Forensic DNA Analyses

7/28/2019 Forensic DNA Analyses

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Forensic DNA: Use, Abuse,

Promise, and Peril


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DNA Identification

Where does DNA come from? 1/2 from mom 1/2 from dad

What is it? Blue print of life

How is DNA different among us?

Common vs Different

What does DNA mean?

Deoxyribonucleic Acid


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Cell Types

Where can DNA be found?

CellBlood

Sweat

Hair RootsSaliva

Various TissueSemen

SAME


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Nucleus

Where is DNA in the body?

Cell


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Nuclear DNA

Where are the types of DNA

found in a cell?

Mitochondrial DNACell


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Maternal

Chromosome

Paternal

Chromosome

Nucleus

Where is DNA in the body?


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Where is DNA packaged in the

body?

Chromosome

DNA


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A =Adenine

T =Thymine

G =Guanine

C =Cytosine

UnitsDoubleHelix

GATC

DNA- What does it look like?


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Sources of Biological Evidence

Blood

Semen

Saliva

Urine

Hair

Teeth

Bone

Tissue


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Types of objects where DNA may

be found

Blood Stains

Semen Stains

Chewing Gum

Stamps & Envelopes

Penile Swabs

Plant Material

Sweaty Clothing

Bone

Hair

Fingernail Scraping

Saliva

Animal Material


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Where DNA Evidence is Found


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Blood

Hair Roots

Saliva

SweatTissue

Chemical

DNA

Isolation of DNA


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Semen stain

Chemical

RemoveEpithelial

DNA

Differential Isolation of DNA

Different

Chemical

SpermDNA

Semen

stain

Epithelial

DNA

Sperm DNA


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DNA

Solution

Amplification

(making copies)


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G

TA

G

A

A T

C

A

T

C

T

Heat

Step one of a single cycle

DENATURE


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T

Step two of a single cycle

ANNEAL


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Step three of a single cycle

T

EXTEND


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1 Cycle2 Cycles

3 Cycles

4 Cycles

5 Cycles

28 Cycles

Amplification

DNA

PCR (Polymerase Chain Reaction)


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Analysis of amplified DNA

Amplified

DNA

DNA

Profile


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Brief History of Forensic DNA Typing

1980 - Ray White describes firstpolymorphic RFLP marker

1985 - Alec Jeffreys discovers multilocusVNTR probes

1985 - first paper on PCR 1988 - FBI starts DNA casework

1991 - first STR paper

1995 - FSS starts UK DNA database

1996 First mtDNA case

1998 - FBI launches CODIS database


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DNA Use in Forensic Cases

Most are rape cases or murders

Looking for match between

evidence and suspect

Must compare victims DNA profile

Mixtures must be resolved

DNA is often degraded

Inhibitors to PCR are often present

Challenges


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Human Identity Testing

Forensic cases -- matching suspect withevidence

Paternity testing -- identifying father

Historical investigations-Czar Nicholas,Jesse James

Missing persons investigations

Mass disasters -- putting pieces back together

Military DNA dog tag

Convicted felon DNA databases


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Sample Obtained fromCrime Scene or Paternity

Investigation Biology

DNAExtraction

DNAQuantitation

PCR Amplificationof Multiple STR markers

TechnologySeparation and Detection of

PCR Products(STR Alleles)

Sample GenotypeDetermination

GeneticsComparison of Sample

Genotype to OtherSample Results

If match occurs, comparisonof DNA profile to population

databases

Generation of CaseReport with Probability

of Random Match

Steps in DNA Sample Processing


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Progression of DNA Typing

Markers RFLP

multilocus VNTR probes

single locus VNTR probes (P32 andchemiluminescence)

PCR DQ-alpha (reverse dot blot)

PolyMarker(6 plex PCR; dots for SNPs)

D1S80 (AMP-FLPs)

singleplex STRs with silver staining multiplex STRs with fluorescent dyes

Mitochondrial DNA sequencing

Multiplex Y-STR with fluorescent dyes


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Blood

Hair Roots

Saliva

SweatTissue

Chemical

DNA

Extraction of DNA


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RFLP Analysis

Enzymes break DNA intorestriction fragments

Measurements taken offragments that vary inlength across people

(length polymorphism)because they containVNTRs

can produce extremely lowrandom match probabilities

requires relatively largefresh samples (>50 ngDNA)

slow and expensive


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Which Suspect, A

or B, cannot be

excluded from theclass of potential

perpetrators of this

assault?

PM DQA1 T t


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PM+DQA1 Test

PCR-based

Extremelysensitive(1ng DNA)

degraded samples

faster and cheaper

than RFLP Statistics less

impressive,particularly withmixed samples

Possible Problems:

interpretation issubjective and can bedifficult

mixtures difficult tointerpret

statisticalcharacterization of mixedsamples is tricky


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DNA in the Cell

Target Region for PCR

chromosome

cell nucleus

Double stranded

DNA molecule

Individual

nucleotides


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Short Tandem Repeats (STRs)

1. CTTA with silver-

stained gel

PCR-based

3 loci for identification

plus sex-typing

Easier interpretation

of mixtures


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2. Gel-based

systems with

Fluorescent

Detection


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3. CapillaryElectrophoresis

AmpFlstr Profiler Plus Groups of amplified STR

products are labeled withdifferent colored dyes(blue, green, yellow)

Electrophoresis anddetection occur in

computer-controlledcapillary device (ABIPrism 310 GeneticAnalyzer)


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Short Tandem Repeats

(STRs)

the repeat region is variable between samples while the

flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another


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Short Tandem Repeat

AGAT AGATAGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

AGAT

6

4

DNA Profile =4,6

TCTA TCTATCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

TCTA

7

5

DNA Profile =5,7

TCTA

TCTA

STR


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Multiplex PCR

Over 10 Markers Can Be

Copied at Once

Sensitivities to levels less

than 1 ng of DNA

Ability to Handle Mixtures

and Degraded Samples

Different Fluorescent Dyes

Used to Distinguish STR

Alleles with OverlappingSize Ranges


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An Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 J OE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTRProfiler PlusKit available from PE Biosystems (Foster City, CA)

9 STRs amplified along with sex-typing marker amelogenin in a single PCR reaction

100 bp 400 bp300 bp200 bp

Size Separation

ColorSeparation

Overview of Steps Involved in DNA


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DNA Quantitation

using Slot Blot

AMEL

D3

TH01

TPOX

Penta D

Penta EFGAD21 D18

CSF

D16D7

D13D5VWA D8

PCR Amplification with Fluorescent STR Kits

and Separation with Capillary Electrophoresis

Blood Stain

Overview of Steps Involved in DNATyping

Genotyping by Comparison to Allelic Ladder

C l l ti f DNA Q titi


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Calculation of DNA Quantities

in Genomic DNAImportant values for calculations:

1 bp = 618 g/mol A: 313 g/mol; T: 304 g/mol; A-T base pairs = 617 g/mol

G: 329 g/mol; C: 289 g/mol; G-C base pairs = 618 g/mol

1 genome copy = ~3 x 109 bp = 23 chromosomes (one member of each pair)

1 mole = 6.02 x 1023 molecules

Standard DNA typing protocols with PCR amplification of STR markers typically askfor 1 ng of DNA template. How many actual copies of each STR locus exist in 1 ng?

1 genome copy = (~3 x 109 bp) x (618 g/mol/bp) = 1.85 x 1012 g/mol

= (1.85 x 1012 g/mol) x (1 mole/6.02 x 1023 molecules)

= 3.08 x 10-12 g = 3.08 picograms (pg)

Since a diploid human cell contains two copies of each chromosome, then

each diploid human cell contains ~6 pg genomic DNA

1 ng genomic DNA (1000 pg) = ~333 copies of each locus (2 per 167 diploid genomes)

Sh t T d R t


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Short Tandem Repeats

(STRs)

the repeat region is variable between samples while the

flanking regions where PCR primers bind are constant

AATG

7 repeats

8 repeats

AATG AATG

Primer positions define PCR product size

Fluorescentdye label


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ABI Prism 310 Genetic Analyzer

capillary

Syringe withpolymer solution

Autosampler

trayOutlet

buffer

Injection

electrode

Inlet

buffer

Ch i t I l d


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Chemistry Involved Injection

electrokinetic injection process importance of sample preparation

(formamide)

Separation

capillary

POP-4 polymer

buffer

Detection fluorescent dyes with excitation and emission

traits

virtual filters (hardware/software issues)

Electrokinetic Injection


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Sample

TubeDNA-

-

Electrokinetic Injection

Process

Electrode

Capillary

-

Q is the amount of sample injected

ris the radius of the capillary

cs is the sample concentration

E is the electric field applied

t is the injection time

s is the sample conductivityb is the buffer conductivityep is the mobility of the sample moleculeseo is the electroosmotic mobility

Rose et al(1988)Anal. Chem. 60: 642-648

Q = sr2cs(ep + eo)Etb


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Separation Issues

Run temperature -- 60 oC helps reduce

secondary structure on DNA and

improves precision

Electrophoresis buffer-- urea in running

buffer helps keep DNA strands

denatured

Capillary wall coating -- dynamic coatingwith polymer

Polymer solution -- POP-4


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DNA Separation Mechanism

+-DNA-

DNA-

DNA-DNA

- DNA-

Size based separation due to interaction of DNA moleculeswith entangled polymer strands

Polymers are not cross-linked (as in slab gels)

Gel is not attached to the capillary wall

Pumpable -- can be replaced after each run

Polymer length and concentration determine the separationcharacteristics

Fluorescent

Emission Spectra for ABI


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ABI 310 Filter Set F

520 540 560 580 600 620 640

WAVELENGTH (nm)

100

80

60

40

20

0

5-FAM JOE NED ROX

Laser excitation

(488, 514.5 nm)

Fluorescent Emission Spectra for ABI

Dyes

L b l d DNA f t

P i i l f S l


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Sample Detection

CCD Panel

ColorSeparation

Ar+

LASER

(488 nm)

Fluorescence ABI Prism

spectrograph

Capillary or

Gel Lane

SizeSeparation

Labeled DNA fragments

(PCR products)

Detection

region

Principles of Sample

Separation and Detection


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15,16 16,17 20,23 12,14 30,30X,Y 13.2,15Evidence

Area 1 Area 2 Area 3 Area 4 Area 5

AREAS OF DNA

SAMPLE Sex Area 6

Ref.Std.2

Ref.Std.1

15,16 16,17 20,23 12,14 30,30X,Y 13.2,15

14,15 17,18 23,24 13,13 30,30X,X 15,1914,15 17,18 23,24 13,13 30,30X,X 15,19


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amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWAD21

FGA

D16

D18 D2

Two

differentindividuals

DNA Size (base pairs)

Results obtained in less than 5

hours with a spot of blood the

size of a pinhead

probability of a random

match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR SGM Plus kit


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PERKIN-ELMERS

PROFILER+ AND COFILER

STATE OF TENNESSEEVERSUS

TAYLOR LEE SMITH


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JUST THE FACTS:


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JUST THE FACTS:

NOT A MIXTURE?1. Sperm Fraction: Eight of thirteen loci have a total

of nine alleles not found in either the victim or the

suspect.

2. Suspect Known: Eight of thirteen loci have a

total of 12 different alleles not found in the sperm

fraction mixture.

3. Victim Known: Ten of thirteen loci have a total of

11 different alleles not found in the sperm fraction

mixture.

COINCIDENCE OR EVIDENCE?


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COINCIDENCE OR EVIDENCE?

The likelihood ratios for producing homozygous genotypes at

four of thirteen STR loci* with DNA from a single individualversus a mixture of DNA from two individuals.

Theta = 0.03 Theta = 0.05

African American 1 in 278,000,000 1 in 43,000,000Likelihood Ratio 16,600 6,500

Caucasian 1 in 183,000,000 1 in 27,500,000

Likelihood Ratio 13,500 5,200

Hispanic 1 in 15,000,000 1 in 3,700,000

Likelihood Ratio 3,900 1,990

*Observed Sperm fraction genotypes: vWA=16, TPOX=8, D5S818=12, and D16S539=10).

Why the Y Chromosome?


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Why the Y Chromosome? Applications

forensic investigations (98% of violent crime by

men)

genealogical purposes

evolutionary studies

Advantages to Human Identity Testing male component isolated without differential

extraction

paternal lineages

Needs population studies to evaluate diversity of

haplotypes

robust assay for accurate characterization of Y

markers


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Y STR Multiplex Assay

100 bp 400 bp300 bp200 bp

DYS19 389II389I

390Primer Amounts Dye

Y19 0.25 M JOEY389 0.125 M FAM

Y390 0.25 M JOE

Prinz et al. 1997(Forensic Sci Int, vol. 85, pp. 209-218)

Quadruplex I

Mitochondrial DNA


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Mitochondrial DNA

What is mtDNATyping?

Database andstatistical issues

A Mitochondrial Exclusion


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A Mitochondrial Exclusion

A Mitochondrial Inclusion


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A Mitochondrial Inclusion


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Mitochondrial Inconclusive?


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The Future of Forensic

DNA

CODIS

SNPs & Chips

FBIs CODIS DNA Database


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FBIs CODIS DNA Database

Combined DNA Index System

Used for linking serial crimes and

unsolved cases with repeat offenders

Launched October 1998 Links all 50 states

Requires >4 RFLP markers

and/or 13 core STR markers Current backlog of >600,000 samples

13 CODIS Core STR Loci


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with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

Cold Hits and Solved Cases


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On August 25, 1979, an 8-year old girl was brutally raped and murdered in

San Pablo, CA. Semen was collected from the body and placed in an

evidence room, where it sat for 22 years. Through this program, a DNA profile

was made and submitted to the state and federal databases. This resulted in

a cold hit identifying Joseph Cordova Jr. as the suspect. Cordova was a

habitual child molester who at the time of the DNA analysis was incarcerated

in a Colorado prison. Cordova was subsequently charged with molesting,

raping and murdering the 8-year old girl.

On November 8, 2000, a 12 year old girl, was kidnapped off of the street in

Rancho Cordova, CA, and driven to Feather River in Sutter County where she

was sexually assaulted and then killed. Nine months later, Justin Weinberger

was stopped for a traffic violation in New Mexico. A check by police revealed

that Weinberger was wanted on a federal warrant for child pornography. He

was detained and voluntarily provided a DNA sample. Analysis of that DNA

sample resulted in a match with evidence identifying Weinberger as the

suspect in this case. Weinberger was subsequently extradited to California

where he was tried and convicted of the murder of the 12-year old girl.

Cold Hits and Solved Cases


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STR Analysis by Hybridization


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STR Analysis by Hybridization

on Microchips

Forensic DNA Analyses

Documents

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