POLYMERASE CHAIN REACTION

Polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it to a large enough amount to study in detail.

Polymerase chain reaction, or PCR, is a technique to make many copies of a specific DNA region in vitro (in a test tube rather than an organism). PCR relies on a thermostable DNA polymerase, Taq polymerase, and requires DNA primers designed specifically for the DNA region of interest.

The polymerase chain reaction has been elaborated in many ways since its introduction and is now commonly used for a wide variety of applications including genotyping, cloning, mutation detection, sequencing, microarrays, forensics, and paternity testing. Typically, a PCR is a three-step reaction.

The number of copies doubles after each cycle. Usually 25 to 30 cycles produce a sufficient amount of DNA. In the original PCR procedure, one problem was that the DNA polymerase had to be replenished after every cycle because it is not stable at the high temperatures needed for denaturation.

The principle of enzymatic replication of the nucleic acids. This method has in the field of molecular biology an irreplaceable role and constitutes one of the basic methods for DNA analysis.

It has been widely used to detect and quantify pathogenic microorganisms that cause various infectious diseases including some arboviruses, STIs, and bacterial infection.

Amplification is achieved by a series of three steps: (1) denaturation, in which double-stranded DNA templates are heated to separate the strands; (2) annealing, in which short DNA molecules called primers bind to flanking regions of the target DNA; and (3) extension, in which DNA polymerase extends the 3′ end of each primer along the template strands. These steps are repeated (“cycled”) 25–35 times to exponentially produce exact copies of the target DNA.

PCR consists of a series of 20–40 repeated temperature changes, called thermal cycles, with each cycle commonly consisting of two or three discrete temperature steps (see figure below). The cycling is often preceded by a single temperature step at a very high temperature (>90 °C (194 °F)), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (T_m) of the primers.

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Regards,
Nicola B
Editorial Team
Journal of  Biochemistry and Biotechnology