This document provides an overview of the history and current methods of forensic DNA analysis. It discusses early methods like RFLP that required large DNA samples, the development of PCR that allowed analysis of smaller samples, and the current standard method using STR analysis of 13-15 loci that can determine a person's profile with a probability of 1 in 1 trillion. It covers DNA mixture interpretation challenges, the CODIS database system, and specialized techniques like mtDNA, Y-STR, and SNP 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
s To link an individual to a crime
scene/criminal act
s To exonerate suspects
s To identify victims of mass
disasters

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

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

5. Mechanisms
for RFLPs

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

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

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

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

11. The Current Method of Choice:
Autosomal Short Tandem Repeats
s Non-coding, tetranucleotide
sequences which vary greatly from
person to person in the number of
repeating units
s Requires <1ng of DNA to type
13-15 STR loci
s 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,
XY

15. STR data (cont’d)

16. STR data (cont’d)
STR TYPING SUMMARY SHEET
Date: DNA Analyst / Serial #: DR #:
9/24/1999 MATTHIES V9780 00-00-00001
Item # AMEL D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 THO1 TPOX
X, Y 17 8, 10
25(S) 15, 17 23, 26 14, 15 26 12, 15 10 9, 13 9, 10 8, 9 9, 10
X, Y 17 8, 10
X 15, 17 28, 11
25(E) 16, 18 19, 26 15 14, 16 8, 13 12 11, 12 7, 8 11
X 15, 17 32.2 11
X 15, 17 28, 11
VICTIM 16, 18 19, 26 15 14, 16 8, 13 12 11, 12 7, 8 11
X 15, 17 32.2 11
X, Y 17 8, 10
SUSPECT 15, 17 23, 26 14, 15 26 12, 15 10 9, 13 9, 10 8, 9 9, 10
X, Y 17 8, 10
“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)
s A database of DNA profiles from
violent felons and crime scene
samples
s Laws concerning who is eligible for
the database vary from state to
state
s 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
s Extremely powerful investigative
tool, linking crimes, and pulling
suspects out of thin air!
s Can prevent, as well as solve
crimes!

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

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

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

29. Mitochondrial DNA (mtDNA)

30. Mitochondrial DNA (mtDNA)
Pros
s Single-cell sensitivity because each cell
contains ~1000 mitochondria
s Especially useful for shed hairs, burnt
remains
s Can be used to establish kinship directly
because entire complement of mtDNA is
maternally inherited

31. Mitochondrial DNA (mtDNA)
Cons
s Single-cell sensitivity because each cell
contains ~1000 mitochondria = very high
contamination risk!
s Heteroplasmy - more than one mtDNA type
manifesting in different tissues in the same
individual
s Lower power of discrimination - maternal
relatives all share the same mtDNA

32. Y-STRs
Problem:
s ~99% of violent crimes are committed by
men
s 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:
s Test for markers found only on the Y-
chromosome. Only male DNA is amplified!

35. Y-STRs
“Khan” Argument
s Lower power of discrimination - paternal
relatives all share the same Y-STR
haplotype (“Wicked Uncle Ernie” Defense)
s 10% of Central Asian males share the same
Y-STR haplotype, thought to belong to
Genghis Khan

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

37. SNPs (cont’d)
s ~50 SNPs provides same power of
discrimination as 13 STR loci
s 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
s Privacy issues - unlike STRs, SNPs can be
correlated with susceptibility/resistance to
diseases
s Requires a relatively large quantity of DNA for
robust assay