1. Polymerase Chain Reaction

2. PCR Repetitive amplification of a piece or region of DNA Numerous uses –Straightforward amplification & cloning of DNA –RT-PCR – reverse transcription coupled with PCR to amplify mRNAs (cDNAs actually are template) –Production of cDNA libraries –Mutagenesis –Sequencing

3. PCR Requirements DNA template –DNA that will be amplified (copied) Oligodeoxynucleotide primers –anneal to template to allow DNA replication thermostable DNA polymerase –DNA polymerase extends the primers to synthesize a copy of the template DNA –thermostable polymerases allow automation and repeated rounds of DNA denaturation deoxynucleotides and appriate reaction conditions –dNTPs are incorporated into synthesized DNA, buffered pH, & Mg 2+ to allow enzyme activity of DNA pol

4. PCR: The Process 1.Begin with a DNA template Insert in vector 1 st strand cDNA Genomic DNA AAAAAAA TTTTTTT

5. PCR: The Process AAAAAAA TTTTTTT 2.Denature template 3.Anneal primers

6. AAAAAAA TTTTTTT PCR: The Process 3.Extend primers with thermostable DNA polymerase Taq Pfu This ends a PCR cycle Additional cycles will repeat these three steps

7. PCR: The Process Beginning of 2 nd cycle 1.Melt newly synthesized DNA from template New strands of DNA are now also available as templates 2.Anneal primers 3.Extend primers 1 & 23

8. PCR: The Process Beginning of 3 rd cycle Melt newly synthesized DNA from template –All new strands of DNA are now also available as templates Anneal primers Extend primers

9. PCR: Yields How much amplification can be achieved? –Each cycle of PCR theoretically doubles the number of template molecules –Therefore the rate of amplification is 2 n Where n is the number of amplification cycles –This will reach a practical maximum yield due to reagent (primer & dNTPs) concentration limits and maximum rate due to limiting enzyme concentrations. This upper limit is about 1x10 6 X amplification.

10. PCR: Yields Example: Starting with 2ng of 5kb DNA template to amplify a 1Kb insert, what is the theoretical yield after 20 cycles? After 30? 1.How many template molecules are there? = 5000bp X 660g bp/mol bp = 3.3x10 6 g template/mol template = 2x10 -9 g template  3.3x10 6 g temp/mol temp = 6x10 -16 mol temp = 6x10 -16 mol temp X 6.02x10 23 molec/mol = 3.64x10 8 molecules 2. How many molecules of insert can be made in 20 cycles? 30? 3.64x10 8 molecules x 2 20 = 3.8x10 14 molecules – 10 6 X 3.64x10 8 molecules x 2 30 = 3.9x10 17 molecules – 10 9 X