PCRPCRPolymerase Chain ReactionPolymerase Chain Reaction

PCRPCR

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

- an in vitro or cell-free method for - an in vitro or cell-free method for synthesizing DNA.synthesizing DNA.

- it was invented in 1985 by Kary Mullis - it was invented in 1985 by Kary Mullis (received the Nobel Prize for chemistry in (received the Nobel Prize for chemistry in 1993).1993).

PCR Machine / ThermocyclerPCR Machine / Thermocycler

PCRPCR

• Components of PCRComponents of PCR– Template DNA– primers– dNTPs (dATP, dTTP, dCTP & dGTP)– Taq DNA polymerase

– MgCl2

– PCR buffer, pH 8

PCRPCR

• Three major phases in PCR: Three major phases in PCR: – Denaturing (94ºC)– Annealing (55ºC)– Extension (72ºC)

• The total time to perform a standard The total time to perform a standard PCR is approximately 4 hours. PCR is approximately 4 hours.

Factors influencing PCRFactors influencing PCR

• Quality of template DNA• Concentration of template DNA• Primers

• Concentration of MgCl2

• Annealing temperature

Quality of template DNAQuality 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 DNAConcentration 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.

PrimersPrimers- 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 MgClConcentration of MgCl22

- MgCl2 concentration is very important.

- excess Mg2+ promotes production of nonspecific product and primer-dimer artifacts.

- insufficient Mg2+ reduces yield.

Annealing temperatureAnnealing 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.