1. iGEM 101: Session 6 4/9/15Jarrod Shilts 4/11/15Ophir Ospovat

2. PCR Principles ▪ In vitro DNA Replication – DNA Polymerase – Primer (for 3’ OH) – dNTP ▪ Specify region to be amplified by primer sequence ▪ Exponential amplification

3. PCR Uses  Amplify specific region of DNA  Increase amount of DNA part  Extract portion out of larger sequence (including genomes)  Test success of reaction by size of product (ex. restriction digest)  Region to be amplified specified by primers  Specialized applications

4. PCR Cycle

5. PCR Primers

6. Primer Design ▪ Around 20 bp ▪ Melting temperature from 52-60 o C – Each bp increases Tm – GC more difficult to melt than AT – Primer Tm no more than 3 o C apart ▪ Avoid secondary structures ▪ GC content 40-60% ▪ Avoid mis-annealing – No long repeated sequences – No primer homology – GC at 3’ end Td = 2°C(A+T) + 4°C(G+C)

7. PCR Reaction

8. Thermocycler Program

9. Special PCR Applications ▪ RT-PCR – Extract dsDNA from RNA ▪ Extension PCR – Add overhang to primers ▪ Overlap PCR – Join sequences together ▪ qPCR – Quantify expression ▪ Allele-specific PCR – Diagnostic tool