PCR Polymerase Chain Reaction

1. PCRPolymerase Chain Reaction

2. PCR - a method for amplifying (copying) small amount of DNA in nearly any amount required, starting with a small initial quantity. - an in vitro or cell-free method for synthesizing DNA. - it was invented in 1985 by Kary Mullis (received the Nobel Prize for chemistry in 1993).

4. PCR Machine / Thermocycler

5. PCR • Components of PCR • Template DNA • primers • dNTPs (dATP, dTTP, dCTP & dGTP) • Taq DNA polymerase • MgCl2 • PCR buffer, pH 8

6. PCR • Three major phases in PCR: • Denaturing (94ºC) • Annealing (55ºC) • Extension (72ºC) • The total time to perform a standard PCR is approximately 4 hours.

9. Factors influencing PCR • Quality of template DNA • Concentration of template DNA • Primers • Concentration of MgCl2 • Annealing temperature

10. - should be free of proteases that could degrade the DNA polymerase. - template DNA with high levels of proteins or salts should be diluted or cleaned up to reduce inhibition of DNA polymerase activity. Quality of template DNA

11. Concentration of template DNA - highly concentrated template DNA may yield nonspecific product or inhibit the reaction. - it is rare that template DNA concentration is too low.

12. - select primers with a random base distribution and GC content similar to template DNA being amplified. - avoid sequences with secondary structure, especially at the 3’ end. - check primers for complementary and avoid primers with 3’ overlaps to reduce primer-dimer artifacts. - design so the base at the 3’ end of the primer is a G or C to enhance specificity. Primers

13. Concentration of MgCl2 - MgCl2 concentration is very important. - excess Mg2+ promotes production of nonspecific product and primer-dimer artifacts. - insufficient Mg2+ reduces yield.

14. Annealing temperature - annealing temperature depends on length and GC content of primers (55ºC good for primers 20 nucleotides long; 50%). - Higher annealing temperatures may be needed to increase primer specificity.