by Debbie Luyo

Created on: July 20, 2008   Last Updated: January 24, 2012

Polymerase Chain Reaction (PCR ) is a technique that utilizes the enzyme DNA polymerase for making copies of a gene, gene fragment, or specific DNA or RNA sequence. DNA polymerase is the enzyme that catalyzes replication and repair of DNA. PCR is a requisite tool in research, clinical, environmental, and forensic laboratories. PCR employs cycles of heating and cooling to first melt and separate the double-stranded DNA containing the sequence of interest, then, with the help of specially designed sequences called primers, locate the target sequence, and exponentially produce new copies. PCR is a versatile and adaptable technique, with many variations and applications. Since its introduction in the mid eighties, PCR has modernized the field of molecular biology. Procedures that once took weeks to produce results can now be accomplished in a few hours.

PCR is often used to amplify an unknown sequence. However, in order to construct complementary primers, some information must be known about sequences bordering the region of interest. Primers are annealed to the sequences that flank the sequence to be amplified, in order to initiate replication of the target DNA. The most commonly used DNA polymerase for PCR is Taq polymerase, isolated from the bacterium Thermus aquaticus, known for its supreme stability at high temperatures. Deoxyribonucleotide triphosphates (dNTPs), the basic units of DNA, are another key component in the PCR mixture. In a small tube the template DNA, water, dNTPs, DNA polymerase, primers, and a salt such as magnesium chloride are briefly centrifuged, along with a control tube with template DNA omitted, and placed in a PCR thermocycler.

A typical PCR run includes a series of 20 to 40 cycles of heating and cooling, separating, then annealing. The first denaturation step brings the reaction mixture to a temperature between 94 and 98C, for 20 to 30 seconds. The heat disrupts the hydrogen bonds between complementary bases on the DNA strand, resulting in two single strands. The second step is the primer annealing step, where the reaction temperature is decreased to between 50 and 65C, for 20 to 40 seconds, to allow annealing of primers to the single-stranded DNA, resulting in a DNA-primer hybrid. Polymerase then binds to the DNA-primer hybrid, and DNA synthesis commences. The amount of target DNA doubles with each subsequent cycle, resulting in exponential amplification of the target DNA.

PCR has many applications. In clinical settings PCR can be

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