DNA polymerase 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. 

Korolev, S., Nayal, M., Barnes, W. M., Di Cera, E., and Waksman, G. (1995). Crystal structure of the large fragment of Thermus aquaticus DNA polymerase I at 2.5 A resolution Structural basis for thermostability. Proc. Natl. Acad. Sd. USA 92, 9264-9268. 

Suzuki, M., Baskin, D., Hood, L., and Loeb, L. A. (1996). Random mutagenesis of Thermus aquaticus DNA polymerase I Concordance of immutable sites in rhtowith the crystal structure. Proc. Natl. Acad. Sci. USA 93, 9670-9675. 

Xia et have developed an activity-based selection method to evolve DNA polymerases with RNA polymerase activity. Stoffel fragment (SF) of Thermus aquaticus DNA polymerase is displayed on a filamentous phage by fusing it to a coat protein, and the substrate DNA template/primer duplexes are attached to other adjacent coat proteins. Phage particles displaying SF polymerases, which extend the attached primer by incorporating ribonucleoside triphosphates and biotinylated UTP, are immobilized on streptavidin-coated beads. After four rounds of screening a SF library, three mutants were isolated and shown to incorporate ribonucleoside triphosphates virtually as efficiently as the wild-type enzyme incorporates dNTP substrates. 

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

Eckert KA, Kunkel TA (1990). High fidelity DNA synthesis hy the Thermus aquaticus DNA polymerase. Nucleic Acids Res., 18 3739-3744. 

Oktem, H. A., Bayramoglu, G., Ozalp, V. C., Arica, M. Y. (2007). Single-step purification of recombinant Thermus aquaticus DNA polymerase using DNA-aptamer immobilized novel affinity magnetic beads. Biotechnol Prog 23, 146-154. 

Tindall KR, Kunkel TA (1988) Fidelity of DNA synthesis by the Thermus aquaticus DNA polymerase. Biochemistry 27 6008-6013... 

Eorterre P. A hot topic the origin of hyperthermophiles. CelL 1996 65 789-92. Korolev S, Nayal M, Barnes WM, Di Cerca E, Waksman G. Crystal structure of a large fragment of Thermus aquaticus DNA polymerase at 2.5A resolution structural basis for thermostability. Proc Nad Acad Sci USA. 1995 92 9264-8. 

Jones MD, Foulkes NS (1989) Reverse transcription of mRNA by Thermus aquaticus DNA polymerase. Nucleic Adds Res 17(20) 8387-8388... 

Examples of enzymes suitable for use in molecular biology are the DNA polymerases from Thermococcus //torafa[74] and P. fiiriosus 15 With respect to fidelity of the polymerase reaction both enzymes surpass the DNA polymerase from Thermus aquaticus ( Taq polymerase ) currently used for DNA sequencing and DNA amplification (via PCR reaction). 

Key elements of dependent promotors are the TATA box, with the consensus sequence TATAAT 10 bp upstream from the transcription initiation site (pos. -10), and the sequence TTGACA at the position -35 (Fig. 1.18). Both sequences are necessary for the recognition of the promotor by a70. Structural analysis of the Thermus aquaticus RNA polymerase holoenzyme bound to DNA shows that all sequence-specific contacts with the core promotor are mediated by the sigma subunit (Murakami et al. 2002). This archaeal RNA polymerase has a subunit structure (a ji/i oxr) similar to that of the eubacterial enzyme. The intervening sequences, as well as other upstream sequences, can also influence the efficiency of transcription initiation. It is not possible to define consensus sequences at these positions. An optimal dependent promotor can be defined as a sequence with the - 35 hexamer as well as the -10 hexamer 17 bp away. The latter lies 7 bp upstream from the transcription initiation site. 

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... 

PGR amplification of a DNA sequence is faciHtated by the use of a heat-stable DNA polymerase, Taq polymerase (TM), derived from the thermostable bacterium Thermus aquaticus. The thermostable polymerase allows the repeated steps of strand separation, primer annealing, and DNA synthesis to be carried out ia a single reactioa mixture where the temperature is cycled automatically. Each cycle coasists of a high temperature step to deaature the template strands, a lower temperature annealing of the primer and template, and a higher temperature synthesis step. AH components of the reaction are present ia the same tube. 

Taq DNA polymerase from Thermus aquaticus) has made it unnecessary to add fresh enzyme for each round of synthesis. Because the amount of target DNA theoretically doubles each round, 25 rounds would increase its concentration about 33 million times. In practice, the increase is actually more like a million times, which is more than ample for gene isolation. Thus, starting with a tiny... 

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

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... 

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