Background Session

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Background Session 5Polymerase Chain Reaction
and Primer Choice May 31, 2008

• Dr. Simona Bartl
• Adjunct Professor
• Moss Landing Marine Labs

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Intro to the Polymerase Chain Reaction (PCR)
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Definition

• A procedure to amplify a specific DNA region
• Using DNA synthesis (DNA replication)
• Yields millions of copies of the target region
• Makes enough DNA for further work
• Is the first step in preparing DNA for
• DNA Sequencing
• Restriction Digestion
• Cloning and Bacterial Transformation

Diagram by Andy Vierstraete 1999
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Amplification steps
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Application Examples

• PCR is commonly used to
• Identify species
• Identify alleles/genotypes to assess variability
  in a population
• Conduct forensic investigations
• Create sequences for phylogenies1 to determine
  taxonomic relationships2
• 1 evolutionary history
• 2 according to scientific classifications

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Non-examples

• PCR is NOT used to
• Amplify RNA or proteins
• Construct traditional genomic or cDNA libraries
• Make monoclonal antibodies
• Conduct stem cell research

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Materials

• PCR is DNA Synthesis
• DNA template
• Can be DNA extracted or purified from an organism
  
• Primers
• Anneal to single-stranded DNA template
• Provide initiation site for extension of new DNA
• Forward primer - Anneals to DNA anti-sense
  strand1
• Reverse primer - Anneals to DNA sense strand2
• 1 template strand for transcription
• 2 identical to mRNA sequence

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Primers
http//www.flmnh.ufl.edu/cowries/PCR.gif
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Materials

• DNA polymerase
• Enzyme that extends growing DNA strand
  complementary to DNA template
• Taq polymerase - thermostable enzyme from
  Thermophilus aquaticus1
• dNTPs
• Nucleotides (Adenine, Cytosine, Guanine, Thymine)
  building blocks for new DNA strands
• Buffer and Ions
• Provides optimal conditions for enzyme
• 1 Bacteria isolated from hot springs

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Amplification Steps
1. Denature double-stranded DNA
2. Anneal primers to single-stranded DNA
3. Extend primers, yielding new double-stranded
DNA
Cycling Repeat steps 1 through 3 (20 - 40 times)
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Equipment
Wikimedia Commons
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PCR Animation - 2D

• PCR Animation - 3D

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Primer choice

• Conserved DNA regions that flank a more variable
  region
• Conserved so primers will anneal (stick) and
  therefore allow DNA synthesis
• Variable so DNA data will be informative

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www-personal.umich.edu