Forensic DNA Analysis

1. Forensic DNA Analysis Criminalist Harry Klann, DNA Technical Leader DNA Detail Los Angeles Police Department Scientific Investigation Division PowerPoint presentation by Criminalist Carl Matthies

2. Objectives of Forensic DNA Testing • To link an individual to a crime scene/criminal act • To exonerate suspects • To identify victims of mass disasters

3. A Brief History • Contemporary Forensic DNA Testing • Casework Applications • Questions?

4. Early 1980s: Restriction Fragment Length Polymorphism (RFLP) • Genetic variation in the distance between restriction enzyme sites • Template DNA digested by enzymes, electrophoresed, detected via Southern blotting • Power of discrimination in the range of 106-108 for a six probe analysis Sir Alec Jeffreys

5. Mechanisms for RFLPs

6. Mid-1980s: The Colin Pitchfork Case • Two young women raped and murdered in Narborough, England • 5,000 local men are asked to provide blood/saliva samples • 1st exoneration and conviction on forensic DNA evidence

7. The Catch: • RFLP testing requires a relatively large amount of HMW DNA (~50ng = thousands of cells) • Not ideal for forensic evidence, in which small, degraded samples are common

8. PCR To The Rescue! • Polymerase Chain Reaction = molecular Xeroxing • Three temperature phases, carried out in a Thermal Cycler, replicate or “amplify” the desired DNA fragment(s) Dr. Kary Mullis Eccentric Genius

10. PCR (cont’d) • First forensic application is the DQα locus, later multi-plexed with Polymarker™ loci using dot-blot detection method • Works with lower quantity (1-2ng), lower quality samples • Power of discrimination goes from 102-106...not good enough for databasing

11. The Current Method of Choice: Autosomal Short Tandem Repeats • Non-coding, tetranucleotide sequences which vary greatly from person to person in the number of repeating units • Requires <1ng of DNA to type 13-15 STR loci • Power of discrimination ranges from 1014-1023. World population is 109 so bring on the database!

12. Applied Biosystems 310 Genetic Analyzer

13. The Process In a Nutshell Amplified DNA samples are injected into a capillary. Fluorescent tags on the DNA fragments are excited by a laser as they pass a window in the capillary, the fluorescence is recorded by a camera, and this signal is converted into a “peak” by the computer software.

14. STR data X, Y, X Y

15. STR data (cont’d)

16. STR data (cont’d) “The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”

17. 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!

18. STR Artifacts -A (“minus A”): Incomplete addition of nucleotide ‘A’ by DNA polymerase; results in a peak that is one base pair smaller than allele peak.

19. STR Artifacts Stutter: Slippage of DNA polymerase; results in a peak that is four base pairs (one repeat unit) smaller than allele peak.

20. STR Artifacts Pull-up: Incomplete filtration of spectral overlap in fluorescent detection system.

21. Pull-up

22. DNA Mixtures When more than one source of DNA is detected in a sample, assignment of genotypes becomes more difficult.

23. Degraded/Trace DNA Samples Larger alleles “drop-out” when template DNA is low in quantity or quality, reducing certainty of genotypes.

24. 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)

25. 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!

26. The Dark Side of CoDIS(What the FBI doesn’t want you to know.) • DNA mixtures and degraded DNA profiles have lead to spurious matches • Stringent laws explicitly permit databasing innocent people • Adding arrestees to database violates presumption of innocence • However, the prosecution rate on case to offender matches is shockingly low! (~10%)

27. LAPD CoDIS Statistics • 177/142 Case-to-Offender matches • 100 Case-to-Case hits with 42 as yet unidentified suspects • 28 DA “rejects” • 9 Convictions; charges filed in 28 more; 4 defendants plead guilty • 280 investigations aided…

28. “Specialized” PCR-based systems • mtDNA • Y-STRs • SNPs

29. Mitochondrial DNA (mtDNA)

30. 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

31. 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

32. 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

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

35. Y-STRs “Khan” Argument • 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

36. Single Nucleotide Polymorphisms (SNPs) • Point mutations (base substitutions) found in 1% or more of the population • 1.8 million identified in human genome • Detected on micro-array plates with fluorescent tags (all or nothing response)

37. SNPs (cont’d) • ~50 SNPs provides same power of discrimination as 13 STR loci • Certain SNPs used as predictors of ancestry/ethnicity by a private sector lab (DNA Witness)

38. Flawed logic in the use of SNPs for predicting ancestry Scenario 1: SNP profile inconsistent with subject’s reported ancestry  subject’s family history is inaccurate  results are indisputable Scenario 2: SNP profile inconsistent with subject’s physical appearance  ad hoc human migration explanation  results are indisputable

39. Other SNP Flaws • Privacy issues - unlike STRs, SNPs can be correlated with susceptibility/resistance to diseases • Requires a relatively large quantity of DNA for robust assay