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(詳細は"Eureka!" moment,〔(Kary Mullis' Nobel Lecture, December 8, 1993 )〕 or as an example of cooperative teamwork between disparate researchers.〔Rabinow P "Making PCR: A Story of Biotechnology" University of Chicago Press (1996) ISBN 0-226-70147-6〕 Following is a list of events before, during, and after its development: ==Prelude== * On April 25, 1953 James D. Watson and Francis Crick published "a radically different structure" for DNA,〔Watson JD, Crick FHC "A Structure for Deoxyribose Nucleic Acid", Nature vol. 171, pp. 737–738 (1953). ()〕 thereby founding the field of molecular genetics. Their structural model featured two strands of complementary base-paired DNA, running in opposite directions as a double helix. They concluded their report saying that "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material". For this insight they were awarded the Nobel Prize in 1962. * Starting in the mid-1950s, Arthur Kornberg began to study the mechanism of DNA replication.〔(Arthur Kornberg's Discovery of DNA Polymerase I) J. Biol. Chem. vol. 280, p. 46. ()〕 By 1957 he has identified the first DNA polymerase.〔Lehman, IR, Bessman MJ, Simms ES, Kornberg A "Enzymatic Synthesis of Deoxyribonucleic Acid. I. Preparation of Substrates and Partial Purification of an Enzyme from Escherichia coli" J. Biol. Chem. vol. 233(1) pp. 163–170 (1958).〕 The enzyme was limited, creating DNA in just one direction and requiring an existing primer to initiate copying of the template strand. Overall, the DNA replication process is surprisingly complex, requiring separate proteins to open the DNA helix, to keep it open, to create primers, to synthesize new DNA, to remove the primers, and to tie the pieces all together. Kornberg was awarded the Nobel Prize in 1959. * In the early 1960s H. Gobind Khorana made significant advances in the elucidation of the genetic code. Afterwards, he initiated a large project to totally synthesize a functional human gene.〔Khorana HG et al. "Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 1. General introduction" J. Biol. Chem. vol. 251(3) pp. 565–70 (1976).〕 To achieve this, Khorana pioneered many of the techniques needed to make and use synthetic DNA oligonucleotides. Sequence-specific oligonucleotides were used both as building blocks for the gene, and as primers and templates for DNA polymerase. In 1968 Khorana was awarded the Nobel Prize for his work on the Genetic Code. * In 1969 Thomas D. Brock reported the isolation of a new species of bacterium from a hot spring in Yellowstone National Park. ''Thermus aquaticus''〔Brock TD, Freeze H "Thermus aquaticus, a Nonsporulating Extreme Thermophile" J. Bact. vol. 98(1) pp. 289–297 (1969).〕 (Taq), became a standard source of enzymes able to withstand higher temperatures than those from ''E. Coli''. * In 1970 Klenow reported a modified version of DNA Polymerase I from ''E. coli''.〔Klenow H and Henningsen I "Selective Elimination of the Exonuclease Activity of the Deoxyribonucleic Acid Polymerase from Escherichia coli B by Limited Proteolysis" Proc Natl Acad Sci vol. 65 pp. 168–75 (1970).〕 Treatment with a protease removed the 'forward' nuclease activity of this enzyme. The overall activity of the resulting Klenow fragment is therefore biased towards the synthesis of DNA, rather than its degradation. * By 1971 researchers in Khorana's project, concerned over their yields of DNA, began looking at "repair synthesis" – an artificial system of primers and templates that allows DNA polymerase to copy segments of the gene they are synthesizing. Although similar to PCR in using repeated applications of DNA polymerase, the process they usually describe〔Panet A, Khorana HG "Studies on Polynucleotides" J. Biol. Chem. vol. 249(16), pp. 5213–21 (1974).〕 employs just a single primer-template complex, and therefore would not lead to the exponential amplification seen in PCR. * Circa 1971 Kjell Kleppe, a researcher in Khorana's lab, envisioned a process very similar to PCR. At the end of a paper on the earlier technique,〔Kleppe K, Ohtsuka E, Kleppe R, Molineux I, Khorana HG "Studies on polynucleotides. XCVI. Repair replications of short synthetic DNA's as catalyzed by DNA polymerases." J. Molec. Biol. vol. 56, pp. 341–61 (1971).〕 he described how a two-primer system might lead to replication of a specific segment of DNA:
:No results are shown there, and the mention of unpublished experiments in another paper〔 may (or may not) refer to the two-primer replication system. (These early precursors to PCR were carefully scrutinized in a patent lawsuit, and are discussed in Mullis' chapters in ''The Polymerase Chain Reaction'' (1994).〔Mullis KB, Ferré F, Gibbs RA "The Polymerase Chain Reaction" Birkhäuser Press (1994) ISBN 0-8176-3750-8〕) * Also in 1971, Cetus Corporation was founded in Berkeley, California by Ronald Cape, Peter Farley, and Donald Glaser. Initially the company screened for microorganisms capable of producing components used in the manufacture of food, chemicals, vaccines, or pharmaceuticals. After moving to nearby Emeryville, they began projects involving the new biotechnology industry, primarily the cloning and expression of human genes, but also the development of diagnostic tests for genetic mutations. * In 1976 a DNA polymerase〔Chien A, Edgar DB, Trela JM "Deoxyribonucleic acid polymerase from the extreme thermophile Thermus aquaticus" J. Bact. vol. 174 pp. 1550–1557 (1976).〕 was isolated from ''T. aquaticus''. It was found to retain its activity at temperatures above 75°C. * In 1977 Frederick Sanger reported a method for determining the sequence of DNA.〔Sanger F, Nicklen S, Coulson AR "DNA sequencing with chain-terminating inhibitors" Proc Natl Acad Sci vol. 74(12) pp. 5463–7 (1977).〕 The technique employed an oligonucleotide primer, DNA polymerase, and modified nucleotide precursors that block further extension of the primer in sequence-dependent manner. For this innovation he was awarded the Nobel Prize in 1980. By 1980 all of the components needed to perform PCR amplification were known to the scientific community. The use of DNA polymerase to extend oligonucleotide primers was a common procedure in DNA sequencing and the production of cDNA for cloning and expression. The use of DNA polymerase for nick translation was the most common method used to label DNA probes for Southern blotting. 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「History of polymerase chain reaction」の詳細全文を読む スポンサード リンク
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