PCR Polymerase Chain Reaction. PCR - a method for amplifying (copying) small amount of DNA in nearly any amount required, starting with a small initial.

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Polymerase Chain Reaction (PCR)

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Presentation transcript:

PCR Polymerase Chain Reaction

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).

PCR Machine / Thermocycler

PCR Components of PCRComponents of PCR –Template DNA – primers –dNTPs (dATP, dTTP, dCTP & dGTP) –Taq DNA polymerase –MgCl 2 –PCR buffer, pH 8

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

Factors influencing PCR Quality of template DNA Concentration of template DNA Primers Concentration of MgCl 2 Annealing temperature

Quality of template DNA - 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.

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.

Primers - 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.

Concentration of MgCl 2 - MgCl 2 concentration is very important. - excess Mg 2+ promotes production of nonspecific product and primer-dimer artifacts. - insufficient Mg 2+ reduces yield.

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.