Polymerase Chain Reaction

1. Polymerase Chain Reaction R P C

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, & Mg2+ to allow enzyme activity of DNA pol

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

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

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

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

8. PCR: The Process • Beginning of 3rd 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 2nWhere 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 1x106 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? • How many template molecules are there? = 5000bp X 660g bp/mol bp = 3.3x106 g template/mol template = 2x10-9 g template  3.3x106 g temp/mol temp = 6x10-16 mol temp = 6x10-16 mol temp X 6.02x1023 molec/mol = 3.64x108 molecules 2. How many molecules of insert can be made in 20 cycles? 30? 3.64x108 molecules x 220 = 3.8x1014 molecules – 106 X 3.64x108 molecules x 230 = 3.9x1017 molecules – 109 X