Forensic DNA profiling workshop

1. Forensic DNA profiling workshop Dan E. Krane, Wright State University, Dayton, OH Keith Inman, Forensic Analytical, Oakland, CA Forensic Bioinformatics (www.bioforensics.com)

2. I: Overview of what DNA tests can do for:A. ProsecutionB. DefenseC. Post-conviction testing

3. DNA Technology in Court • Criminal Prosecution • Unprecedented sensitivity and specificity for typing biological samples • Growing use of databanks and dragnets to identify suspects • Rapidly becoming cheaper and faster

4. Possible DNA Sources

5. DNA Technology in Court • Criminal Defense • Unprecedented sensitivity and specificity for typing biological samples • Potential support for alternative theories of the case

6. DNA Technology in Court • Post-conviction exonerations (208 in US) based on DNA evidence have revealed problems with the justice system

7. II: The evolution of DNA technology

8. Three generations of DNA testing RFLP AUTORAD Allele = BAND DQ-alpha TEST STRIP Allele = BLUE DOT Automated STR ELECTROPHEROGRAM Allele = PEAK

9. Two relatively new DNA tests Mitochondrial DNA mtDNA sequence Sensitive but not discriminating Y-STRs Useful with mixtures Paternally inherited

10. Phenotyping: DNA WitnessTM • New test by DNA Print Genomics (Florida) • Tests SNPs (single nucleotide polymorphisms) • Identifies ‘genetic heritage’ of sample • Provides percentages of makeup: • Sub-Saharan African • East-Asian • Indo-European • Native-American • Latest versions infer hair and eye color • Has been used in assisting investigations

11. Lab-on-a-chip • Currently in R&D • Use microdevices to • Extract DNA • Quantify DNA • PCR amplify DNA • Capillary electrophoresis • All on small, integrated glass or plastic chip • Quick (test in half hour?) • Very small samples? • Portable?

12. Basic terminology: Genetics • DNA Polymorphism (“many forms”) • Regions of DNA which differ from person to person • Locus (plural = loci) • Site or location on a chromosome • Allele • Different variants which can exist at a locus • DNA Profile • The combination of alleles for an individual

13. Basic terminology: Technology • Amplification or PCR (Polymerase Chain Reaction) • A technique for ‘replicating’ DNA in the laboratory (‘molecular Xeroxing’) • Region to be amplified defined by PRIMERS • Can be ‘color coded’ • Electrophoresis • A technique for separating molecules according to their size

14. Automated STR Test

15. STR • Short tandem repeat • Describes a type of DNA polymorphism in which: • a DNA sequence repeats • over and over again • and has a short (usually 4 base pair) repeat unit • A length polymorphism -- alleles differ in their length 3 repeats: AATG AATG AATG 4 repeats: AATG AATG AATG AATG 5 repeats: AATG AATG AATG AATG AATG 6 repeats: AATG AATG AATG AATG AATG AATG

16. Crime Scene Samples & Reference Samples Differential extraction in sex assault cases separates out DNA from sperm cells • Extract and purify DNA

17. Extract and Purify DNA • Warm soapy water • Releases biological material • Organic Extraction • Ethanol Precipitation

18. PCR Amplification Groups of amplified STR products are labeled with different colored dyes (blue, green, yellow)

19. The ABI 310 Genetic Analyzer

20. Detector Window ABI 310 Genetic Analyzer: Capillary Electrophoresis • Amplified STR DNA injected onto column • Electric current applied • DNA pulled towards the positive electrode • DNA separated out by size: • Large STRs travel slower • Small STRs travel faster • Color of STR detected and recorded as it passes the detector

21. SGM+ raw data

22. D3 vWA FGA D8 D21 D18 Amelogenin D5 D13 D7 Reading an electropherogram • ALLELE CALLS • Loci arranged by size and color • Allele designation given as a number • NUMBER OF PEAKS • 1 peak = homozygous • 2 peaks = heterozygous • 3 or more peaks = mixed sample (?) • HEIGHT OF PEAK • Proportional to amount of allele (approx) • RFU (relative fluorescent units) • Amelogenin • Sex of sample • XY = Male • X = female

23. Allelic Ladder

24. Evidence (Bloodstain) Suspect reference Comparing electropherograms DIFFERENT

25. Victim reference Evidence (Bloodstain) Comparing electropherograms SAME

26. 4 PEAKS Victim reference X>Y Evidence (swab) Suspect reference MIXTURE

27. x 0.222 x 2 Statistical estimates: the product rule 0.222 = 0.1

28. x x 1 in 111 1 in 20 1 in 22,200 x x 1 in 100 1 in 14 1 in 81 1 in 113,400 x x 1 in 116 1 in 17 1 in 16 1 in 31,552 Statistical estimates: the product rule 1 in 10 = 0.1 1 in 79,531,528,960,000,000 1 in 80 quadrillion

29. What more is there to say after you have said: “The chance of a coincidental match is one in 80 quadrillion?”

30. What more is there to say after you have said: “The chance of a coincidental match is one in 80 quadrillion?” • Two samples really do have the same source • Samples match coincidentally • An error has occurred

31. DNA match probability • Random Match Probability (RMP) • What is the chance of finding a random, unrelated person in a given population that has a given DNA profile? • NOT the probability that the defendant is guilty • NOT the probability that someone other than the defendant committed the crime

32. What can we make of the DNA profile at this locus?

33. Combined probability of inclusion (CPI; reciprocal of CPE) What fraction of a population cannot be excluded as a possible contributor to a given mixture?

34. Combined probability of inclusion (CPI) What fraction of a population cannot be excluded as a possible contributor to a given mixture? For a locus with n alleles A1 through An CPIlocus = (A1+ A2+…+ An)2 Total CPI with 8 loci CPItotal = CPI1x CPI2x…x CPI8

35. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134

36. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134 (p + q + r)2 = CPI 0.289 = CPI

37. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134 (p + q + r)2 = CPI 0.289 = CPI • What statistic should be applied to “Tom”? • SuspectFGA • Tom 21, 22

38. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134 (p + q + r)2 = CPI 0.289 = CPI • What statistic should be applied to “Dick”? • SuspectFGA • Tom 21, 22 • Dick 22, 22

39. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134 (p + q + r)2 = CPI 0.289 = CPI • What statistic should be applied to “Harry”? • SuspectFGA • Tom 21, 22 • Dick 22, 22 • Harry 22, 25

45. Combined probability of inclusion (CPI) What fraction of a population cannot be excluded as a possible contributor to a given mixture?

46. p(21) = 0.185 q(22) = 0.219 r(23) = 0.134 (p + q + r)2 = CPI 0.289 = CPI • What statistic should be applied to “Franklin”? • SuspectFGA • Tom 21, 22 • Dick 22, 22 • Franklin 22, 25

47. Combined probability of inclusion (CPI) What fraction of a population cannot be excluded as a possible contributor to a given mixture? What portion of the population cannot be excluded from contributing their DNA to a given mixture if they are allowed to not match at two loci? X

48. Matching profiles in NIST database n = 257 African Americans, 302 Caucasians (1 in 21 African Americans; 1 in 38 Caucasians)

49. Combined probability of inclusion (CPI) What fraction of a population cannot be excluded as a possible contributor to a given mixture? What portion of the population cannot be excluded from contributing their DNA to a given mixture if they are allowed to not match at two loci? X

50. Ignoring loci with “missing” alleles Labs often claim that this is a “conservative” statistic Ignores potentially exculpatory information “It fails to acknowledge that choosing the omitted loci is suspect-centric and therefore prejudicial against the suspect.” Gill, et al. “DNA commission of the International Society of Forensic Genetics: Recommendations on the interpretation of mixtures.” FSI. 2006.