Steps of Polymerase Chain Reaction (PCR) are given below. Arrange them in the correct sequence.

A. Annealing of primers to the template DNA

B. Isolation of gene of interest

C. Extension of primer end on the template DNA using Taq polymerase

D. Denaturation of template DNA by heating

Choose the correct answer from the options given below.

B, D, A, C

The correct answer is Option (2) -B, D, A, C

The correct sequence of steps in Polymerase Chain Reaction (PCR) is:

B. Isolation of gene of interest

D. Denaturation of template DNA by heating

A. Annealing of primers to the template DNA

 C. Extension of primer end on the template DNA using Taq polymerase

Polymerase Chain Reaction (PCR) is a technique of synthesizing multiple copies of the desired gene (or DNA) in vitro. This technique was developed by Kary Mullis in 1985. It is based on the principle that a DNA molecule, when subjected to high temperature, splits into two strands due to denaturation. These single stranded molecules are then converted to original double stranded molecules by synthesizing new strands in presence of enzyme DNA polymerase. Thus, a double stranded molecule of DNA is duplicated and multiple copies of the original DNA sequence can be generated by repeating the process several times.

The basic requirements of PCR are:

(i) DNA template. The desired segment of the target DNA molecule that is to be amplified.

(ii) Two nucleotide primers. Two nucleotide primers, usually 10-18 nucleotide long and complementary to the sequences present at the 3' ends of the target DNA segment.

(iii) Enzyme. High temperature (more than 90°C) stable DNA polymerase (usually Taq polymerase), for synthesis of new DNA molecules, are required.

Working Mechanism of PCR:

(1) First of all, the target DNA (DNA segment to be amplified) is heated to high temperature (94°C). Heating results in the separation of two strands of DNA. Each of the two strands of the target DNA now act as template for synthesis of new DNA strand. This step is called denaturation. Denaturation is the separation of the two strands of DNA by breakdown of hydrogen bonds on heating.

(2) Denaturation is followed by annealing (Anneal = Join). During this step, two oligonucleotide primers hybridize to each of single stranded template DNA in presence of excess of synthetic oligonucleotide. Annealing is carried out at lower temperature (40° – 60°C).

(3) Third and final step is extension. During this step, the enzyme DNA polymerase synthesizes the DNA segment between the primers. Usually Taq DNA polymerase, isolated from a thermophilic bacterium Thermus aquaticus, is used in most of the cases. The two primers extend towards each other in order to copy the DNA segment tying between the two primers. This step requires presence of deoxynucleoside triphosphates (dNTPs) and Mg²⁺ and occurs at 72^°C.

The above mentioned three steps complete the first cycle of PCR. The second cycle begins with denaturation of extension product of first cycle and after completing the extension step, two cycles are completed. If these cycles are repeated many times, the DNA segment can be amplified to approximately billion times, i.e., one billion copies of desired DNA segment are made. If required, the amplified fragment can be ligated with a vector for further cloning.

 Polymerase chain reaction (PCR) : Each cycle has three steps: (i) Denaturation; (ii) Primer annealing; and (iii) Extension of primers