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DNA STRUCTURE
* Deoxyribose Sugar *
* Phosphate *
* Nitrogen Base *
Guanine
“Purines”
Adenine
Thymine
“Pyrimidines”
Cytosine
= “Nucleotide”
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Polymerase Chain Reaction
• Makes amplification of very small samples
of DNA possible
• Technique is indispensible in medical
testing and forensics along with other
fields.
Development of PCR
In the 1960s Thomas Strock discovered
Thermus aquaticus (aka “Taq”)
Molecular Biologist, Kary Mullis, a North
Carolina native developed the technique
in 1983 while .
In 1993, he was awarded the Nobel Prize
in Chemistry for this achievement.
How DNA Is Collected
Hair
Tissue
Blood
Teeth
Semen
Bone
Urine
Saliva
What Is Needed for PCR?
• Template (the DNA you want to amplify for the study)
• Sequence-specific primers flanking the target
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sequence:
Forward primer
Reverse primer
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Target sequence
• Nucleotides (dATP, dCTP, dGTP, dTTP)
• Magnesium ions (enzyme cofactor)
• Buffer, containing salt
• Taq polymerase
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How Does PCR Work?
• Heat (94°C) to denature DNA strands
• Cool (60°C) to anneal primers to template
• Warm (72°C) to activate Taq polymerase,
which extends primers and replicates DNA
• Repeat multiple cycles
Heat causes DNA strands to separate
Denaturing
Template
DNA
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Denaturation of DNA at 94°C
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Annealing Primers
• Primers bind to the template sequence
• Taq polymerase binds to double-stranded
substrate
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Primers anneal at 60°C
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Taq Polymerase Extends…
• Taq polymerase extends primer
• DNA is replicated
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Extends at 72°C
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Exact-length Target Product is Made in the Third Cycle
Cycle 1
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Cycle 2
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Cycle 3
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Milestones in Forensic DNA Analysis
1985
Alex
Jeffreys
develops
RFLP.
1992
FBI initiates
STR work.
1995
OJ Simpson trial
focuses public
attention on DNA
evidence.
2001
Many remains from the World Trade Center
disaster in New York are identified using a
combination of DNA profiling approaches.
2004
Indian Ocean
tsunami:
Interpol and
other world
agencies to
use DNA
profiling to
identify victims.
1990
PCR analysis
using single
locus STR
begins.
1994
DNA Identification
Act provides funding
for national DNA
database.
1998
FBI starts CODIS
database: All remains
from Swissair disaster
were identified using
STR DNA profiling.
2004
California’s Proposition 69
provides funding to maintain
a DNA database.
Special Considerations for PCR in Forensics
• PCR amplifies ALL DNA, especially
susceptible to contamination
– DNA from person doing the collection
– DNA from lab tech
– Residual DNA on lab equipment
– Contaminated reagents
• ALWAYS RUN A NEGATIVE
CONTROL!!!!