IDENTITY TESTING AND FORENSIC SCIENCE Chapter 11

1. IDENTITY TESTING AND FORENSIC SCIENCE Chapter 11 Donna C. Sullivan, PhD Division of Infectious Diseases University of Mississippi Medical Center

2. Objectives Compare and contrast different types of polymorphisms. Define restriction fragment length polymorphisms. Describe short tandem repeat structure and nomenclature. Describe gender identification using the amelogenin locus. Illustrate the use of STR for bone marrow engraftment monitoring. Define single nucleotide polymorphisms. Discuss mitochondrial DNA typing.

3. Polymorphism A DNA polymorphism is a sequence difference compared to a reference standard that is present in at least 1�2% of a population. Polymorphisms can be single bases or thousands of bases. Polymorphisms may or may not have phenotypic effects.

4. Polymorphic DNA Sequences Polymorphisms are found throughout the genome. If the location of a polymorphic sequence is known, it can serve as a landmark or marker for locating other genes or genetics regions. Each polymorphic marker has different versions or alleles.

5. Types of Polymorphic DNA Sequences RFLP: restriction fragment length polymorphisms VNTR: variable number tandem repeats (8 to >50 base pairs) STR: short tandem repeats (1�8 base pairs) SNP: single-nucleotide polymorphisms

6. DNA DIFFERS PERSON TO PERSON All individuals genetically unique (except identical twins) Differences in DNA sequence detected by RFLP using large quantities intact DNA PCR in conjunction with various methods Single nucleotide polymorphisms (SNPs)

7. TYPES OF ANALYSIS RFLP ANALYSIS VNTR analysis RE digestion, gel electrophoresis, Southern Blot SNP analysis PCR, gel electrophoresis Allele specific PCR Ligase chain reaction PCR ANALYSIS STR analysis PCR, electrophoresis SNP analysis PCR, gel electrophoresis Allele specific PCR Ligase chain reaction

8. DNA Profiles from12 Unrelated Individuals At ONE Polymorphic Locus

9. VNTR: Variable Number Tandem Repeats Short sequence of DNA repeated in head-to-tail fashion Occur at specific chromosomal locations Interspersed throughout human genome Number of repeated units vary between individuals

10. MULTIPLE ALLELES AT VNTR LOCI VNTR loci flanked by conserved DNA sequences Restriction enzyme cleavage sites in conserved DNA flank VNTR alleles Alleles vary in length between enzyme cuts

11. Multiple Alleles at VNTR loci The most common forensic method to characterize VNTRs is using Southern Hybridization as described for Problem 1. If the DNA flanking a VNTR is cut with a restriction endonuclease, the size of the resulting DNA fragment can vary, resulting in RFLPs, or "restriction fragment length polymorphisms". This is shown diagrammatically in the figure, where the red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. In this diagram, only three different variants (alleles) are illustrated for the VNTR locus, but 50 or more different alleles are often found at human VNTR loci.The most common forensic method to characterize VNTRs is using Southern Hybridization as described for Problem 1. If the DNA flanking a VNTR is cut with a restriction endonuclease, the size of the resulting DNA fragment can vary, resulting in RFLPs, or "restriction fragment length polymorphisms". This is shown diagrammatically in the figure, where the red boxes represent the repeat unit and the blue lollipops represent cut sites for a restriction endonuclease. In this diagram, only three different variants (alleles) are illustrated for the VNTR locus, but 50 or more different alleles are often found at human VNTR loci.

12. Restriction Fragment Length Polymorphisms Restriction fragment sizes are altered by changes in or between enzyme recognition sites.

13. Restriction Fragment Length Polymorphisms The presence of RFLP is inferred from changes in fragment sizes.

14. Restriction Fragment Length Polymorphisms The presence of RFLP is inferred from changes in fragment sizes.

15. RFLP ANALYSIS: Detect Alleles by Southern Blot Hybridization Analysis of VNTR locus by Southern blot most commonly results in two-band pattern, comprised of a band inherited from each parent Single band patterns possible with 2 alleles of same size For 3 different alleles, six different genotypes possible

16. Distinguish Individuals by Combination of Alleles at VNTR Loci

17. RFLP Analysis: Detect Alleles by Southern Hybridization

18. CALCULATION OF POSSIBLE GENOTYPES For N different alleles, total possible genotypes: (n X (n+1)) 2 For 3 alleles, 6 genotypes For 4 alleles, 10 genotypes For 5 alleles, 15 genotypes

19. Restriction Fragment Length Polymorphisms RFLP genotypes are inherited. For each locus, one allele is inherited from each parent.

20. Parentage Testing by RFLP Which alleged father�s genotype has the paternal alleles?

21. Short Tandem Repeat Polymorphisms (STR) STR are repeats of nucleotide sequences. AAAAAA� - mononucleotide ATATAT� - dinucleotide TAGTAGTAG� - trinucleotide TAGTTAGTTAGT� - tetranucleotide TAGGCTAGGCTAGGC� - penta nucleotide Different alleles contain different numbers of repeats. TTCTTCTTCTTC - four repeat allele TTCTTCTTCTTCTTC - five repeat allele

23. Short Tandem Repeat Polymorphisms STR alleles can be analyzed by fragment size (Southern blot).

24. Short Tandem Repeat Polymorphisms STR alleles can also be analyzed by amplicon size (PCR).

25. Short Tandem Repeat Polymorphisms Allelic ladders are standards representing all alleles observed in a population.

26. Evidence Testing by STR-PCR Which suspect�S1 or S2�was at the crime scene? (V = victim, E = crime scene evidence, M = molecular weight standard)

27. Evidence Testing by STR-PCR Which suspect�S1 or S2�was at the crime scene? (V = victim, E = crime scene evidence, AL = Allelic ladder)

28. Short Tandem Repeat Polymorphisms Multiple loci are genotyped in the same reaction using multiplex PCR. Allelic ladders must not overlap in the same reaction.

31. Short Tandem Repeat Polymorphisms by Multiplex PCR

32. Amelogenin Locus, HUMAMEL The amelogenin locus is not an STR. The HUMAMEL gene codes for amelogenin-like protein. The gene is located at Xp22.1�22.3 and Y. X allele = 212 bp Y allele = 218 bp Females (X, X) - homozygous Males (X, Y) - heterozygous

33. Analysis of Short Tandem Repeat Polymorphisms by PCR STR genotypes are analyzed using gel or capillary gel electrophoresis.

34. STR-PCR STR genotypes are inherited. One allele is inherited from each parent.

35. Parentage Testing by STR-PCR Which alleged father�s genotype has the paternal alleles?

36. Short Tandem Repeat Polymorphisms: Y-STR The Y chromosome is inherited in a block without recombination. STR on the Y chromosome are inherited paternally as a haplotype. Y haplotypes are used for exclusion and paternal lineage analysis.

37. Chimerism Testing Using STR Allogeneic bone marrow transplants are monitored using STR.

38. Chimerism Testing Using STR There are two parts to chimerism testing: pretransplant informative analysis and post-transplant engraftment analysis

39. Chimerism Testing Using STR:Informative Analysis STR are scanned to find informative loci (donor alleles differ from recipient alleles). Which loci are informative?

40. Chimerism Testing Using STR:Informative Analysis There are different degrees of informatively. With the most informative loci, recipient bands or peaks do not overlap (stutter) in donor bands or peaks. Stutter is a technical artifact of the PCR reaction in which a minor product of n-1 repeat units is produced.

41. Examples of Informative Loci (Type 5) [Thiede et al., Leukemia 18:248 (2004)]

42. Examples of Non informative Loci (Type 1)

43. Chimerism Testing Using STR:Informative Analysis Which loci are informative?

44. Chimerism Testing Using STR:Engraftment Analysis Using informative loci, peak areas are determined in fluorescence units or from densitometry scans of gel bands. A(R) = area under recipient-specific peaks A(D) = area under donor-specific peaks

45. Chimerism Testing Using STR:Engraftment Analysis Formula for calculation of % recipient or % donor (no shared alleles).

46. Chimerism Testing Using STR:Engraftment Analysis Calculate % recipient DNA in post-1 and post-2:

47. Chimerism Analysis of Cellular Subsets Cell subsets (T cells, granulocytes, NK cells, etc.) engraft with different kinetics. Analysis of cellular subsets provides a more detailed description of the engrafting cell population. Analysis of cellular subsets also increases the sensitivity of the engraftment assay.

48. Chimerism Analysis of Cellular Subsets T cells (CD3), NK cells (CD56), granulocytes, myeloid cells (CD13, CD33), myelomonocytic cells (CD14), B cells (CD19), stem cells (CD34) Methods Flow cytometric sorting Immunomagnetic cell sorting Immunohistochemistry + XY FISH

49. Chimerism Analysis of Cellular Subsets Detection of different levels of engraftment in cellular subsets is split chimerism.

50. Single Nucleotide Polymorphisms (SNP) Single-nucleotide differences between DNA sequences. One SNP occurs approximately every 1,250 base pairs in human DNA. SNPs are detected by sequencing, melt curve analysis, or other methods. 99% have no biological effect;60,000 are within genes.

51. SNP Detection by Sequencing

52. SNP Haplotypes SNPs are inherited in blocks or haplotypes.

53. Applications of SNP Analysis SNPs can be used for mapping genes, human identification, chimerism analysis, and many other applications. The Human Haplotype Mapping (HapMap) Project is aimed at identifying SNP haplotypes throughout the human genome.

54. Top Ten Ways to Know that You are a Forensic DNA Scientist 10.You have your children�s DNA profiles framed on your desk instead of their pictures. 9.When your children hurt themselves, you are more interested in collecting their blood to generate a DNA profile than getting them cleaned up� 8.Your pockets are full of napkins with DNA sequences written on them. 7.You want to name your first four kids: Adenine, Thymine, Guanine, and Cytosine. 6.You wonder how jello would work as a separation medium�and have tried it when no one else was around�but were too afraid to publish the results.

55. Top Ten Ways to Know that You are a Forensic DNA Scientist 5.You want to start a paternity testing business for all those who appear on the Jerry Springer show. 4.You know that DNA stands for more than the National Dyslexics Association. 3.You want to do a study on the genetics of inbreeding�and have selected the Reservoir deer population for a case study.

56. Top Ten Ways to Know that You are a Forensic DNA Scientist 2.You know that �scientists� on the popular TV show CSI: Crime Scene Investigation cannot possibly get their DNA results within the timeframe of a single commercial break. 1.Your license plate reads: OJ DID IT!

57. LOCARD TRANSFER THEORY �When two objects come in contact, traces from one will be transferred from one to another, and in both directions.�

58. FORENSCI SCIENCE CONCEPTS Identification Classification: characteristics shared by items (Firearms, Shoes) Groups (Same type of gun, same size shoe) Individualization Created by random acts (rifling on bullets) Fingerprints (influenced by environmental, developmental factors) Traits that are so rare, alone or in combination, that they can not be duplicated by chance

59. DNA Identification Applications Sexual assault Homicide and other violent crimes Exculpate wrongly accused suspects Identify serial crimes Identify human remains Sex offender tracking Parentage testing

60. DNA Analysis Overview Forensic testing (Criminal) ~10,000 cases per year 75% involve sexual assault Paternity testing (Civil) ~200,000 cases per year ~130,000 cases use DNA Exclusions equal ~30% of all test results

61. In The Beginning Alec Jeffreys, geneticist at University of Leicester, UK Coined the term �DNA fingerprinting� RFLP analysis Demonstrated that forensic samples, even dried stains several years old, contained sufficient DNA to yield conclusive results

62. Finding A Pitchfork In The Haystack: Midlands Rapes Two British schoolgirls raped and murdered Kitchen porter at an insane asylum confessed to one of the murders Were both crimes committed by the same man? Did the kitchen porter do it?

63. Finding A Pitchfork In The Haystack: Midlands Rapes Semen samples from rape/murder scenes: Both crimes committed by same man Blood sample from kitchen porter: Did not match crime scene evidence Police felt that the murderer was a local Tested DNA from every man between the age of 17-34 (4,582 men) in the local area No matches found

64. Finding A Pitchfork In The Haystack: Midlands Rapes Local baker over-heard some colleagues in a pub Colin Pitchfork, another baker, had convinced another employee to give blood in his place Police arrested Pitchfork (he immediately confessed) Pitchfork�s DNA was perfect match to crime scene evidence (4,583rd man tested)

65. COMMERICAL DEVELOPMENT OF DNA TESTING: US Began in 1987 Cellmark (O.J. Simpson tests done by this company) Lifecodes First US case: Florida vs. Tommy Lee Andrews Convicted of rape, Nov. 6, 1987 Prosecution cases won time after time (�Aura of infallibility�, �You can�t argue with science�)

66. New York vs Castro: The Chink in the Armor Vilma Ponce, seven months pregnant, found raped and stabbed 60 times Her two year old daughter found also repeatedly stabbed Police interviewed Jose Castro, janitor in neighboring building Detective noticed bloodstain on Castro�s watch and asked to have it examined

67. New York vs Castro: The Chink in the Armor Lifecodes analyzed dried blood, identified it as Vilma Ponce�s blood (1:189,000,000) Defense demanded examination of genetic analyses Expert witnesses for both the prosecution and the defense agreed that Lifecodes quality control was poor MIT mathematician Eric Lander disputed the genetic frequency numbers

68. THE FBI AND THE MOUNTIES FBI and NIH established collaborative research project to establish DNA identification techniques The RCMP established their own lab as well (both systems based on English model) Standardization of techniques Detailed protocols, validation studies Instituted quality controls for labs nationwide

69. National Research Council Report: Few Answers, More Questions Proposes Ceiling Principle (use most conservative method of calculating frequencies) No population geneticists or statisticians on panel Suggested studies were unsound Internally inconsistent Does not address paternity or PCR Growing body of papers refute NCR report NCR convened new panel

73. COLLECTION OF EVIDENCE Collect stained item directly Clothing, fabric, pieces of tissue Remove stain, transfer to more suitable substrate Cotton swabs, scraping from hard surfaces Controls must be collected at the same time from an adjacent object/area not obviously stained

75. Preservation Of Evidence SHOULD BE DRY! If it is wet, dry it immediately If you have to wet it to collect it, dry it ASAP SHOULD BE FROZEN! If it is dry, this is not as critical If it is a large piece of material, freeze ASAP

76. Evaluation Of Evidence Establish type of biological material present in sample Is it blood? Catsup? Is it human blood? �State of the DNA� How much degradation of the DNA is there? How much total DNA is present? How much DNA is human?

78. Forensic Blood Typing ABO groups Phosphoglucomutase (PGM) Erythrocyte acid phosphatase (EAP) Haptoglobin Adenylate kinase (AK)

79. Semen Can Be Identified With Several Laboratory Tests Acid Phosphatase - a presumptive test Anti P30 - a quantitative and qualitative test that identifies the presence of the protein P30 found only in human semen Spermatozoa search - a microscopic analysis visually observing the presence of sperm cells

80. Mixed Vs Contaminated Samples MIXED SAMPLES Contain DNA from more than one individual Mixing occurs before or during commission of a crime CONTAMINATED SAMPLES Materials deposited during collection, preservation, handling or analysis

81. HOW MUCH SAMPLE DO YOU NEED?

82. How Much DNA Do You Have? Slot Blot

83. How Much DNA Do You Have? Yield Gel

84. Gender Identification Amelogenin gene for tooth pulp found on sex chromosomes ( one on X, one on Y) Different size PCR products from X and Y copies of the gene: X Chromosome 106 bp PCR product Y Chromosome 112 bp PCR product �Right side primers� Located to right of amelogenin gene, included amelogenin gene X chromosome 549 bp (set 1), 977 bp (set 2) Y Chromosome 360 bp (set1), 788 bp (set2)

85. Gender Identification:Amelogenin locus

87. Types Of DNA Analysis:RFLP Restriction Fragment Length Polymorphism Minimum amount of DNA 10-50 ng Must be high molecular weight (>20-23 kb), minimum degradation DNA must be digested to completion

88. RFLP Characteristics Determines variation in length of a defined DNA fragment Highest degree of discrimination per locus (Power of Discrimination, Pd) Many loci have been mapped for use (>15) Many variations at each loci Use of multiple restriction enzymes/sample Usually two bands/individual (one from each parent) Occasionally, three or more bands due to VNTRs (variable number tandem repeats)

89. RFLP Analysis Restriction enzymes are usually 4 base cutters (HaeIII, HinfI) Complete digestion of sample DNA required Partial digestion results in some fragments of the correct size, some slightly larger Use a test gel to determine whether partial digestions have occurred �Star Activity� refers to digestion of DNA at non-standard sites (due to changes in standard digestion conditions, ie, salt conc.)

90. Types Of DNA Analysis:PCR Can be performed on degraded DNA Minimum DNA required 0.2-0.5 ng Contaminants must be removed Dyes from denim Microorganisms

92. HLA DQ?/HLA DQA1 System HLA: human lymphocyte antigens Determine whether patient has antibodies against a potential donor�s HLA antigens HLA DQ ? is historic name for this region A 242 bp region with variation detected with specific probes for subregions

93. HLA DQ?/HLA DQA1 System Original test could detect 6 common DQ ? alleles, 21 possible genotypes Subsequent analysis, renaming by geneticists resulted in DQA1 test, increased number of subtypes detected (up to 28 detectable types) Cheap, fast color reactions

94. Reverse Dot Blot There are several steps in a DQ alpha PCR test: 1.DNA from 50 or more cells is extracted. Notice that this test requires fewer cells than the RFLP test. Sensitivity (the number of cells needed) is the main advantage of PCR tests. However, the increased sensitivity also makes PCR tests more vulnerable to trace contaminants, i.e., DNA from unexpected sources. 2.The DNA from the sample is copied over and over resulting in amplification of the original target sequence. The copying or amplification is accomplished in a machine specially designed for this purpose. This machine is called a thermal cycler. 3. The amplified DNA is next treated with a variety of probes that are bound to a blot (Note: In RFLP, the target DNA is bound to the blot and the probe DNA is added. For the DQ alpha dot blot, the probe DNAs are bound to a small blot strip and the target DNA is added). Each probe is found in a specific "dot" on the blot strip (which is known as a "dot blot"). A chemical reaction causes the dot to darken and become noticeable when DNA containing a particular allele (variant) is present. There are several steps in a DQ alpha PCR test: 1.DNA from 50 or more cells is extracted. Notice that this test requires fewer cells than the RFLP test. Sensitivity (the number of cells needed) is the main advantage of PCR tests. However, the increased sensitivity also makes PCR tests more vulnerable to trace contaminants, i.e., DNA from unexpected sources. 2.The DNA from the sample is copied over and over resulting in amplification of the original target sequence. The copying or amplification is accomplished in a machine specially designed for this purpose. This machine is called a thermal cycler. 3. The amplified DNA is next treated with a variety of probes that are bound to a blot (Note: In RFLP, the target DNA is bound to the blot and the probe DNA is added. For the DQ alpha dot blot, the probe DNAs are bound to a small blot strip and the target DNA is added). Each probe is found in a specific "dot" on the blot strip (which is known as a "dot blot"). A chemical reaction causes the dot to darken and become noticeable when DNA containing a particular allele (variant) is present.

95. Reverse Dot Blot Analysis Commercially available strips containing oligonucleotides corresponding to specific genes/expressed sequence tags (ESTs) Sample is amplified by PCR and hybridized to strip Color detection system used to identify positive reaction between amplified DNA and test oligonucleotides

98. The new U.S. federal legislation Authorizes the U.S. Department of Justice to collect DNA samples from federal arrestees. This could potentially involve an estimated 250,000 new DNA profiles annually. Allows federal DNA grant money under the 2005 President�s DNA Testing Initiative (Justice for All Act) and other federal legislation to be used for forensic DNA testing of arrestees.

99. The new U.S. federal legislation Allows States to upload DNA profiles from arrestees into the federal CODIS database. Previously, convicted felons were essentially the only DNA profiles included in the CODIS database as previous protocols made it difficult to include arrestees. This provision could potentially result in millions of individuals being added to the CODIS database in the coming years. Safeguards in the legislation also enable arrestees who are exonerated or never charged with the crime to request that their DNA profiles be expunged from the database. Authorizes DNA samples to be taken from immigrants trying to cross the U.S. border illegally. This provision could potentially affect an estimated 300,000 individuals each year.

100. The Combined DNA Index System (CoDIS) A database of DNA profiles from violent felons and crime scene samples Laws concerning who is eligible for the database vary from state to state Database currently contains about 2,038,470 felons and 93,956 crime scene profiles (19,00 hits so far)

101. The Mystical Power of CoDIS Extremely powerful investigative tool, linking crimes, and pulling suspects out of thin air! Can prevent, as well as solve crimes!

102. Different Systems, Different Markers

105. Capillary Electrophoresis System

106. Capillary Electrophoresis System

107. CODIS Profile

112. How Are These Astronomical Figures Derived? The product rule: combined probability of a series of independent events is determined by multiplying the probabilities of each event. STR loci are inherited independently (unlinked) Homozygous loci: p2 (same allele inherited from mother and father) Heterozygous loci: 2pq (either allele could be inherited from either parent) p(17)2 x 2p(15)q(17) x 2p(23)q(26)�. (.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalent to a probability of one in 76,000 using just 3 of the 13 loci!

113. Genotype Probability at any STR Locus Part of the work of forensic DNA analysis is the creation of population databases for the STR loci studied. Probability calculations are based on knowing allele frequencies for each STR locus for a representative human population (and showing Hardy-Weinberg equilibrium for the population by statistical tests).

114. POPULATION DATABASES:STR Analysis Probability calculations based on knowing allele frequencies for a representative population For a heterozygous individual, if the two alleles have frequencies of p and q in a population, probability (P) of having both alleles at a single locus P=2pq

116. Calculations of Probability of a Match for Four Single Locus Probes in a Sexual Assault Case

117. PROBABILITY FOR A DNA PROFILE Product rule calculations now accepted in court Multiply individual probabilities for each locus to obtain the expected frequency of a given DNA profile in a reference population Probability (P) is product of probability of each locus Profile Probability= (P1)(P2)�.(Pn)

118. PROBABILITY FOR DNA PROFILE WITH 13 CODIS LOCI Probability can be an extremely low number when all 13 CODIS loci are included in DNA profile A typical profile may be no more frequent than 1 in 5 quadrillion individuals (5 million billion)

119. Mitochondrial DNA (mtDNA)

120. Mitochondrial DNA Polymorphisms Sequence differences in the hypervariable regions (HV) of the mitochondrial genome.

121. Mitochondrial DNA Polymorphisms Mitochondria are maternally inherited. There are an average of 8.5 base differences in the mitochondrial HV sequences of unrelated individuals. All maternal relatives will have the same mitochondrial sequences. Mitochondrial typing can be used for legal exclusion of individuals or confirmation of maternal lineage.

122. Mitochondrial DNA (mtDNA) Pros Single-cell sensitivity because each cell contains ~1000 mitochondria Especially useful for shed hairs, burnt remains Can be used to establish kinship directly because entire complement of mtDNA is maternally inherited

123. Mitochondrial DNA (mtDNA) Cons Single-cell sensitivity because each cell contains ~1000 mitochondria = very high contamination risk! Heteroplasmy - more than one mtDNA type manifesting in different tissues in the same individual Lower power of discrimination - maternal relatives all share the same mtDNA

124. Y-STRs Problem: ~99% of violent crimes are committed by men DNA Mixtures of male suspect and female victim can pose an analytical challenge, especially when the female contribution is much greater than the male = preferential amplification

125. Y-STRs Solution: Test for markers found only on the Y-chromosome. Only male DNA is amplified!

127. Y-STRs Lower power of discrimination - paternal relatives all share the same Y-STR haplotype (�Wicked Uncle Ernie� Defense) 10% of Central Asian males share the same Y-STR haplotype, thought to belong to Genghis Khan

128. Forensic Case Issue Checklist: Evidence collection Item Description Item Packaging Item Labeling Photographs Diagrams of location site

129. Forensic Case Issue Checklist: Evidence Transportation and Storage From Scene To and from Storage To and from Laboratory To and from Court Back to storage or laboratory Elsewhere

130. Forensic Case Issue Checklist: At Laboratory Intake Lab Number Description of Evidence Item Weight Diagrams Photos Manipulation of evidence item Cutting Staining Packaging (compare with original)

131. Forensic Case Issue Checklist Testing Quality of documentation Description of test Preparation of solutions or other testing material Procedures followed Positive and Negative Controls Amount of evidence used Preliminary results Peer review Repackaging Reporting INTERPRETATION Specificity Accuracy

132. Forensic Quality Assurance Programs Lab inspection and accreditation ASCLD (American Society of Crime Laboratory directors) and AABB (American Association of Blood Banks) Personnel qualifications, training, and certification ABC (American Board of Criminalistics), general knowledge and specialties (I.e., DNA/serology) Proficiency testing CTS (Collaborative Testing Services) and CAP (College of American Pathologists)

133. Attorney�s Discovery Request Primary data RFLP autoradiographs PCR-color photos, strips All case and bench notes (including chain of custody documents) Laboratory protocols (including summary of population data) Proficiency test results of analyst

134. What Is An Expert Witness? Undergraduate and graduate degrees in relevant field of expertise Specialized training in the subject area Some training in forensics Professional licenses or certifications required by professional groups Evidence of experimentation, teaching and publication within the specialty area Prior disciplinary evidence directly relevant to the issues being considered

135. Defense Lines Of Attack: General No quality assurance required of labs Conflict of interest of experts Personnel no adequately trained Insufficient materials provided in response to discovery

136. Defense Lines Of Attack: Chain of Custody Documentation not adequate Samples improperly stored or transported Not all people/technicians who handle evidence are testifying Fraud or evidence tampering

137. Defense Lines Of Attack: Technical Lab procedures not followed correctly Contamination prior to or following evidence submission Mixed stains Non-human DNA present Other substances inhibitory to testing PCR products Samples switched inadvertently Band shifting and ethidium bromide Inadequacy of validation studies Procedures not peer reviewed

138. Defense Lines Of Attack: Statistical Population studies not adequate Inappropriate database used Undetected substructure excessive Database not adequately tested or peer reviewed Product rule not applicable Locus does not follow Hardy-Weinberg Loci are in linkage disequilibrium Error rate not known or incorporated into calculations

139. PROSECUTION TACTICS Educate the jury Detailed explanation of the underlying scientific principles and techniques involved in DNA testing �K.I.S.S.� principle Present evidence in ~30 min, non-technical Experience of expert, basic goal of DNA testing, widespread usage and acceptance

140. �K.I.S.S.� IN ACTION Who are you and what is it that you do? Why are you qualified to do it? Do you and others perform DNA testing often? What was the result of DNA testing in this case? Have your DNA test results ever excluded a suspect

141. THE �K.I.S.S.� OF DEATH If someone disagrees with your result, is there a scientific way to check if you got the right answer?

142. Levels Of Proof Civil: Substantial evidence Clear and convincing Preponderance of evidence (>50%) Criminal: Proof beyond a reasonable doubt Scientific: Proof to a scientific or medical certainty

143. Serial Killer Case Sept. 15, 1990, Prague: woman found strangled, apparently sexually assaulted Jan. 1991-April 1992, Austria: bodies of 7 prostitutes discovered in wooded areas in different parts of Austria Missing between 16-353 days Same cause of death, pattern of disposal and positional posing of bodies No relevant biological evidence suitable for DNA analysis

144. Serial Killer Case May, 1991, Austria: potential suspect identified by retired homicide detective Similar to 1975 case in Germany in which 18 year old girl strangled with her own brassier June-July, 1991, Los Angeles: bodies of 3 prostitutes discovered Strangled with their own brassieres Linked all three based on characteristics of cuts to bras, knots used

145. Serial Killer Case Suspect arrested in German case in 1975, convicted, sentenced to life in prison (which means he got out on parole in May, 1990) While in prison, man became an author and journalist Police able to tract suspect�s movements, match with times of murders in Prague, Austria, Los Angeles

146. Serial Killer Case Circumstantial evidence: Travel activity No alibi Similarity to Prague murder for which he had been convicted Physical evidence (June, 1992) Several head hairs found on the seat of suspect�s BMW (driven in Sept. 1990, sold, scraped--except the seats, which remained in a garage for 18 months)

147. Hair Found On Suspect�s Car Seat

148. Serial Killer Case Isolated 9 nanograms human DNA from hair June, 1993: 2 ng for HLA-DQA typing (1:13 match to Prague victim) Amplitype PM PCR typing: confirmation of match and frequency of 1 in 2,300 April, 1994: 2 ng for STR analysis (3 loci) June, 1994: 1 ng for Amelogenin locus

149. Serial Killer Case

150. Serial Killer Case Discrimination to 1 in 2.1 million women Confirmed identity of individual contributing the hair sample recovered from car seat by reverse paternity Suspect convicted in Austrian court system, sentenced to life in prison for murder of nine women, including the Prague murder Suspect committed suicide by hanging himself in jail

151. Child Molestation Case

152. Example of Actual DNA Evidence Presented to a Jury

153. The results of RFLP analysis of one VNTR locus in a sexual assault case. DNA from suspects 1 and 2 are compared to DNA extracted from semen evidence. You can see in this sample that suspect 1 and the sperm DNA found at the scene match. Suspect 2 has a profile totally different from the semen sample; his DNA fragments have run much farther down the gel, meaning that they are shorter. You can also tell that he is a homozygote because there is only one, darker band indicating the presence of two copies of the same fragment. The other samples tested come from heterozygotes, because they have two bands of distinct sizes in each lane. DNA isolated from the victim as well as a human DNA (K562) that serves as a standard size reference are included as controls. The results of RFLP analysis of one VNTR locus in a sexual assault case. DNA from suspects 1 and 2 are compared to DNA extracted from semen evidence. You can see in this sample that suspect 1 and the sperm DNA found at the scene match. Suspect 2 has a profile totally different from the semen sample; his DNA fragments have run much farther down the gel, meaning that they are shorter. You can also tell that he is a homozygote because there is only one, darker band indicating the presence of two copies of the same fragment. The other samples tested come from heterozygotes, because they have two bands of distinct sizes in each lane. DNA isolated from the victim as well as a human DNA (K562) that serves as a standard size reference are included as controls.

154. Bungling Burglar

155. DNA Profile Warrant

156. Investigations And Prosecutions Aided By DNA Evidence California �cold hits identified a rapists (1999 crime; 1998 crime which also helped bring charges for a 2003 rape). Failure to follow up on a cold hit report resulted in the preventable victimization of a California child. Colorado � a cold hit on the DNA database identified a rapist already named through a John Doe warrant (2001 case). Connecticut � a cold hit linked a sex offender to a Florida murder (1996 crime). Florida � a cold hit identified a burglar (evidence from a bag); and a murder suspect (2004 crime). Georgia � a cold hit with Indiana identified a murder suspect (on database for murder). Illinois � DNA identified a hit-and-run murder suspect (evidence on airbag); a 1991 murder suspect (cigarette butts collected by Michigan police); a 2006 murder (evidence on knife handle); and helped secure a guilty plea in a carjacking case and in an unrelated assault case (evidence from cap in both cases). Michigan � a rapist was sentenced to life after DNA identified him (1996 crime); a beastiality case was closed through DNA tests. Missouri �a cold hit helped convict a murderer (1980 crime). Montana � DNA identified a murder suspect and eliminated several other suspects (1976 case). Ohio �cold hits helped to convict a serial burglar and a murderer (1999 case). Oregon � a cold hit identified a suspect in four burglaries who eventually confessed to 200 burglaries (DNA from a beer bottle). Texas � DNA is key at a murder trial (evidence on a belt); cold hits linked the same man to three murders (on database for burglary). Virginia � a cold hit identified a murder suspect (1977 case). Federal � DNA in a missing persons cases helped to bring a gang-related murder conviction.

157. How Do You Identify Someone Who Has Never Been Genotyped? Need genetic information from their family members Parents: Half of the missing persons alleles must be present in one parent, half in the other parent Spouses and children: Half of the genes from child will be from spouse, half from missing parent

158. MISSING FATHER: What is his genotype?

159. MISSING MOTHER: What is her genotype?

160. Using Personal Effects to Identify Remains

161. Using Family Members to Identify Remains

162. PRENATAL DIAGNOSIS OF RAPE VICTIMS Study looked at 10 victims of sexual assault who became pregnant Prior to testing they were interviewed about continuing the pregnancy All women had a consensual partner who was also potential father of unborn child

163. PRENATAL DIAGNOSIS OF RAPE VICTIMS Inclusion: match between husband and fetus Exclusion: no match between husband and fetus (Rule out, rule in. In criminal cases, may obtain inconclusive result)

164. PRENATAL DIAGNOSIS OF RAPE VICTIMS

165. CASE RESULTS: In Utero Paternity Testing

166. Snowball the Cat: Prince Edward Island, Canada In 1994, Shirley Duguay, a 32-year-old mother of five, disappeared. Her body was found in a shallow grave a few months later. Among the chief suspects in the murder was the woman�s estranged common-law husband, Douglas Beamish, who was living nearby in his parents� home. Royal Canadian Mounted Police had no evidence linking Beamish to the crime. During the search for the victim�s body, however, the Mounties discovered a plastic bag containing a leather jacket with blood stains that matched the victim�s blood.

167. Snowball the Cat: Prince Edward Island, Canada The jacket also contained 27 strands of white hair, which forensic investigators determined were from a cat. The Mounties remembered a white cat named Snowball living in Beamish�s parents� home. The trick was to prove the cat hair found in the jacket was Snowball�s.

168. �Meow Plex�: Cat STR

169. Angiosperm Witness for the Prosecution A young woman was murdered in Phoenix, Arizona, and a pager found at the scene of the crime led the police to a prime suspect. He admitted picking up the victim, but claimed she had robbed him of his wallet and pager. The forensic squad examined the suspect's pickup truck and collected pods later identified as the fruits of the Palo Verde tree (Cercidium spp.).

170. Angiosperm Witness for the Prosecution One detective went back to the murder scene and found several Palo Verde trees, one of which showed damage that could have been caused by a vehicle. The detective's superior officer innocently suggested the possibility of linking the fruits and the tree by using DNA comparison. A geneticist at the University of Arizona in Tucson agreed to take on the case. Established evidence that individual Palo Verde trees have unique patterns of DNA. It was then a simple matter to link the pods from the suspect's truck to the damaged tree at the murder scene and obtain a conviction.

171. Viral DNA/RNA Evidence Convicts Doctor Dr. Richard Schmidt had an extramarital affair with his nurse, Janice Trahan. The relationship terminated when the doctor refused to leave his wife. The doctor and nurse continued their working relationship, with the nurse allowing the doctor to continue to administer a series of Vitamin B-12 shots.

172. Viral DNA/RNA Evidence Convicts Doctor The nurse�s health began to deteriorate shortly after one treatment and she received notification that she had contracted HIV and hepatitis. Analysis of HIV strain from nurse closely resembled that of a strain from one of the doctor�s patients. The doctor was charged and convicted to attempted second degree murder. He was sentenced to 50 years of hard labor.

173. The Remains Of The Evangelist Luke Luke was born in Antioch, Roman province of Syria Died in Thebes, Greece, at age of 84, around 150 AD Buried in Thebes but moved to Constantinople around 338 AD Body moved to Padua, Italy, sometime before 1177 AD

174. The Wandering Saint

175. Analysis Of The Remains Removed two teeth and extracted DNA Isolated control region of mitochondrial DNA and cloned it Compared sequences from body to sequences of DNA from Ancient and modern Greeks Ancient and modern Turks Ancient and modern Syrians

176. Teeth Removed From The Religious Relic: The body of St. Luke?

177. Is This St. Luke? The body is right age The body is not Greek Highest probability is that the body belonged to a Syrian but it is very difficult to tell Syrians from Turks

178. Influence of the Media GALLOP Poll March 17, 2000 How much have you heard about the use of DNA evidence in criminal trials: a great deal, a moderate amount, only a little, or nothing at all? A great deal A moderate Only a little Nothing No opinion amount at all 41% 40% 16% 3% < 0.5%

179. GALLOP Poll March 17, 2000 From what you have read or heard, do you think that DNA evidence is completely reliable, very reliable, only somewhat liable, or not reliable at all? Completely Very reliable Somewhat Not reliable No opinion reliable reliable at all 20% 57% 20% 1% 2% Skepticism appears to be highest among racial minorities, as only 58% of nonwhites believe DNA testing is completely or very reliable, compared to 81% of whites.

180. GALLOP Poll March 17, 2000 Compared with fingerprint evidence, do you think that DNA evidence is more reliable, about as reliable, or less reliable? More reliable About as reliable Less reliable No opinion 50% 41% 7% 2%

181. GALLOP Poll March 17, 2000 As you may know, DNA test have only been recognized as evidence in the past few years. Do you think that prisoners who were convicted before DNA tests were ever conducted should-- or should not-- be allowed to get DNA tests now--if such test might show they were innocent? Should be allowed Should not be allowed No opinion 92% 6% 2%

182. DATABANKS, DATABASES AND THE LEGAL SYSTEM Database is computer repository of DNA profiles Databank includes Enabling legislation Specification for sample collection, analysis and search parameter Specifications for processing �hits� Database

183. ELEMENTS OF A SUCCESSFUL DATABANK Legislation What crimes merit DNA data collection? Collection of samples Offenders (State or county, parole or probation) Cases (Urgency, case going to trial tomorrow, statute of limitations) Analysis of samples Choice of markers Offenders (all in one databank?) Cases (Got a suspect? How old is the case?) Analysis of Data Computer programs Communication between agencies Privacy

184. SEARCHING DATABANKS Stringency (High, moderate, low) Minimum number of loci required to match Include candidate specimens that match all but one loci Indexes to search (Violent offenders, sexual predators, burglars) Confirmation of a match

185. DNA STATUES BY STATE Mississippi DNA identification system Convicted sex offender must provide blood sample Louisiana Relevant evidence database (DNA, blood, saliva testing to prove identity in criminal setting) DNA detection of sexual and violent offenders Arkansas Proceeding concerning illegitimate juveniles Alabama Samples taken from a child Convicted sex offenders Colorado: NONE Virginia Most comprehensive, considered the Gold Standard

186. National DNA Index System

187. Summary Four types of polymorphisms are used for a variety of purposes in the laboratory: RFLP, VNTR, STR, and SNP. Polymorphisms are used for human identification and parentage testing. Y-STR haplotypes are paternally inherited. Polymorphisms are used to measure engraftment after allogeneic bone marrow transplants.

188. Summary Single-nucleotide polymorphisms are detected by sequencing, melt curve analysis, or other methods. SNPs can be used for the same applications as other polymorphisms. Mitochondrial DNA typing is performed by sequencing the mitochondrial HV regions. Mitochondrial types are maternally inherited.