Polymerase chain reaction

(PCR); a technique developed in 1984 by Kary Mullis (and in 1993 honored with the Nobel Prize in Chemistry), to amplify exponentially a stretch of DNA situated between two known, relatively short stretches of sequence

Description
PCR usually employs a heat-stable DNA polymerase and exploits the double-strandedness and complementary base-pairing of DNA, in combination with the directionality of DNA synthesis from 5' to 3' to achieve specific amplification of a DNA template. As a minimum, dNTPs as building blocks for synthesis and a high excess of two primers flanking the target stretch of DNA (and facing each other with their 3' ends) are included in the reaction. Following a melting step at high temperature and annealing at a temperature appropriate for the template and primers used, most template DNA will have annealed to the abundant primers instead of re-annealing to the complementary template strand, allowing the extension of the primers along the template strand in a subsequent elongation step. Through multiple repeats of the procedure, both strands of the DNA fragment between and including the primers are amplified and serve as additional templates in subsequent amplification steps. DNA fragments only amplified by one of the primers (for instance by unspecific binding) are amplified linearly with every repeat of the procedure, those amplified by both primers are amplified exponentially, instead. In summary, PCR relies of three basic steps for exponential amplification.
 * 1) At the denaturation step, all double-stranded (ds) DNA is melted (denatured) into single strands (usually at 95 &degree;C).
 * 2) At the annealing step, single-stranded DNA re-anneals to complementary sequences and forms dsDNA (usually at between 50 and 65 &degree;C, depending on the primers used)
 * 3) At the polymerization step, the re-annealed primers are extended by the polymerase alongside the original and additional subsequently synthesized stretches of DNA template (usually at between 65 and 72 &degree;C, depending on the produt length desired and the polymerase used).

With the inclusion of internally annealing fluorescent probes of different colors and the combination of different primer pairs in a single reaction tube (so-called multiplexing), the amplification and specific detection of multiple DNA fragments are possible. All in all, PCR is one of the most versatile methods in use in molecular biology today, with critical roles in the detection of trace amounts of DNA material in pre-implantation diagnosis, paternity testing and forensics, in genotyping and haplotyping more generally, in molecular cloning, in the indirect quantification of mRNA by quantitative RT-PCR and in myriad other applications.