Understanding Forensic DNA
Testing:
There’s Nothing To Fear
Eric J. Carita
Forensic DNA Consultant
DNA 101
Basic Genetics
What is DNA?
DNA is…
Deoxyribonucleic Acid
The inherited genetic material that makes us
what we are
One set of
22 autosomes
(plus X)
One set of
22 autosomes
(plus X or Y)
Paternity Testing
Two alleles* for each
autosomal genetic marker
Pattern Comparisons
• Between Known & Evidentiary DNA
Profiles
• Similar to fingerprint comparisons
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Individual
nucleotides
Base Pairing of DNA Strands
Hydrogen bonds
3’
5’
A=T
G  C hybridized
strands
Phosphate-sugar
T=A
backbone
CG
A=T
A
T
A
T
G
C
C denatured G
C 5’
strands
3’G
5’
A=T
GC
3’
T=A
CG
A=T
T=A
CG
CG
3’
GC
5’
Butler, J.M. (2001) Forensic DNA Typing, Figure 2.2, ©Academic Press
Mendel’s Ist Law—Equal Segregation
During the formation of gametes (sperm & egg), the two
members of a gene pair (allele) separate into the gametes,
so that half of the gametes carry one allele and half carry the
second allele.
We inherit half of our nuclear genes (one allele from each
locus) from each parent.
Mendel’s 2nd Law—Independent Assortment
During gamete formation, the segregation of one gene pair is
independent of other gene pairs.
Thus, the segregation of alleles at one locus is independent of
the segregation of alleles at other (unlinked) loci.
Source of tremendous genetic variation that can be exploited
for forensic purposes.
Human Genome
~3 billion base pairs of DNA
30,000-35,000 genes
Population-each gene has multiple forms
Allelic variation-basis of forensic DNA typing
Dozens of polymorphic loci validated for forensic use
DNA - Unique, Yet the Same
1. Of the 3,000,000,000 DNA bases,
about 0.3% is not conserved:
~1 million bases
2. Forensic STR analysis looks at the
length of 13-15 areas of DNA.
Characteristics of DNA
•
DNA is inherited from parents
(half from mother, half from father).
• No two individuals have the
same DNA profile; except for
identical twins.
• Genes have multiple forms. This
variation is the basis of forensic DNA
typing.
Forensic Science:
the application of natural
sciences to matters of
the law.
Criminalistics:
The recognition, identification,
individualization, and evaluation of
physical evidence using the
methods of the natural sciences in
matters of legal significance.
Forensic DNA Testing is
Nothing
Just
Just like CSI!
DNA Casework
1. Forensic Analysis (Criminal).
-179 labs conducting DNA analysis in 50 states.
~ 40,000 cases/year received.
~25,000 analyzed.
~80% sexual assaults.
2. ~30% of the time the suspect is excluded by
DNA.
3. ~ 300,000 paternity cases per year.
Where can we get DNA
from?
Sources of Biological Evidence
•
•
•
•
•
•
•
•
•
Blood
Semen
Saliva
Urine
Hair
Teeth
Bone
Tissue
All cells – except RBC
Other Possible items for DNA Testing:
1. cigarette butts
2. gloves, bandanas, ski masks, baseball caps
general clothing
3. condoms (inside vs. outside)
4. stains on furniture, pillows, sheets
5. hair clips, lipsticks
6. letters, envelopes, and stamps
7. plant and animal sources of evidence
Locard’s Principle of Exchange
Anytime there is contact
between two
surfaces, there will be a
mutual exchange of matter
across the contact
boundary
Transfer of DNA Evidence
• Locard’s Theory
• Biological fluids
– Saliva
– Semen
– Blood
• Epithelial (Skin) Cells
– Touch DNA
– Fingerprints
• Primary vs. Secondary Transfer
– Dependent upon the source of DNA
How much do you need?
What we need today
“Touch DNA”
What is Touch DNA?
1.
2.
3.
4.
DNA left on evidence by the “handler”.
Usually no visible stain.
Usually very small amounts of DNA are deposited.
Significant increase in number of touch DNA cases
in last 2 years.
Associated with many crimes including:
Property Crimes
Threatening letters
Robbery
Homicide
Assault/Sexual Assault
Vandalism
Common Sources of Touch
DNA
From Hands:
Gloves
Knife handles
Weapon handles
Firearm grips
Plastic bag handles
Steering Wheels
Rope
Shoe laces
Electrical cords
Common Sources of Touch
DNA continued…
Wearer:
Baseball caps
Sweatbands
Shirt/jacket collars
Socks
Waistbands of pants
Eyeglasses
* Mixtures v. Single Source
TOUCH DNA CASES
Commonly swabbed areas
Counter
* NY hat
* Gun
*
TOUCH DNA EVIDENCE : WHAT
DOES IT MEAN…….?
Shows linkage or association but….
• DNA recovered from an object may not be from the
last person to touch it. Factors include:
-Length of contact
-Good cell shedder or not
-Vigorous contact vs. incidental
• DNA profiles recovered from touch evidence are
often mixtures – multiple individuals
-Elimination known(s)
-Lawful owner
-Crime scene personnel, officers
COLLECTION OF TOUCH DNA FROM
EVIDENCE
When:
- At the Crime Scene by Law Enforcement
(e.g. door knobs, counters, windows)
- Forensic Laboratory by analyst
(if other testing is needed)
Where:
- Areas of contact
(e.g. grips, slide, trigger, magazine, cartridge cases; fired vs. unfired)
- Any touched object…
(but be cautious regarding objects accessible to the general public)
DNA Profiles from Weapons and the
DNA Database
The weapon must be associated with a crime
- seized vs. surrendered.
The weapon cannot be seized from the suspect’s
person or property.
Cannot use a “possession” sample as an alternate
way to get a suspect’s known profile into the
DNA Database.
TOUCH DNA EVIDENCE :
COLLECTION SUGGESTIONS…….
Proper collection of “touch” DNA evidence:
Collection protocol:
-Wear latex gloves (change frequently)
-Disposable face masks/supplies
- Clean instruments with bleach and alcohol
How :
-Swab using sterile swab/solution
Collection of Touch DNA Evidence
1. Contamination is a significant possibility.
2. Impact of contamination is false exclusion of
suspect or artificial mixtures.
How to minimize:
Gloves, Masks, Disposable Instruments,
procedure (no talking over evidence!!!)
Identification of Contamination:
Know the DNA profiles of:
First Responders, Major Crime Squads,
and Laboratory Personnel
Forensic DNA Testing
How do we go from this . . .
. . . To this?
Evidence Collection
With increasingly sensitive DNA tests, proper collection
protocols are more critical.
Standard measures: Gloves, disposable supplies, etc.
consider masks—especially for low yield samples.
Clean any non-disposable instruments with bleach and alcohol.
Elimination swabs from people at the scene answers
standard defense question.
Collect evidence to avoid/minimize mixtures especially with
certain samples.
DNA in the Cell
chromosome
cell nucleus
Double stranded
DNA molecule
Target Region for PCR
Polymorphic
Region
DNA Amplification with the
Polymerase Chain Reaction (PCR)
5’
3’
5’
3’
3’
3’
5’
5’
Starting DNA
Template
Separate
strands
(denature)
Forward primer
5’
3’
5’
3’
Make copies
Add primers
(extend
primers)
5’ (anneal)
3’
3’
5’
Reverse primer
PCR Copies DNA Exponentially through
Multiple Thermal Cycles
Original DNA target region
Thermal cycle
In 32 cycles at 100% efficiency, 1.07 billion
copies of targeted DNA region are created
Short Tandem Repeats (STRs)
AATG
7 repeats
8 repeats
Repeat number varies between alleles. PCR
primers bind to flanking regions that are constant.
Homozygote = Two copies of same allele.
Heterozygote = Two different alleles.
Multiplex PCR
• 15 STR Markers Can Be
Amplified in 1 reaction.
• Sensitivity = less than 250
pg of DNA.
• Ability to Handle Mixtures
and Degraded Samples.
• Different Fluorescent Dyes
Used to Distinguish STR
Alleles with Overlapping
Size Ranges.
Example of Forensic STR Multiplex Kit
AmpFlSTR® Identifiler™
Kit available from PE Biosystems (Foster City, CA)
200 bp
100 bp
300 bp
400 bp
Color
Separation
Size Separation
D8
D21
D3
THO1
D13
D19
vWA
TPOX
A
D5
D7
CSF
D16
D2
VIC
NED
D18
FGA
FAM-6
FGA
PET
15 STRs amplified along with sex-typing marker amelogenin in a single PCR
reaction.
LIZ
LIZ-internal lane standard
Fluorescent STR Analysis
-Fluorescent
dye tags on the primers
-Visualize emitted light with a
digital camera.
-Collect and analyze data with
computer.
Capillary Electrophoresis
B.
E.
A.
C.
D.
D.
A. Capillary Array
B. Polymer Delivery
Pump
C. Pump block
D. Buffer reservoirs
E. Detection Block
(camera)
Forensic DNA Analysis
Forensics: Pattern Comparisons
Forensic DNA Analysis
Evidentiary DNA profile(s) are generated from samples
submitted to Forensic Lab.
Known profile(s) of suspect/victim (blood or buccal) are
compared to DNA profiles from instant case.
Evidentiary profiles entered into CODIS database. Suspect’s
profile is not entered into CODIS database.
DNA MIXTURES
•Common in Forensic DNA testing.
Sexual Assault samples-intimate swabs, clothing.
•Mixtures of victim & suspect(s).
-How many people?
-Previous consensual partners?
-Contamination: scene, collection, lab?
•Mixture not always detected at all tests.
DNA
DNA Profile
Profile Detection
Detection
Profile
ProfileAADetected
Detected
Factors:
Factors:
1.
1. Quantity
Quantityof
of DNA
DNA
2.
2. Quality
Quality of
of DNA
DNA
Profile
Profile BB Detected
Detected
Mixture Detection?
25:1
Only Profile B Detected
Factors:
1. Quantity
2. Quality
3. Ratio
1:1
Profiles A and B Detected
Stochastic Fluctuation
•Stochastic = chance.
•Result of PCR founder effect and chance allele
sampling.
If you amplify small amounts of DNA (LCN PCR), can see
stochastic effects.
The Meaning of a DNA Match?
1. Person A is the source of the DNA profile from the evidence.
2. The identical twin of person A is the source of the DNA
profile.
or
3. Another person who coincidentally has the same profile
as person A is the source of the DNA profile from the
evidence.
= the random match probability
DNA Conclusions
1. Included – source or contributor
2. Excluded – source or contributor
3. CBE – source or contributor
4. Inconclusive
5. Insufficient data
Y-DNA Typing
Y Chromosome Testing
• Paternal inheritance.
• Detects male component of a mixture.
• Less discriminating than standard DNA
testing. Statistics = counting method
(linkage).
• Important for detecting the semen donor in
sexual assault mixtures.
When to Use Y-STR Testing
• Sexual assaults by vasectomized or azoospermic males (no sperm left
behind for differential extraction)
• Extending length of time after assault for recovery of perpetrator’s
DNA profile (greater than 48 hours)
• Male-female mixtures
• Other bodily fluid mixtures (blood-blood, skin-saliva)
• Gang rape situation to include or exclude potential contributors
• When you want to double the amount of DNA for the PCR Reaction.
Y-STRs
“detects male component of a mixture”
SCENARIO #1
SCENARIO #2
F
F
F
F
F
Y-STRs
X:Y = 30:1
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X X
X
X
X
X
X
X X
Y
X
X
X
Y Profile Detected
Disadvantages of the Y-Chromosome
• Loci are not independent of one another and therefore rare
random match probabilities cannot be generated with the
product rule; must use haplotypes (combination of alleles
observed at all tested loci)
• Paternal lineages possess the same Y-STR haplotype
(barring mutation) and thus fathers, sons, brothers, uncles,
and paternal cousins cannot be distinguished from one
another
• Not as informative as autosomal STR results
– More like addition (10 + 10 + 10 = 30) than multiplication (10 x
10 x 10 = 1,000)
Forensic Advantages of Y-STRs
• Male-specific amplification extends range of cases accessible to obtaining
probative DNA results (e.g., fingernail scrapings, sexual assault without
sperm)
• Technical simplicity due to single allele profile; can potentially recover
results with lower levels of male perpetrator DNA because there is not a
concern about heterozygote allele loss via stochastic PCR amplification;
number of male contributors can be determined
• Courts have already widely accepted STR typing, instrumentation, and
software for analysis (Y-STR markers just have different PCR primers)
• Acceptance of statistical reports using the counting method due to
previous experience with mtDNA
• Double the Genomic DNA within the PCR Amplification reaction.
A Haplotype
• Although 17 loci are typed
• They are linked and are treated as one “super” locus
• A haplotype essentially is an allele
• The more alleles at a locus, generally the lower the
effect of substructure on statistical calculations
Y-STRs can permit simplification of male DNA
identification in sexual assault cases
No signal observed
Female Victim
DNA Profile
Male Perpetrator
DNA Profile
DNA Profile from
Crime Scene
Autosomal STR
Profile
Y-Chromosome STR
Profile
Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.2, ©Elsevier Science/Academic Press
( p)(1  p)
p  1.96
N
p2
+ 2pq +
q2
=1
1/N
1-
Forensic DNA Statistics
AA + 2AB +2AC+ BB+ 2BC + CC = 1
P = .5 x .5 x .5 x .5 x .5 = 1/32
STR Allele Frequencies
45
Frequency (%)
40
35
Locus: TH01
30
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
25
20
15
10
5
*Proc. Int. Sym. Hum. ID
0
(Promega) 1997, p. 34.
6
7
8
9
9.3
Number of repeats
10
The Meaning of a DNA Match?
1. Person A is the source of the DNA profile from the evidence.
2. The identical twin of person A is the source of the DNA
profile.
or
3. Another person who coincidentally has the same profile
as person A is the source of the DNA profile from the
evidence.
= the random match probability
Random Match Probability
Is not:
Defense Fallacy.
A) Therefore, everyone else with the same genotype has
an equal chance of being guilty.
B) Therefore, every possible genotype in a mixture has an
equal chance of having committed the crime.
Random Match Probability
Is not:
Prosecutor’s Fallacy.
A) There is only a 1 in 100 million chance that the DNA profile
came from someone else.
B) There is only a 1 in 100 million chance that the defendant is
not guilty.
RMP is not:
1. The probability that someone else is guilty.
2. The probability that someone else left the DNA.
3. The probability that the defendant is not guilty.
United States Court of Appeals, 9th Circuit
(Troy Don Brown v. Farwell and State of Nevada)
Statements of the DNA Analyst:
1. 1 in 3 million randomly selected people would match the
DNA found in victim’s underwear.
2. 99.99967 percent chance that the DNA found in the
underwear was from Brown.
Source probability ≠ Random match probability.
Combined Probability of
Inclusion Calculations
in DNA Mixtures
Definition
Combined Probability of Inclusion calculation estimates
the portion of the population that would be expected to
be included as possible contributors to an observed
DNA mixture (i.e. all alleles from individual observed in
the DNA mixture profile).
Calculation of CPI estimates requires no assumptions
about the number or identity of contributors individuals.
CPI estimates are generally thought to be conservative.
Calculation to Convey to the Court
• A Y STR haplotype (evidence sample) is searched in a
reference database(s) of unrelated individuals
• The number of times the haplotype is observed in a
database and divided by total samples in the database
• The size of a database can be and is often limited
• With databases (e.g., n = 100 to 3000), many possible
haplotypes will not be observed and there will be
sampling error
Likelihood Ration
• 60,000 times more likely that it’s the suspects
DNA profile mixed with two unknown persons
as compared to three unknown individuals
• Makes assumptions to the number of
contributors.
• Primarily used in Europe
• Very few labs use the LHR in the United States
(3)
• Valid & Reliable…but is it the Best stat to
use???
Math in the Courtroom
• “Mathematics, a veritable Sorcerer in our computerized
society , while assisting the trier of fact in the search for
the truth, must not cast a spell over him.”
-People v. Collins, 438 P. 2d 33, Cal. 1968
___________
• Trial by Mathematics, L. Tribe, 84 Harv. L. Rev.1329:
• “directness and precision may overshadow basic concepts
and fundamental bases of the trial system.”
• “significance, appropriateness, and dangers of mathematical
proofs may depend dramatically on whether such proof is
meant to bear on occurrence, identity, or frame of mind.”
__________
• “Without the probability assessment, the jury does not know
what to make of the fact that the patterns match: the jury does
not know whether the patterns are as common as pictures
with two eyes, or as unique as the Mona Lisa.”
- US v. Yee, 134 FRD 161, 181 (ND Ohio, 1991)
“We … hold that DNA evidence is only admissible
when both the evidence of a match and the statistical
significance of the match are admissible. Thus we reject
the State’s overly simplistic argument that statistics go
simply to the weight, not the admissibility of the DNA
matching evidence.”
State of Michigan Court of Appeals, People v. Coy II. Calhoun
Circuit Court, LC No. 98-001925 FC. November 17, 2000.
LCN PCR Testing
What is LCN?
1. < 100 pg amplifications
2. < 200 pg amplifications
3. Additional PCR Cycles (32-34 vs. 28)
4. Multiple amps of same sample
– composite profiling
5. Post PCR cleanup
– remove ions for CE injection
6. Nested PCR (2 rounds of PCR)
Budowle, et al. Validity of Low Copy Number Typing and Applications to Forensic Science, Croat. Med J. 2009; 50: 210-17 fig. 3
LCN - Uses
Budowle, et al. Validity of Low Copy Number Typing and Applications to Forensic Science, Croat. Med J. 2009; 50: 210-17 fig. 2
Common Challenges to LCN
1. Not Generally Reproducible
2. Confidence: Contaminant v. True profile
3. No Universal Interpretational Guidelines
- Drop-in, Drop-out, Increased Stutter, Stochastic Effects,
Heterozygote Peak Height Imbalance, Controls, etc.
4. Not Generally Accepted w/in Forensic Comm.
5. NDIS – Does not accept profiles from “LCN” samples
General Defense Challenges
1. Interpretation
- Included v. Exclusion v. CBE v. Inconclusive
2. Statistics
- 95% CI, CPI, Major/Minor SS stat
3. Collection
4. Procedure / Methodology
- Admissibility (Daubert, Frye, etc.)
- Reliability, Validity, Reproducibility, General Acceptance within the
Relevant Scientific Community, etc.
5. Primary v. Secondary Transfer
6. Possible logical scenarios for DNA being on a piece of evidence
What You Will Need
Forensic Links
www.dnaresource.com (Smith Alling Lane)
www.denverda.org
www.cstl.nist.gov/div831/strbase
www.ndaa.org/apri/index.html
www.fbi.gov/hq/lab/fsc/current/index.htm
(Forensic Science Communications)
www.fbi.gov/hq/lab/html/codis1.htm (CODIS Database)
www.ornl.gov/sci/techresources/
Human_Genome/elsi/forensics.shtml
www.dnapolicy.net
www.scientific.org
www.corpus-delicti.com/forensic_fraud.html
www.nlada.org/Defender/forensics/for_lib/Index/DNA#DNA
Any Questions…
Eric J. Carita
Forensic DNA Consultant
83 Kapitulik Rd
North Grosvenordale, CT 06255
1-860-377-4067
ejcarita@yahoo.com