Question

Describe the steps of PCR technique.

Explain different steps involved in PCR technique.

Answer 1

1. Step i: The reaction mixture is heated to a temperature (90-98°C) to separate two strands of desired DNA. This is called denaturation.
2. Step ii: The mixture is allowed to cool (40-60°C) that permits pairing of the primer to the complementary sequences in DNA. This step is called annealing.
3. Step iii: 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.

DNA replication through polymerase chain reaction

Answer 2

The three essential steps of the PCR technique are:

1. Heat denaturation: This step involves the heating of DNA at about 91°C. The heating breaks the hydrogen bonds to make ssDNA. The DNA molecule with more G-C pairs needs a higher temperature.

2. Annealing: It is the pairing of primers to the ssDNA segment. The primers have to be designed as per the requirement. This step requires temperature at about 55°C

3. Polymerisation: The temperature is raised to 72°C. The Taq polymerase adds dNTPs behind the primer on the ssDNA. These three steps constitute one cycle of the reaction (3-5 mins). The process is carried out for about 28-30 cycles beyond which its reliability decreases.