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
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Mechanisms for RFLPs
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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
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PCR (contd)

• First forensic application is the DQa 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 lt1ng of DNA to type 13-15 STR loci
• Power of discrimination ranges from 1014-1023.
  World population is 109 so bring on the database!

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Applied Biosystems 310 Genetic Analyzer
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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
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STR data (contd)
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STR data (contd)
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
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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.
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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 doesnt 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)

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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

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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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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 (contd)

• 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 subjects
  reported ancestry ? subjects family history is
  inaccurate ? results are indisputable
• Scenario 2
• SNP profile inconsistent with subjects
  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