Polymerase Chain Reaction (PCR)
Introduction
The polymerase chain reaction (PCR) is a revolutionary technique in molecular biology that is used to amplify (cloning) a single gene or a piece of DNA into thousands to millions of copies.It is based upon in vitro replication process which is carried out by DNA polymerase enzyme. In this technique DNA polymerase is compelled to polymerize a given piece of DNA again and again so that multiple copies are produced, thus, the technique is known as Polymerase chain reaction (PCR).
Components of the PCR technique
The following are the components required for carrying out a PCR reaction;- Template DNA
- Deoxiribo-nucleoside triphosphate (dNTPs)
- Primers
- Taq polymerase
1. Template DNA or Target DNA
It is the piece of DNA which to be cloned or amplified. It may be a useful gene found in the genomic DNA or the piece of DNA of infecting organisms.2. Deoxiribo-nucleosidetri-phosphate (dNTPs)
These are free nucleotides that act as raw material for the synthesis of new DNA fragments. There are four different types of dNTPs (dATP, dGTP, dCTP, dTTP) required in this process.3. Primers
DNA polymerase is unable to initiate polymerization unless primers are attached. Two sets of primers viz forward primer and the backward primer are used in this technique that select 3' ends of the target DNA fragment by annealing with its complementary sequence.4. Taq polymerase
The Taq DNA polymerase is a temperature tolerant enzyme isolated from Thermus aquaticus, a bacterium found in hot springs. This enzyme is stable and active at near-boiling temperatures.These are basic components required for the assembly of a PCR reaction. These components are dissolved along with Tris HCl (pH 8.3) and MgCl2 to form a mixture called PCR mixture or reaction mixture. The PCR mixture is placed in an instrument called a thermocycler or PCR machine. Thermocycler regulates the temperature during various steps of PCR reaction according to the need.
Mechanism or procedure
Typically PCR consists of a series of 20-40 repeated temperature changes, called cycles, with each cycle, commonly consisting of three discrete temperature steps. A PCR-amplification cycle consists of three basic steps. They are denaturation, primer annealing, and extension or polymerization. Its time duration and sequence of the steps have to be programmed in the thermocycler.A. Denaturation
It is the denaturation step, the template is heated to 94oC for one minute or up to five minutes. At this high temperature, the DNA undergoes complete denaturation and the double-stranded DNA (dsDNA) becomes single-stranded (ssDNA). Every single ssDNA acts as a template for the in vitro DNA synthesis.B. Primer annealing
The next step is 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 54oC and the approximately the time required for this step is 2 minutes.C. Extension or polymerization
The final step in each cycle is the primer extension or polymerization in which the Taq DNA 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 72oC. This step takes just one minute to be completed.At the end of the first cycle, one target DNA molecule is converted into two molecules. The second cycle immediately starts with the denaturation by heating at 94oC, so that all the newly synthesized DNA are also denatured to single strands, which again act as templates. It will again be followed by the continuous resulting in the amplification of the selected DNA sequence at an exponential rate i.e., the number of existing DNA molecules becomes double after each cycle.
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