The correct sequence of steps involved in polymerase chain reaction (PCR) are:

A. DNA polymerase is used to extends the primers using oligonucleotides.

B. Desired DNA fragments are denatured.

C. Amplified fragments are ligated with the vector for cloning.

D. Oligonucleotides are added

Choose the correct answer from the options given below:

B, D, A, C

The correct answer is Option (4) -B, D, A, C

B. Desired DNA fragments are denatured.

D. Oligonucleotides are added

A. DNA polymerase is used to extends the primers using oligonucleotides.

C. Amplified fragments are ligated with the vector for cloning.

The PCR involves three major cyclic reactions:

Denaturation

Annealing

Extension

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