Thermostable DNA 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. 

Saiki, R. K., etal. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239,487-491. 

Taq polymerase is a thermostable DNA polymerase which was originally isolated from the bacterium Thermus aquaticus, which lives in hot springs. 

Automated programmable instruments that can carry out the repeated thermal cycles necessary for PCR and that can accommodate multiple samples simultaneously are now widely available. The procedure is usually performed with thermostable DNA polymerases. PCR is widely used to facilitate detection of minute amounts of viral DNA. The technique can also be used to detect specific point mutations, provided the approximate site of mutation is known. One limiting feature of this approach arises from the fact that the bacterial polymerases frequently make errors when synthesizing new strands and so can introduce mutations that are not present in the original sample. 

Several of the enzymes involved in the processes of repheating, transcription and reverse transcription are available commercially and are used by molecular biologists in the manipulation of nucleic acids. One of the most important of these is Taq polymerase (Taq), which is a thermostable DNA polymerase named after the thermophihe bacterium Thermus aquaticus from which it was originally isolated. This enzyme is especially important, as it is central to the technique known as PCR, which allows sophisticated, targeted in vitro amplification and manipulation of sections of DNA or RNA. DNA... 

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... 

A high profile example of biocatalysis patenting is that of Taq polymerase, the thermostable DNA polymerase enzyme isolated from the thermophilic microorganism... 

The PCR is a three-step cyclic process that repeatedly duplicates a specific DNA sequence, contained between two oligonucleotide sequences called primers (154,155). The two primers form the ends of the sequence of DNA to be amplified and are normally referred to as the forward and reverse primers. The forward primer is complementary to the sense strand of the DNA template and is extended 5 to 3 along the DNA by DNA polymerase enzyme (Fig. 27). The reverse primer is complementary to the antisense strand of the DNA template and is normally situated 200-500 base pairs downstream from the forward primer, although much longer sequences (up to 50 kbase) can now be amplified by PCR. The process employs a thermostable DNA polymerase enzyme (such as the Taq polymerase from Thermus aqualicus BM) extracted from bacteria found in hot water sources, such as thermal pools or deep-water vents. These enzymes are not destroyed by repeated incubation at 94 °C, the temperature at which all double stranded DNA denatures or melts to its two separate strands (155). 

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

DNA synthesis (step ) is catalyzed by the thermostable DNA polymerase (still present). 

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]. 

Various thermostable DNA polymerases are available commercially from various vendors. The first thermostable DNA polymerase enzyme that became available commercially was Taq DNA polymerase, isolated from T. aquaticus. This enzyme lacks 3 -to-5 proofreading exonuclease activity and hence has a higher error rate than those enzymes that possess this proofreading activity, such as pfu enzyme. For most routine purposes any thermostable DNA polymerase should suffice, irrespective of its error rate during PCR. 

Haff LA, Smirnov IP. Single-nucleotide polymorphism identification assays using a thermostable DNA polymerase and delayed extraction MALDI-TOF mass spectrometry. Genome Res 1997 7 378-388. 

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. 

PCR reactions should be performed using a thermostable DNA polymerase with proofreading function, i.e., Vent, New England Biolabs Beverly, MA, or Pfu, Stratagene, La Jolla, CA. The protocol used will vary with the source of target DNA and enzyme used. 

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. 

PCR reactions include the cyclical use of high temperatures which can lead to the denaturation of thermolabile enzymes for this reason a thermostable DNA polymerase, Taq polymerase, is typically used. 

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