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The polymerase chain reaction (PCR) is a technology in molecular biology used to amplify a single copy or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence. Developed in 1983 by Kary Mullis,〔(US4683195 )〕 PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications. These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensic sciences and DNA paternity testing); and the detection and diagnosis of infectious diseases. In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith for his work on PCR.〔(Kary Mullis Nobel Lecture, December 8, 1993 )〕 The method relies on thermal cycling, consisting of cycles of repeated heating and cooling of the reaction for DNA melting and enzymatic replication of the DNA. Primers (short DNA fragments) containing sequences complementary to the target region along with a DNA polymerase, which the method is named after, are key components to enable selective and repeated amplification. As PCR progresses, the DNA generated is itself used as a template for replication, setting in motion a chain reaction in which the DNA template is exponentially amplified. PCR can be extensively modified to perform a wide array of genetic manipulations. Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase (an enzyme originally isolated from the bacterium ''Thermus aquaticus''). This DNA polymerase enzymatically assembles a new DNA strand from DNA building-blocks, the nucleotides, by using single-stranded DNA as a template and DNA oligonucleotides (also called DNA primers), which are required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample through a defined series of temperature steps. In the first step, the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting. In the second step, the temperature is lowered and the two DNA strands become templates for DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions. ==Principles and procedure== PCR amplifies a specific region of a DNA strand (the DNA target). Most PCR methods typically amplify DNA fragments of between 0.1 and 10 kilo base pairs (kbp), although some techniques allow for amplification of fragments up to 40 kbp in size. The amount of amplified product is determined by the available substrates in the reaction, which become limiting as the reaction progresses. A basic PCR set up requires several components and reagents.〔 Chapter 8: In vitro Amplification of DNA by the Polymerase Chain Reaction〕 These components include: * ''DNA template'' that contains the DNA region (target) to amplify * Two ''primers'' that are complementary to the 3' (three prime) ends of each of the sense and anti-sense strand of the DNA target * ''Taq polymerase'' or another DNA polymerase with a temperature optimum at around 70 °C * ''Deoxynucleoside triphosphates'' (dNTPs, sometimes called "deoxynucleotide triphosphates"; nucleotides containing triphosphate groups), the building-blocks from which the DNA polymerase synthesizes a new DNA strand * ''Buffer solution'', providing a suitable chemical environment for optimum activity and stability of the DNA polymerase * ''Bivalent cations'', magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be used for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis * ''Monovalent cation'' potassium ions The PCR is commonly carried out in a reaction volume of 10–200 μl in small reaction tubes (0.2–0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect, which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube. 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「Polymerase chain reaction」の詳細全文を読む スポンサード リンク
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