Forensic DNA Typingor

Did you kill (rape…) that person?

How DNA can “definitively” say.

Adapted from:

National Institutes of Science & Technology

http://www.cstl.nist.gov/div831/strbase/intro.htm

Brief History of Forensic DNA Typing

• 1980 - Ray White describes first polymorphic RFLP marker (Restriction Fragment Length Polymorphism [alleles]).

Different RFLP for different people• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of

tandem repeats) probes.• 1985 - first paper on PCR• 1988 - FBI starts DNA casework• 1991 - first STR paper (renaming of VNTR– could be larger repeats, STR 4-6 bp’s.

now using mostly 4 bases )

• 1995 - FSS (Forensic Science Service-UK) starts UK DNA database• 1998 - FBI launches CODIS (Combined DNA Information Service)

Now FBI use 13 loci: PCR identifies it: in the quadrillions – except for identical. Except for police mistakes, it’s done deal.

RFLP’s: Sickle Cell hemoglobin

Case 1: Screening for the sickle-cell gene Sickle cell disease is a genetic disorder in which both genes in the patient encode the amino acid valine (Val) in the sixth position of the beta chain (betaS) of the hemoglobin molecule. "Normal" beta chains (betaA) have glutamic acid at this position. The only difference between the two genes is the substitution of a T for an A in the middle position of codon 6. This •converts a GAG codon (for Glu) to a GTG codon for Val and •abolishes a sequence (CTGAGG, which spans codons 5, 6, and 7) recognized

and cut by one of the restriction enzymes.         

Brief History of Forensic DNA Typing

• 1980 - Ray White describes first polymorphic RFLP (Restriction Fragment Length

Polymorphism) marker—detect to transferring to membrane. Probe w southern blot (radiological). Diff. RFLP for dif. People. Single rflp

• 1985 - Alec Jeffreys discovers multilocus VNTR (variable number of tandem repeats) probes (stat. very impressive identical 4-6 bp that are spec. 7 and 9 repeat, one from mom and dad, on chrom. 1- nowadays use pcr- but flanking sequence that is unique to chromo1)). Jeffreys almost ident. Typing. Now use PCR.

• 1985 - first paper on PCR (Kerry Mullis)• 1988 - FBI starts DNA casework • 1991 - first STR paper ( renaming of VNTR– could be larger

repeats, STR 4-6 bp’s. now using mostly 4 bases )• 1995 - FSS (Forensic Science Service-UK) starts UK DNA

database• 1998 - FBI launches CODIS (Combined DNA Information

Service) database. Now FBI use 13 loci: PCR identifies it: in the quadrilians – except for identical.

DNA Use in Forensic Cases

• Most are rape cases (>2 out of 3)

• Looking for match between evidence and suspect

• Must compare victim’s DNA profile

•Mixtures must be resolved

•DNA is often degraded (stored wet- have mold, nuclease)

•Inhibitors to PCR are often present

Challenges

Human Identity Testing

• Forensic cases -- matching suspect with evidence

• Paternity testing -- identifying father

• Historical investigations• Missing persons investigations• Mass disasters -- putting pieces back together

• Military DNA “dog tag”• Convicted felon DNA databases

Sample Obtained from Crime Scene or

Paternity Investigation Biology

DNAExtraction

DNAExtraction

DNAQuantizatio

n

DNAQuantizatio

n

PCR Amplificationof Multiple STR

markers

PCR Amplificationof Multiple STR

markers

TechnologySeparation and Detection of

PCR Products(STR Alleles)

Sample Genotype

Determination

GeneticsComparison of Sample

Genotype to Other Sample Results

Comparison of Sample Genotype to Other

Sample Results

If match occurs, comparison of DNA profile to population databases

If match occurs, comparison of DNA profile to population databases

Generation of Case Report with Probability

of Random Match

Generation of Case Report with Probability

of Random Match

Steps in DNA Sample Processing

Sources of Biological Evidence

• Blood• Semen• Saliva• Urine• Hair• Teeth (useful in fires).

• Bone (there are cells. Decalcify it.

100,000 year old- has DNA. Has Dinosaur!)

• TissueAll felony arrests- cheek swab.

DNA in the Cell

Target Region for PCRTarget Region for PCR

chromosome

cell nucleus

Double stranded DNA molecule

Individual nucleotides

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 (Polymerase Chain Reaction) Copies DNA Exponentially through Multiple Thermal Cycles

Original DNA target region

Heat

Cool

Heat

DNA Poly.dUTP

Oligo’s

1 copy2 copies

Cool 4 copiesHeat …

Short Tandem Repeats (STRs) (say chromo 3)

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

Identical in all people

Identical in all people

170 bp170 bp195 bp195 bp

Different primer sets produce different PCR product sizes for the same STR allele

TCAT repeat unitTCAT repeat unit

Diff. PCR primers sets, can amplify the same region. Different companies sell different kits.

Choosing which STRs:Significant statistical variation – but not too many. Freq. that are measured in pop. : Loc 1 -10%. Loc 2 – 10%; locus 1+2 -1/100. Random

match with 13 primers 1/1013.

Variation Among STRs

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 Overlapping Size Ranges

Most rxns: require 2 PCR (tubes) 7 or 8 primer pairs in one tube– need total of about 2 tubes for 13 different STRs.

$20-$25 per rxn in lab. $150 incl labor. Cost for forensic up to $1000.

An Example Forensic STR Multiplex Kit

D3 FGAvWA 5-FAM (blue)

D13D5 D7 NED (yellow)

A D8 D21 D18 JOE (green)

GS500-internal lane standard

ROX (red)

AmpFlSTR® Profiler Plus™Kit 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 bpSize Separation

Col

or S

epar

atio

n

Available Kits for STR Analysis

• Kits make it easy for labs to just add DNA samples to a pre-made mix

• 13 CODIS core loci– Profiler Plus and COfiler (PE Applied Biosystems)– PowerPlex 1.1 and 2.1 (Promega Corporation)

• Increased power of discrimination– CTT (1994): 1 in 410– SGM Plus™ (1999): 1 in 3 trillion– PowerPlex ™ 16 (2000): 1 in 2 x 1017

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

5 min from inj. to output.

Close-up of ABI Prism 310 Sample Loading Area

Autosampler Tray

Sample Vials

Electrode

Capillary

See Technology section for more information on CE

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Tw

o di

ffer

ent i

ndiv

idua

ls

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

D tells chromosome 21—happens to be down’s syndrome. 2 peaks cause heterozygotic

Amelogenin amel protein that happens to be on sex chromosome, tooth enamel– top: 2 peaks: x and y. (universal that two diff. people.) Bottom only 1 peak cause they have two X chromosomes.

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)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

FBI’s CODIS DNA Database

Combined DNA Index System --all 50 states can upload their convicted felony and seq. of unsolved cases…. In Florida to convicted felon.

• 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 with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

The End