eric j. carita forensic dna consultant understanding forensic dna testing: there’s nothing to fear

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

1-860-377-4067ejcarita@yahoo.com

Any Questions…