Question

Give the steps in PCR or polymerase chain reaction with suitable diagrams.

Answer 1

Polymerase Chain Reaction (PCR) is the process of in vitro amplification of the gene of interest using a PCR machine.

i. PCR can generate a billion copies of the desired segment of DNA or RNA, with high accuracy and specificity, in a few hours.

ii. The process of PCR is completely automated and involves automatic thermal cycles for denaturation and renaturation of double-stranded DNA.

iii. The device required for PCR is called a thermal cycler.

iv. Requirements for polymerase chain reaction:

1. DNA containing the desired segment to be amplified
2. several molecules of four deoxyribonuclueoside triphosphates (dNTPs)
3. excess of two primer molecules
4. heat-stable DNA polymerase and
5. appropriate quantities of Mg⁺⁺ ions.

Mechanism of PCR:

At the start of PCR, all the requirements are mixed together in ‘Eppendorf tube’ and the following operations are performed sequentially:

Step i: Denaturation

The reaction mixture is heated to a temperature (90–98oC) to separate two strands of desired DNA. This is called denaturation.

Step ii: Annealing

The mixture is allowed to cool (40–60oC) that permits the pairing of the primer to the complementary sequences in DNA. This step is called annealing.

Step iii: Primer extension / Polymerization

The temperature (70–75°C) allows thermostable Taq DNA polymerase to use single-stranded DNA as a template and adds nucleotides. This is called primer extension. It takes around two minutes duration.

v. One cycle takes around 3 to 4 minutes.

vi. To begin the second cycle, DNA is again heated to convert double-stranded DNA into single strands.

vii. In an automatic thermal cycler, the above three steps are automatically repeated 20-30 times. Thus, at the end of ‘n’ cycles, 2n copies of DNA segments are produced.

viii. The machine performs the entire operations automatically and precisely.

DNA replication through a polymerase chain reaction.