1. 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

2. 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)

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

4. 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 for a six probe analysis
Sir Alec Jeffreys
“Please just call me Alec.”

5. 4/15/2017
Mechanisms
for RFLPs

6. 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

7. 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

8. 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

9. 4/15/2017

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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 not good enough for databasing

11. 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 STR loci
Power of discrimination ranges from World population is 109 so bring on the database!

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

13. 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.

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STR data
X, Y,
X Y

15. 4/15/2017
STR data (cont’d)

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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?
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) = , which is equivalent to a probability of one in 76,000 using just 3 of the 13 loci!

18. 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.

19. 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.

20. 4/15/2017
STR Artifacts
Pull-up: Incomplete filtration of spectral overlap in fluorescent detection system.

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Pull-up

22. 4/15/2017
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
4/15/2017
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)
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)

25. 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!

26. 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.

27. 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

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

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

30. 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

31. 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

32. 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

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

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35. 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

36. 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)

37. 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)

38. 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.

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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