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

Background Session #5 Polymerase Chain Reaction and Primer Choice May 31, 2008. Dr. Simona Bartl Adjunct Professor Moss Landing Marine Labs. Intro to the Polymerase Chain Reaction (PCR). Definition. A procedure to amplify a specific DNA region Using DNA synthesis (DNA replication)

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

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  1. Background Session #5Polymerase Chain Reaction and Primer Choice May 31, 2008 Dr. Simona Bartl Adjunct Professor Moss Landing Marine Labs

  2. Intro to the Polymerase Chain Reaction (PCR) May 31st Workshop

  3. 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 May 31st Workshop

  4. Amplification steps May 31st Workshop

  5. 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 May 31st Workshop

  6. Non-examples • PCR is NOT used to: • Amplify RNA or proteins • Construct traditional genomic or cDNA libraries • Make monoclonal antibodies • Conduct stem cell research May 31st Workshop

  7. 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 May 31st Workshop

  8. Primers http://www.flmnh.ufl.edu/cowries/PCR.gif May 31st Workshop

  9. Materials • DNA polymerase • Enzyme that extends growing DNA strand complementary to DNA template • Taq polymerase - thermostable enzyme from Thermophilus aquaticus1 • dNTP’s • 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 May 31st Workshop

  10. 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) May 31st Workshop

  11. Equipment Wikimedia Commons May 31st Workshop

  12. PCR Animation - 3D PCR Animation - 2D May 31st Workshop

  13. 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 May 31st Workshop

  14. www-personal.umich.edu May 31st Workshop

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