Polymerase chain reaction thermostable polymerases

While many diseases have long been known to result from alterations in an individual s DNA, tools for the detection of genetic mutations have only recently become widely available. These techniques rely upon the catalytic efficiency and specificity of enzyme catalysts. For example, the polymerase chain reaction (PCR) relies upon the ability of enzymes to serve as catalytic amplifiers to analyze the DNA present in biologic and forensic samples. In the PCR technique, a thermostable DNA polymerase, directed by appropriate oligonucleotide primers, produces thousands of copies of a sample of DNA that was present initially at levels too low for direct detection. 

Polymerase chain reaction (PCR) The process by which a specific sequence of DNA can be amplified (copied many times) in vitro. It requires a pair of primers and template DNA, thermostable DNA polymerase (e.g. Taq polymerase), deoxynucleotide triphosphates and a thermocycler. The process can amplify large... 

The polymerase chain reaction (PCR) is an important procedure in genetic engineering that allows any DNA segment to be replicated (amplified) without the need for restriction enzymes, vectors, or host cells (see p. 258). However, the nucleotide sequence of the segment has to be known. Two oligonucleotides (primers) are needed, which each hybridize with one of the strands at each end of the DNA segment to be amplified also needed are sufficient quantities of the four deoxyribonucleo-side triphosphates and a special heat-tolerant DNA polymerase. The primers are produced by chemical synthesis, and the polymerase is obtained from thermostable bacteria. 

Polymerase Chain Reaction DNA Amplification with a Thermostable Enzyme... 

Fig. 27. The polymerase chain reaction. DNA amplification with a thermostable DNA polymerase enzyme.

The polymerase chain reaction utilizes a thermostable DNA polymerase to amplify DNA through a series of temperature cycle steps. The key to the specificity of the reaction is the selection of oligonucleotide primers that hybridize to the opposite strands of the DNA being tested, about 400-2000 bp apart. If the sequence of the primers is unique within the genome, and the primers hybridize to the target DNA at a high enough temperature to avoid close matches (various... 

Polymerase chain reaction (PCR) is a method for amplifying DNA from a small amount of DNA catalyzed by thermostable DNA polymerase under appropriate reaction conditions with a pair of primers (oligonucleotides) that are complementary to DNA. K. Mullis, who invented the technique in the 1980s, was awarded a Nobel prize in 1994. Since its invention, various refinements and modifications have been described, and several review articles and books have been written on the subject [17-20]. 

PCR The polymerase chain reaction that utilizes a thermostable DNA polymerase to make many copies of the same piece of DNA. This allows specific amplification of rare pieces of DNA. 

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. 

Polymerase chain reaction (PCR) Target DNA polymerase (thermostable) Yes... 

When the amount of target nucleic acid is increased by synthetic in vitro methods, target amphfication is said to occur. The polymerase chain reaction (PCR) is the best known and most widely applied of the target amplification methods. Because of the commercial availability of thermostable DNA polymerases, kits, and instrumentation, this method has been widely adopted in research and is also rou-tmely used in the clmical laboratory. 

The evolution of a transaminase from Arthrohacter citreus to a thermostable transaminase with increased specific activity and decreased inhibition by the amine product was accomplished using error prone polymerase chain reaction (PCR) [64] The reaction of substituted tetralone 75 and isopropylamine to produce substituted (S) aminotetralin 76 was carried out at greater than 50 °C to facilitate the removal of the acetone by product and drive reaction equilibrium (Figure 14.43). 

Olive, D. M., Detection of enterotoxigenic Escherichia coli after polymerase chain reaction amplification with a thermostable DNA polymerase. J. Clin. Microbiol. 27, 261-265 (1989). 

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... 

Describe the polymerase chain reaction. Explain the roles of the primer and thermostable DNA polymerase in amplifying the target DNA sequence. Appreciate the practical applications of the PCR technique. 

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. 

More recently, DNA amplification by the polymerase chain reaction with thermostable Taq polymerase (Saiki et al. 1988) has replaced cloning to provide substrate for direct analysis of variation. Once a segment of DNA is amplified, variation can be revealed by restriction enzyme digestion (either 4- or 6-cutter) followed by gel electrophoresis and ethidium bromide staining, or by DNA sequencing. 

Development of the polymerase chain reaction has been to a large extent facilitated by the availability of thermostable DNA polymerases, enzymes that catalyze the elongation of the primer DNA strand. So far, this is the largest application of thermophilic enzymes. 

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