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Eric J. CaritaForensic DNA Consultant
Understanding Forensic DNA Testing:
There’s Nothing To Fear
DNA 101Basic Genetics
What is DNA?
DNA is…
Deoxyribonucleic Acid
The inherited genetic material that makes us
what we are
One set of22 autosomes
(plus X)
One set of22 autosomes(plus X or Y)
Two alleles* for eachautosomal genetic marker
Paternity Testing
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
A = TG C
T = A
A = T
C G
T
CCA
GG
TA
G C
T = A
T = A
C G
A = T
A = TG C
5’
3’
5’
3’
5’ 3’
3’ 5’denatured
strands
hybridizedstrands
Hydrogen bonds
C G C
G
G C
Phosphate-sugar backbone
Butler, J.M. (2001) Forensic DNA Typing, Figure 2.2, ©Academic Press
Mendel’s Ist Law—Equal SegregationDuring 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 thesecond allele.
We inherit half of our nuclear genes (one allele from eachlocus) from each parent.
Mendel’s 2nd Law—Independent Assortment
During gamete formation, the segregation of one gene pair isindependent 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 exploitedfor 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
1. Of the 3,000,000,000 DNA bases, about 0.3% is not conserved:
~1 million bases
2. Forensic STR analysis looks at thelength of 13-15 areas of DNA.
DNA - Unique, Yet the Same
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
like CSI!JustJustNothing
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 capsgeneral 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. DNA left on evidence by the “handler”.2. Usually no visible stain.3. Usually very small amounts of DNA are deposited.4. Significant increase in number of touch DNA cases
in last 2 years.
Associated with many crimes including:Property CrimesThreatening lettersRobberyHomicideAssault/Sexual AssaultVandalism
From Hands:GlovesKnife handlesWeapon handlesFirearm gripsPlastic bag handlesSteering WheelsRopeShoe lacesElectrical cords
Common Sources of Touch Common Sources of Touch DNADNA
Common Sources of Touch Common Sources of Touch DNADNA continued…continued…
Wearer:
Baseball capsSweatbandsShirt/jacket collarsSocksWaistbands of pantsEyeglasses
* Mixtures v. Single Source
TOUCH DNA CASESTOUCH DNA CASES
Commonly swabbed areas
* Counter* NY hat* Gun
TOUCH DNA EVIDENCE : WHAT TOUCH DNA EVIDENCE : WHAT DOES IT MEAN…….?DOES IT MEAN…….?
Shows linkage or association but….Shows linkage or association but….• DNA recovered from an object may not be from the DNA recovered from an object may not be from the
last person to touch it. Factors include:last person to touch it. Factors include:-Length of contact-Length of contact-Good cell shedder or not-Good cell shedder or not-Vigorous contact vs. incidental-Vigorous contact vs. incidental
• DNA profiles recovered from touch evidence are DNA profiles recovered from touch evidence are often mixtures – multiple individualsoften mixtures – multiple individuals
-Elimination known(s)-Elimination known(s)-Lawful owner-Lawful owner-Crime scene personnel, officers-Crime scene personnel, officers
COLLECTION OF TOUCH DNA FROM COLLECTION OF TOUCH DNA FROM EVIDENCEEVIDENCE
When:When: - - At the Crime Scene by Law At the Crime Scene by Law EnforcementEnforcement
(e.g. door knobs, counters, windows) (e.g. door knobs, counters, windows)
- - Forensic Laboratory by analystForensic Laboratory by analyst (if other testing is needed)(if other testing is needed)
Where:Where: - Areas of contact - Areas of contact (e.g. grips, slide, trigger, magazine, cartridge cases; fired vs. unfired)(e.g. grips, slide, trigger, magazine, cartridge cases; fired vs. unfired)
- Any touched object…- Any touched object…(but be cautious regarding objects accessible to the general public)(but be cautious regarding objects accessible to the general public)
DNA Profiles from Weapons and the DNA Profiles from Weapons and the DNA DatabaseDNA 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.
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
TOUCH DNA EVIDENCE : TOUCH DNA EVIDENCE : COLLECTION SUGGESTIONS…….COLLECTION SUGGESTIONS…….
Collection of Touch DNA Evidence
1. Contamination is a significant possibility.
2. Impact of contamination is false exclusion ofsuspect 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 answersstandard defense question.
Collect evidence to avoid/minimize mixtures especially withcertain samples.
DNA in the Cell
Target Region for PCRTarget Region for PCR
chromosome
cell nucleus
Double stranded DNA molecule
Polymorphic
Region
Make copies (extend primers)
Starting DNA Template
5’
5’
3’
3’
5’
5’
3’
3’
Add primers (anneal) 5’3’
3’5’
Forward primer
Reverse primer
DNA Amplification with the
Polymerase Chain Reaction (PCR)
Separate strands (denature)
5’
5’3’
3’
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created
PCR Copies DNA Exponentially through Multiple Thermal Cycles
Original DNA target region
Thermal cycleThermal cycleThermal cycle
Short Tandem Repeats (STRs)
Repeat number varies between alleles. PCR primers bind to flanking regions that are constant.
7 repeats
8 repeats
AATG
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
LIZ-internal lane standard
LIZ
AmpFlSTR® Identifiler™Kit available from PE Biosystems (Foster City, CA)
15 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction.
100 bp 400 bp300 bp200 bp Size Separation
Col
or
Sep
arat
ion
FAM-6 D8 D21 D7 CSF
D3 THO1 D13 D16 D2 VIC
D19 vWA TPOX D18 NED
PET A D5 FGA FGA
-Fluorescent dye tags on the primers
Fluorescent STR AnalysisFluorescent STR Analysis
-Visualize emitted light with a digital camera.-Collect and analyze data with computer.
Capillary Electrophoresis
A. Capillary ArrayB. Polymer Delivery PumpC. Pump blockD. Buffer reservoirsE. Detection Block (camera)
A.
B.
C.
D. D.
E.
Forensic DNA Analysis
Forensics: Pattern Comparisons
Forensic DNA Analysis
Evidentiary DNA profile(s) are generated from samplessubmitted to Forensic Lab.
Known profile(s) of suspect/victim (blood or buccal) arecompared to DNA profiles from instant case.
Evidentiary profiles entered into CODIS database. Suspect’sprofile 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 Profile Detection
Profile A Detected Profile B Detected
Factors:1. Quantity of DNA2. Quality of DNA
DNA Profile Detection
Profile A Detected Profile B Detected
Factors:1. Quantity of DNA2. Quality of DNA
Mixture Detection?
Only Profile B Detected Profiles A and B Detected
25:1 1:1
Factors:1. Quantity2. Quality3. Ratio
Stochastic Fluctuation
•Stochastic = chance.•Result of PCR founder effect and chance allelesampling.
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 DNAprofile.
or3. 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 contributor2. Excluded – source or contributor3. CBE – source or contributor4. Inconclusive5. 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”
F
FF
F
F
SCENARIO #1 SCENARIO #2
Y-STRs
Y
Y Profile Detected
30:1
X
XX X
XX
X
X
XX
X
XX
X
X
X
X
X
X
X
XX
X
X X
X
XX
XX
X:Y =
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
Autosomal STR Profile
Y-Chromosome STR Profile
Female Victim DNA Profile
Male Perpetrator DNA Profile
DNA Profile from Crime Scene
No signal observed
Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Figure 9.2, ©Elsevier Science/Academic Press
Y-STRs can permit simplification of male DNA identification in sexual assault cases
Forensic DNA Statistics
1-1/N p2 + 2pq + q2 = 1
P = .5 x .5 x .5 x .5 x .5 = 1/32
AA + 2AB +2AC+ BB+ 2BC + CC = 1
N
ppp
)1)((96.1
STR Allele Frequencies
0
5
10
15
20
25
30
35
40
45
6 7 8 9 9.3 10
Caucasians (N=427)
Blacks (N=414)
Hispanics (N=414)
Locus: TH01
*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34.
Number of repeats
Fre
qu
ency
(%
)
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 DNAprofile.
or3. 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
Is not:
Defense Fallacy.
A) Therefore, everyone else with the same genotype hasan equal chance of being guilty.
B) Therefore, every possible genotype in a mixture has anequal chance of having committed the crime.
Random Match Probability
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 isnot 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 theunderwear 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 computerizedsociety , while assisting the trier of fact in the search forthe 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 conceptsand fundamental bases of the trial system.”
• “significance, appropriateness, and dangers of mathematicalproofs 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 knowwhat to make of the fact that the patterns match: the jury doesnot 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
1. < 100 pg amplifications2. < 200 pg amplifications3. Additional PCR Cycles (32-34 vs. 28)4. Multiple amps of same sample
– composite profiling5. Post PCR cleanup
– remove ions for CE injection6. Nested PCR (2 rounds of PCR)
What is LCN?
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
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
Common Challenges to LCN
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
General Defense Challenges
What You Will Need
Forensic Links
www.dnaresource.com (Smith Alling Lane)www.denverda.orgwww.cstl.nist.gov/div831/strbasewww.ndaa.org/apri/index.htmlwww.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.shtmlwww.dnapolicy.netwww.scientific.orgwww.corpus-delicti.com/forensic_fraud.htmlwww.nlada.org/Defender/forensics/for_lib/Index/DNA#DNA
Eric J. CaritaForensic DNA Consultant
83 Kapitulik RdNorth Grosvenordale, CT 06255
Any Questions…
