Question

Explain briefly:

PCR

Answer 1

Polymerase chain reaction (PCR) is a technique for producing numerous copies of a desired gene (DNA) in vitro. Kary Mullis developed this approach in 1985. It is based on the fact that when exposed to high temperatures, a DNA molecule separates into two strands due to denaturation. These single-stranded molecules are subsequently transformed into double-stranded molecules by synthesising additional strands in the presence of the enzyme DNA polymerase. Repeating the process several times produces copies of the original DNA sequence. The basic prerequisites of PCR are a DNA template, two nucleotide primers (typically 20 nucleotides long), and an enzyme DNA polymerase that is stable at high temperatures (commonly Taq polymerase). The mechanism of PCR is as follows:

1. First, the target DNA (DNA segment to be amplified) is heated to a high temperature (94-96° C). Heating separates two strands of DNA. Each of the two strands of target DNA now serves as a template for synthesising a new DNA strand. This procedure is known as denaturation.
2. Denaturation is followed by annealing. During this stage, two oligonucleotide primers hybridise with each single-stranded template DNA in the presence of an excess of synthetic oligonucleotides. Annealing is done at a lower temperature (40° to 60°C).
3. The third and last step is extension. In this stage, the enzyme DNA polymerase synthesises the DNA segment between the primers. Taq DNA polymerase, which was obtained from the thermophilic bacterium Thermus aquatics, is commonly utilised in most circumstances. The two primers extend towards each other to duplicate the DNA segment located between them. This phase requires the presence of deoxynucleoside triphosphates (dNTPs) and Mg²⁺ and takes place at 72°C.

The above three procedures finish the first PCR cycle. The second cycle begins with the denaturation of the previous cycle's extension product, and two cycles are finished after the extension phase is completed. If these cycles are performed several times, the DNA segment can be amplified roughly a billion times, yielding one billion copies of the target DNA segment.