1. A program of ITEST (Information Technology Experiences for Students and Teachers) funded by the National Science Foundation Background Session #5 Polymerase Chain Reaction and Primer Choice May 31, 2008 Dr. Simona Bartl Adjunct Professor Moss Landing Marine Labs

2. May 31st Workshop2 Intro to the Polymerase Chain Reaction (PCR)

3. May 31st Workshop3 Diagram by Andy Vierstraete 1999 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

4. May 31st Workshop4 Amplification steps

5. May 31st Workshop5 Application Examples  PCR is commonly used to…  Identify species  Identify alleles/genotypes to assess variability in a population  Conduct forensic investigations  Create sequences for phylogenies 1 to determine taxonomic relationships 2 1 evolutionary history 2 according to scientific classifications

6. May 31st Workshop6 Non-examples  PCR is NOT used to:  Amplify RNA or proteins  Construct traditional genomic or cDNA libraries  Make monoclonal antibodies  Conduct stem cell research

7. May 31st Workshop7 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 strand 1 Reverse primer - Anneals to DNA sense strand 2 1 template strand for transcription 2 identical to mRNA sequence

8. May 31st Workshop8 Primers http://www.flmnh.ufl.edu/cowries/PCR.gif

9. May 31st Workshop9 Materials  DNA polymerase Enzyme that extends growing DNA strand complementary to DNA template Taq polymerase - thermostable enzyme from Thermophilus aquaticus 1  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

10. May 31st Workshop10 Amplification Steps Cycling: Repeat steps 1 through 3 (20 - 40 times) 3. Extend primers, yielding new double-stranded DNA 2. Anneal primers to single-stranded DNA 1. Denature double-stranded DNA

11. May 31st Workshop11 Equipment Wikimedia Commons

12. May 31st Workshop12 PCR Animation - 3D PCR Animation - 2D

13. May 31st Workshop13 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

14. May 31st Workshop14 www-personal.umich.edu