POINTS AND AUTHORITIES IN REPLY TO PEOPLE’S MOTION TO ADMIT DNA EVIDENCE

I  INTRODUCTION

The People have posed the question of whether the prong one issues in this case were resolved in People v. Allen (1999) 72 Cal.App.4th 1093, 85 Cal.Rptr.2d 655, and hence no prong one hearing is necessary in this case.^[1]

Attached to this brief as Exhibit A is a declaration from Dr. Christie Davis, a molecular biologist at Lexigen Science and Law Consultants, Inc., based in San Francisco. Lexigen Science and Law was a consultant in the Allen case, and is also consulting in this case. Therefore Dr. Davis is in a unique position to discuss the characteristics of the testing method used in these two cases to show how different they really are.

The test in the Allen case was done using the method contained in the Promega GenePrint Silver Stain STR kit (hereafter Promega’s CTT) as analyzed using gel electrophoresis and a straightforward visual inspection of the resulting patterns. On the other hand, two of the methods used in the Nawi case are the Perkin Elmer Applied Biosystem’s AmpFlSTR Green I and Profiler Plus STR kits, as analyzed using capillary electrophoresis, the ABI Prism 310 Genetic Analyzer (ABI 310), and an extremely complicated computer software program called Genotyper. ^[2]

An analogy might be helpful to focus in on the significance of the differences between these two methods. Suppose that Honda wanted to determine at what speed in a collision would an Accord be in danger of having the gas tank explode. They could do actual testing using an Accord in crash tests to actually observe at what speed, angle of impact, etc. resulted in the gas tank exploding (in Allen the method used was relatively simple and the DNA was directly visualized).

Now suppose that Volkswagen (the method used in Allen was designed by Promega, in Nawi the method was developed by Perkin-Elmer) wanted to answer the same question as to the Volkswagen Beetle (in Allen, the loci^[3] tested were different from the Profiler Plus loci tested in Nawi). Volkswagen could use a different method to answer the same question. They could try a computer simulation method where they did not crash cars at all, but they programmed in various relevant parameters (ie. tensile strength of gas tank, effect of air resistance, mass of cars involved in collision, etc.), generating a simulated result for the Beetle (the Nawi STR methods are very complex, involving the use of several computer programs to analyze the data, and does not involve actual visualization of the DNA).

No-one would reasonably argue that the method used by Honda could be used to validate the accuracy of the method developed by Volkswagen. While both methods are attempting to get to the same result, the steps involved are completely different, as are the methods for visually presenting the data.

The various Forensic DNA analysis techniques in use today are modular in nature. That is they consist of multi-step procedures to go from an item of evidence on one hand to a final analysis on the other. Some of the methods share certain techniques in common, others contain techniques unique to each test. For those methods that share common steps, if the particular step is identical with each test, then that step need not be revalidated for the new test. However, if there is any variation in the step from one test to the other, then there must be new validation studies conducted of the variation to make sure its works.

If a particular step is unique to one method, then validation studies of a different method are useless and irrelevant. The differences between the two methods being considered here, as described in the Davis declaration, are so striking that no-one can reasonably argue that validation of one method serves to validate the other.

II   AN APPELLATE RULING APPROVING ONE DNA TESTING METHOD UNDER PRONG ONE OF KELLY DOES NOT PRECLUDE A PRONG ONE CHALLENGE WHERE A DIFFERENT TESTING METHOD WAS USED

Once a trial court has admitted evidence based upon a new scientific technique, and that decision is affirmed on appeal by a published appellate decision, the precedent so established may control subsequent trials, at least until new evidence is presented reflecting a change in the attitude of the scientific community. (People v. Kelly (1976) 17 Cal.3d 24, 549 P.2d 1240; 130 Cal. Rptr. 144)^[4] This principle would only apply where DNA testing using a particular methodology had already been litigated in a trial court and passed muster in a court of appeal. However, if there is any Amaterial scientific distinction@ between the two tests involved, then a Kelly prong one hearing is required for both tests. (People v. Venegas, supra., 18 Cal.4th at 54) The declaration from Dr. Davis makes it patently obvious that the methods used in Allen are materially scientifically different from the methods used in Nawi.

The courts have long recognized that there is far more involved in assessing the admissibility of DNA evidence than merely showing that DNA is capable of being used to assist in establishing the source of evidentiary material derived from biological sources. The spate of appellate court opinions discussing the various different kinds of tests available, and the various steps within each test, attests to the fact that different DNA methods require their own independent validation. If it were otherwise, a single appellate ruling admitting all DNA testing results would have silenced the protestations of DNA critics years ago. As the National Research Council^[5] has observed, "(I)t is meaningless to speak of the reliability of DNA typing in general‑without specifying a particular method." (National Research Council, DNA Technology in Forensic Science, (1992) (hereafter NRC I) at page 8.). Thus, the focus on the specific methods and procedures used in the particular case has been universal in Kelly- Frye litigation, as demonstrated by the numerous cases cited by the defense at pp. 18-20 of its Opposition.

Even where the same method is used, a Kelly prong- one challenge is required in some circumstances. Illustrative is People v. Brown (1985) 40 Cal. 3d 512, 528, 534, a case where the Supreme Court, noting that electrophoretic tests of crime‑scene stains had previously been upheld, ruled that "[T]he prosecution failed to satisfy the Kelly/Frye rule by demonstrating at trial the scientific acceptance of the tests performed." (Brown, supra, at 528) In distinguishing other forms of electrophoretic testing that had been previously accepted, the Court pointed out that the Aelectrophoretic method itself is apparently performed under substantial chemical and electrical variations, and considerable training and experience are necessary to interpret the visual results." (Brown, supra, at 534) Thus, if there are substantial variations in the way the method is used, a new Kelly first prong hearing is required, even though the methods are the same. Obviously, where the two methods are different, a Kelly prong one hearing is mandated.

III   THE METHOD OF TESTING USED IN NAWI IS DIFFERENT FROM THE METHOD USED IN ALLEN

  Dr. Davis of Lexigen Science and Law Consultants, Inc. lists her credentials in her declaration and her attached C.V. They will not be repeated here.

She lists the various testing methods that have been used since the beginning of forensic analysis of DNA evidence in criminal cases in the late 1980's. They include RFLP (the only one not to use PCR amplification as one of its steps), DQA1/PM, DQa, D1S80, Green I, Blue, Cofiler, AmpFlSTR Profiler Plus, Promega CTT, and Yellow tests. These tests are all different methods used to isolate and examine different fragments of DNA using different techniques. Each method must be separately validated before it can be used in casework. (Davis declaration para. 3)

Dr. Davis is very familiar with the two different methods used in the Allen case and the Nawi case as Lexigen Science and Law Consultants, Inc. was and is involved in both cases. As stated above the test in the Allen case was done using the method contained in the Promega GenePrint Silver Stain STR kit (Promega’s CTT) analyzed by gel electrophoresis. The method used in the Nawi case is the Perkin Elmer Applied Biosystem’s AmpFlSTR Green I and Profiler Plus kits, analyzed by the ABI Prism 310 Genetic Analyzer (ABI 310). (Davis declaration para. 6)

The method used in the Allen case can be described as DNA extraction, amplification of CTT using the Promega GenePrint kit, separation of the DNA fragments by Polyacrylmide gel electrophoresis and visualization of the DNA by silver staining. The testing done in the Nawi case, AmpFlSTR Green I/ Profiler Plus/ABI 310, is a different method from the Promega test done in the Allen case. It can be described as DNA extraction, amplification of 13 loci using PE Applied Biosystem’s AmpFlSTR Green I and Profiler Plus kits, separation of the DNA fragments by electrokinetic injection and capillary electrophoresis. The DNA is not visualized, but captured as fluorescent emissions by a camera and represented as digitized fluorescent activity that is analyzed by computer. (Davis declaration para. 7)

Both methods utilize the polymerase chain reaction (PCR). The PCR process is not a test in itself, but a way to increase the numbers of copies of one or more genetic markers. The reaction is complex and each reaction is different, depending on its makeup of primers needed to recognize the genetic markers of interest. A well known reaction mechanism of the PCR process is that optimal conditions for the amplification of a DNA sequence vary significantly according to the DNA sequence of interest and the primers required to achieve multiple copies of that sequence. If more than one sequence is to be copied at the same time, the primers needed must be engineered in such a way as to not interact with each other. The sequences of primers used to amplify the three loci analyzed in Allen, are significantly and materially different from the sequences of primers used to amplify the thirteen loci analyzed in the AmpFlSTR Green I and Profiler Plus kits. Both methods utilize a thermocycler to drive the amplification, but the thermocycler timing and temperature conditions are different, the chemicals used in the amplification process are different and each must be separately validated for this step of the test to work properly. (Davis declaration para. 8)

It is important to note that the first method to be developed, which took advantage of the Polymerase chain reaction (DQ Alpha), was referred to as Athe@ PCR test. Prosecutors routinely tried to argue that since the DQ Alpha test has passed Kelly first prong analysis, therefore, all other newer methods, which have PCR as one of its steps, should be considered as generally accepted. This argument has been routinely rejected in the courts. See, Opposition at pp. 18-20. (citing numerous cases). PCR is simply a method of taking a very small amount of DNA and multiplying it so there is enough DNA to analyze. 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.@)

The same problems of nomenclature arise with respect to the ASTR@ tests. The People in this case vaguely and misleadingly assert that Athere is nothing unique about PCR STR versus any PCR.@ (Motion at p. 3) But STR is not a method of analysis. The term is used to define a type of DNA sequence configuration. STRs are fragments of DNA defined as containing 2‑7 basepair repeats in tandem. Several different methods have been developed in an attempt to characterize the genetic information contained in various STR fragments or genes. It is these different methods that are subject to Kelly prong one analysis, not the fact that STR genes are capable of being used for human identification. In the words of knowledgeable scientists from the federal government’s National Institute of Standards and Technology, A(b)efore a new STR system or STR mulltiplex may be routinely employed in human identity testing it should be extensively validated to insure reliability of results.@ NIST, Validation Studies on STR Systems (2000), http://www.cstl.nist.gov/div831/strbase/valid.htm.

It is also important to note that even though the PCR amplification process is used in all PCR type tests, this does not mean that once the PCR step is held to be admissible in one test, it is therefore approved for all tests. This is because the components of the chemical soup used in the amplification process is different for different tests. There are different primers^[6] used depending on which fragments are to be amplified. A Different number of cycles^[7] may be used in different tests.  Any time there is an adjustment in the chemical soup or the number of cycles, new validation studies are necessary to determine the faithfulness of the amplification step (see earlier discussion regarding the thermocycler and primers unique to each testing method).

It is the process of validation that is required before any particular method should be used in criminal casework. According to NRC I, "Each DNA typing method must be rigorously characterized with respect to the type of possible artifacts, the conditions under which they are likely to occur, the scientific controls for detecting their occurrence, and the steps to be taken when they occur..."  (NRC I at 54.)  The TWGDAM guidelines and the DAB guidelines (further described below) affirm the importance of basing forensic tests on publicly available methods and reagents to ensure that the relevant scientific community has the opportunity to review the data and deem whether the test is reliable for use on forensic samples.

TWGDAM (Technical Working Group for DNA analysis methods) is a group of government and private forensic scientists.  In 1995, TWGDAM published its guidelines  to ensure the Aquality, integrity, and reliability of the DNA typing data and its presentation through the implementation of a detailed quality assurance (QA) program.@  TWGDAM, Guidelines for a Quality Assurance Program for DNA Analysis, (April 1995) 22 Crime Lab Digest 21, 22)  According to NRC II^[8], Athese guidelines define currently accepted practice.@ Id p 24

The DNA Identification Act of 1994(42 USC Section 14131) established a federal framework for setting national standards on quality assurance and proficiency testing.  (NRC II, at 24). These standards were to be developed by a DNA Advisory Board, appointed by the FBI from a list of nominations made by the National Academy of Sciences and professional societies representing the forensic community  The DNA Advisory Board (hereafter DAB) was established and on July 15, 1998, the Quality Assurance Standards For Forensic DNA Testing Laboratories was approved by the Director of the FBI, to take effect on October 1, 1998. Both the DAB Standards and TWGDAM guidelines set out minimal requirements for various laboratory activities and personnel.

No developmental validation studies have yet been published for the AmpFlSTR Green I kit, the Profiler Plus kit, capillary electrophoresis ,  the ABI Prism 310 Genetic Analyzer, or Genotyper, and it is this decisive fact which led to the recent exclusion of the test  results in California (Bokin), Colorado (Schreck), and Vermont (Pfenning). See also, State v. Anderson (Del. Super. 2000) 2000 WL 303319(AThe Court will hold a Daubert hearing before the commencement of trial to determine whether the underlying reasoning and methodology of the STR testing method of the DNA samples is scientifically valid and can be properly applied to the facts of this case, if the State intends to use such evidence.@) The NRC I report, the DAB guidelines and the TWGDAM guidelines affirm the importance of basing forensic tests on publicly available methods and reagents to ensure that the relevant scientific community has the opportunity to review the data and deem whether the test is reliable for use on forensic samples. Completion of this review according to the guidelines of TWGDAM and DAB has not taken place for the AmpFlSTR Green I or Profiler Plus kits, the method of amplification, capillary electrophoresis, electrokinetic injection, capture of data by the ABI 310 Genetic Analyzer and analysis through the use of computer software.^[9] (Davis declaration para. 19)

It is the very fact that Perkin Elmer has attempted to draw a line in the sand by refusing to make its primer sequences and validation studies public that has led to an explosion of litigation with respect to  their kits.^[10] As stated forcefully by the court in Colorado v. Schreck, supra, A PE has resisted releasing its developmental data claiming that the data was unavailable, that it had never been systematically recorded, that it was scattered throughout various departments at the company and its collection at this time would be unduly burdensome.@ (Exhibit B at p.19) And as the court continued,

The Court finds it unbelievable that a research and development company does not record developmental data as part of its standard operating procedures. The failure to systematically organize and manage this data while successfully resisting multiple  subpoenas duces tecum forces courts and the collective scientific community to simply take PE’s claim at face value, an unacceptable position for both.  (Id. at n. 32)

One crucial requirement established by all guidelines and standards is that forensic DNA tests must be developmentally validated through extensive empirical studies. See, DAB Standard 8.1( AThe 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 reproducibility of the procedure.@); TWGDAM Guidelines 4.1.2 (A Validation studies must have been conducted by the DNA laboratory or scientific community prior to the adoption of a procedure by the DNA laboratory.@).

Paragraphs eight through nineteen of the Davis declaration discuss the many differences between the method used in Allen and the method used in Nawi and need not be outlined here. Suffice it to say that, referring to the earlier analogy, Honda’s method of analysis of no use in validating Volkswagen’s newer, more sophisticated, method - they are apples and oranges. A detailed analysis of the methods used in Allen and Nawi vividly shows that these two tests are apples and oranges as well.

Also attached to the Davis declaration are sample outputs from each of the two testing methods. This further demonstrates the tremendous differences in the technologies involved.

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. See, Opposition, Exhibit E, Declaration of Dr. Davis at & 11.   Thus one piece of evidence analyzed with the Green I  kit can have a different genetic profile than one analyzed with the Promega 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 exculpatory, and then another lab retested the same evidence with Perkin Elmer’s kit and the results were inculpatory, then under the logic of ASTRs are STRs@, 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.  Exhibit E, 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 Aaddress 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 18-18 and with Geneprint 16-18. A 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 A 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. AOur 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 A[t]he (Green I) 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 Aa 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’ declarations 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^[11] kits are generally accepted as reliable.

VI  CONCLUSION

      No appellate court decisions evaluate the use of laser‑fluorescent capillary electrophoresis with the Profiler Plus and Green I primer systems.  Allen is the first California case making a reference to "STR's," and addressed solely the CTT Silver Stain Gel Electrophoresis test for STR loci, the only such test available at that time.  Failing to anticipate the new technologies which would examine similar allele patterns at multiple loci,  the opinion rather improvidently referred to the tests as "STR tests," which merely names the object of such tests, the comparison of alleles at STR loci, rather than the particular method, which is the true subject of the court's opinion.   Allen in no way authorizes admission of evidence regarding DNA testing which utilizes the new laser fluorescent instruments and unique new primer sets.  These technologies were not at issue in Allen,  and they present very substantial differences, from a Kelly perspective. Venegas makes clear that a court must evaluate each step of the process separately, and that general acceptance of  materially different steps in those processes must be proved at a hearing because 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." (Venegas, supra, at p. 54.)

The ruling in Allen should be deemed to have no precedential or binding effect on this case.

  Dated: June 19, 2000                        

  Respectfully submitted,

  MICHAEL N. BURT

COUNSEL FOR ROBERT NAWI

                    DECLARATION OF CHRISTIE T. DAVIS, Ph.D.

I, CHRISTIE T. DAVIS, Ph.D. declare under penalty of perjury that the following statements are true and correct:1. I, Christie T. Davis, Ph.D., am a molecular biologist at Lexigen Science and Law Consultants, Inc., based in San Francisco, California;2. I have a Ph.D. in Microbiology and Immunology from The Medical College of Pennsylvania, a B.S. in Biology from Virginia Commonwealth University and twenty years of experience of working with DNA technology. I have held research and teaching appointments at major US universities (see curriculum vitae, attached hereto);3. I have served as a forensic DNA consultant for the past 2.5 years. During the past year and a half, I have qualified to testify in Court in eight state cases and two federal cases, encompassing the areas of DNA, serology, evidence handling, and proper forensic testing procedures. These cases involved PCR based tests DQA1/PM, DQa, D1S80, Green I, Blue, COfiler and AmpFlSTR Profiler Plus. I have worked on other cases encompassing RFLP, Promega CTT, and Yellow tests. These tests are all different methods used to isolate and examine different fragments of DNA using different techniques. Each method must be separately validated before it can be usedin casework;4. I have worked on over one hundred cases involving DNA evidence analyzed by the following laboratories, among others: Cellmark Diagnostics, LabCorp, Federal Bureau of Investigation DNA Analysis Unit, SERI, Oregon State Police Crime Lab, Alaska State Crime Lab, Ohio Bureau of Criminal Investigation, Arizona Department of Public Safety, Illinois State Crime Lab, Utah Department of Public Safety, Genelex, Forensic Science Associates, Reliagene, Santa Clara County District Attorneyos Lab, Technical Associates, Forensic Analytical, Sacramento Regional Crime Lab, Helix Biotech, California Department of Justice Sacramento Lab, San Francisco Police Department Crime Lab, San Bernardino Sheriffos Department Crime Lab, LosAngeles Police Department Crime Lab, Orange County Sheriffos Crime Lab, Washoe County Crime Lab, California Department of Justice Santa Rosa Lab,and California Department of Justice DNA Laboratory;5. I have reviewed reports, bench notes, autoradiograms, lumigrams, electropherograms, electronic data, test gels, test strips and other data and records generated by these labs in the course of performing such tests. In some cases I have monitored testing done at a laboratory. I have also reviewed protocols, and other research records generated by these labs to validate their DNA typing procedures. I have reviewed and am familiar with the guidelines for forensic testing written by the Technical Working Group on DNA Analysis Methods (TWGDAM) also known as the Scientific Working Group on DNA Analysis Methods (SWGDAM), the DNA Advisory Board (DAB) and the reports on DNA typing produced by the National Research Council of theNational Academy of Sciences in 1992 (NRC I) and 1996 (NRC II);6. I have been asked by the attorney for Robert Nawi to render an opinion on the material scientific differences between the PE Applied Biosystem’s AmpFlSTR Green I and Profiler Plus kits, analyzed by the ABI Prism 310 Genetic Analyzer (ABI 310) and the Promega GenePrint Silver Stain STR kit (Promegaos CTT) analyzed by gel electrophoresis. The AmpFlSTR Green I and Profiler Plus/ABI 310 tests were used in People v Nawi and the Promega CTT/gel test was used in People v Allen (1999) 72 Cal. App. 4th 1093;7. I am aware that the Promega CTT/gel test was utilized in the Allen case because Lexigen Science and Law Consultants, Inc was a consultant in the case. The method used in the Allen case can be described as DNA extraction, amplification of 3 loci (CSF1PO, THO1, TPOX)(CTT) using the Promega GenePrint kit, separation of the DNA fragments by Polyacrylamide gel electrophoresis and visualization of the DNA by siver staining. The testing done in the Nawi case, AmpFlSTR Green I and ProfilerPlus/ABI 310 is a different method from the Promega test done in the Allen case. These can be described as DNA extraction, and amplification of CTT by use of the Green I kit or 9 STR loci using PE Applied Biosystem’s AmpFlSTR  Profiler Plus kit, separation of the DNA fragments by electrokinetic injection and capillary electrophoresis. The following methods hold true for use of either Green I or Profiler Plus. The DNA is not visualized, but captured as fluorescent emissions by a camera and represented as digitized fluorescent activity that is analyzed by computer. These methods are explained below in greater detail.

8. All tests, mentioned in paragraph 6 utilize the polymerase chain reaction (PCR). The PCR process is not a test in itself, but a way to increase the numbers of copies of one or more genetic markers. The reaction is complex and each reaction is different, depending on its makeup ofprimers needed to recognize the genetic markers of interest. A well known reaction mechanism of the PCR process is that optimal conditions for the amplification of a DNA sequence vary significantly according to the DNA sequence of interest and the primers required to achieve multiple copies of that sequence. If more than one sequence is to be copied at the same time,the primers needed must be engineered in such a way as to not interact with each other. The sequences of primers used to amplify the three loci analyzed in People v. Allen, are significantly and materially different from the sequences of primers used to amplify the 3 loci analyzed in AmpFlSTR Green I kit or the 9 loci in the AmpFlSTR Profiler Plus. Both methods utilize a thermocycler to drive the amplification, but the thermocycler timing and temperature conditions are different, the chemicals used in the amplification process are different and each must be separately validated for this step of the test to work properly;

9. The Promega CTT kit was designed to amplify three particular Short Tandem Repeats (STRs), THO1, TPOX and CSF1PO. STRs are not in themselves a test. The term is used to define a type of DNA sequence configuration. STRs are segments of DNA defined as containing 2-7 basepair repeats in tandem. Different numbers of repeats define the allele calls. The forensic community has adopted 4 basepair repeat STRs. There are three types analyzed in the Profiler Plus kit; simple, complex and compound, depending on the DNA sequences present.The three STRs analyzed in the Promega CTT kit were all simple 4 basepair repeat STRs. These 3 STRs are the same 3 analyzed in the AmpFlSTR Green I kit The primers used for amplification were designed by Promega and are different from the primers used for the PE Applied Biosystem tests. Each set of primers must be separately validated. The amplified DNA from the CTT kit was separated on a gel and visualized by silver staining. Silver staining is a method where the gel is added to a compound containing silver grains. The silver binds to the DNA fragments, turning them brown, so that one can see the DNA bands with the naked eye (see Exhibit A). The loci were chosen and the primers designed to produce allelic products that wouldnot overlap, so that upon visualization by silver staining, DNA bands of allelic products from different loci could be visually analyzed. Both strands of DNA were visualized. The separation of the DNA fragments was accomplished by gel electrophoresis through a vertical, denaturingPolyacrylamide gel. Denaturing means that the double strands of the DNA are kept apart so that each strand runs separately. Under these electrophoretic conditions because of the base sequence differences between two strands of the same fragment, the two strands will run through the gel at different rates. Depending on the size of the fragments, both strands can be resolvedby silver staining. One fragment will stain as two bands, rather than one. This banding pattern is called a doublet (see Exhibit A, area labeled H). Protocols had to be developed to handle interpretation of data involving doublets. The AmpFlSTR Green I and Profiler Plus tests were developed using fluorescent dye tagging of only one DNA strand from each fragment to eliminate doublets.

 10. DNA testing in this case consisted of two kits produced by Perkin-Elmer (PE Applied Biosystems), a competitive company to Promega. Several materially different approaches from the Promega kit are utilized in these tests. The amplified products, some of which overlap in size, are labeled with fluorescent dyes, separated by capillary electrophoresis under denaturingconditions so that the DNA is single stranded, captured as digitized representations and analyzed by computer programs. The DNA itself is not visualized. Only one strand of each fragment is captured for representation. The Green I kit contains primers attached with one of two dyes, green or red. For the Profiler Plus kit, four different dyes are used representing four colors; blue, green, yellow and red. Each has a slightly different spectral emission. Ten loci are multi-plexed into one amplification. The loci are assigned by color so that the amplified product from any single color will not overlap another product in that same color, but it may overlap amplified products from the loci tagged with other colors. For example, a product from a locus assigned to blue will not overlap with any other blue tagged product, but may overlap with a product tagged with green and/or yellow. The same theory holds true for green and yellow. In the detection process, millions of copies of green, blue and yellow tagged product may be detected almost simultaneously. Size determination of the DNA fragments is dependent on the ability of the software to separate the dye colors from each other. In addition, the dyes used do not have unique spectral emission footprints, but overlap each other. The spectral overlap is handled by a complex mathematical matrix as part of the computer analysis. None of the above described steps were involved in the testing method used in the Allen case;

11. Electrophoresis is a technique that can be applied to DNA to cause separation of fragments. DNA is a uniformly negatively charged molecule, meaning that regardless of size all fragments will migrate toward the anode (positive charge) at the same rate. DNA fragments can be separatedaccording to size if the DNA is applied simultaneously to a sieving matrix and an electric current. The method used in Allen, described in detail elsewhere in this declaration, involved gel electrophoresis, where the DNA migrates through a flat acrylamide gel. The method used in Nawi was capillary electrophoresis, described more fully below. This method involvesmigration of DNA fragments through a small capillary tube through the use of both electric current and osmotic flow.

12. None of the loci amplified in the AmpFlSTR Profiler Plus kit are contained in the Promega kit, and the primers are very different. The loci analyzed in the AmpFlSTR Green I kit are the same loci analyzed in the Promega CTT kit but the primers used are very different. The protocol used in the Promega kit cannot be used in the Perkin-Elmer test. The kit parts from Promega cannot be substituted for those from Perkin-Elmer. The thermal cycler program, including temperature and cycle number, are different in all three kits. One program cannot be exchanged for another program. They are completely separate tests with material differences between them. The Promega CTT kit was not widely adopted by forensic labs and for all intents and purposes has been abandoned in favor of the fluorescent technology, such as AmpFlSTR Profiler Plus or the Green I kit analyzed with the ABI 310;

13. The AmpFlSTR Profiler Plus kit produced by Perkin-Elmer contains 9 STR loci D3S1358, vWA, FGA, D8S1179, D21S11, D18S51, D5S818, D13S317 and D7S820; all are different from those used in People v. Allen and have not been previously used in testing which has been involved in a published court decision. TWGDAM requires that extensive studies be done to analyze each locus used in forensic testing and that additional strenuous studies be donewhen loci are combined together for analysis;

14. Capillary Electrophoresis (CE) used as a means of DNA separation in this case is an entirely different mechanism than gel electrophoresis used in People v. Allen. CE relies on polymer chemistry, using a liquid polymer. The polymer molecule must approximate the size of the DNA molecules being separated. The polymer is a hydrophilic molecule that is held in a specificosmotic buffer, producing a "viscous buffer". The viscosity of the buffer is due to the percentage of polymer used. At a certain percentage, the polymer approaches an entanglement threshold. The polymer entangles with itself and with the DNA. The percentage of the polymer can change the dynamics of the entanglement, which increases or decreases the "pore size" that the DNA hasto work through. The polymer is injected into a fused silica capillary that is coated with a negative charged ion. DNA fragment separation is dependent on both electrical current and osmotic flow. Negatively charged DNA molecules, neutral molecules and positive charged molecules will move through the capillary. Any cation (positive charge) can bind to thecapillary wall, and in turn can bind DNA molecules. If enough molecules bind to the wall, the electroosmotic flow will be interrupted, which can cause the capillary to fail. This interference can decrease the resolution of detected peaks, causing problems in data interpretation;

15. CE, used with the AmpFlSTR Green I and Profiler Plus kits is defined as a physical sieve. The polyacrylamide gel electrophoresis used in People v. Allen is defined as a chemical sieve. It relies on a solid matrix made by cross linking acrylamide monomers, not a polymer. The chemical cross linking produces a three dimensional, semi-solid fixed pore size sieve. The movement of molecules through the gel is wholly dependent on electric current. There is no osmotic flow. The DNA is applied to the top of the gel and relies on electric current to move the molecules into the gel. In CE, the DNA is applied to the capillary by electrokinetic injection. An electrode is placed into the DNA solution along with the capillary. The end of the electrode is just below the end of the capillary. A strong negative charge is applied, which repulses the DNA from the electrode into the end of the capillary. The injection also depends upon the osmotic difference between the buffer that the DNA is in and the buffer that the polymer is in. This difference is important to the initial stacking of DNA into the capillary;

16. Electric current requirements are very different between the two systems and cannot be interchanged. The gel used with the Promega kit cannot sustain the high voltage used in CE. The electrophoresis buffer required for current flow is different in CE than gel electrophoresis. The two buffers cannot be interchanged. CE is materially scientifically different from the separationapproach used in People v Allen;

17. CE occurs in an automated fashion using a machine called an ABI Prism 310 Genetic Analyzer which was used in this case. This machine loads the samples, and is the body in which visualization and fluorescent capture occurs. This automation and visualization is materially scientifically different from that of the testing in People v Allen. The DNA molecules are labeled with a fluorescent tag attached to the primer. The tagged molecules pass through a detection box that contains a laser and a charged coupled device (CCD camera). The laser excites the tag, which emits a light that is captured by the CCD camera. This capture is recorded as an analog event that is provided to GeneScan in a digitized form for computer analysis (see Exhibit B). The final product produced is not a photograph or x-ray film copy of a silver stained gel as was produced in People v Allen, but a printout of an electropherogram where DNA is represented by a peak of activity reported by the GeneScan software that has a specific relative fluorescent unit (rfu) value and a specific size. (see Exhibits C through F). Exhibits C through F are representatives of GeneScan processed data. Exhibits C & D are an electropherogram containing all peaks from one sample and the tabular data for that sample. Exhibits E & F areelectropherograms of the same sample, but the four colors have been separated, each into its own electropherogram. The attending tablular data are the same as that found in Exhibits C and D. A second computer program, GenoTyper, presents the sized data in an electropherogram that reports the rfu of a peak as well as the allele call. Exhibits G through I are GenoTyper printouts for Blue, Green and Yellow for the same sample as analyzed in Attachments C through F. These attachments include the allelic ladder supplied by the manufacturer for each of the loci tagged with the different colors and the electropherogram of the analyzed sample. The boxesunder each peak contain either the allele call or the peakos rfu value.

18. Analysis of the test in People v. Allen was very simple; silver stain of the gel followed by direct visual examination of the DNA. The genotype was determined by visual comparison of the sample DNA to a series of known, sized DNA bands run on the same gel. The test run in this case is more complex. The DNA is represented in an electropherogram as peaks, defined byrelative fluorescence versus time from beginning of the run to detection. One cannot actually visualize the DNA. Fluorescent emissions are processed and normalized through two computer programs costing about $14,500. All emissions are analyzed and presented as peaks, whether they are from DNA or from artifacts. This system is known to produce many artifacts.Developmental validation studies of these software programs have not been published;

19. The developmental validation studies, for the ABI Prism 310 Genetic Analyzer and the AmpFlSTR Green I and Profiler Plus kits used in testing in this case have not been published. The NRC I report, the DAB guidelines and the TWGDAM guidelines affirm the importance of basing forensic tests on publicly available methods and reagents to ensure that the relevant scientific community has the opportunity to review the data and deem whether the test is reliable for use on forensic samples. Completion of this review according to the guidelines of TWGDAM, and DAB has not taken place for the AmpFlSTR Green I or Profiler Plus kits, the method of amplification, CE, electrokineticinjection, capture of data by the ABI 310 Genetic Analyzer and analysis through the use of computer software;

20. TWGDAM Guidelines were originally established in 1991 in recognition that changes in the quality assurance standards for DNA testing would be necessary to accommodate evolving technology and laboratory practices. A committee headed by the FBI was established to develop minimal guidelines for crime labs to follow. These guidelines include recommendationsregarding validation of new DNA Analysis procedures. Each different procedure or method requires its own process of validation. TWGDAM defines validation as the process used by the scientific community to acquire the necessary information to assess the ability of a procedure to reliably obtain a desired result, determine the conditions under which such results can beobtained and determine the limitation of the procedure. The validation process identifies the critical aspects of a procedure which must be carefully controlled and monitored.