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.
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