POLYMERASE reaction or PCR is the in

POLYMERASE
CHAIN REACTION (PCR)

 

INTRODUCTION

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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 are basically serve as the replication primers.

3.        
A DNA polymerase enzyme. The primers are extended by this enzyme and
polymerizes 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 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
indispensible tool in forensic science, particularly in DNA fingerprinting,
Genetic mapping, genetic testing, tissue typing etc.