Review of Polymerase Chain Reaction (pcr) Procedure

What is PCR?

Polymerase chain reaction or PCR is the in vitro technique for the synthesis of the DNA or we can say that it is an in vitro DNA replication procedure. This technique is generally used in case of molecular biology for the amplification of a single copy or a few copies of DNA segment. After the completion of this procedure, it leads to the generation of thousands or million copies of that particular DNA segment from the very few copies of DNA. This PCR technique has an elegant simplicity.

Components Which Are Required In PCR:

1. DNA template containing the target region.
2. Two primers of DNA. The synthetic oligonucleotides (primers, are prepared) should be complementary to the sequences on the opposite strands of the target DNA at positions defining the ends of the segment to be replicated. These primers basically serve as the replication primers.
3. A DNA polymerase enzyme. The primers are extended by this enzyme and polymerize the new DNA strand. In PCR, since a high temperature is required, so, for the polymerization of new DNA strands, thermophilic DNA polymerases are used. As for example Taq DNA polymerase, Deep vent R DNA polymerase.
4. Deoxynucleoside triphosphates or dNTPs (dATP, dGTP, dCTP and dTTP).
5. A buffer solution to maintain the appropriate chemical environment. This is required for the optimum polymerase enzymatic activity and its stability.
6. Mg2+, a bivalent cation.

Procedure

Thermal cycling is involved in this polymerase chain reaction. This thermal cycling consists of repeated heating and cooling cycles of the reaction for melting and enzymatic replication of the DNA. It sets a chain reaction motion in which exponential amplification of the DNA template takes place.

The isolated DNA which contains the segment to be replicated is first heated briefly to denature it. After heating it, it is then cooled in the presence of the large excess of the synthetic oligonucleotide primers. After that, 4 dNTPs along with the Taq DNA polymerase are added to it and the primed DNA segment is replicated selectively in vitro. The cycle of the heating, cooling, and replication is reaped 25 to 30 times over a few hours in an automated process. The basic three steps of the PCR are: Denaturation (94-98°C); Annealing (50-65°C) and extension (72°C). This whole process can be described through the below diagram.

Importance of PCR

This powerful technique is used in case of the (i) Medical diagnosis; (ii) Forensics and (iii) The studies of molecular evolution. Valuable diagnostic information can be provided by the PCR in medicine. Bacteria and viruses can be readily detected with the use of specific primers. As for example, certain cancers are early detected by the promising PCR method. Mutations of certain growth-control genes (ras-genes) can be identified as well by the PCR. genetic testing, tissue typing, identifying oncogenes, etc. Addition to all of these, PCR is used as an indispensable tool in forensic science, particularly in DNA fingerprinting, Genetic mapping, genetic testing, tissue typing etc.