Polymerase Chain Reaction (PCR) Technique

The polymerase chain reaction (PCR) is a technique by which small samples of DNA can be quickly amplified, that is, increased to quantities that are large enough for analysis.

Starting with just one gene-sized piece of DNA, PCR can be used to make literally billions of copies in only a few hours.

• Each strand of the target DNA will serve as a template for DNA synthesis.
• To this DNA is added a supply of the four nucleotides (for assembly into new DNA) and the enzyme for catalyzing the synthesis, DNA polymerase. Short pieces of nucleic acid called primers are also added to help start the reaction. The primers are complementary to the ends of the target DNA and
• will hybridize to the fragments to be amplified .
• Then, the polymerase synthesizes new complementary strands.
• After each cycle of synthesis, the DNA is heated to convert all the new DNA into single strands. Each newly synthesized DNA strand serves in turn as a template for more new DNA.

Polymerase Chain Reaction (PCR)

As a result, the process proceeds exponentially. All of the necessary reagents are added to a tube, which is placed in a thermalcycler. The thermalcycler can be set for the desired temperatures, times, and number of cycles. Use of an automated thermalcycler is made possible by the use of DNA polymerase taken from a thermophilic bacterium such as Thermus aquaticus; the enzyme from such organisms can survive the heating phase without being destroyed. Thirty cycles, completed in just a few hours, will increase the amount of target DNA by more than a billion times.

The amplified DNA can be seen by gel electrophoresis. In real-time PCR, the newly made DNA is tagged with a fluorescent dye, so that the levels of fluorescence can be measured after every PCR cycle (that's the real time aspect) .Another PCR procedure called reverse-transcription PCR uses viral RNA or a cell's mRNA as the template. The enzyme, reverse transcriptase, makes DNA from the RNA template, and the DNA is then amplified.

Note that PCR can only be used to amplify relatively small, specific sequences of DNA as determined by the choice of primers. It cannot be used to amplify an entire genome.

PCR can be applied to any situation that requires the amplification of DNA. Especially noteworthy are diagnostic tests that use PCR to detect the presence of infectious agents in situations in which they would otherwise be undetectable.