Enzymes Taq polymerase

Enzyme Taq polymerase (or some other enzyme) adds new deoxyribonucleotides during strand elongation. Taq is added to the assay at 1 unit per 50 qL of reaction mixture. 

It is practical to use PCR reagents in 10 x working solutions that will be diluted by a factor of ten for final concentration in the PCR samples. An exception is the thermostable enzyme Taq polymerase, which is stored at a much higher concentration. All reagents are available from numerous companies. 

There is another important factor that could affect the amphfication step that should be taken into consideration in the analysis and leads also to false negative results This is the presence of different inhibitors of the enzyme Taq polymerase, used in the PCR reaction, such as proteins, fats, polysaccharides, polyphenols and other compoimds that may be present in DNA extracted from many different food matrices [14-16] In order to avoid false negative results and to evaluate the possibihty to amphfy the extracted DNA, it is necessary to first amphfy a reference gene present in the genome of the analyzed species [17]. 

Extremophiles are a source of evolution-hardened enzymes and principles, such as thermostable enzymes (Taq polymerase for PCR) and cold-adapted enzymes with applications in the detergent and food industries, fine chemicals production, bioremediation as well as broader applicable mechanisms for then- high catalytic efficiency based on stractural X-ray studies. 

Molecular methods used to uncover mutations are subject to several variables. The anticoagulants used for blood collection can affect digestion with restriction enzymes and amplification reactions. The type of detergent used in cell lysis can affect amplification of DNA by inhibiting the DNA-amplifying enzyme such as the taq polymerase used in the polymerase chain reaction (116). The control of contamination is crucial in ensuring the quality of results obtained by molecular analysis (117). 

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... 

Make a PCR master mix as described containing excess amonnt of dNTP and Taq polymerase enzyme. For instance, we fonnd that if mnltiplexing for amplification of promoters of five genes has to be performed at the same time, 0.2 pL 10 pM dNTP and 1.25 U Taq DNA polymerase (Invitrogen) for each gene in the reaction are reqnired. For each gene, consider lOpL of this PCR master mix. 

Preparation of Taq polymerase-Taq-start antibody complex 1 mix 1 1 Taq-start antibody Taq polymerase enzyme and incubate at 22°C for 10 min. 

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). 

A sample of double-stranded DNA is denatured. One of the resulting single strands is used as a template to direct the synthesis of a complementary strand of radioactive DNA using a suitable DNA polymerase. The "Klenow fragment" of E. coli, DNA polymerase I, reverse transcriptase from a retrovirus, bacteriophage T7 DNA polymerase, Taq polymerase, and specially engineered enzymes produced from cloned genes have all been used. 

Other Class A polymerases. The Thermus aquati-cus (Taq) polymerase is best known for its widespread use in the polymerase chain reaction (PCR Fig. 5-47). Like E. coli I the enzyme is a large multidomain protein. The structure of the catalytic domains of the two enzymes are nearly identical, but the Taq polymerase has poor 3 -5 editing activity.276 The enzyme has been carefully engineered to improve its characteristics for use in the PCR reaction.277... 

The association of Taq polymerase with the ends of the PCR product stencally hindering the restriction enzyme... 

Figure 6.6. Amplification of DNA by PCR. Target DNA sequence from a complex genome can be amplified by heat denaturation, providing appropriate conditions for the enzyme (Taq DNA polymerase) that allow it to cause exponential amplification of a particular DNA segment. Among components besides the enzyme that are essential for amplification process are oligonucleotide primers in opposite orientation to each other, shown by dotted arrows, deoxynucleotide triphosphates (dNTPs), Mg2+, and buffer. A 30-cycle amplification leads to a many-million-fold amplification of the discrete DNA segment, flanked by oligonucleotide primer sequences. (Reproduced from Short Protocols in Molecular Biology, 4th ed., F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds., Wiley, New York, 1999, p. 15-1.)...

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