Polymerase Chain Reaction (PCR)
The technique used to amplify (clone) a single gene or a piece of DNA into thousands to millions of copies by means of in vitro replication process is called polymerase chain reaction (PCR). In this technique DNA polymerase is compelled to polymerize (polymerase reaction) a given piece of DNA repeatedly, so that multiple copies are produced, thus, the technique is known as polymerase chain reaction (PCR). A special DNA polymerase, the Taq polymerase is used in PCR technique, which is specialized temperature-tolerant enzyme isolated from Thermus aquaticus, a bacterium found in hot springs. This enzyme is stable and active at near-boiling temperatures. In order to perform PCR, template DNA (DNA to be amplified), free nucleotides (deoxyribo-nucleoside triphosphates or dNTPs), primers and Taq polymerase are dissolve in suitable buffer to make PCR mixture or reaction mixture. The PCR mixture is placed in an instrument called thermocycler or PCR machine. Thermocycler regulates the temperature during various steps of PCR reaction according to the need. Mechanism of PCR Reaction PCR cycle consists of three steps: denaturation, primer annealing, and extension or polymerization each requires a specific temperature. The time duration, temperature and sequence of the steps have to be programmed in the thermocycler. DENATURATION In the denaturation step, the template is heated to 94°C for one minute. At this high temperature the DNA undergoes complete denaturation and the double-stranded DNA (dsDNA) becomes single-stranded DNA (ssDNA). Each single ssDNA can act as the template for the in vitro DNA synthesis. PRIMER ANEALING The next step is the primer annealing. In this step the two primers, the forward primers and the backward primers, anneal or hybridize to the single-stranded template DNA at its complementary regions. Annealing is usually carried out at a lower temperature depending on the length and sequence of the primers. In standard cases it is 54°C and approximate time required for this step is 2 minutes. EXTENSION OR POLYMERIZATION The final step in each cycle is the primer extension or polymerization in which the Taq polymerase synthesizes new DNA strands to the 3′ ends of primers using dNTPs. The optimum temperature for carrying out the primer extension reaction or polymerization of dNTPs is standardized at 72°C. This step takes just one minute to be completed. At the end of first cycle one target DNA molecule is converted in to two molecules. The 2ND cycle immediately starts with the denaturation by heating at 94°C, so that all the newly synthesized u are also denatured to single strands, which again act as templates. It will again be followed by the Primer annealing and extension and thus the cycle of denaturation, primer annealing, and extension continues resulting in the amplification of selected DNA sequence at an exponential rate i.e. the number of existing DNA molecules becomes doubled after each cycle.
