PCR Amplification of DNA

1. PCR Amplification of DNA

2. PCR • PCR or polymerase chain reaction that was developed by Kary Mullis in the mid-1980’s. • 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. The idea is that in each cycle of melting, annealing and DNA synthesis the amount of the DNA segment is doubled. • This gives an exponential increase in the amount of the specific DNA as the cycles proceed. • After 10 cycles the DNA is amplified 103 fold and after 20 cycles the DNA will be amplified 106 fold. • Usually amplification is continued until all of the nucleotide precursors are incorporated into synthesized DNA.

14. Exponential Amplification

15. Materials: • sterile water • 10X amplification buffer with 15mM MgCl2 • 10 mM dNTP • 50 μM oligonucleotide primer 1 • 50 μM oligonucleotide primer 2 • 5 unit/μl Taq Polymerase • template DNA (1 μg genomic DNA, 0.1-1 ng plasmid DNA) in 10 μl • mineral oil (for thermocyclers without a heated lid )

16. 1. Combine the following for each reaction (on ice) in a 0.2 or 0.5 ml tube: 

17. 2. Prepare a control reaction with no template DNA and an additional 10 μl of sterile water. 3. If the thermocycler does not have a heated lid, add 70-100 μl mineral oil (or 2 drops of silicone oil) to each reaction. 4. Place tubes in a thermal cycler preheated to 94 degrees C. 5. Run the following program: • 94 degrees C 1 min • 55 degrees C 1 min or annealing temperature appropriate for particular primer pair • 72 degrees C 1 min (if product is <500 bp), 3 min (if product is >500 bp) for 30 cycles. Program a final extension at 72 degrees C for 7 min.