Thermostable DNA polymerases, use

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... 

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 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. 

A method for synthesizing and amplifying a specific DNA sequence based on the use of specific oligonucleotide primers and unique DNA polymerases such as the thermostable DNA polymerase from Thermus aquaticus... 

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. 

In addition to predictable improvements in speed, simplicity, and automation of PCR diagnostic tests, there are several improvements in the procedure that will find use in the reference laboratory prior to their incorporation into kits. First, it has been shown that considerable improvement in target specificity and concomitant improvements in specific product yield and test sensitivity can be achieved by adding or activating the thermostable DNA polymerase before the first cycle at a temperature at or above the primer-annealing temperature (FI). This activation can be accomplished in several ways (1) by adding the enzyme after the reaction has reached an elevated temperature and (2) by sequestering the primers by... 

Prior to the use of thermostable DNA polymerases in PCR, scientists had to supplement the reaction laboriously with new enzyme (such as Klenow or T4 DNA polymerase) after each denaturation cycle at 95°C. The identification of thermostable DNA polymerases revolutionized PCR because of their ability to withstand high denaturation temperatures. The use of thermostable DNA polymerases also allowed higher annealing temperatures, which improved the stringency of primer annealing. 

TABLE 2.4 Characteristics of Commonly Used Thermostable DNA Polymerases... 

Today, hot-start PCR is most common technique used to reduce nonspecific amplification due to the assembly of amplification reactions at room temperature or on ice. At room temperature, PCR primers can anneal to template sequences that are not totally complementary. Since thermostable DNA polymerases have activity at these low temperatures, the polymerase can extend misannealed primers. This newly synthesized DNA is 100% complementary to the DNA template, allowing primer extension and the polymerization of undesired amplification products. However, if the reaction is heated to temperatures above 60°C before polymerization begins, the stringency of primer annealing is increased, and subsequent synthesis of undesired PCR products is avoided or reduced. 

PCR has a great potential for differentiation between phylogenetically closely related species. Also, the entire PCR chemistry and recombinant thermostable DNA polymerase can be synthesized and manufactured in unlimited amounts and constant quality (Holzhauser et al., 2006). This is not always the case regarding ELISA tests, for which the quality may vary considerably between batches, particularly for tests based on polyclonal antibodies. In addition, the use of an amplification control for each individual sample in the PCR vessel enables the measurement of potential adverse effects on the PCR reaction, in contrast to the reaction between target, antibodies, and conjugate in an ELISA reaction. 

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