Forensic DNA Analysis Criminalist Harry Klann, DNA Technical Leader 4/15/2017 Forensic DNA Analysis Criminalist Harry Klann, DNA Technical Leader DNA Detail Los Angeles Police Department Scientific Investigation Division PowerPoint presentation by Criminalist Carl Matthies

Objectives of Forensic DNA Testing 4/15/2017 Objectives of Forensic DNA Testing To link an individual to a crime scene/criminal act To exonerate suspects To identify victims of mass disasters Mass disasters can be either manmade (9/11 act of terrorism) or natural (2005 Indian Ocean Tsunami, 295,000 dead)

Contemporary Forensic DNA Testing Casework Applications Questions? 4/15/2017 A Brief History Contemporary Forensic DNA Testing Casework Applications Questions?

Early 1980s: Restriction Fragment Length Polymorphism (RFLP) 4/15/2017 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 “Please just call me Alec.”

4/15/2017 Mechanisms for RFLPs

Mid-1980s: The Colin Pitchfork Case 4/15/2017 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 “The Blooding” by Joseph Wambaugh, retired LAPD Sergeant

4/15/2017 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

PCR To The Rescue! Polymerase Chain Reaction = molecular Xeroxing 4/15/2017 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

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4/15/2017 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

The Current Method of Choice: Autosomal Short Tandem Repeats 4/15/2017 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!

Applied Biosystems 310 Genetic Analyzer 4/15/2017 Applied Biosystems 310 Genetic Analyzer

The Process In a Nutshell 4/15/2017 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.

4/15/2017 STR data X, Y, X Y

4/15/2017 STR data (cont’d)

4/15/2017 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…”

How are these astronomical figures derived? 4/15/2017 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!

4/15/2017 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.

4/15/2017 STR Artifacts Stutter: Slippage of DNA polymerase; results in a peak that is four base pairs (one repeat unit) smaller than allele peak.

4/15/2017 STR Artifacts Pull-up: Incomplete filtration of spectral overlap in fluorescent detection system.

4/15/2017 Pull-up

4/15/2017 DNA Mixtures When more than one source of DNA is detected in a sample, assignment of genotypes becomes more difficult.

Degraded/Trace DNA Samples 4/15/2017 Degraded/Trace DNA Samples Larger alleles “drop-out” when template DNA is low in quantity or quality, reducing certainty of genotypes.

The Combined DNA Index System (CoDIS) 4/15/2017 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)

The Mystical Power of CoDIS 4/15/2017 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!

The Dark Side of CoDIS (What the FBI doesn’t want you to know.) 4/15/2017 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%) DNA mixtures can be tricky; requiring close examination of the electronic data, the questioned profile, and the offender “match” profiles. For example, an unknown mixture profile with three alleles at the D21S11 locus, ex. (28, 30, 31) would “hit” on (28, 28), (28, 30), (28, 31), (30, 30), (30, 31), and (31, 31). A total of 6 genotypes. If we designate an obligate or required allele (+), (28, 30+, 31)* would now hit only on the following genotypes: (28, 30), (30, 30), and (30, 31). We have reduced the number of hits at the D21S11 locus by half. Assigning obligate alleles at as many loci as possible can reduce the number of spurious hits to the offender database. * In this example, which could be a vaginal swab taken from the victim of a sexual assault, the victim was determined to be a (28, 31) at D21S11. Therefore, the 30 allele is foreign to the victim and presumed to be from the suspect.

LAPD CoDIS Statistics 177/142 Case-to-Offender matches 4/15/2017 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… 177 cases matched to 142 offenders California State DNA Index System (SDIS): 304,817 offender STR profiles 9,633 forensic STR profiles

“Specialized” PCR-based systems 4/15/2017 “Specialized” PCR-based systems mtDNA Y-STRs SNPs

Mitochondrial DNA (mtDNA) 4/15/2017 Mitochondrial DNA (mtDNA)

Mitochondrial DNA (mtDNA) 4/15/2017 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

Mitochondrial DNA (mtDNA) 4/15/2017 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

Y-STRs Problem: ~99% of violent crimes are committed by men 4/15/2017 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

4/15/2017 Y-STRs Solution: Test for markers found only on the Y-chromosome. Only male DNA is amplified!

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Y-STRs “Khan” Argument 4/15/2017 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

Single Nucleotide Polymorphisms (SNPs) 4/15/2017 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)

4/15/2017 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)

Flawed logic in the use of SNPs for predicting ancestry 4/15/2017 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 Scenario 2: LAPD Detective Tom M., SNPs 62% European, but appears SE Asian/Indian.

4/15/2017 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