3-2.3 Basic Protocol for Polymerase Chain Reaction:
3-2.3.1 Components and reagents:
A basic PCR set up requires the following essential components and reagents :
1. Template DNA containing the DNA region (target) to be amplified.
2. Primers that are complementary to the 5' ends of each of the sense (Forward primer) and anti-sense strand of the DNA target (Reverse primer).
3. Taq polymerase or other thermostable, high fidelity DNA polymerase (Pfu polymerase isolated from Pyrococcus furiosus ).
4. Deoxyribonucleotide triphosphates (dNTPs), which are the building-blocks for a newly synthesized DNA strand.
5. Buffer solutions to provide a suitable chemical condition for optimum activity and stability of the DNA polymerases.
6. Divalent cations (eg. magnesium or manganese ions). They act as a co-factor for Taq polymerase which increases its polymerase activity. Generally Mg2+ is used, but Mn2+ can be applied to achieve PCR-mediated DNA mutagenesis. This is because higher Mn2+ concentration leads to higher error rate during DNA synthesis.
3-2.3.2 P rocedure:
Typically, PCR is designed of 20-40 repeated thermal cycles, with each cycle consisting of 3 discrete temperature steps: denaturation, annealing and extension. The thermal cycles are often proceeded by a temperature at a high range (>90°C), and followed by final product extension or brief storage at 4 degree celsius. In PCR cycles, the temperatures and the duration of each cycle is determined based on various parameters like the type of DNA polymerase used, the melting temperature (Tm) of the primers, concentration of divalent ions and dNTPs in the reaction etc. The various steps involved are:-
a) Initial Denaturation
b) Denaturation
c) Annealing
d) Extension
e) Final extension