Polymerase Chain Reaction (PCR) is the process of amplification of fragments of DNA for further analysis in biomedical research and in Forensics. PCR was invented by Karry Mullis in 1983.After the extraction of DNA, DNA is amplified using PCR. There are three different steps in PCR- Denaturation, Annealing and Extension. Amplification process takes place inside the thermal cycler. After adding the reagents required for PCR along with the DNA, PCR was processed inside the thermal cycler with suitable temperatures.There are different steps in a PCR reaction.
The components of PCR reaction are DNA template, Taq Buffer, Taq Polymerase, Forward Primer, Reverse Primer, Di nucleo tri phosphate (DNTP),Magnesium Ions, Dimethyl sulfoxide (DMSO), Bovine Serum Albumin (BSA). Two primers (Forward and reverse primers) are complementary to 3′ end of the sense and antisense strands of target DNA. Polymerase aids in adding new nucleotides to the growing DNA chain.DNTP’s are the building blocks of nucleotides from which DNA polymerase synthesize new strands. Buffer provides optimum activity of DNA polymerase by providing suitable chemical environment. Magnesium ions particularly Magnesium chloride provide specificity for the activity of DNA polymerase. DMSO is used if the Guanine- cytosine content is high and aids in reducing the melting temperature of DNA. BSA aids in preventing the adhesion of enzymes to the surfaces and tubes.
Initialization is a process in which the polymerase requires heat activation and the temperature was set up to 94-98 C for 1-10 minutes depending upon the polymerase. Denaturation is a process in which the double stranded DNA is denatured and the temperature is between 94-98 C for 20-30s.Annealing is a process in which the primer anneals to the DNA in producing new DNA strands and the temperature should be between 50-65C for 20-40s.Extension is a process in which the optimum activity of DNA polymerase takes place between 72-80C by adding DNTP for forming DNA strands.
There are different types of PCR that was performed during my research.Real time PCR, Conventional PCR, and Length Heterogeneity PCR (LH-PCR).In Real time PCR, the amount of product formed can be observed in real time.In LH-PCR the DNA is amplified using fluorescent labeled primers and the length heterogenity was obtained using Capillary Electrophoresis.