Describe the steps of PCR technique.
One cycle of Polymerase Chain Reaction involves three basic steps:
a. Denaturation b. Annealing c. Polymerization
The desired DNA is heated to a high temperature of about 91°C and forms a single stranded DNA. It results in the separation of the two strands of DNA, each of which would function as a template for the synthesis of a new molecule of DNA.
1. It is the process in which the two primers (oligonucleotides) hybridise to each of the strands of DNA. It requires a temperature of 55°C.
In this step, the Taq polymerase carries out the synthesis of DNA region between the two primers by using deoxyribonucleotides (dNTPs) and Mg2+. The optimum temperature for this polymerization reaction is 72°C.
The three essential steps of PCR technique are -
1) Heat denaturation: This step involves 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 the higher temperature.
2) Annealing: It is 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.