Forensic DNA Analysis PCR Polymerase Chain Reaction Forensic DNA Analysis

Forensic DNA Analysis

PCR

Polymerase Chain Reaction

Forensic DNA Analysis

Human Identity Testing• Forensic cases -- matching suspect with

evidence

• Paternity testing -- identifying father

• Mass disasters -- putting pieces back together

• Historical investigations• Missing persons investigations• Military DNA “dog tag”• Convicted felon DNA databases

Involves generation of DNA profiles usually with the same core STR (short tandem repeat) markers

Involves generation of DNA profiles usually with the same core STR (short tandem repeat) markers

Basis of DNA Profiling

The genome of each individual is unique (with the exception of identical twins) and is inherited from parents

Probe subsets of genetic variation in order to differentiate between individuals (statistical probabilities of a random match are used)

DNA typing must be performed efficiently and reproducibly (information must hold up in court)

Current standard DNA tests DO NOT look at genes – little/no information about race, predisposal to disease, or phenotypical information (eye color, height, hair color) is obtained

O

Base(A, T, C, or G)

HO

1’

3’ 2’

4’

5’

CH2OP

H

HH

H

O

O-

HO

O

Base(A, T, C, or G)

HOH

1’

3’ 2’

4’

5’

CH2OP

H

HH

H

O-

HO

5’end|

Phosphate

| Sugar—Base…

|Phosphate

| Sugar—Base…

| 3’end

A) B)

5’

3’

Figure 2.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

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

Figure 2.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

2 repeats

3 repeats

--------AGACTAGACATT-------

--------AGATTAGGCATT-------

---------(AATG)(AATG)(AATG)----------

---------(AATG)(AATG)----------

(B) Length polymorphism

(A) Sequence polymorphism

Figure 2.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

63

4 5

Homologous pair of chromosomes

Homologous pair of chromosomes

Locus A

Locus B

Allele 1 Allele 2

Allele 2Allele 1

Figure 2.6, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

ORGANIC FTA PaperCHELEX

Blood stain

PUNCH

WASH Multiple Times with extraction buffer

PERFORM PCR

PCR Reagents

SDS, DTT, EDTA and

proteinase K

INCUBATE (56 oC)

Phenol,chloroform,

isoamyl alcohol

QUANTITATE DNA

Apply blood to paper and allow

stain to dryBlood stain

VORTEX

(NO DNA QUANTITATION TYPICALLY PERFORMED WITH

UNIFORM SAMPLES)

Water

INCUBATE (ambient)

5% Chelex

INCUBATE (100 oC)

REMOVE supernatant

INCUBATE (56 oC)

QUANTITATE DNA

PERFORM PCR

PERFORM PCR

Centrifuge

Centrifuge

Centrifuge

Centrifuge

REMOVE supernatantTRANSFER aqueous (upper) phase to new tube

CONCENTRATE sample (Centricon/Microcon-100 or ethanol

precipitation)

Centrifuge

TE buffer

Figure 3.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Evidence (female fraction)

Evidence (male fraction)

Suspect

Victim

male

female

male

female

The four samples typically associated with a forensic DNA case…

20 ng

10 ng

5 ng

2.5 ng

1.25 ng

0.63 ng20 ng

10 ng

5 ng

2.5 ng

1.25 ng

0.63 ng

Calibration standards

Calibration standards

Unknown Samples

~2.5 ng

Figure 3.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Importance of DNA Quantitation DNA amount

(log scale)

0.5 ng

-A

+A

Too much DNA Off-scale peaks Split peaks (+/-A) Locus-to-locus

imbalance

100 ng

10 ng

1 ng

0.1 ng

0.01 ng

2.0 ng

Too little DNA Heterozygote peak

imbalance Allele drop-out Locus-to-locus imbalanceStochastic effect when amplifying low

levels of DNA produces allele dropout

STR Kits Work Best in This Range

High levels of DNA create interpretation challenges (more artifacts to review)

Well-balanced STR multiplex

PCR is based on DNA Replication and Sanger DNA Sequencing

• Kary Mullis invented PCR in 1983.• Awarded Nobel Prize in Chemistry for this

discovery.• His clever contribution was to use two sets of

primers to make specific short pieces of DNA.• Original PCR was based on Kornberg’s E. coli

DNA polymerase I.• Others added the contribution of Thermal Stable

DNA polymerases. Without that, you had to add fresh polymerase with each thermal cycle.

PCR Reaction Requires:• dATP, dGTP, dCTP, dTTP (dNTPs).• DNA Polymerase enzyme which tolerates high

temperatures. This is a Pol I enzyme not the Pol III enzyme.

• Target DNA- from the genomic DNA or any other source.

• DNA Primers- short synthetic DNA which are complimentary to the target DNA made chemically on a machine.

• Correct pH buffer.• MgCl2 (needed by the enzyme)

PCR Reaction Requires:• If you leave even one item out of the

tube, it will not work.

• Heating to make single strand DNA is critical, since DNA Polymerase doesn’t unwind double stranded DNA.

• Temperature of hydrogen-bond formation step is very critical.

How Does PCR Work?

• In the Polymerase Chain Reaction, a DNA template is repetitively:– Denatured into single stranded molecules

(95 degrees C).

– Primes hybridized to specific oligonucleotide primers (one specific primer per strand).

– DNA polymerase replicates the target DNA from the 3’ end of the primers (extension).

PCR

Heat DNA to separate DNA into single strands so

hydrogen bonds can form between the primers

and the DNA (often called annealing).

This works because you add MUCH more of the

primers than target DNA:

1x10-20 Moles template vs. 1x10-12 Moles

primer:100,000,000 fold excess!

The hydrogen bonding between target and primer is controlled by adjusting the temperature of the solution.

At 55° C, primers of 18 nucleotides in length efficiently form H-bonds to a DNA template.

Adjustments in temperature and time are made to account for the G/C vs. A/T richness of the primer and its length.

Hybridization Temperature Is Critical

• Exam analogy: Too easy and every one gets 100. Too hard and no one passes.

• Temperature too low, and the primers work less specifically- false positives.

• Temperatures too high and the primers don’t work at all- false negatives.

Longer primers can form more stable H-bonds.

Primers with a high G/C % can H-bond at higher temperatures.

Primers with a high A/T % can H-bond with lower temperatures.

PCR uses a Thermalcycler• The Thermalcycler is a machine that changes

temperatures of the tubes.

• When you program the Thermalcycler, you specify a

series of temperatures and times:

Temperature Time– 94 C 30 seconds– 55 C 30 seconds– 72 C 60 seconds

You specify the number of times this series should be repeated for example, 35 times).

94 oC

60 oC

72 oC

Time

Te

mp

era

ture

Single Cycle

Typically 25-35 cycles performed during PCR

94 oC 94 oC 94 oC

60 oC60 oC

72 oC72 oC

The denaturation time in the first cycle is lengthened to ~10 minutes when using AmpliTaq Gold to perform a “hot-start” PCR

Figure 4.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Primer DesignGood PCR primer pairs:• Both have similar hybridization temperatures

because they have similar G/C%.• Do not prime each other into amplifying (Primer-

Dimers).• Don’t have hairpins (self-priming).• Are long enough to be unique so they don’t

prime the wrong DNA by accident.

Primer DimersCaused by designing primers with some self-complementary DNA sequence. Need a free 3’ OH to build onto and a template.

Amplification Is Exponential

• The number of fragments of DNA is going up exponentially- so with 20 replication cycles, you would have 220 pieces of amplified DNA, almost all 25 nucleotide in length.

Why Use a Thermal Stable Polymerase?

• When E. coli polymerase I proteins are heated to almost boiling temperature, they become inactivated.

• The DNA needs to be heated to separate strands of DNA.

• Polymerase I purified from bacteria that live in hot springs are thermostable and still are active after being heated.

(A) Simultaneous amplification of three locations on a DNA template

Locus A Locus CLocus B

(B) Resolution of PCR products with size-based separation method

A

CB

small large

Figure 4.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Sample Obtained from Crime Scene or Paternity

Investigation

DNAExtraction

DNAExtraction

DNAQuantitation

DNAQuantitation

PCR Amplificationof Multiple STR markers

PCR Amplificationof Multiple STR markers

Biology

Separation and Detection of PCR Products(STR Alleles)

Technology

Sample Genotype Determination

Genetics

Comparison 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

Figure 1.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

VNTR DNA

• VNTR stands for "variable number of tandem repeats.“

• STR stands for short tandem repeat.• A tandem repeat is a short sequence of

DNA that is repeated in a head-to-tail fashion at a specific chromosomal location or “locus”.

• Tandem repeats are interspersed throughout the human genome.

VNTR and STR DNA

• Some STR and VNTR sequences are found at only one site -- a single locus -- in the human genome.

• For many tandem repeats, the number of repeated units vary between individuals. Each variant STR or VNTR is called an allele.

• A person can only have TWO alleles: one each from the father and mother.

Six possible VNTR combinations are possible with 3 different VNTR alleles.

Allele Frequencies • The chance that an individual might

have a particular pattern of 2 VNTR alleles is:

2pq• where p and q are the frequencies of

the two alleles in the appropriate comparison population.

Allele Frequencies

Example:• p is 0.1 (10% of the reference

population has this VNTR allele) • q is 0.05 (5% of the reference

population has this VNTR allele)• 2pq frequency is 0.01 (or 1% of the

reference population).

Allele Frequencies

• The frequencies of different VNTR alleles in many different populations of racially and ethnically diverse peoples is being determined.

• Scientists do DNA typing on a statistically significant random group in the population and calculating the allele frequencies.

The frequency of each allele is 33.3%.

PowerPlex® 1.1 PowerPlex® 2.1505 nm scan 585 nm scan

505 nm scan 585 nm scan

FGA

TPOX

D8S1179

VWA

Penta E

D18S51

D21S11

TH01

D3S1358

D16S539

D7S820

D13S317

D5S818

CSF1PO

TPOX

Amelogenin

TH01

VWA

9.3/10

ladders ladderssamples

Fluorescein-labeled TMR-labeled

Figure 14.8, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Which child is not likely to be theoffspring of the father?

Applications of VNTR AnalysisCasualties of Accidents or War• DNA typing can be done of relatives of the

victims to identify their remains (TWA flight 800).

• Armed Forces take blood sample of all enlisted and do DNA analysis if needed.

• Exhumed bones of missing have a mitochondial DNA test if VNTR doesn’t work (more copies of this DNA).

Applications of VNTR/STR Analysis

Forensic Analysis• DNA is purified from blood, bone, semen or

other biological material in a crime scene.• VNTR pattern is determine for as many as 16

different loci from victim and suspects.• Compare patterns of suspects/victims with

patterns at crime scene.

Applications of VNTR/STR Analysis

Forensic Analysis• Databases are assembled of VNTR/STR

allele patterns from criminals.• Databases can be checked to find “John Doe”

suspects based on their DNA• Police can get blood using a warrant for

testing DNA.• DNA data can be used to clear suspects.

DNA test froma vaginal swab ofa rape victim.

Is the defendant’s DNA the same as in the male fraction?

Autosomal (passed on in part, from all ancestors)

Y-Chromosome(passed on complete,

but only by sons)

Mitochondrial (passed on complete, but only by daughters)

Lineage Markers

Figure 9.1, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Mitochondrial DNA

• Not all DNA is in the nucleus

• mtDNA is in the Mitochondria

• Important to Anthropology

• Use to understand possible human ancestry

Mitochondrial Inheritance

• Each cell has thousands of mitochondria

• Mitochondria contain circular DNA, thought to be derived from an ancient symbiosis with bacteria

• Thus the egg contributes both genetic material in the nucleus and genetic material from the mitochondria

Mitochondrial Inheritance

• All mitochondria are inherited from the mother in meiotic cell division.

• The sperm have very little cytoplasm and therefore, virtually no mitochondria.

• The egg is a big cell with lots of mitochondria.

• Mutations in mitochondrial DNA lead to mosaicism for mitochondrial DNA, called heteroplasmy

Advantages of Mitochondrial DNA in Forensics

• Nuclear DNA results up to about 5 years.

• Mitochondrial DNA at least 25 years- smaller and circular. Enzymes that degrade DNA work well on the broken ends.

• There are more copies of mitochondrial DNA than genomic DNA per cell.

1 2

3 54

1211109

6 7 8

181715 16

13 14

MtDNA Haplotype Groups:1

2,3,6,8,11,13,15,164,9,10

57

1214,17,18

MtDNA Haplotype Groups:1

2,3,6,8,11,13,15,164,9,10

57

1214,17,18

A B

B C

C C

D B

B

B

B

B B

E

F

G

G

G

Figure 10.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Mitochondrial DNA was sequenced from 7 females with a missing soldier and identified Michael Blassie as the unknown.

3’-TAAATGATTCC-5’

ATT

ATTTACTAA

ATTTACT ATTTAC

ATTTATTTA

AT

ATTTACTA

ATTTACTAAGATTTACTAAGG

A

DNA template5’ 3’

Primer anneals Extension produces a series of

ddNTP terminated products each one base different in length

Each ddNTP is labeled with a different color fluorescent dye

Sequence is read by noting peak color in electropherogram (possessing single base resolution)

Figure 10.5, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

The Y Chromosome

• Y chromosomes are inherited essentially intact from father to son

• Because of the usual lack of recombination of the Y chromosome, it can be used to track male migration patterns (e.g., Cohens, Jeffersons)

?uncle 3rd cousin

Figure 9.3, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Female-Male Mixture Performance with Autosomal vs. Y-Chromosome DNA Markers

Female Victim DNA Profile

Male Perpetrator DNA Profile

DNA Profile from Crime Scene

Autosomal STR Profile

Y-Chromosome STR Profile

No signal observed

Figure 9.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Thomas Jefferson II

Field Jefferson Peter Jefferson

President Thomas Jefferson

Eston Hemings Thomas Woodson

Different Y Haplotype

Same Y Haplotype

Jefferson Y Haplotype

Jefferson Y Haplotype

?

Figure 9.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Historical Investigation DNA Study

Modern Use of Y-STR TestingCaptured December 13, 2003

Is this man really Sadaam Hussein?

Uday and Qusay Hussein

Killed July 22, 2003

Matching Y-STR Haplotype Used to Confirm Identity

(along with allele sharing from autosomal STRs)

Butler, J.M. (2005) Forensic DNA Typing, 2nd Edition, Box 23.1, p. 534

Compare with database to determine haplotype frequency

Extract mtDNA from evidence

(Q) sample

PCR Amplify HV1 and HV2 Regions

Sequence HV1 and HV2 Amplicons

(both strands)

Confirm sequence with forward and reverse strands

Note differences from Anderson (reference) sequence

Compare Q and K sequences

Performed separately and preferably after

evidence is completed

Extract mtDNA from reference

(K) sample

PCR Amplify HV1 and HV2 Regions

Sequence HV1 and HV2 Amplicons

(both strands)

Confirm sequence with forward and reverse strands

Note differences from Anderson (reference) sequence

Figure 10.4, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

Definitions• Homozygous gene: a gene in which both alleles

are the same: AA or aa.• Heterozygous gene: a gene in which the two

alleles are different.