Presentation for Kevin Fitzgerald

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Polymerase Chain Reaction
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3 end
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.

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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.
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How DNA Is Collected
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What Is Needed for PCR?

• Template (the DNA you want to amplify for the
  study)
• Sequence-specific primers flanking the 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 (94C) to denature DNA strands
• Cool (60C) to anneal primers to template
• Warm (72C) to activate Taq polymerase, which
  extends primers and replicates DNA
• Repeat multiple cycles

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Denaturing Template DNA

• Heat causes DNA strands to separate

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Denaturation of DNA at 94C
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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 60C
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Taq Polymerase Extends

• Taq polymerase extends primer
• DNA is replicated

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Extends at 72C
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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
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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!!!!

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