Polymerase chain reaction thermus aquaticus

Tunis. M.A., K.B. Myambo, D.H. Gelfand and M.A. Brow 1988. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85 9436-9440. 

The polymerase chain reaction uses (1) a thermostable DNA polymerase, such as Taq polymerase derived from the bacterial thermophile Thermus aquaticus, (2) a DNA template which is to be amplified, (3) two primers, each typically of around 20 nucleotides, which anneal to distinct parts on the complementary strands of the target and serve as sites for commencing DNA polymerase action, (4) a solution including the four deoxynucleoside triphosphates dATP, dCTP, dGTP and dTTP, Mg2+, salts and pH buffer. 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

Holland, P.M. Abramson, R.D. Watson, R. Gelfand, D.H. Detection of specific polymerase chain reaction product by utilizing the 5 to 3 exonuclease activity of Thermus aquaticus. Proc. Natl. Acad. Sci. 1991, 88, 7276-7280. 

A bacterium originally isolated from a hot spring in Yellowstone National Park provides the key to a powerful molecular tool for the study of DNA. Polymerase chain reaction (PCR) allows scientists to produce unlimited amounts of any gene of interest and the bacterium Thermus aquaticus produces a heat-stable DNA polymerase (Taq pol)unerase) that allows the process to work. 

Using a heat-stable DNA polymerase produced by the bacterium Thermus aquaticus and specific DNA primers, polymerase chain reaction allows the amplification of DNA sequences that are present in small quantities. This technique is useful in genetic screening, diagnosis of viral or bacterial disease, and forensic science. 

A number of smaller enzyme-producing companies focus on thermophilic micro-organisms (and other extremophiles) to identify and produce new types of thermostable enzymes Unitika, Pacific Enzymes, Genis, Diversa (formerly Recombinant BioCatalysis), and others. One extremozyme that has already found commercial application is the heat-stable DNA polymerase from Thermus aquaticus (Taq-polymerase) that gave rise to the polymerase chain reaction (PCR). Using PCR, nucleic acids or segments of DNA can by amplified in vitro without having to replace the enzyme after each amplification cycle when the DNA template is denatured by heat. A number of new hyperthermophilic enzymes with temperature optima between 75 and 118°C have been described in the past few years [81], such as... 

EXAMPLE 8.16 Often only trace amonnts of a specific DNA sample of interest are available. Larger amonnts can be prodnced nsing the polymerase chain reaction (PCR) that is dependent on the availability of thermostable DNA polymerases, e.g., Taq polymerase, that is prodnced by the thermophilic bacterium Thermus aquaticus. It is purified in large quantities and is available commercially. 

These extremophiles are not typical of the life forms that make up the majority normally encountered by biological engineers. But, they serve as a raninder that life can exist in very harsh conditions, and that there are organisms with competitive advantage to inhabit very unlikely spots. In fact, it is an enzyme from one of these extranophiles Thermus aquaticus, living in the hot springs of Yellowstone National Park in the United States) that is used in the polymerase chain reaction (PGR) to amplify the small samples of DNA (see Section 5.3.4). 

Nelson RM, Long GL (1989) A general method of site-specific mutagenesis using a modification of the Thermus aquaticus polymerase chain reaction. Anal Biochem 180 147-151... 

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