experiments reported here show that certain terminal nucleotides can either inhibit or enhance adenine addition by Taq and that PCR primer design can be used to modulate this activity. The methods we propose can substantially improve allele-calling for problematic microsatellite markers when using GENOTYPER software.
4. Cline J, Braman JC, Hogrefe HH Stratagene Cloning Systems PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 1996 Sep 15;24(18):3546-3551:Replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNApolymerases were compared using a PCR-based forward mutation assay. Averageerror rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6))< < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimizationexperiments indicated that Pfu fidelity was highest in the presence of 2-3 mMMgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions,the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) thanthe error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the errorrate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfuincreased approximately 9-fold. An increase in error rate with pH has also beennoted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow,suggesting that the parameters which influence replication error rates may besimilar in pol l- and alpha-like polymerases. Finally, the fidelity of 'longPCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNApolymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to beless than the error rate of Taq DNA polymerase, but approximately 3-4-foldhigher than the error rate of Pfu DNA polymerase.
5. Hughes MS, Beck LA, Skuce RA Department of Agriculture for Northern Ireland, Stromont, Belfast. J Clin Microbiol 1994 Aug;32(8):2007-2008 Identification and elimination of DNA sequences in Taq DNA polymerase.This study confirms that different preparations of Taq DNA polymerase are contaminated with eubacterial DNA. The contaminants appeared to represent more than one strain or species but were not identified as Thermus aquaticus or Escherichia coli. Differences in microcentrifuge tube composition appeared to affect elimination of the contaminants.
6. Cline J, Braman JC, Hogrefe HH Stratagene Cloning Systems PCR fidelity of pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res 1996 Sep 15;24(18):3546-3551:Replication fidelities of Pfu, Taq, Vent, Deep Vent and UlTma DNApolymerases were compared using a PCR-based forward mutation assay. Averageerror rates (mutation frequency/bp/duplication) increased as follows: Pfu (1.3x 10(-6)) < Deep Vent (2.7 x 10(-6)) < Vent (2.8 x 10(-6)) < Taq (8.0 x 10(-6))< < exo- Pfu and UlTma (approximately 5 x 10(-5)). Buffer optimizationexperiments indicated that Pfu fidelity was highest in the presence of 2-3 mMMgSO4 and 100-300 microM each dNTP and at pH 8.5-9.1. Under these conditions,the error rate of exo- Pfu was approximately 40-fold higher (5 x 10(-5)) thanthe error rate of Pfu. As the reaction pH was raised from pH 8 to 9, the errorrate of Pfu decreased approximately 2-fold, while the error rate of exo- Pfuincreased approximately 9-fold. An increase in error rate with pH has also beennoted for the exonuclease-deficient DNA polymerases Taq and exo- Klenow,suggesting that the parameters which influence replication error rates may besimilar in pol l- and alpha-like polymerases. Finally, the fidelity of 'longPCR' DNA polymerase mixtures was examined. The error rates of a Taq/Pfu DNApolymerase mixture and a Klentaq/Pfu DNA polymerase mixture were found to beless than the error rate of Taq DNA polymerase, but approximately 3-4-foldhigher than the error rate of Pfu DNA polymerase.
7. Herber B; Herold K DNA typing of human dandruff J Forensic Sci, 1998 May, 43:3, 648-56:Dandruff particles contain a considerable portion of nucleated cells within the aggregates of nuclei-free corneocytes. The nuclei could be partially degraded due to epidermal differentiation processes. To test the suitability of DNA from dandruff for forensic application, a study of 35 subjects and two crime cases has been carried out using different STR polymorphisms. In 90% of the samples DNA genotyping could be easily performed indicating that DNA from dandruff is at least suitable for STR analysis. DNA quantity per dandruff particle varies extremely within a range from 0.8 to 16.6 ng DNA for the tested subjects. Genotyping was also possible for a single dandruff particle if DNA extraction volume had been reduced. In mixed samples from dandruff coextracted with bloodstains or semen stains dandruff genotype was detectable. in some cases. These results demonstrate the relevance of dandruff in forensic casework either as an additional sample or as a source of inadvertent contamination.
8.Lorente M, Entrala C, Lorente JA, Alvarez JC, Villanueva E, Budowle B Dandruff as a potential source of DNA in forensic casework J Forensic Sci 1998 Jul; 43(4):901-902 :Dandruff is a clinical alteration of the skin that consists histologically of orthokeratotic clumps with minute parakeratotic foci found in inflammatory pathologies such as seborrheic dermatitis and psoriasis. Therefore, some nucleated cells should be found in dandruff and hence there is a possibility that forensically typeable DNA could be extracted from dandruff. Because of a particular case in which we were involved, a study was carried out to determine whether or not DNA could be extracted from dandruff, and if the two most widely used extraction techniques (Chelex and organic) would be applicable. Results show that sufficient quantities of DNA (more than 30 to 40 ng) can be obtained from as little as 1.0 to 1.5 mg of dandruff. Both methods yield DNA, although the organic procedure seems to yield more (72.5 ng Chelex vs. 183.3 ng organic). All the DNA samples extracted were typed correctly for the loci HUMTH01 and HUMvWA. Therefore, dandruff can be considered a potential source of DNA for forensic identification.
9. Toledano T; Quarino L; Leung S; Buffolino P; Baum H; Shaler RC, An assessment of DNA contamination risks in New York City Medical Examiner facilities J Forensic Sci, 1997 Jul, 42:4, 721-4: DNA evidence holds an important position in criminal investigations and proceedings. The polymerase chain reaction (PCR) is often utilized to amplify polymorphic regions of DNA which are subsequently typed to produce distinct genotypes. The sensitivity of PCR-based techniques provides a major advantage over other DNA or conventional serological typing systems. Samples containing quantities of DNA in the picogram range are often typed. However, the unprecedented sensitivity of PCR is often cited as a criticism. One concern is that the interpretation of PCR typing can be affected by DNA contaminants from foreign sources. In this report, the level of DNA contamination in New York City Medical Examiner facilities and its potential affects on HLA-DQA1 typing were assessed. Two related studies conducted over a five week period measured and typed HLA-DQA1 from accumulated DNA on autopsy room and Forensic DNA Laboratory structures. The potential for DNA contamination from airborne sources was also evaluated in the autopsy suites. This study demonstrated the presence of small amounts of DNA on structural surfaces, but little evidence of airborne DNA contamination.
10.Riley,DE Compromised optimal hybridization temperatures may explain PM plus DQA1 anomalies Biochem Mol Biol Int1998 Jan.44(1):59-68:The multiplex DNA typing test kit, PM plus DQA1 relies on 36 different oligonucleotides that all must hybridize correctly for accurate DNA typing. The G + C contents of the probe oligonucleotides range from 47% to 83%. Since G + C content directly affects optimal hybridization temperatures, a wide range of optimal hybridization temperatures is expected. This was verified by two empirically tested formulas. The recommended hybridization temperature is 55 degrees C. The control probe has an estimated optimal temperature of 75 degrees C indicating low stringency of the control. The B allele probe of the GC locus has an estimated optimal hybridization temperature of 70 degrees C. These discrepancies are unexplained by GC clusters or sequence palindromes. The departures from temperature optima may be responsible for observed PM plus DQA1 artifacts and for previously observed, low tolerance of this system to temperature variation. Since optimal temperature and cation concentration are related variables, careful attention to these is recommended as are positive controls demonstrating amplification of all alleles monitored by PM plus DQA1.
11. Crouse CA, Vincek V, Caraballo BK Analysis and interpretation of the HLA DQ alpha "1.1 weak-signal" observed during the PCR-based typing method J Forensic Sci 1994 Jan; 39(1):41-51 The Perkin-Elmer (PE) AmpliType DQ alpha Forensic Kit is currently available for amplification and typing of a polymorphic region in the Human Leukocyte Antigen (HLA) DQ alpha DNA sequence. Following amplification of the DQ alpha region with the PE kit, typing strips are processed. These strips contain immobilized DNA probes designed to distinguish six possible HLA DQ alpha alleles. It has been observed in this laboratory and others that in a single source DNA sample, it is possible to detect a weak signal on the 1.1 specific allele dot when the samples' genotype clearly does not contain the 1.1 allele. It has been suggested that a potential source of this weak-signal is the non-specific amplification of a HLA DX alpha gene sequence. To demonstrate the relationship of the DX alpha gene to the 1.1 nonspecific signal, we designed biotinylated DX alpha PCR primers specific for a 178 bp region in which the amplified product spans the homologous DQ alpha region encompassing the DNA probes present on the typing strips. DX alpha DNA sequences from various DQ alpha genotypes were amplified and hybridized to DQ alpha typing strips. We have demonstrated that DX alpha PCR products do not always hybridize to the 1.1 probe on the typing strips. Sequence analysis of DX alpha PCR products show that this region is polymorphic which may explain why the occurrence of the "1.1 weak-signal" is unpredictable. We have further analyzed the effect of DNA template concentration for the DQ alpha amplification protocol and have shown that regulation of PCR input DNA optimizes the amplification and typing protocols for HLA DQ alpha alleles and minimizes the potential observation of the "1.1 weak-signal
12. Grow M, Phillips V, Reynolds R Post-amplification primer extension of heat-denatured AmpliType PCR products: effects on typing results J Forensic Sci 1996 May;41(3):497-502:Alleles of the HLA, DQA1, LDLR, GYPA, HGBB, D7S8 and GC loci, which are amplified using the AmpliType(R) PM PCR Reaction Mix and Primer Set, can be detected using sequence-specific oligonucleotide probes immobilized on a nylon membrane strip. Using reagents supplied in AmpliType PCR Amplification and Typing Kits, patterns of blue dots corresponding to particular alleles are visualized on the DNA probe strips. Frequently, the correct interpretation of typing results is dependent not only on the presence of probe signals but also on their relative intensities. The relative probe signal intensities obtained from an undegraded DNA sample extracted from a single individual will be different from those obtained from degraded DNA and from samples containing DNA from more than one source. Because probe signal intensity is an essential consideration for interpretation, factors that can influence it need to be identified. Clearly, the time and temperature of the assay steps and the salt concentration in the typing solutions can affect probe signal intensity. Also, if heat-denatured PCR products are allowed to cool for several minutes, the strands will reanneal and become unavailable for binding to the probes immobilized on the strips. However, the selective loss of GC B and HLA DQA1 4.1 probe signals observed after shorter cooling times cannot be explained by these factors. We demonstrate that following heat denaturation of PM PCR products there is sufficient residual Taq DNA polymerase activity to extend primers as the solution cools and that this primer extension occurs at a more rapid rate than PCR product reannealing. Primer extension across probe binding sites will prevent hybridization of the PCR product to complementary probes on the strip. The extent of signal reduction is dependent on the position of the probe binding site relative to the 3' ends of the primers and on the strand to which the probe is complementary. We recommend a simple modification to the AmpliType typing protocol to ensure all probe binding sites will be available for hybridization to PM and HLA DQA1 DNA probe strips.
13. Walsh PS, Erlich HA, Higuchi R Preferential PCR amplification of alleles: mechanisms and solutions PCR Methods Appl 1992 May; 1(4):241-250 :The preferential PCR amplification of one allele relative to another in a heterozygous sample could result in an incorrect or ambiguous genetic typing of that sample. There are several mechanisms that could potentially lead to such preferential PCR amplification. First, preferential amplification can result from significant GC% differences between alleles if the conditions of the reaction (denaturation temperature (Tden), duration at the Tden' salt and co-solvent concentrations, etc.) allow the denaturation of one allele but not the other (differential denaturation). For example, the DQa1.1, -1.2, and -1.3 alleles of the HLA-DQa locus do not amplify at a Tden < 89 degrees C; these same conditions still allow amplification of the DQa2, -3, and -4 alleles. However, no differences in amplification efficiency were found between the different HLA-DQa alleles when the Tden was set at the recommended Tden of 94 degrees C, even after as many as 102 cycles of amplification. Second, for PCR-based genetic typing systems in which the PCR products from different alleles differ in length, preferential amplification of the shorter allelic product can occur. Experiments in which the variable number tandem repeat (VNTR) marker D17S5 (YNZ22) was amplified under various conditions suggest that the smaller allelic products are amplified preferentially when Taq polymerase is limiting. Preferential amplification of VNTR alleles can also occur if the target DNA is sufficiently degraded. Third, when the initial number of genomes sampled is very small, stochastic fluctuation in the number of copies of each allele can result in what appears to be preferential amplification. Finally, less efficient priming of DNA synthesis of one allele versus another can occur because of mismatches between the primer and the specific allelic template, resulting in preferential amplification of the other allele. General strategies to avoid preferential amplification are discussed
14.Pai CY, Chou SL, Tang TK, Wei YH, Yang CH Prevention of false results from preferential PCR amplification of VNTR alleles. J Formos Med Assoc 1996 Jan;95(1):69-72:In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework.
15. Lee HC, Ladd C, Scherczinger CA, Bourke MT Forensic applications of DNA typing: part 2: collection and preservation of DNA evidence. Am J Forensic Med Pathol 1998 Mar;19(1):10-18 : The initial stages of physical evidence examination are pivotal to the successful resolution of criminal investigations. Recent cases clearly reinforce the notion that methods of evidence collection and preservation will continue to be rigorously scrutinized and challenged in court. This article reviews forensic applications of DNA typing, focusing on the collection and preservation of biological evidence. Topics addressed include physical evidence collection at the crime scene, the forensic laboratory, and the autopsy room. Specific concerns pertaining to different sources of DNA evidence are discussed, as are special collection methods associated with various substrates on which the evidence is deposited.
16. McCormack JM, Sherman ML, Maurer DH Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods. Hum Immunol 1997 Apr 15;54(1):82-88 : Quality control for DNA contamination in laboratories using PCR-based class II HLA typing methods. control (QC) in laboratories performing molecular histocompatibility class II typing often includes a polymerase chain reaction (PCR) approach for monitoring DNA contamination. An oligonucleotide primer set was designed, (RBQBf/RBQBr), which is specific for nonpolymorphic regions of the DR-B, DQ-B, and DP-B consensus sequences with an expected PCR product size of 81 bp. RBQBf/RBQBr detected genomic DNA from reference cell lines LWAGS and BM21 (50 to 100 picograms) as well as DR-B, DP-B, and DQ-B amplicon (1 copy). Additionally, RBQBf/RBQBr detected SSP products from routine DR-B and DQ-B typings. Validation studies employing controlled DNA contamination of laboratory surfaces revealed that increasing amounts of wipe test samples (5% to 20% v/v) were inhibitory to the wipe test PCR, whereas lower amounts (1% to 2%), or alternatively, a diluted wipe test sample, increased the sensitivity of the test and optimized the results. Collectively, this study describes a primer set, RBQBf/RBQBr, which detects both genomic DNA and DR-B, DQ-B, or DP-B amplicon and furthermore illustrates the necessity of routine testing for potential inhibitory factors that may be introduced into the wipe test PCR.
16. Meldgaard M, Morling N Detection and quantitative characterization of artificial extra peaks following polymerase chain reaction amplification of 14 short tandem repeat systems used in forensic investigations Electrophoresis 1997 Oct;18(11):1928-1935 :Detection on automated DNA sequencers of polymerase chain reaction (PCR) products of tetra- and penta-nucleotide short tandem repeat (STR) loci frequently reveals one or more extra peaks along with the true, major allele peak. The most frequent extra peak pattern is a single smaller peak which is one repeat unit shorter than the true allele peak. The existence of such artificial peaks is of special importance when the methods are used for forensic investigations because the artificial extra peaks may simulate true alleles when samples containing mixtures of DNA from different individuals are analyzed. We have investigated the relative levels of formation of extra peaks in 14 STR marker systems. We found that not only the parameters of the PCR but also factors determining the stringency during the post-PCR and pre-electrophoresis handling of samples were of importance for the formation of extra peaks. In our hands, the amounts of extra peaks were reduced (i) if the samples were effectively denatured immediately before loading, (ii) if they contained substantial amounts of formamide (i.e. > or = 50%), and (iii) if the temperature of the electrophoresis gel was above a certain level (i.e. > or = 43 degrees C). The results suggest that extra peaks may in part be due to re-annealing of the PCR product under suboptimal conditions.
17. Schmitt C, Schmutzler A, Prinz M, Staak M High sensitive DNA typing approaches for the analysis of forensic evidence: comparison of nested variable number of tandem repeats (VNTR) amplification and a short tandem repeats (STR) polymorphismForensic Sci Int 1994 Jun 3;66(2)129-141: The approach of using nested primers for the APO B variable number of tandem repeats (VNTR) increases the sensitivity of the polymerase chain reaction (PCR) to single cell level. Different experiments and a comparison to the short tandem repeats (STR) system VWA were carried out, to determine the applicability of this method to forensic samples. Nested amplification of the Apo B VNTR was affected by a strong tendency towards preferential amplification of the shorter alleles. This phenomenon was observed for DNA quantities as low as 100 pg and impaired, depending on the allele length, the results for mixed samples. As expected, VWA polymorphism showed less preferential amplification. The high sensitivity of both PCR systems is accompanied by an increased susceptibility to contamination. Using artificially contaminated bloodstains, the bloodstain genotype, the contamination or both genotypes could be found on one piece of evidence. Here a single analysis can lead to an incorrect result. Therefore a strategy for obtaining reliable results should consist of multiple stain extractions and the amplification of different stepped dilutions of the DNA solution.
18.Wu H, Zhang S, Xiao C Shadow bands in PCR amplification of trinucleotide repeats and their elimination Chung Hua I Hsueh I Chuan Hsueh Tsa Chih 1998 Feb 10;15(1):42-45:OBJECTIVE: To explore a method for eliminating the shadow bands in PCR amplification of trinucleotide repeats. METHODS: CTG trinucleotide repeat sequence in the 3'-untranslated region of the human myotonin protein kinase gene was used as templates DNA in PCR amplification, and the effects of Taq DNA polymerase alone and its mixture with Pwo DNA polymerase on occurrence of the shadow bands' were compared. The PCR products containing(CTG)5, both from Taq DNA polymerase and from Taq+Pwo polymerase amplification, were cloned into pBluecript KS and sequenced on ALF express TM DNA sequencer respectively. RESULTS: The PCR of DNA sequence containing CTG repeats frequently produced a main band (usually darker) and a shadow band (lighter) that differed from the main band by 3 base pairs when Taq DNA polymerase was used alone. However, when the mixture of Taq DNA polymerase and Pwo DNA polymerase was used, the shadow bands usually disappeared. In the 4 clones containing PCR products amplificated by Taq DNA polymerase, one clone contained only 4 CTG repeats. However, this kind of decrease of repeat copy was not observed in the 5 clones containing PCR products amplified by using mixture of Taq+Pwo DNA polymerase. CONCLUSION: The results of this experiment demonstrate that shadow bands occur during the PCR. Pwo DNA polymerase in known to possess not only DNA polymerase activity, but also 3'-5' exonuclease activity, or proofreading ability. The mixture of Taq DNA polymerase and Pwo DNA polymerase has higher processivity than Taq DNA polymerase itself. This explain the effect on eliminating or reducing the occurrence of shadow bands in polymerase chain reaction
19. Schmerer WM, Hummel S, Herrmann B Reproducibility of aDNA typing Anz 1997 Jun;55(2):199-206 : The reproducibility of short tandem repeat (STR) amplification of aDNA extracts is limited by means of formation of artifacts during PCR. One of these artifacts, the so called allelic-dropout (failure of the amplification of alleles), may result in false-homozygote typing of a sample, if genotyping is based on the analysis of a single amplification product. 30 tooth- and bone samples collected from 17 individuals of three burial sites were investigated in a blind test. Using the polymerase chain reaction (PCR) the two independent segregating STR-loci HUMVWA and HUMTH01 were amplified. Corresponding to a mathematical estimation, multiple amplifications of each sample and genelocus were carried out. The results of this genotyping were compared intraindividually.
20.Gallo JC, Thomas E, Novick GE, Herrera RJ Effects of subpopulation structure on probability calculations of DNA profiles from forensic PCR analysis. Genetica 1997;101(1):1-12: DNA typing for forensic identification is a two-step process. The first step involves determining the profiles of samples collected at the crime scene and comparing them with the profiles obtained from suspects and the victims. In the case of a match that includes the suspect as the potential source of the material collected at the crime scene, the last step in the process is to answer the question, what is the likelihood that someone in addition to the suspect could match the profile of the sample studied? This likelihood is calculated by determining the frequency of the suspect's profile in the relevant population databases. The design of forensic databases and the criteria for comparison has been addressed by the NRC report of 1996 (National Research Council, 1996). However, the fact that geographical proximity, migrational patterns, and even cultural and social practices have effects on subpopulation structure establishes the grounds for further study into its effects on the calculation of probability of occurrence values. The issue becomes more relevant in the case of discrete polymorphic markers that show higher probability of occurrence in the reference populations, where several orders of magnitude difference between the databases may have an impact on the jury. In this study, we calculated G values for all possible pairwise comparisons of allelic frequencies in the different databases from the races or subpopulations examined. In addition, we analyzed a set of 24 unrelated Caucasian, 37 unrelated African-American, and 96 unrelated Sioux/Chippewa individuals for seven polymorphic loci (DQA1, LDLR, GYPA, HBGG, D7S8, GC, and D1S80). All three sets of individuals where sampled from Minnesota. The probability of occurrence for all seven loci were calculated with respect to nine different databases: Caucasian, Arabic, Korean, Sioux/Chippewa, Navajo, Pueblo, African American, Southeastern Hispanic, and Southwestern Hispanic. Analysis of the results demonstrated marked differences in the probabilities of occurrence when individuals were compared to the different populations and subpopulation databases. The possible genetic and forensic consequences of subpopulation structu re on probability calculations are discussed.
21.Budowle B, Koons BW, Moretti TR Forensic Science Research and Training Center, FBI Laboratory, FBI Academy, J Forensic Sci 1998 May;43(3):657-660 Subtyping of the HLA-DQA1 locus and independence testing with PM and STR/VNTR loci.. Allele and genotype frequencies for six loci (HLA-DQA1 and PM loci) were determined in African Americans, United States Caucasians, and Southwestern Hispanics. The data include allele frequencies of the HLA-DQA1 4 subtypes. The HLA-DQA1 4 allele subtyping affords greater power of discrimination in African Americans and Southwestern Hispanics than in Caucasians, due to the relatively lower 4.2/4.3 allele frequency in Caucasians. Based on the exact test, all loci, except the GYPA locus in the African American sample (p = 0.011), meet Hardy-Weinberg expectations. There were two examples of significant departures from expectations of independence between alleles of the HLA-DQA1 and PM loci (HBGG/Gc in African Americans, p = 0.30; LDLR/DQA1 in Caucasians, p = 0.023). The HLA-DQA1 and PM loci also were tested for associations with three STR loci and the DIS80 locus. There were four examples of significant departures from expectations of independence (TPOX/D7S8 and THO1/HBGG in African Americans, p = 0.035 and 0.028, respectively; THO1/LDLR in Caucasians, p = 0.028; and GYPA/D1S80 in Hispanics, p = 0.046). The HLA-DQA1 and PM allele frequency data were compared with previously reported data on other sample populations of the same population categories from our laboratory; the allele frequencies at all loci, except the D7S8 locus in Hispanics (p = 0.028), were statistically similar. The frequency data can be used in forensic analyses and paternity tests to estimate the frequency of a multiple locus DNA profile in various general United States populations.
22. Mueller,L Commentary on Budowle et. al. validation and population studies of the loci LDLR, GYPA. HBGG,D7S8, and GC(PM loci), and HLA-DQA (1998) 43 J Forensic Sci 446-447: " A major conclusion of the (1995) paper by Budowle et. al., was that ‘ all loci meet Hardy- Weinberg expections and there is little evidence for associations of alleles between the loci.'.... The genotypes of all individuals in the FBI's four data bases was provided to me by the FBI through discovery in a criminal case....It appears that Budowle et. Al. altered the original data in the Caucasian-HBGG data base when performing (statistical tests)." Retesting showed " a highly significant departure from the Hardy- Weinberg equilibrum" for the HGBB locus.
23. Kline, MC Amplication of a vWA Allele (1998) 43 J Forensic Sci 250: "While typing 600 population samples... our laboratories...encountered an African American sample that typed differently at the v WA locus depending on the primers used. The two STR kits initially used were Promega... Powerplex...and Perkin-Elmer-Applied Biosystem's AmpF1STR Blue... Subsequently this sample was correctly) amplified with monoplex vWA primers. The PE-ABI monoplex ... primers were designed to amplify a shorter product than the vWA primers of the Amp F1STR Blue multiplex syste. Therefore, these primers do not bind to the same location on the genome.PE-ABI is aware of this problem and they are actively pursuing an explanation for this allelic dropout by sequencing the sample.
24.Harashima N, Liu C, Katsuyama Y, Ota M, Fukushima H Department of Legal Medicine, Shinshu University School of Medicine.Sequence variation of allele 27 at the D1S80 locus Int J Legal Med 1997;110(1):22-26: In 180 unrelated Japanese individuals 18 examples of allele 27 were detected at the locus D1S80 (MCT118). On 6% polylacrylamide gels 5 out of these 18 alleles were found to migrate between allele 26 and allele 27, but closer to allele 27, and thus were labelled variants of allele 27. All 18 examples of allele 27 were sequenced and the results were compared. Although all had the same number of base pairs (578 bp) the five variants could be subdivided into three types. V1, V2 and V3. The variants and the standard allele were composed of the same kinds of repeat units, but the order of arrangement was different. We investigated whether it was possible to distinguish the standard allele 27, and the variants V1, V2, and V3 by PCR-RFLP. EcoRII and MspI which have restriction sites within the repeat units were adopted as restriction enzymes. The variants could be discriminated from each other after treatment of the PCR fragments with EcoRII or MspI, followed by PAG electrophoresis.

3.THE ABSENCE OF A REGULARLY MAINTAINED PROFICIENCY TESTING PROGRAM WHICH INCLUDES EXTERNAL PROFICIENCY TRIALS PRECLUDES VAIDATION AND GENERAL ACCEPTANCE OF THE PCR METHODS USED IN THIS CASE

The importance of external blind proficiency testing on samples that replicate case work is discussed in sections II E and II F in the context of developing a reliable method to determine the error rate of a laboratory. External blind trials, however, also serve a critical role in establishing the reliability of a new method, or validating that a laboratory can reliably apply a generally accepted method. The NRC I was unequivocal on this point:
Most important, there is no substitute for rigorous external proficiency testing via blind trials. Such proficiency testing constitutes scientific confirmation that a laboratory's implementation of a method is valid not only in theory, but in practice. No laboratory should let its results with a new DNA typing method be used in court, unless it has undergone such proficiency testing via blind trials.

NRC 1 at 55 (emphasis added).
The Crime Lab has not undergone external proficiency testing via truly blind trials with any of the PCR based methods. Ironically, it was established in Bokin that the same Lab does employ blind proficiency testing for the much less demanding narcotics testing program. Given the controversy about the reliability of forensic PCR methods among the leading scientists in the field, the NRC's bright line injunction has particular force: None of the PCR testing should be used in this case because the Crime lab has not demonstrated its reliability through external blind proficiency testing, or, for that matter, through a regularly maintained open proficiency testing program that includes the specific kits used in this case. In this regard, the federal auditors documented in December 1998 that
The record keeping system (of the SFPD) does not facilitate ascertaining when Analysts have been proficiency tested, for what methods, and with what results. There is no documentation that the Laboratory Manager has reviewed the proficiency test results or that the Analyst has seen the results. In the absence of a Quality Assurance Manager, the Laboratory Manager must document that proficiency tests have been successfully completed and that the Analysts have seen their results....Ralph Whitten's most recent proficiency test result is for 1997. He should be tested at least every 180 days.
(Exhibit B at 12-13)
Also, it must again be pointed out that Judge Dondero specifically found in Bokin that "(f)amiliarity with the record involving past efforts at proficiency testing by Keel suggests lapses in meeting deadlines." Exhibit A at 14
The NRC I warned that forensic PCR testing "poses even more serious issues of proficiency, control, and technology transfer than RFLP typing." NRC Report, at 70. It decried reliance upon the commercially distributed PCR "kits" that are being used in this case and observed that "[i]nformation on the extent of the contamination problem in PCR analysis and the differential amplification of mixed samples needs to be further developed and published." Id.
Basic methodological issues in the forensic PCR testing remain unexplored or in controversy. Despite the admonitions of the NRC and leading scientists, forensic laboratories have not responded to the fact that techniques for the collection, preservation, and handling of crime scene samples is an essential part of forensic PCR testing methodology because of the special problem of sample contamination. Although such organizations as the American Society for Histocompatibility and Immunogenetics has, for PCR techniques, detailed "Standards for Histocompatibility Testing," there is no comparable set of guidelines for forensic PCR testing. In this envirornment of an unregulated industry operating without objective standards, a regularly maintained proficiency testing program is one of the few safeguards available to ensure correct scientific results. The absence of such a program in this case precludes a finding of general acceptance of the Crime Lab's methods and procedures.
B. THE DQ ALPHA+PM TEST KIT USED BY THE SF CRIME LAB IS NOT GENERALLY ACCEPTED IN THE SCIENTIFIC COMMUNITY

As discussed above, neither Morganti nor Wright consider the general acceptance of the DQ Alpha+PM test used in this case. This multiplex kit was not in use at the time of Morganti or Wright. This kit is a substantial scientific departure from the kits used in Morganti and Wright. TWGDAM 4.4.1.6 specifically requires as part of the developmental validation process that " (w)here more than one locus is amplified in one sample mixture, the effects of such amplification on each system (alleles) must be addressed and documented." The defense submits that the DQ AlphaPM test is not generally accepted as reliable because of a multitude of problems in the following areas of concern: the quality and specificity of amplification,quantification problems,inhibition, contamination, false positives,and the subjective nature of determining genotypes when dealing with mixed samples.
a. Quality and Specificity
Amplification conditions can affect the quality and specificity of the process. Critical parameters include the temperatures and times period of the amplification and the number of cycles of amplification; the composition of the amplification mixture from primer to the chemical reagents use; and the amount and nature (single stranded or double stranded) of the DNA from the sample. NRC 1, at 63-64. The thermocycler (the machine used in the amplification process to control the temperature of the reaction) can effect the results based on simple variations in temperature between manufacturers, or from sample well to sample well, or simply from failure to adequate calibrate and monitor the accuracy of the thermocycler. Id. at 64. Additionally, for each assay precise amplification conditions must be established to ensure reliability and accuracy. NRC 1, at 64.
b.Quality and Quantity
The quality and quantity of the amplified DNA is also a major concern. Under certain conditions, it is known that DNA polymerase, the enzyme used to amplify DNA, may "misincorporate" in a random or a specific fashion. Id. Misincorporation means that during the process of amplification, incorrect bases are added to the DNA being amplified resulting in a DNA that has a different sequence than the starting DNA. Therefore, when the amplified DNA is typed based on its sequence such as with the PM and DQ tests, there will be errors in the genotype due to misincorporation.
When the DNA to be amplified contains tandem repeated units, the DNA polymerase may slip during the amplification resulting in additions or deletions of the repeat, which creates a heterogeneous collection of amplified DNA, making the interpretation difficult. Id. Therefore, each new assay, or each new gene/marker, must be studied for DNA polymerase misincorporation or slipping prior to its use in the forensic setting.
Amplification may also be affected by the efficiency of the amplification of different alleles of a marker. Id. For instance, a sample may contain a 50:50 mixture of two alleles but yield a 90:10 ratio of amplified DNA. This "differential amplification" may be the result of a number of mechanisms, including the length of the sample DNA (larger fragments amplify less efficiently); the difference in G-C nucleotide content of a sequence which affects the denaturation efficiency (more G and C nucleotides in a sequence results in lower denaturation); and the temperature of the reaction. Id. In order to ensure that differential amplification does not occur for a given allele, there must be safeguards in place for each test and in every laboratory.
Differential amplification is a particular problem where there is a mixed sample. For example, where the DNA is typed and four alleles are seen, with alleles 1 and 2 being stronger than 3 and 4. The conclusion that the alleles 1 and 2 come from one source while 3 and 4 from a second could only be justified if it has been demonstrated that the amplification process for that gene yielded amplified DNA in direct proportion to the starting DNA. Id. at 65. If there was differential amplification, then no conclusion as to which allele came from the same source would not be justified.
Finally, differential amplification is seen where the primer amplifies DNA that does not come from the target gene but from some other random sequence of DNA. Id. This occurs when the number of amplification cycles is long, therefore, it is necessary to determine the limit of cycles for each allele.
c.Inhibition
Forensic samples may contain factors that inhibit the amplification. This happens by the factor binding to the DNA or by inhibiting the DNA polymerase. Id. at 65. This is seen in DNA from old blood stains and from specific fabrics or material, for example, blue denim. Id.; Del Rio, et al, PCR-Based Human Leukocyte Antigen (HLA) DQ Typing of Blood Stained Light and Dark Blue Denim Fabric, (1996) 40(3) 490. "Each PCR system should be thoroughly characterized on a range of simulated and known forensic samples, to document any effect on reliability. NRC 1, at 65.
d.Contamination
As discussed at length above, contamination of samples can limit the reliability of any PCR amplification. Contamination of samples equally affects the reliability of the new PM+DQA1 kit used in this case.

e. False Positives
As indicated above, one recent article regarding the combined DQ Alpha +PM kit found that attempts at typing samples using this multiplex kit resulted in failed hybridization and allelic dropout. Donald E. Riley, Compromised Optimal Hybridization Temperatures May Explain PM Plus DAQ1 Anomalies, 44 Biochemistry and Molecular Biology International 59, 63 (1998). Surveying prior studies, Dr. Riley states that "three independent studies reported false typings with either DQ Alpha and Polymarker. In two of these studies, the false typings were confirmed by other techniques such as DNA sequence data or by comparisions with nonmultiplex systems."

f. The PM+DQA1 Test Kit is Not Reliable and Therefore Not Generally Accepted Because of the Subjective Nature of Determining Genotype with Mixed Samples

No matter the procedure used to detect amplified DNA, there are risks that the resulting genotype will be misinterpreted. Id. at 68.
Reverse dot blotting is used in the test for DQ and PM. The absence of a dot theoretically means that the allele is not present. Id. The presence of a dot generally means that an allele is present. However, the presence of a intermediate intensity dot, can be the result of a number of events. There could be contamination from another sample or the technician, or there could be technical variations in the condition of the particular test, or the weaker dot shows a true heterozygote. This type of problem can be resolved by repeating the experiment or comparing the sample at a number of loci or markers. "Rarely, the origin of an intermediate hybridization signal might remain unresolved; if the type of the sample is still questionable, the data should be discarded." Id.
An illustration of the problems encountered with determining a genotype from reverse dot blot strips is seen in a study in which field tests were conducted by seven laboratories using the PM+DQA1 kit. Fildes, Nicola, and Reynolds, Rebecca, Consistency and Reproducibility of AmpliType® PM Results Between Seven Laboratories: Field Trial Results, Journal of Forensic Sciences (March 1995) 40:279. Of the 287 "DNA-containing samples" tested for the PM markers, there were 7 samples in which no results were reported or the results were "uninterpretable." Of the 224 samples tested for DQ , 14 samples were reported as either no results or uninterpretable results. Id. at 281. The "no result reported" are those samples in which there may have been some typing dots visible, but no control dot was visible. Id. The "uninterpretable result reported" samples were those in which the "interpretation of the strip was prevented by extraneous dots on either DNA probe strip or by imbalanced dots on the PM DNA probe strip that were greater in intensity than the ‘S' and/or ‘C' dots." Id. at 282. An "extraneous dot" is one that indicates the presence of more than two alleles, or a mixed sample. In general, these results reflected poor separation of sperm and epithelial cells in "postcoital swab" extracts, however, there were at least eight strips that exhibited contamination from external sources. Id. at 282, 284.
The laboratories in the study were requested to repeat the tests for the simulated sexual assault samples. Id. Two of the six laboratories that repeated the tests did not reproduce the same results. Id. One laboratory obtained 100% reproducibility in the PM test, but only 82% in the DQ test. The second laboratory achieved on 91% reproducibility in the PM test and 82% reproducibility in the DQ test. Id.
The PM test is designed so that the dot intensities are balanced, so that if there is a mixture, then the darker dots are paired and the lighter dots are paired. The PM test is sensitive to variations in the alleles of certain markers and to assay conditions. The GYPA locus has variants that affect the intensity of the dots, making it difficult to type the strips due to the color imbalance. Id. at 284. If the conditions of the assay fall outside the parameters of the method, such as differences in the water bath temperature, salt concentrations of solution or times of incubation, the balance of the dot intensities may be affected. Id. The imbalance caused by the conditions of the test also affects the balance of the "S" control dot, making it difficult to interpret the results of a mixed sample.
In 1996, a study was published that outlined the problem of reduced signal intensity at the GC B allele and the DQ 4.1 allele that could not be attributed solely to renaturation of the DNA prior to hybridization to the strip. Grow et al., Post-Amplification Primer Extension of Heat-Denatured AmpliType® PCR Products: Effects on Typing Results, Journal of Forensic Sciences (May 1996) 41:497. The study concluded that after the amplification was completed, these two alleles continued to be extended resulting in a decreased signal on the strip, and that the heat treatment recommended was not sufficient to prevent the decrease. Id. at 500.
5. THE D1S80 TEST KIT USED BY THE CRIME LAB IS NOT GENERALLY ACCEPTED AS RELIABLE IN THE SCIENTIFIC COMMUNITY.

The D1S80 testing in this case was performed using a AmpliFLP D1S80 PCR Amplification and Typing Kit ("D1S80 kit") marketed by Perkin-Elmer. These kits first appeared in 1993.
The kit was specifically designed to attempt to type DNA for forensic identification purposes. The manufacturer specifies that the kit is for forensic or research use only and is not for use in diagnostic procedures. See Perkin-Elmer AmpliFLP D1S80 Allelic Ladder Kit Insert. Thus, the manufacturer recognizes that the kit is unsuitable for medical and clinical uses, which require certification, which D1S80 has not achieved. It can only be distributed in the unregulated, non-certified crime-lab community. This produces the paradoxical result that "clinical laboratories must meet higher standards to be allowed to diagnose strep throat than forensic laboratories must meet to put a defendant on death row." Eric Lander, DNA Fingerprinting on Trial, 399 Nature 501, 505.
As discussed above, California appellate cases have no considered or approved the D1S80 procedure. Many labs do not even attempt to test crime scene evidence with the D1S80 kit.
a. D1S80 was Commercially Developed and had No Independent Testing or Approval Before Being Marketed and Used

The use of a commercially distributed D1S80 kit does not mean that the test is valid or reliable for use for crime scene evidence. There is no regulatory monitoring or control of this kit. It is not officially or independently tested or approved before being introduced commercially for use in crime laboratories. The NRC has recommended that kits be tested and approved by and independent government agency before they are put to use by the crime labs. NRC 1 at 69; see also Randolph H. JonaKait, Forensic Science: The Need for Regulation, 4 Harvard J. of Law and Technology 109-140 (1991); Office of Technology Assessment, Genetic Witness (1990).
b. D1S80 Testing of Mixed Crime Scene Evidence Samples from Unknown Sources is not Generally Accepted as Valid and Reliable

Applying DNA testing methods to crime scene evidence is much more difficult than simply using the methods developed for research purposes with known and undegraded or uncontaminated samples. The techniques are more demanding with crime scene evidence than with other applications of the same techniques. "It is generally accepted that applying DNA tests to forensic samples, especially criminal evidence, potentially presents more difficulties than analyzing samples in basic research or clinical diagnosis. Samples from crime scenes are frequently small and might be of poor quality because of exposure to a spectrum of environmental onslaughts. Unlike paternity samples, where each sample is from an identified source, the contributor to evidence from a crime scene is often unknown." Office of Technology Assessment, supra at 59. See also Lander, supra, at 501 (use of DNA test analysis for medical diagnosis does not mean the technique is reliable for forensic identification).
The D1S80 method, as applied to testing crime scene evidence, involves additional critical steps that do not arise in other applications of the technology of amplifying non-crime scene samples. NRC 1 at 77. For example, the NRC 1 report noted that the scientific community was uniformly alarmed about the special dangers of contamination in crime scene evidence. NRC 1 at 65-66.
As indicated above, to be accepted under Kelly, the D1S80 method must be accepted as reliable as it is applied in the specific forensic application. For the D1S80 typing kit used in this case, there have been published validation studies for the D1S80 kit. Nevertheless, the experience with this kit has shown that there are serious reliability problems which prevent the kit from being generally accepted in the community of molecular biologists.

1. the microbial contamination problem
The most serious problem to emerge is the inability of the kit to distinguish between human DNA and certain forms of microbial DNA commonly found in crime scene samples. All experts for both sides in Bokin agreed that this is a serious concern. On this issue, the People's Department of Justice witness, Dr. John Tonkyn, testified as follows:
b. You said that the TWGDAM guidelines do not specifically call for microbial studies?

a. Not for internal validation, no.

1. But for developmental validation it's widely understood in the forensic community that nonhuman studies, including microbial studies, are very important when it comes to STR typing?

1. That's correct.

17. As well as D1S80 or any PCR system?

1. Yes.

Q. And the reason for that is it's known generally that microbial DNA can produce bands that, in some situations, can mimic human bands?

1. I think the reason that that study is included in TWGDAM is that forensic samples are often contaminated with microbial DNA, and you want to determine if the system that you're using can produce bands that you might mistake for those created from human DNA.

(R.T. 5400)

Although the People's own witness thus acknowledged the importance of the microbial contamination problem, the People in Bokin cited to pre-1996 D1S80 validation studies that have generally ignored or failed to comprehensively consider this issue. The People did acknowledge a 1996 study, Fernandez-Rodriguez, et al., Microbial DNA Challenge Studies of PCR-Based Systems Used in Forensic Genetics, Advances in Forensic Haemogenetics, 16th Congress of the Interational Society for Forensic Haemogenetics 177 (1996), which was the first to document the microbial contamination problem, but the People failed to appreciate the significance of the study. The study found that certain microbes commonly found in crime scene samples do in fact mimic human DNA. A 1997 study by the same authors repeated and expanded the first study to include additional microbes.(Defense Exhibit TT) The People in Bokin did not cite the more recent study, but they did provide a faxed letter by the authors of the study written after the first study, but before the second, indicating that Dr. Bruce Budowle of the FBI, under unknown circumstances, prevailed upon the authors, who are themselves law enforcement personnel engaged in routine D1S80 forensic casework, to opine that their first study should not preclude forensic use of D1S80 testing methods under certain circumstances, including quantification of human and total DNA, and " double typing by native and denaturing PAGE." The more recent study found additional microbes that mimiced human DNA and also suggested that the problem could be solved by double typing by native and denaturing PAGE.
The short answer to any reliance on the faxed letter is that it is irrelevant because Allen Keel does not in fact utilize the suggested remedial procedure. More broadly, as in People v Brown, 40 Cal 3d at 533, the People cannot seriously contend that the hearsay opinion letter of forensic technicians is sufficient to state the views " of the relevant community of impartial scientists."(emphasis in original) Those scientists, including Drs. Reilly and Libby, are of the strong opinion that the microbial contamination problem precludes the unmodified D1S80 kit methodology from being generally accepted within the community of molecular biologists.
The Kelly-Frye admissibility hearings conducted to date on the D1S80 kit have focussed heavily on the microbial contamination problem. In Riverside County, following a lengthy hearing, Superior Court Judge Bailey declared that results of the D1S80 test were inadmissible because " (t)he Court finds that there is a genuine controversy, difference of opinion and lack of consensus in the relevant scientific community as to the general acceptance and effectiveness of the PCR based D1S80 testing procedure." People v. Scott, Ruling of Jan. 23, 1997, submitted as Exhibit C in this hearing, at p. 6. Central to the Court's ruling was the finding that the microbial contamination problem identified in the Fernandez- Rodriquez study " has not been adequately provided for in the D1S80 kit now distributed by Roche Molecular Systems and appears not to be adequately addressed by others doing the D1S80 testing procedure." (Id.)
In San Francisco, the D1S80 test was addressed in Bokin, but not in McClanahan. Judge Dondero opted to treat the microbial contamination problem as a prong three issue rather than a prong one issue as was done in Scott. He wrote: " The fact the (Spainish) researchers found continued evidence of microbial DNA which mimicked himan DNA does not cause this Court toexclude such evidence, especially when the problem can be controlled by certain measures int he particular laboratory. Any forensic witness subpeoned by the prosecution would be expected to consider and deal with this observation by the Madrid group." Ruling of May 6, 1999, submitted as exhibit A in this hearing. Therefore, whether the issue is treated under prong 1 or prong 3 of Kelly- Frye, it is clear that the microbial contamination problem requires some showing by the People that the problem has been resolved by adopting the procedures suggested in the Spainish studies or some other equally effective procedure. No court yet, however, has held that the problem can simply be ignored on the say so of a particular lab technician who believes, contrary to the weight of scientific opinion, that the problem does not merit solving.

2. Other Reliability Problems
Moreover, there are also numerous other reliability problems with the D1S80 typing kit which precludes its general acceptance. The peer reviewed scientific articles marked as defense exhibits in Bokin highlight a few of those problems, including that " many variants (or interalleles) exist in the D1s80 locus that do not match the allelic ladder, which makes genotype identification difficult"(Exhibit 57), that the effectiveness of the method is " often reduced by the occurrence of PCR artifacts, such as bogus alleles and allelic dropout" (exhibit 58), that " more recent studies indicate that it is possible to obtain ambiguous and even incorrect results from moderately degraded DNA samples" (Exhibit 64), that, in the words of the kit insert, "[a]t certain levels of DNA degradation, only the smaller allele of a heterozygous sample may be detected (and) [s]ome amplified D1s80 alleles in the size range of the ...Allelic Ladder do not line up with a ladder band" (Exhibit 65), and that " preferential amplification, anomalous products , and length limitations may be incountered" (Exhibit 66), and finally, in the words of Dr. Holt and her colleages, that " [w]ith regard to the D1S80 kit, the discrimination potentail of this locus can be very informative, yet the allele size range is rather large,resulting in potential for preferential amplification." (People's Exhibit 23). These problems go beyond " issues and system limitations" as the People euphemistically put it in their brief in Bokin. (Brief, p 9-10) They go to "the crucial issue:whether for the typing categor(y)...at issue here, current methodology, employed by qualified techniquians, can discriminate reliably between testable and untestable samples and between accurate and inaccurate results." People v. Brown , 40 Cal. 3d at 534 (emphasis in original) The literature and the testimony before the Court is that there is currently no scientific consensus on this crucial issue.
c. The D1S80 Test Does Not Contain Basic Scientific Controls
To be scientifically accepted, a test must incorporate controls to identify internal failures and contamination and otherwise assure that the test procedures are working properly and producing correct results. There are a number of types of controls designed to detect different potential problems. Positive controls show whether the test correctly produces positive results when it should. Negative controls show whether the test correctly produces negative or not results when it should. Substrate controls show whether or not a sample has been contaminated by its substrate and/or handling.
The scientific method and principles of validity and reliability dictate that no meaningful conclusions can be drawn from a test unless all appropriate controls have been included. As the National Research Council noted, "[f]orensic DNA analysis should be governed by the highest standards of scientific rigor in analysis and interpretation. Such high standards are appropriate for two reasons: the probative power of DNA typing can be so great that it can outweigh all other evidence in a trial; and the procedures for DNA typing can be so great that it can outweigh all other evidence in a trial; and the procedures for DNA typing are complex, and judges and juries cannot properly weigh and evaluate conclusions based on differing standards of rigor. NRC 1 at 52. See also Randolph N. Jonakait, Will Blood Tell? Genetic Markers in Criminal Cases, 31 Emory L.J. 833.
D1S80 lacks all the minimum controls that "should be processed at the same time as the evidence samples." Technical Working Group for DNA Analysis Methods, Guidelines for a Quality Assurance Program for DNA Analysis (1995) Crime Lab Digest §7.5 [hereinafter TWGDAM Guidelines]. Controls required by TWGDAM and not included in D1S80 testing are:
1. Negative controls: reagent blank and amplification blank. TWGDAM Guidelines, §7.5.1.1;
2. Positive Controls: known human DNA "carried through the… typing…. TWGDAM Guidelines §7.5.1.2;
3. Substrate Controls, which "should be collected from the evidence, including unstained areas adjacent to stained areas …and should be processed at the same time as the evidence samples." TWGDAM Guidelines §7.5.1.3;
4. The TWGDAM guidelines further dictate that "the sample should be split for duplicate analysis as early as possible prior to the amplification" as a further control. TWGDAM Guidelines §7.5.1.4.

d. Research Testing of D1S80 has Revealed Many False Positives
Many false results have been documented in the scientific literature. See, e.g., Kloosterman, et al., PCR Amplification and Detection of the Human D1S80 Locus, 105 J. Legal Med. 257, 263 (1993). Among the problems that may cause false identification are:
1. Differential Amplification:
c. The technician my falsely interpret the results as homozygous;
d. Missing alleles in a mixes sample will result in false typing;
2. Degraded DNA will result in false results by failing to resolve all alleles through electrophoresis, and/or through the addition of false bands in the results;
3. Mixed samples will likely result in varying band intensity and a false interpretation;
4. Certain strains of bacteria produce false bands on the human allelic measurement ladder;
5. There are additional alleles that have not been identified;
6. Insufficient or excessive quantity of DNA can result in a false signal, and thus a false result that is undetected by the analyst. Rebecca Reynolds et al CITE.

e. Preferential Amplification
A known and generally accepted principle of the PCR-amplification process is that smaller alleles are amplified preferentially over larger ones. TWGDAM Guidelines require that "the potential for [problems with] differential [preferential] amplification must be addressed. TWGDAM Guidelines §4.4.1.5. A serious scientific defect of the D1S80 test is its inability to specifically identify all alleles.
Preferential amplification proses and insurmountable problem when attempting to interpret a mixture. An analyst cannot determine whether one of the larger alleles was "out-competed" in the amplification process. What the analyst sees in interpreting a mixture is potentially incorrect. Furthermore, the test contains no internal control or other device to identify when or if preferential amplification has occurred.
Similarly, there are no controls for "off ladder variants," those alleles that do not fall with the core-repeats allelic ladder.
One study that attempted to validate the D1S80 test, reported numerous problems in achieving reliable results. In a paper released following a presentation at the 14th Congress of the International Society for Forensic Haemogenetics, the authors concluded that using the D1S80 test should only be undertaken and interpreted "with great care," and that "more recent studies indicate that it is possible to obtain ambiguous and even incorrect D1S80 types from moderately degraded D1S80 samples." Marco A Scarpetti & Stephanie A. Turek, Validation of the AmpliFLP D1S80 Amplification Kit for Casework in the Detroit Police Crime Lab, Sixth International Symposium on Human Identification (Promega Conference) Scottsdale, Arizona, October, 1995, Poster Abstract.
The authors of this paper also stated that "[a]mbiguous types are obtained when the smaller of two observed alleles is amplified to a significantly greater extent than the other allele. This type of result can arise from amplification of a partially degraded DNA sample or from amplification of one of a variety of mixed samples." Id. The authors went on to warn that "[i]ncorrect types are obtained when the degraded DNA sample can support amplification of products in the size range of the smaller allele but not in the size range of the larger allele. As a result, a sample from a heterozygous individual can be analyzed incorrectly as a homozygous type." Id. The authors discussed the inadequacy of the D1S80 ladder and detailed the ongoing development and release of new component ingredients.

6. THE GREEN I AND PROFILER PLUS STR TEST KITS, USED IN CONJUNCTION WITH THE 310 GENETIC ANALIZER AND AUTOMATED CAPILLARY ELECTRPHORESIS,ARE NOT GENERALLY ACCEPTED IN THE SCIENTIFIC COMMUNITY
In their pleadings in Bokin and McClanahan, the People provided a list of 34 "articles" that purport to support their contention that STR typing of forensic samples is generally accepted in the scientific community. Although "the People are not aware of any publications questioning the scientific reliability of PCR-based DNA analyses and databases" Opp. at 76, several of the articles included in their list of publications detail many of the problems that arise from amplification and typing STR loci. See, e.g., T.M. Clayton et al., Further Validation of a Quadruplex STR DNA Typing System: A Collaborative Effort to Identify Victims of a Mass Disaster, For. Sci. Int'l 17-25 (1995) (finding that only 37% of samples were successfully typed when four STR loci were amplified simultaneously and a full 44% of samples yielded no result); K.A. Micka, et al., Validation of Multiplex Polymorphic STR Amplification Sets Developed for Personal Identification Applications, 41 J. For. Sci 582-590 (1996) (noting problems in typing mixtures); P. Gill, et al., Development of Guidelines to Designate Alleles Using an STR Multiplex System, 89 For. Sci. Int'l 185-97 (1997) (describing problems with stutter bands and non-specific artifacts).
Most remarkably, not one of the articles listed by the People in their pleadings verify that the methods used by the Crime Lab in their STR typing of TPOX, THO1 and CSFIPO loci or the nine Profiler Plus loci are appropriate for forensic use. Such verification is required by the National Research Council, and discussed in detail, in section I(D)(5), supra.
Not one of the articles listed in the People's motion verify that the tests used and protocols followed by the Crime lab in their typing of the thirteen STR loci in this case are "valid not only in theory, but also in practice." Indeed, most of the articles listed are completely irrelevant to the reliability of the testing conducted by the Crime Lab. See, e.g., J.L. Weber & P.E. May, Abundant class of Human DNA Polymorphisms Which Can be Typed Using the Polymerase Chain Reaction, 44 Am. J. Hum. Gen. 388 (1989); P. Gill, et al., Automated Short Tandem Repeat (STR) Analysis in Forensic Casework-A Strategy for the Future 16 Electrophoresis 1543 (1995); Walsh, et. Al Development of the AmpF1STR Blue PCR Amplification Kit (Perkin-Elmer product literature).
The People attempt to show general acceptance and reliability by illustrating that research is conducted in the area of STR typing. Clearly, abundance of research and general acceptance and reliability are not the same. Without a doubt, there is , for example, an abundance of research on the topic of cold fusion, but obviously, there is no general acceptance of that theory.
Another crucial concept neglected by the People and their witness is the concept of validation. TWGDAM Guideline 4.1.2 states: "Validation studies must be conducted by the DNA laboratory or scientific community prior to the adoption of the procedure by the DNA laboratory." Contrary to all other witnesses, both NRC reports, and the TWGDAM and DNA Advisory Board Guidelines, Dr. Cydne Holt began her testimony in Bokin with the dubious assertion that validation studies "do not overlap with the idea of general acceptance of STR's necessarily," and that she did not need to rely, as she had so extensively done in McClanahan, on Perkin Elmer's Green I validation study. ( R.T. 46) This shift of position, of course, was dictated solely by the company's desire to maintain trade secrets and Judge Dondero's ruling upholding the priviledge but denying to the People the use of the company's validation study. But even Dr. Holt had to admit on cross examination that somebody somewhere has to validate a system before it is used forensically. (R.T. 96)
Importantly though, Dr. Holt also maintained that the way the validation process works is that "the manufacturers ...release the kit...and...by definition the validation studies are performed by the forensic scientist on the specific commercially available kit, post-release from the manufacturer." (R.T. 114) Fast forward then to the testimony of Dr. John Tonkyn, who maintained that the DOJ never did a full developmental validation study on the Green I kit because "[t]he developmental validation had already been carried out at Perkin Elmer, which markets this kit." (R.T. 5340) Like Holt, Tonkyn testified that "[t]he kit requires 4.1, developmental validation, before it can be utilized. In addition, each laboratory that uses this must comply with Section 4.5, internal validation." (R.T. 5456) Significantly, Tonkyn conceded on cross examination that "a validation study which dealt, for instance, with the Promega CTT system, using the Promega primers in a silver staining system, would not validate developmentally or internally the Green I Kit, which uses an entirely different set of primers, an entirely different software, and entirely different parameters." (R.T. 5477) The People's own witness thus completely undermines the People's reliance in its brief in Bokin on validation studies such as those contained at Tabs 2, 6-7, 9-10, and 20 of the People's post-hearing brief, all of which are subject to the same criticism leveled by Tonkyn against a damaging stutter study brought to his attention by the defense.
This leaves only Tab 8, The Perkin Elmer Manual that the Court ruled the People could not rely upon because of Perkin Elmer's refusal to disclose the underlying data. When Dr. Tonkyn was specifically asked to cite all of the validation studies he was aware of concerning the Green I kit, the only one he could cite was Perkin Elmer's Green I Manual. (R.T. 5477 ) The Manual, in turn, cites environmental abuse and nonhuman specimen studies supposedly conducted by DOJ which Tonkyn swears DOJ never conducted. (R.T. 5341, 5383,5394-5397) Despite the fact the Manual cites studies that were never conducted, and that the data underlying the Manual has never been examined by DOJ or anyone else, Tonkyn testified that when the DOJ first performed casework in September of 1997 utilizing the Green kit and the 377 Sequencer (DOJ never did casework using the 310; R.T. 5479) "we relied on the developmental validation that had gone into that User's Manual to cover Section 4.1 of TWGDAM." (R.T. 5454)
Another disturbing aspect of the testimony of Dr. Holt, representing perhaps the best example of her bias and downright propensity to fabricate testimony, is her testimony in McClanahan and Bokin about the American Academy of Forensic Sciences abstract entitled TWGDAM Validation of AmpFLSTR Green I and AmpFLSTR Profiler I by Dr. John Tonkyn ( People' Exhibit 80.)
In McClanahan, the People introduced the abstract as one basis for Dr. Holt's broad assertion that "multiple laboratories have done our experiments over again and have validated the green and all the other multiplexes that we've developed." (R.T. 831) She specifically testified that this abstract was "definitely peer reviewed" (R.T. 834) and "I've read it and I was there when he presented it..." (R.T. 916) She continued: "Dr. Tonkyn is representing the California Department of Justice in this talk and their validation found Green I and Profiler to be useful for forensic investigations. They now use these systems routinely in their laboratory. They performed experiments to address all the TWGDAM guideline that we've been going over." (R.T. 915)
In the consolidated cases, Holt was again called upon by the People to emphasize the importance of Dr. Tonkyn's abstract:
17. And does that particular abstract have any significance in terms of your opinion that STRs are generally accepted and reliable for human identification?

1. Sure.

17. Are there any parts in particular without reading the entire abstract?

1. This scientist validated, as the titles says, according to TWGDAM guidelines the Green I and the Profiler Kit for forensic casework, both of those kits contain the STR loci that we have been talking about, CTT, using the same exact reaction conditions and primer pairs. They address the TWGDAM guidelines. I won't read them all out unless the court would like to hear those again. Some of the particular results that they were presenting are effects of environmental abuse, such as semen and bloodstains to several temperatures and sunlight, effect of degraded DNA, and effects of PCR inhibitors, and in all cases, the correct genotype was obtained. ... And they are using a minimum signal threshold of 150 like we have been talking about. And go on to generalize: "that based on the results of our validation study, guidelines were developed which take into account signal strength, the presence of stutter bands and peak height rations to assist in the interpretation of casework and offender databank results and the statistical analysis of genotype frequency."

q. So would it be fair to say that the California Department of Justice advertise validated the Green I Kit for forensic use?

a. Yes.

(R.T. 963)

On cross examination, Dr. Holt further gilded the lily by claiming that the DOJ abstract concerning Green I and Profiler I was not a work in progress, but rather, and more significantly, "a report of their validation studies. They had finished those when they presented it.... They validated the system. Their efforts or their experiments--It was already finished when they presented their work." (R.T. 965) She even testified that she "remembered Dr. Tonkyn presenting their guidelines relative to quantitating the stutter and peak height ratios for interpreting mixtures." (R.T. 988)
Judge Dondero soon learned that most, if not all, of this testimony is an utter fabrication. Following Dr. Holt's testimony, and in direct contradiction to it, the Attorney General inadvertently undermined Dr. Holt's testimony by presented the declaration of DOJ Lab Director Lance Gima, which declares under penalty of perjury that "I am specifically aware of the validation studies for the AmpFISTR Green I and... Profiler I conducted by DOJ analysts....These validation studies are internal rather than developmental. As such, they are primarily pertinent to this laboratory's assessment of the effectiveness of our own procedures and methodology. The studies have not, as yet, been peer reviewed....Further, with respect to AmpFISTR Profiler I, DOJ cannot provide a complete validation study since our laboratory did not complete the study; instead, our laboratory began using...Profiler II during the development of our multiplex protocols, and it was that data which was presented at the February 1998 American Academy of Forensic Sciences proceedings. Profiler II employs different loci than does Profiler I." (Defense Exhibit 7K)
Then, apparently forgetting or choosing to ignore the testimony of Dr. Holt, the People presented the rebuttal testimony of Dr. Tonkyn to the effect that the DOJ Green I study was not in fact a developmental validation study at all, but only an internal one, and that it was done not on a capillary electrophoresis platform using a 310 Genetic Analyzer, but on a gel-based 377 DNA Sequencer using a 1996 pre-release kit and "in-house procedures" that were significantly modified from the protocol in the 1997 Green I User's Manual.(R.T. 5340-5341; 5421-5424, 5432, 5453). As an example, Tonkyn stated that the study was conducted with a stutter cut-off of twenty percent, not the seven/ten percent cut-offs which are currently used, which means that the study was eliminating calls in the ten to nineteen percent range which the current program would call as true alleles. (R.T. 5360) He also admitted that the study was flawed because of "a mistake that I had made in interpreting a peak as stutter, when, upon reanalysis, we are positive that this represents a spillover artifact from one lane of a gel into the next lane." He further stated that the study had found a number of atypical results, including stutter peaks in excess of the ten/seven cut-off, and one stutter peak as high as twenty two percent (which he now attributed to his carelessness in causing contamination and improper interpretation), as well as a null allele, imbalanced alleles, and alleles falling below the expected 150 RFU cut-off. (R.T. 5374, 5387-5392)
There were other problems as well. In the mixture portion of the study, the samples were "overlapped each other so much that the whole experiment was sort of a wash. You couldn't really draw any conclusions." (R.T. 5368) Also, "Perkin Elmer had not finalized the protocol which included the particular number of cycles of PCR." (Id). Moreover, there was a major problem with the quantification of the test samples, as a DOJ analyst "had quantified or quantitated the samples at a much earlier time point. And what we have seen when you have DNA stored at very low concentrations, the concentration will sometimes go off." (R.T. 5399) Along the same lines, in the population database portion of the study, for some samples "the actual quantitation of the DNA was done incorrectly or the DNA degraded during shipping, possibly due to not being frozen during shipping." (R.T.5391)
Dr. Tonkyn also admitted that, contrary to the information in the abstract about the Green I aspect of the study, the DOJ had not in fact conducted nonhuman specimen studies or certain environmental abuse studies. (R.T. 5341, 5383, 5394) Dr. Tonkyn further testified that when he gave his oral presentation, he did not discuss with his audience the DOJ's finding of a number of atypical and unexplainable results, or the finding of excessive stutter peaks, including one in excess of 20 percent, but he did testify that,
"when I wrote that abstract in August of ‘97, we had been planning on validating Profiler I. But in between that time, we changed our minds and decided to validate the Profiler Plus, as that had just come out. And at the meeting before I started my presentation, I made an announcement that what was in the proceedings was not exactly correct, in that we were going to present validation of Profiler Plus, not Profiler I." (R.T. 5382)

He was adamant that "when I gave the presentation, I prefaced my prefaced my presentation with the fact that it was not correct; we were not going to be talking about validation of Profiler I, but Profiler Plus." (R.T. 5469) He also testified that,

"when I gave the presentation, I stated that the developmental validation work that we did or experiments that are covered under developmental validation guidelines of TWGDAM were not complete, and they weren't the same for both the Green I and Profiler Plus, and I had listed which ones had been done with which system." (R.T. 5469)

He also testified with respect to stutter that,

"I believe what I stated during my talk was that we were in the process of collecting stutter data for all of the loci and that we had not found any stutter peaks that differed greatly from what Perkin Elmer had listed in their User's Manual, so we might have seen some that were just slightly by a percentage or two higher than the cut-offs, and that we were continuing to collect data and we possibly might modify our stutter cut-offs based on our own findings."
(R.T. 5386)

Unless, therefore, Perkin Elmer employee Holt is an incredibly bad listener, she could not possibly have heard Tonkyn attest to stutter guidelines or "experiments to address all the TWGDAM guidelines," and she certainly could not have heard him talk or present data on a completed validation study for Profiler I, since no such study was ever conducted and the audience was so informed. Dr. Holt's confident testimony to the contrary is compelling evidence of her bias and her tendency to distort the truth to fit her own (or her company's) goals.
In the face of this record, the People made the audacious claim in Bokin that the "widespread use of STRs in forensic casework attests to the general acceptance of STRs because each laboratory must first, before commencing casework: (1) generally rely upon the published Developmental Validation studies, and (2) specifically test the reliability of the typing kit through Internal Validation studies." (People's Post Hearing Brief, p. 6) Of course, if the solitary developmental validation study has not been published or if it is based on false information, or if the internal validation work relates to different protocols or is of such a shoddy nature that it cannot reasonably be relied upon for any purpose, then the logic of the People's position crumbles.
In the end, the Court in Bokin was inevitably lead by the People's own witnesses in a giant circle which begins and ends with Perkin Elmer's so-called validation study and the refusal of the company to disclose the data underlying it, coupled with an absence of any other relevant validation studies, and the admission of the People's own witness that validation specific to the kit must be performed as a prerequisite to reliable use.
All the defense experts were of the opinion that without the publication and critical peer review of Perkin Elmer's Green I data and the appearance of additional extensive validation, there can not be, and has not been, any general acceptance of the reliability of the Green I kit in the molecular biology or forensic science communities. The opinions of these experts is persuasive, particularly in light of a number of reliability problems which have already surfaced in the brief history of forensic STR use, including, most recently, the inconsistency between Perkin Elmer and Promega multiplex STR test kit results discovered by NIST's Dr. Kline and the New York Medical Examiner in 1997 (Defense Exhibit G). As Exhibit G indicates, Perkin Elmer has attempted to downplay this problem, but even the company admits that "non-concordant results will always be a possibility when the same DNA sample is amplified using different pairs of primers." (Id.) But if that is the case, then STR multiplex typing kits have no place in an adversary system in which each side would be free to choose competing kits that produce diametrically opposite results and to then make the claim, based on Dr. Holt's and Perkin Elmer's theory, that both kits are reliable. As a group of Canadian forensic researchers has observed in the 1997 Promega Proceeding abstract put into evidence by the People, it is a fundamental tenet of even the forensic practitioners of DNA typing that "[c]leary, for the PCR-based STR loci, the type at a given locus for a given individual should not change as a function of the test method as was the case in the enzyme dependent RFLP test." (People's Exhibit 37, p 154; R.T. 180) Even Dr. Holt subscribed to this obvious principle. (R.T. 181) Yet there is every indication, by Perkin Elmer's own admission in Exhibit G, that the problem, apparently caused by a primer binding site mutation, is inevitable and will reoccur in the future.
There are also a whole range of other reliability problems ably outlined in Defendant's B70 in Bokin, the NIST survey, Validation Studies Performed on STR Systems. As there outlined, these problems include: species specificity problems (the one study conducted to date, Defendant's Exhibit 80 and People's Tab 20, was conducted on Promega's silver stain platform), mixture interpretation problems (see, Watts v. State (1999) __ Miss.__ , 1999 WL 33867 (noting expert testimony cautioning against extensive reliance upon mixed PCR DNA samples and stating "[g]iven that evidence, one would have to question the reliability of any statistical evidence that might be derived therefrom."), contamination problems stemming from the extreme sensitivity of PCR, stutter band problems, null alleles, variant alleles, nontemplate addition of nucleotides, and possible linkage of STR systems to genetic disease. Ironically, the People included the NIST survey as one of their prime exhibits (Tab 10) in Bokin, and they cite it for the proposition that "the articles in peer-reviewed journals unanimously declare that STRs are forensically reliable." (People's Brief, p. 5) This is a gross overstatement. The preface to the NIST survey, written in June 1998, states: "Before a new STR system or STR multiplex may be routinely employed in human identity testing it should be extensively validated to insure reliability of results. We have included below many of the validation studies which have appeared in the literature. We hope this material will help future researchers design their validation studies and allow those who use common STR systems to review what studies have been performed previously." One of the specific statements in the survey is that "[s]purious PCR products may arise from non-templated nucleotide addition of adenine or from stutter bands and thus provide contaminating peaks in an electropherogram." Clearly, the thrust of the survey is that we are at the beginning of the validation process for STR systems, not at the end as the People would have the Court believe. Significantly, not one of the forty six studies cited in the NIST survey deals specifically with the Green I or Peofiler Plus kits using capillary electrophoresis and the 310 Genetic Analyzer. More to the point in Mr. Nawi's case, at least one study conducted bvy the Department of Defense DNA Registry has concluded that successful typing of "aged" (8 year old) fingernails is not possible. See, The Extraction of DNA From Human Nail Material, Proceedings From the Seventh International Symposium on Human identification (Promega Corp. 1996) p. 133, attached hereto as Exhibit F.
If Judge Dondero's experience with the disclosure of the data underlying the DOJ abstract proves anything, it is, in the words of the first NRC report, that "[i]f a new DNA typing method (or a substantial variation on an existing one) is to be used in court, publication and scientific scrutiny are very important." NRC I at 56 "Extensive empirical characterization must be undertaken. Results must be published in appropriate scientific journals. Publication is the mechanism that initiates the process of scientific confirmation and eventual acceptance or rejection of a method." (Id.) ( emphasis added)
The defense at this stage is not complaining of a failure of discovery but rather a failure to sustain the burden of proof on the prong one issue. The court in People v. Axell (1991) 235 Cal. App. 3d 836, 856, citing State v. Schwartz (Minn. 1989) 447 N.W. 2d 422, declared that "admissibility of specific test results in a particular case hinges on the laboratory's compliance with appropriate standards and controls, and the availability of their testing data and results." The specific RFLP test results were admissible in Axell, in part because "Cellmark did comply in this case with defense requests for discovery and defense experts visited Cellmark's laboratory." Id. p 857 See also, People v. Barney (1992) 8 Cal. App. 4th 798, 813 (contention that general scientific acceptance is precluded by the failure of the FBI and Cellmark to publish in peer review journals and otherwise share their data and methodology is foreclosed because "there have been numerous published articles on DNA analysis as performed by the FBI, of which we have taken judicial notice.")
By contrast, in State v. Schwartz, supra at 427, the Minnesota Supreme Court held as follows:
Even if a laboratory has followed reliable procedures to ensure accurate test results, constitutional concerns may prevent the admissibility of such evidence. The fair trial and due process rights are implicated when data relied upon by a laboratory in performing tests are not available to the opposing party for review and cross examination....
Access to laboratory information generally is significant for another reason. The validity of testing procedures and principles is assessed in the scientific community by publishing the data in peer review journals. The TWGDAM, FBI and CACLD standards stress that publication of a laboratory's work product and data used in DNA analysis, as well as independent replication and validation studies, are essential prerequisites to reliability. Efforts to assess the reliability of the commercial laboratories' methodology consequently have been hindered because this information has not yet been made fully available. For example, Cellmark has not yet published data regarding its methodology and its probes are only selectively available.
While we agree with the trial court that forensic DNA typing has gained general acceptance in the scientific community, we hold that admissibility of specific test results in a particular case hinges on the laboratory's compliance with appropriate standards and controls, and the availability of their testing data and results. ... Because the laboratory in this case did not comport with these guidelines, the test results lack foundational adequacy and, without more, are thus inadmissible.

The same result should follow here. The People and Perkin Elmer cannot in fairness be allowed to have their witnesses cite Perkin Elmer's study as the sole source of developmental validation and then turn around and ask the Court to uphold a trade secret privilege as to the underlying data. It is a matter not only of fairness, but of general acceptance as well, since all of the defendant's experts, as well as the NRC reports and the TWGDAM and DAB Guidelines are of one mind in declaring that particular DNA methods are generally accepted as reliable within the broad scientific community if, but only if, the methods have been subjected to extensive validation and peer review. Judge Dondero's ruling could not be clearer on this point: " the sum of this record is that the government has not provided the Court with enough to conclude that STRs identified by using Green One with ABD Genetic Analyzer 310 satisfies TWGDAM requirements pertaining to developmental validation. There was no discovery of developmental validation conducted by Perkin-Elmer. The exhibits referenced by the prosecution in its post hearing brief deal with peer review of other methods involving STR identifications." Exhibit A at 9

B. THE DNA EVIDENCE IS INADMISSIBLE BECAUSE THERE ARE NO GENERALLY ACCEPTED STATISTICAL METHODS THAT ADDRESS BOTH THE PROBABILITY OF A COINCIDENTAL MATCH BETWEEN TWO PEOPLE WHO SHARE COMMON GENETIC CHARACTERISTICS AND THE PROBABILITY THAT A MATCH WOULD MISTAKENLY BE REPORTED DUE TO LABORATORY ERROR.

1. The Failure To Account For Lab Error Rate

Evidence of a DNA "match" between two samples is impossible to evaluate without reliable information on the likelihood that a match would be declared if the samples are from different individuals. Most commentators consider the ability to express this probability to be crucial to the admissibility of DNA-derived evidence: "without being informed of such background statistics, the jury is left to its own speculations." McCormick, Evidence, 655 (Cleary, Ed.).
As indicated at the outset of this opposition, the need for background statistics to show the meaning of a DNA match is firmly established in California law. People v. Venegas 18 Cal 4th at 82 ( " A determination that the DNA profile of an evidentiary sample matches the profile of a suspect establishes that the two profiles are consistent, but the determination would be of little significance if the evidentiary profile also matched that of many or most other human beings."). It is also established that the statistical calculation phase of DNA analysis "requires Kelly screening to assure both that the statistical methodology used is generally accepted in the scientific community, and that the calculations in the particular case followed correct scientific procedures." People v. Soto , 21 Cal. 4th at 519.
Since DNA testing methods have been employed in courts, the issue of statistics, the calculation of genotype frequencies and the proper use of such statistics in the court room has been an area of contention and debate. See generally, Symposium: The Evaluation Of Forensic DNA Evidence, 37 Jurimetrics J. 395 (1997); Devlin, et al., Comments on the Statistical Aspects of the NRC's Report on DNA Typing, (1994) Journal of Forensic Sciences, 39(1):28.
Issues surrounding the use of statistical analysis and population genetics in the forensic arena involve the manner in which a particular database is constructed and the underlying assumptions in the statistical analysis. In order for the "product" or "multiplication" rule, which is used in PCR based testing, to be valid the each gene that is used must be independent of the other. This means that there is no actual linkage between two genes that cause them to be inherited together. One common example of such a linkage is hair and eye color. If one were to take the frequency of blond hair and then separately determine the frequency of blue eyes, and multiply them together as is done with the PM and DQ tests, the resulting frequency of blue eyed-blonde haired individuals in the population would be much smaller than the actual occurrence in the population. Blond hair may occur in 10% (0.10) of the population and blue eyes in 15% (0.15) of the population. The resulting frequency would be 0.10 x 0.15 = 0.015 or 1.5%. This number however, fails to account for the fact that the most people with blond hair have blue eyes and therefore, the actual frequency for blond hair and blue eyed individuals would be much higher than calculated. This type of linkage or lack of independence is often called substructure. The issue of whether substructures actually occurs in human populations is subject to much debate that has yet to be resolved for PCR based systems.
In its second report, the NRC devoted two chapters to the issue of statistical analysis of DNA evidence. NRC 2, at 89-166. Soon after the release of the second NRC report, a symposium was held and papers published from that symposium on many issues, chief among them the recommendations of the NRC's second report. See, D.H. Kaye, Dna, Nas, Nrc, Dab, Rflp, Pcr, And More: an Introduction to The Symposium of The 1996 Nrc Report on Forensic Dna Evidence, Jurimetrics, (Summer 1997) 37:395.
In particular, some commentators recommend using error rates taken from proficiency tests conducted by laboratories to "correct" the genotype frequencies calculated in a specific case. Johnathan Koehler, Why Dna Likelihood Ratios Should Account For Error (Even When a National Research Council Report Says They Should Not), Jurimetrics, (Summer 1997) 37:425. Others argued that error rates of laboratories be presented to the jury so that the jury can assess the weight of the genotype frequencies in light of the relatively high error rates most laboratories have. William C. Thompson, Accepting Lower Standards: The Nation Research Council's Second Report on Forensic Evidence, Jurimetrics, (Summer 1997) 37:405.
Finally, information has recently surfaced that a database used for the DQ Alpha + polymarker test kit that was published in a peer reviewed journal and which is used by forensic laboratories to calculate genotype frequencies was altered to conform the database to the requirements of Hardy-Weinberg equilibrium. Had the database not been altered, the database would not have fit within the Hardy-Weinberg assumption which underlies the multiplication rule. This result calls into question all of the databases currently in use for forensic testing.
In the midst of these controversies, some consensus has been reached. It is now broadly recognized that a false "match" between samples can occur in two ways.
Interpretation of DNA typing results depends not only on population genetics, but also on laboratory error. Two samples might show the same DNA pattern for two reasons: two persons have the same genotype at the loci studied, or the laboratory has made an error in sample handling, procedure, or interpretation.

NRC 1 at 88.
Thus, to evaluate DNA evidence, the jury needs statistics that address the probability of both events that could cause a false match. To provide statistics that reflect the probability of one event that could cause an innocent person to match, and not the other, would leave the jury to speculate about the meaning of DNA evidence.
Especially for a technology with high discriminatory power, such as DNA typing, laboratory error rates must be continually estimated in blind proficiency testing and must be disclosed to juries. For example, suppose the chance of a match due to two persons' having the same pattern were 1 in 1,000,000, but the laboratory had made one error in 500 tests. The jury should be told both results; both facts are relevant to a jury's determination.

NRC 1 at 89.
The same point was made by Professor Daniel Hartl in an expert's report filed in U.S. v. Yee. Although his criticism was directed specifically at the FBI's DNA testing procedures, his point applies to any PCR-based DNA test:
Once the FBI has declared a match, they treat this declaration as if there were no operator error and no measurement error. The technology simply is not up to this standard....In my judgment, experimental error will turn out to be a very significant term, and perhaps the dominant term, in any valid estimation of the true probability of a match, and any statistical calculation that fails to take this into account is simply meaningless.

Daniel L. Hartl, Expert's Report in the Case of United States v. Yee, at 4, U.S. v. Bonds, 12 F.3d 540 (6th Cir. 1993), aff'g U.S. v. Yee, 134 F.R.D. 161 (N.D. Ohio 1991) [emphasis added].
Other experts have recently echoed this conclusion:
Statisticians and geneticists involved in the controversy over DNA testing have understandably been fascinated by and mostly written on disputes regarding the statistical and genetic issues that DNA identification raise, but laboratory error places the most serious limits on the evidentiary import of reported DNA matches. If justice is the mutual goal of those involved in the debates over DNA identifications--and I believe it is everyone's concern--the possibility of error must be honestly faced, and it must be incorporated into estimates of the incriminatory power of DNA matches.
Richard Lempert, Comment: Theory and Practice in DNA Fingerprinting, 9 Statistical Science 255, 257 (1994).Accord, Laurence D. Mueller, The DNA Controversy And NRC II, in Statistical Methods in the Health Sciences:Genetics, ME Halloran & S. Geiser (eds., 1999) pp.17-18; Jonathan Koehler, Why DNA Likelihood Ratios Should Account For Error (Even When A National Research Council report Says they Should Not) 37 Jurimetrics 425(1997), R. Lempert, After The DNA Wars: Skirmishing With NRC II, 37 Jurimetrics 439(1997); William Thompson, Accepting Lower Standards: The National Research Council's Second Report On Forensic DNA Evidence, 37 Jurimetrics J. 405-424 (1997); David Balding, Errors and Misunderstanding In the Second NRC Report, 37 Jurimetrics J. 469 (1997). In Bokin,the Court has before it the consensus statement of twenty seven prominent statisticians and population genetists attesting to the fact that lab error rates must be accounted for in the statistical computation. ( Bokin Exhibit 105).
Finally, the courts are beginning to heed the well-reasoned statistical arguments of these experts.With respect to Dr. Koehler, who has written most prolifically on the issue, it is significant that the Mississipi Supreme Court very recently declared in Watts v. State (1999) __ Miss.__ , 1999WL 33867 that "as... Dr. Koehler's articles sugges[t], the introduction of statistical evidence can be meaningless without any evidence of the testing laboratory's error rate." The Court also described Dr. Koehler's standing in the scientific community: " Dr. Koehler, adjunct professor at the University of Texas School of Law and assistant professor at the University's Buisness, is one of the leading authorities on the use (and misuse) of statistics in the presentation of DNA evidence." Id at n. 2
When DNA evidence was first introduced in the courtroom, the primary concern was the likelihood of a coincidental match. Little attention was devoted to the likelihood of false positives, perhaps because there was a widespread misperception that false positives are impossible in RFLP-based DNA tests. This misperception was generated and sustained by self-serving claims of DNA test promoters that DNA tests are infallible, fail-safe, and error-free. Professor Jonathan Koehler has suggested that DNA test promoters "engaged in a sinister semantic game" in which they were able to issue misleading denials of the possibility that a DNA test could make an "error" by excluding consideration of human error in administering or interpreting the test. Koehler, Error and Exaggeration, supra, at 24. Needless to say, the effort to distinguish "human error" from "test error" is pointless and misleading when humans are necessarily involved in administration and interpretation of the test, when occasional human errors are inevitable, and when it is necessary to know the overall rate of error (from whatever cause) to evaluate the test results. "For juries it is of little significance what causes an innocent person to match, what matters is how often such matches might be expected." Laurence Mueller, The Use of DNA Typing in Forensic Science, 3 Accountability in Research 55, 56 (1993); see also Thompson, Lessons, supra, at 92.
In any case, the potential for false positives due to laboratory error in DNA testing is now beyond dispute. "Laboratory errors happen, even in the best laboratories and even when the analyst is certain that every precaution against error was taken." NRC 1 at 88-89. See also Koehler, DNA Matches and Statistics, 76 Judicature 222, 229 ("[B]ased on the little evidence available to date, a reasonable estimate of the false positive error rate is 1-4 percent."); Koehler, Error and Exaggeration, supra, at 26 (proficiency testing shows error rate of 1-4%); Donald Berry, Comment, 9 Stat. Sci. 252, 253 (1994)("Only the frequency and type of errors are at issue."); R.C. Lewontin, Comment: The Use of DNA Profiles in Forensic Contexts, 9 Stat. Sci. 259 (1994)(discussing sources of error); William C. Thompson, Comment, 9 Stat. Sci. 263, 265 (1994)(discussing data on laboratory error); Cf. Dan L. Burk, DNA Identification: Possibilities and Pitfalls Revisited, 31 Jurimetics 53, 80 ("Bald statements or broad hints that DNA testing is infallible...are not only irresponsible, they border on scientific fraud").
Indeed, most experts have long believed that having an accurate estimate of the false positive rate is more important than having an accurate estimate of the probability of a coincidental match because the rate of false positives is likely to be much greater than the rate of coincidental matches, at least for RFLP-based tests. Paul J. Hagerman, DNA Typing in the Forensic Arena, 47 Am.J.Hum.Genet. 876 (high false positive rate makes probability of coincidental match irrelevant); Richard Lempert, Some Caveats Concerning DNA As Criminal Identification Evidence: With Thanks to the Reverend Bayes, 13 Cardozo L.Rev 303, 325 (the probability of a coincidental match between people who have the same DNA profile "is usually dwarfed by the probability of a false positive error"); Mueller, The Use of DNA Typing in Forensic Science, supra, at 58 (exact probability of a coincidental match "should hardly matter" to jury given much greater likelihood of false positive); Richard Ostrowski & Daniel Krane, Unresolved Issues in Forensic Use of DNA Profiling, 3 Accountability in Research 47 (1993).
A central premise of California appellate cases is that evidence of a DNA match is inadmissible unless accompanied by statistics that can tell the trier of fact what the match means. Venegas, Axell, Barney, and Wallace all recognize this principle. Now that the scientific community has recognized that error rates must be taken into account in order to make a meaningful evaluation of DNA evidence, the logic of those cases requires that juries be given statistics on the probability of laboratory error; without such statistics, evidence of a DNA match is inadmissible because it is impossible to evaluate.
Venegas expressly left this issue open (18 Cal. 4th at 95 n. 42), and Soto does not mention the issue. The 1996 NRC Report recommends without persuasive analysis that the issue should be simply ignored see, Richard Lempert, After the DNA Wars: Skirmishing With NRC II, (1997) 37 Jurimetrics J. 439. But it would be absurd for courts to insist on valid quantitative estimates of the probability of a coincidental match, without also requiring valid estimates of the rate of false positives due to laboratory error, when the scientific community has determined that the latter is more important than the former to a rational evaluation of DNA evidence. If DNA evidence is "meaningless" without statistical estimates of the probability of a coincidental match, it is also "meaningless" without statistical estimates of the probability of a false positive.
Professor Eric Lander, one of the earliest and most influential scientific commentators on DNA evidence, and a member of the 1992 NRC panel, explained the matter succinctly:
it is simply crazy and scientifically unacceptable to agonize over the exact population frequencies, which might be one in a million, or one in a hundred thousand, or one in ten thousand for the frequency of a genotype in a population, and yet not have actual data for the accuracy, the proficiency of a laboratory's handling of samples...[T]he scientific acceptability of DNA evidence depends on the proficiency of a laboratory being tested such that one can know what the error rate is likely to be, or at least have an upper bound on that error rate.

Prof. Eric Lander, Testimony as a Court's Witness in U.S. v. Porter, District of Columbia Crim. Docket 3F-6277-89, p. 46, July 28, 1994. In Soto, the Supreme Court placed great weight on the views of Dr. Lander in rejecting a challenge to the effect of population substructuring on RFLP statistical computations. 21 Cal.4th at 538
At this point, there is broad agreement among scientists with the NRC's position on error rates: DNA evidence cannot be evaluated without knowing the rate of false positives due to laboratory error, error rates must therefore be estimated and these estimates must be disclosed to juries. There is some disagreement, however, about the best way to present error rate statistics.
The NRC Report recommends that jurors be given two numbers, one indicating the frequency of matching genotypes and the other indicating the rate of laboratory error. "The jury should be told both results; both facts are relevant to a jury's determination." NRC 1 at 89.
Other scientists believe that the error rate and frequency statistics should be combined into a single number that reflects the overall likelihood that the laboratory would declare a match between samples from different people. For example, Professor Hagerman suggested that "[r]esults of DNA typing should always be reported as the sum of the laboratory error rate and the estimated frequency of recurrent band patterns in the relevant population." Hagerman, DNA Typing in the Forensic Arena, 47 Am. J. Hum. Genet. 876 (1990).
Some experts have gone so far as to suggest that jurors be told only the false positive rate; they reason that the probability of a false positive is so much greater than the probability of a coincidental match (at least for multi-locus RFLP matches) that the latter probability has little bearing on the value of the evidence. For example, if the probability of a coincidental match were .000001 (one chance in one million), and the probability of a false positive were .01 (one chance in one hundred), then the overall probability of a match between samples from different people would be approximately .010001, a number that conveniently rounds off to .01 (one in 100). So why not just tell jurors the false positive rate and avoid the risk that they will be confused or unduly swayed by an impressive number (one in one million) that has little meaning or value relative to the false positive rate?
The rate of false positives defines a practical lower bound on the probability of a match, and probability estimates based on population data that are smaller than the false-positive rate should be disregarded.

R.C. Lewontin & Daniel Hartl, Population Genetics in Forensic DNA Typing, 254 Science 1745, 1749 (1991).
Professor Richard Lempert specifically cites the danger of confusion and prejudice as a reason for presenting only the error rate statistic in cases where the probability of a false positive greatly exceeds the probability of a coincidental match.
jurors provided with a laboratory's false positive rate and with information about the likelihood, assuming no testing error, of a match if the DNA evidence was not the defendant's, are likely to be hopelessly confused about the weight to accord the testimony because ordinary people are not very good at working with conditional probabilities. Thus, jurors ordinarily should receive only the laboratory's false positive rate as an estimate of the likelihood that the evidence DNA did not come from the defendant.
Lempert, Caveats, supra, at 325.
Another issue that is being debated is what to tell jurors about the error rate of a laboratory that has performed relatively few blind proficiency tests and therefore has limited data on which to base an error rate estimate. Suppose a laboratory had made no false positive errors, but had participated in only ten proficiency tests in which a false positive might have occurred. It would obviously be quite misleading to tell the jury that the laboratory's error rate is zero. If the true false positive rate were one in 100, or one in 50, or even one in 10, there is a very good chance an error would fail to occur in the first ten trials.
The best solution to this problem is to use a statistical device known as a confidence interval: the number presented to the jury is not the actual number of errors, but a number that can be said with a given degree of confidence (by convention 95% or 99%) to be less than or equal to the true rate of error. For example, if a laboratory had completed 1000 tests without error, one could say, with 95% confidence, that the laboratory's true rate of error is less than or equal to .003 (three in 1000), because there is a 95% chance that at least one error would have been made in 1000 trials if the error rate exceeded .003. Laurence D. Mueller, The DNA Controversy And NRC II, in Statistical Methods in the Health Sciences:Genetics, ME Halloran & S. Geiser (eds., 1999) pp.18(" (L)aboratories should use the upper 95% confidence interval for their own individual estimates of error when they have a perfect record, or if the number of tests they have done is very small the laboratory may use an estimate taken from the industray in general.");Mueller, The Use of DNA Typing in Forensic Science, 3 Accountability in Research 55 (1993); Saks and Koehler, What DNA 'Fingerprinting' Can Teach the Law About the Rest of Forensic Science, 13 Cardozo L. Rev. 361-372, 369-70; Koehler, DNA Matches and Statistics: Important Questions, Surprising Answers, 76 Judicature 222, 228 (1993).
The advantage of the confidence interval approach is that it is conservative. It minimizes the risk that the number reported to the jury will understate the true error rate and thereby be detrimental to the interests of the defendant. There is only a five percent chance, for example, that a number reported with 95% confidence would understate the true error rate. By contrast, there is a very good chance that the actual frequency of errors in a limited number of tests will understate the true error rate, to the detriment of the defendant. Basic principles of fairness and due process therefore require the use of the confidence interval approach.
Continuing debate is to be expected over how best to determine the rate of laboratory error and how best to present error rate data to the jury. The continuing scientific debate about how to determine and express the error rate for forensic DNA laboratories reflects a broad consensus that laboratory error rates are a crucial factor affecting the value of DNA evidence. The fundamental point recognized by the first NRC Report -- that the error rate must be known in order to evaluate DNA evidence -- is not in dispute. Consequently, unless the People can show that they have accounted for this issue in some scientifically accepted manner, this court should hold that all DNA evidence is inadmissible under Kelly and Venegas.
2. There Is No Generally Accepted Method For Computing The Frequency of Multi-Locus Profiles.
Based on the report of Allen Keel in this case, it is evident that the prosecution is seeking to introduce an impressively high random match statistical probability which combines the six loci in the DQ Alpha test kit with the one locus in the D1S80 kit and the thirteen STR loci in the Green I and Profiler-Plus kits. As discussed above, the DQ Alpha +Polymarker test allows DNA from six different loci (areas of the genome) to be "amplified" via PCR and then "typed." The D1S80 kit allows amplification and typing at an additional locus,and the STR loci adds thirteen more loci to the mix. TheDQ Alpha + polymarker loci have varying numbers of alleles (types), as does the D1S80 locus and the STR loci. A "match" on all of the loci in four kits means that two samples have the same allele (type) at each loci.
Evidence of such a match is meaningless, of course, unless one knows the probability of finding a match between samples from different people. This probability depends in part on the likelihood of a false positive in the tests, which is completely unknown and impossible to estimate in the absence of rigorous blind proficiency testing (which has not yet been done). The probability of a match between different people also depends on the likelihood of a coincidental match, which depends, in turn, on the rarity of the set of matching alleles. A set of alleles, from different loci, is called a multi-locus genotype. It is the method for estimating the frequency of multi-locus genotypes that is discussed here.
The major problem with the method is that it makes use of the product rule (multiplication of allele frequencies) without the necessary demonstration that the alleles are statistically independent. The use of the product rule assumes that the various alleles are inherited independently, that there is no reason for persons with a given allele at one loci to have a preferential probability of having a particular allele at another loci, and that there is no population substructure. See NRC 1, Chapter 3. In Soto, the Supreme Court ruled that the debate regarding the effect of population substructuring on RFLP calculations had been resolved empirically by "extensive literature in peer reviewed journals." 21 Cal. 4th at 540 But the debate regarding the effect of such substructuring on the independence of PCR loci continues unabated in scientific journals. Indeed, not even the San Francisco Crime Lab thinks that it can employ the unmodified product rule to PCR loci. In his report on Mr. Nawi's case, Mr. Keel notes that

" (t)he THO1 and HGBB loci are on chromosome 11 and are sufficiently close to one another that they are considered to be linked. However, it is not known whether or not that linkage is sufficiently close to create associations between these two loci. Similarly, FGA and GYPA are neighbors on chromosome 4, and D7S8 and D7S820 are neighbors pon chromosome 7. Until the question of any association between systems on the same chromosome is addressed in the scientific literature, it is appropriate to employ frequency information from only one of each of these neighboring pair of loci.
Report p. 8 n. 8

This reasoning is sound as far as it goes, but it fails to recognize that all of the loci used in the calculation must be empirically demonstrated to be be independent. As the NRC I has noted, "[t]he key question underlying the use of the multiplication rule is whether actual populations have significant substructure for the loci used for forensic testing." NRC 1 at 79 [emphasis added]. As defendant's statistical experts testified in Bokin, without proof of the absence of substructure for the DQ Alpha +polymarker, D1S80, and particular STR loci used in this case, the assumption of statistical independence that underlies the use of the product rule is based entirely on speculation. In light of this testimony, the two NRC Reports, and the scientific furor that has arisen over the use of the product rule for RFLP-based tests, it is clear that the scientific community does not accept its use for PCR based tests without proof of the independence of the alleles.
Thus, as pointed out above at page 42, nothing in the Soto decision impacts on the issues in this case. First, it was an RFLP case in which the Court attached the "greatest significance" to NRC II. 21 Cal. 4th at 539. The NRC II stresses the tentative nature of our current state of knowledge with respect to PCR-based systems, as well as a fundamental distinction between RFLP (VNTR) loci and PCR loci.
The lack of information about population substructure also raises severe doubts about the representativeness of the small convenience samples that serve as data bases for PCR statistics. There appears to be little information upon which to judge whether the frequency of particular PCR alleles in one ethnic group, or one geographic area, is likely to be the same as in another. Thus, the relevance of small unrepresentative data bases appears questionable even for estimating the frequency of individual alleles, let alone estimating the frequency of multi-locus genotypes.
Assuming it should be applied at all, there can be no doubt that some modification of the product rule must be made in this case, because the Supreme Court has already found in People v. Venegas, supra that " [t]here has been significant scientific controversy over whether constraints should be imposed on the use of this rule in light of certain population genetics theories." 18 Cal. 4t at 65 The existence of this controversy for PCR testing means that there is presently no general scientific consensus to use the product rule in its unmodified form for PCR loci. 18 Cal 4th at 84
Moreover, our Supreme Court clearly broke new ground in Venegas, when it "upheld the trial court's finding of a scientific consensus that the NRC's ‘modified ceiling' approach or method-used to calculate the statistical probabilities of a match between the evidentiary samples and the DNA of an unrelated person chosen at random from the general population-is forensically reliable, in that from the scientifically based range of probabilities it selects the figures that most favor the accused, and therefore cannot furnish a basis on which to invalidate the admissibility of such evidence on motion of the accused." 18 Cal.4th at 55(emphasis added) Although the term "forensically reliable" may look and sound like the same statistical artifice invented by Dr. Bruce Budowle and used by others to downplay the statistical significance of empirically found departures from Hardy Weinberg or linkage equilibrium, the concept as used in Venegas is in fact exactly the opposite of that used by Budowle et al., who employ the concept to justify numbers that disfavor the accused. As applied to this case, the principle of forensic reliability as used in Venegas clearly dictates a modification of the product rule for scientific and due process reasons.18 Cal. 4th at 84 ( modified ceiling principle must be adopted because it is "forensically reliable" in that " it resolves any imprecision in the statistical calculations in a way that preserves the constitutional presumption of the suspect's innocence")
The scientific reasons for such a modification were explained in numerous defense exhibits and in the testimony of defense witnesses Drs. Laurence Mueller, and Sandy Zabell in Bokin. In general, the exhibits and the testimony of Drs. Mueller and Zabell established, in the words of the court in Venegas, that " a figure for the statistical probabibility of a DNA profile derived from modifying the probabilities of its individual allele frequencies is accurate only insofar as the multiplied frequencies are statistically independent from each other." 18 Cal. 4th at 85 See also, United States v. Shea (D.N.H. 1997) 957 F.Supp. 331, 336, ( "The product rule can be applied reliably ... only if the estimate of allele frequencies is reasonably accurate and the conditions in the population approximate what are known as Hardy-Weinberg equilibrium and linkage equilibrium.")(PCR case) ; United States v. Lowe (D. Mass. 1996) 954 F. Supp. 401,418 ("The validity of using the product rule in this context depends upon the independence , or lack of association between the alleles or loci examined.")(PCR case)
These same witnesses also established, in the words of the court in State v. Copeland (1996) 130 Wash. Sd 244, 922 P.2d 1304, 1317, that "potential problems could result from ‘genetic drift' resulting in small populations having distinct differences, too small a database, lack of randomness of the samples, and most importantly, lack of a truly mixed population such that each locus is in Hardy-Weinberg equilibrium as well as linkage equilibrium." Recent population studies submitted by the defense, including even studies conducted by Drs.Budowle and Brenner, two experts heavily relied upon by the People, have demonstrated a consistent pattern of statistically significant departures from both HW and linkage equilibium for some of the very databases used by the San Francisco Police Department.

2. There Is No Generally Accepted Method For Constructing And Using DNA Databases.
The defense established through the testimony of Mr. Keel in Bokin that the San Francisco Police Department is currently using an FBI STR database made up of less than two hundred samples.The People's own witness, Dr. Holt, testified that an adequate database would require "hundreds" of database samples. (R.T. 250) See also, People v. Venegas, 18 Cal. 4th at 64 ("[T]he FBI and Cellmark...and other forensic laboritories use one or more population databases containing measurements of the DNA fragments of several hundred persons at each loci...");United States v. Lowe (D. Mass. 1996) 954 F. Supp. 401,407(quoting NRC II for proposition that an adequate database must be " at least several hundred persons."); Id. p. 409n. 10( " the government's experts testified , and Lowe's expert did not dispute, that a database of several hundred individuals is sufficient."); Franson v. Micelli (1994) 269 Ill. App. 3d 20, 645 N.E. 2d 404,406 vacated on other grounds, 666 N.W. 2d1188 (Statistical expert "testified that the ‘Caucasian' data base used by Genetic Design was too small and that an appropriate random sampling should include at least 100,000 subjects. He did admit, however, that some statisticians believe that 5,000 is a valid starting point.") The utilization of an STR database containing 197 samples undoubtedly introduces imprecision into the statistical calculation, and defendants experts so testified.

4. Conclusion
The statistical issues identified by the defense witnesses have lead our Supreme Court and many other courts to declare the unmodified product rule inadmissible on a variety of statistical or due process grounds. See, People v. Venegas, 18 Cal. 4th at 84 ( modified ceiling principle must be adopted because it is "forensically reliable" in that " it resolves any imprecision in the statistical calculations in a way that preserves the constitutional presumption of the suspect's innocence"), People v. Barney, supra; State v. Carter, 246 Neb. 953, 524 N.W.2d 763, 780-83 (1994) (statistical probability calculation used in PCR DNA analysis which was based on the Blake database is not generally accepted within the scientific community) Com. v. Curnin (1991) 409 Mass. 218, 221-227 , 565 N.E.2d 440, 442-445(there is currently no general acceptance as to the statistical calculation process, and thus DNA analysis evidence is inadmissible.); see also Caldwell v. State (1990) 260 Ga. 278 [393 S.E.2d 436, 444] [laboratory's statistical evidence inadmissible because state failed to show data bases were in Hardy- Weinberg equilibrium]; State v. Schwartz (Minn. 1989) 447 N.W.2d 422, 428-429 [exclusion of statistical probability evidence necessary because of potentially exaggerated impact on jury].)
Under the Kelly standard, the appropriate approach to a modification of the product rule is to adopt a method of calculation which is both generally accepted by the scientific community and forensically reliable within the meaning of Venegas. In this regard, it is significant that in Venegas, based on the Court's review of both NRC reports, the Court concluded that " the modified ceiling method ... has been generally accepted in the relevant scientific community, and that satistical probability calculations of random matches of DNA profiles computed under that methodology are admissible under the Kelly test." 18 Cal, 4th at 89
At the very least, therefore, this Court must follow Venegas and rule that in the absence of the type of statistical showing made in Soto, the modified ceiling method is one approriate and mandated modification of the product rule. Further, the Court should decide an issue expressly left open in Venegas and rule along with the Mississippi Supreme Court that "as... Dr. Koehler's articles sugges[t], the introduction of statistical evidence can be meaningless without any evidence of the testing laboratory's error rate." Watts v. State (1999) __ Miss.__ , 1999WL 33867
The People will no doubt scream that Venegas was an RFLP case. But not even the critics of Venegas would claim that the unmodified product rule can be reliably applied in a PCR case. In this regard, it is telling that the leading proponent of reverse forensic reliability, Dr. Bruce Budowle, has recently taken the positon that in light of his own persistent findings of statistically significant departures from HW in PCR databases, the correct rule is that "[b]y applying the recommendations of the NRC II Report, these allele frequency data( DQ A1, PM,D1s80, CSF1PO, TPOX,and THO1) can be used to estimate the rarity of a multiple locus profile." (Bokin Exhibit 82, People's Tab 26) See also,United States v. Lowe (D. Mass. 1996) 954 F. Supp. 401,407 ("(T)he 10x factor, or confidence interval, is now generally regarded as a suitable alternative for dealing with the uncertainty that underlies the statistical approach utilized in DNA analysis.")(PCR case)
Even under this approach, which is foreclosed by Venegas, the Court would be obligated to adopt all of the recommendations of NRC II concerning the product rule, including the use of theta, the 2P rule, and the 10x factor. Use of all these statistical devices would be necessary in order to adjust for the several sources of statistical error outlined in NRC II and by the witnesses in Bokin. Although there might be some room for debate on whether adjustments like the 2P rule should be applied to the particular loci at issue here, the defendants would be required to be given the benefit of the doubt on this issue pursuant to the principle of forensic reliability established in Venegas. But again, that same principle mandates use of the modified ceiling approach in lieu of the recommendations of NRC II.
If any distinction at all is to be made between this cases and Venegas, it is that in view of the substantial departures from HW and linkage equilibrium found by Dr. Mueller in PCR databases ( see, Laurence D. Mueller, The DNA Controversy And NRC II, in Statistical Methods in the Health Sciences:Genetics, ME Halloran & S. Geiser (eds., 1999); Mueller, L Commentary on Budowle et. al. validation and population studies of the loci LDLR, GYPA. HBGG,D7S8, and GC(PM loci), and HLA-DQA (1998) 43 J Forensic Sci 446-447), and in light of his testimony in Bokin that departures from linkage equilibrium cannot be corrected by following the recommendations of the NRC, this case is one in which the product rule cannot be applied at all because there is presently a lack of scientific consensus on how to account for such disequilibrium in a PCR case. This result would be fully justified here because in addition to the testimony of Drs. Mueller and Zabel, the Court in Bokin had before it the consensus statement of twenty seven prominent statisticians and population genetists attesting to the fact that " many [scientists] oppose the product rule,asserting that there are as yet insufficient data to determine the effect of population substructure on probabilty calculations." (Defendants Exhibit 105).Dr Mueller, it should be noted, is the expert that our Supreme Court relied upon most heavily in Venegas in finding a prong three statistical violation. In no event, therefore, is the unmodified product rule justified in this case.


C. FAILURE OF THE TESTING LABORATORIES TO USE A GENERALLY ACCEPTED METHOD FOR DETERMINING FALSE POSITIVE ERROR RATES RENDERS THE PROSECUTION'S DNA EVIDENCE INADMISSIBLE.

1. Essential Elements of an Acceptable Method for Error Rate Determination.
As noted earlier, the scientific community now recognizes that evidence of a DNA match cannot meaningfully be evaluated without knowing the rate of laboratory error. Consequently, under the logic of Venegas, Axell, Barney and Wallace, evidence of a DNA match cannot be admitted without statistics on the error rate. To comply with the requirements of Kelly, the method used to determine the error rate must be generally accepted as reliable within the relevant scientific community.
Scientific commentary makes it clear that, in order to be accepted as reliable by the scientific community, the method for determining error rate must involve externally administered blind proficiency testing on samples that replicate casework. A blind proficiency test is one in which the analyst is not aware he or she is being tested. The NRC Report declares that "...laboratory error rates must be continually estimated in blind proficiency testing." NRC 1 at 89. The proficiency tests must be "truly representative of case materials (with respect to sample quality, accompanying description, etc.)." Id. The NRC notes that "[t]ests based on pure blood samples would probably underestimate an error rate." Thus, it appears that such tests are not accepted as a reliable way to determine the error rate of a forensic test.
Other commentators agree. For example, Professor Jonathan Koehler declares that, "[t]he best way to measure the rate of false positive error associated with a laboratory or an individual technician is through an ongoing series of blind, external proficiency tests conducted under realistic conditions." Koehler, Error and Exaggeration, supra, at 28. Others who endorse this position are R.C. Lewontin, supra, at 260 ("there must be frequent independent and unannounced inspections and tests"); Mueller, supra, at 57 (noting with approval the NRC's call for blind proficiency tests); Hagerman, supra (noting importance of proficiency testing to error rate determination).
The NRC Committee emphasized the importance of proficiency testing in a prefatory statement to its report:
We regard the accreditation and proficiency testing of DNA typing laboratories as essential to the scientific accuracy, reliability, and acceptability of DNA typing evidence in the future. Laboratories involved in forensic DNA typing should move quickly to establish quality assurance programs. After a sufficient time for implementation of quality assurance programs has passed, courts should view quality control as necessary for general acceptance.
NRC 1 at x.
In recent testimony, Professor Eric Lander, a member of the NRC Committee, explained that the Committee did not wish to call a moratorium on all forensic DNA testing but wanted to see proficiency testing programs set up as soon as possible because the Committee members unanimously believed "that proficiency testing is an essential component of the scientific reliability and acceptability of [DNA] evidence," U.S. v. Porter, supra, at 43. In saying that laboratories should move "quickly" to establish such programs, the Committee chose the term with care "to be faster than with all deliberate speed, for example," Id. at 80, and to clearly signal courts that failure to do proficiency testing would not be acceptable for long.
Lander also explained the reason the Committee demanded blind proficiency testing: only blind testing gives an adequate measure of the likelihood of error.
An adequate [method of proficiency testing] must surely be blind. If you know that you are working on test samples rather than case samples, you will, even if you don't intend to, be more careful. Thus, open proficiency testing where the examiner knows that they are being examined, does not provide an adequate measure of proficiency. Blind proficiency testing, where samples are worked in normal case flow, provides a good measure of that.

Id., at 50.
In light of these very powerful pronouncements demanding realistic blind proficiency testing, it is clear that there are scientists significant in number and expertise who would accept no less. It would therefore appear impossible for any method of error rate estimation to meet the requirements of Kelly unless it incorporated blind proficiency testing on samples simulating casework.
2. The SFPD Crime Lab Has Not Employed a Generally Accepted Method to Estimate Lab Error Rate.
The evidence introduced in Bokin showed that the SFPD Crime Lab has not completed any external blind proficiency testing on realistic samples to provide a meaningful estimate of its error rate. Further, the evidence showed that the open proficiency tests conducted to date have been done in a haphazard way and in violation of established protocol. The true error rate of the Lab and its analysts is therefore unknown and unknowable based on currently available data. Consequently, the value of the DNA evidence them Lab offers is impossible to evaluate and therefore inadmissible.

CONCLUSION
For the above-stated reasons, defendant Robert Nawi respectfully requests that the People's Motion to Admit DNA Test Results be denied in the absence of a Kelly-Frye hearing at which the People are required to prove all three prongs of the Kelly- Frye test. Defendant further requests that the Court take judicial notice of the record and ruling in People v. Bokin and hold that DNA testing using the Green I kit is inadmissible as a matter of law.
DATED: December 23,1999

MICHAEL N. BURT

Attorneys for Defendant
Robert Nawi

______________________
By: MICHAEL N. BURT
DEPUTY PUBLIC DEFENDER

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