Question

describe the steps involved in PCR DNA amplification?

Answer 1

Answer-

1. Polymerase chain reaction (PCR) is technique in which DNA is synthesized in thermocycler. PCR is use to amplify template DNA in controlled temperature inside the thermocycler. In the reaction mixture of PCR target DNA to be amplified, dNTPs, Taq DNA polymerase, forward and reverse primers, buffer and Mg2+ ions are added in proper concentration. Then polymerization is done in a machine known as thermocycler and the Steps of PCR are-

1. DENATURATION- This is the first step of PCR in which PCR reaction mixture is heated between temperature 90-98°C for 2min to denature DNA so that this step is known as denaturation. At the end of this step two strands of dsDNA gets separated and then in next step primer can anneal with both separated DNA strands.

2. ANNEALING- in this second step of PCR, temperature of reaction mixture is cooled to 40-60°C for 1min. This allows annealing of primer to the complementary sequence of target DNA which is present at 3' end of both DNA strands. In this step primer anneals with DNA so that this step is known as annealing.

3. PRIMER EXTENSION- this is third and last step of PCR in which DNA polymerase synthesizes new DNA strand complementary to template DNA using 3'-OH of primer and DNA is synthesized in 5' to 3' direction by adding new nucleotides at 3' end of primer. Extension occurs at 72°C for 2min . In this step primer is extended by DNA polymerase so that this step is known as primer extension.

After certain number of cycles, DNA is amplified and multiple copies of DNA are synthesized in reaction.