Vent DNA polymerase

An especially intriguing pair of products obtained from marine organisms in recent years are Vent and Deep Vent DNA polymerase. These products are used in DNA research studies. Their special feature is that they are at least 10 times as efficient as other similar products in polymerase chain reactions because they can tolerate temperatures just below the boiling point of water, a characteristic that comparable research tools lack. Vent and Deep Vent DNA polymerases are obtained from the bacterium Thermococcus litoralis, which is found around deep-sea hydrothermal vents at the bottom of the ocean. 

Corresponding lOx polymerase buffer with MgCl2 (for Taq DNA polymerase) or MgSC>4 (for Vent DNA polymerase)... 

Kong, H., R.B. Kucera and W.E. Jack 1993. Characterization of a DNA polymerase from the hyperthermophile archaea Thermococcus litoralis Vent DNA polymerase, steady state kinetics, thermal stability, processivity, strand displacement, and exonuclease activities. J. Biol. Chem. 268 1965-1975. 

Byrappa S, et al. (1995). A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase. Genome Res., 5 404 07. 

As for polymerization and processivity measurements, SD activity is also influenced by the reaction conditions employed. In particular, archaeal Family B DNA polymerases exhibit SD in a temperature dependent manner. For example, primer extension by Vent DNA polymerase T. litoralis) is completely blocked by downstream primers at 55°, but increasingly larger SD products are observed as the reaction temperature is raised from 63° to 12° Greater SD activity has also been noted for exo versions of Vent and 9°N-7 DNA polymerases, possibly because the substrate spends less time in the exonuclease active site. ... 

DNA polymerase A wide range of DNA polymerases is available for PCR. A cloned heat-stable DNA polymerase from Thermus aquaticus Taq DNA polymerase) is the most commonly used. Other enzymes such as DNA polymerase from Pyrococcus furiosus Pfu DNA polymerase) or Thermoccocus litoralis (Vent DNA polymerase. New England Biolabs) have been reported to have a lower error rate... 

For final assembly PCR, we recommend using AmpliTaq DNA polymerase which in our hands works better than Deep Vent DNA polymerase. 

X ThermoPol reaction buffer 100 mM KCl, 100 mM (NRiIjSO, 200 mMTris-HCl pH 8.8,20 mM MgSO, 1% Triton X-100 (supplied with Deep Vent DNA polymerase)... 

Of the modifications to purine analogues, the majority of publications involve guanine derivatives, in particular guanine lesions. For adenosine analogues, incorporation of V -oxidised adenosine into a DNA duplex resulted in a decrease in thermal stability, but translesion bypass using Klenow fragment (exo ) or Vent DNA polymerase (exo ) resulted in incorporation of dTMP. ... 

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). 

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. 

DNA polymerase is required for synthesis. There are several different polymerases available, each with its 6Wn characteristics that will affect its efficacy in the PCR. Since many labs have studied the properties of the different polymerase in current use, much is known about their efficiencies in PCR and rate of base misincorporation (Table 7.1). The thermostable polymerases (Tag, Vent) are preferred in PCR over the heat-labile enzymes... 

The DNA polymerases of T. littoralis and P. furiosus have been marketed for use in DNA amplification by the polymerase chain reaction (PCR) method as the Vent and pfu DNA polymerases, respectively. These enzymes are more accurate in vitro than the Thermus aquaticus (Taq) DNA polymerase, both in classical fidelity tests [132] and in PCR [133,134]. Indeed, they have an associated 3 to 5 exonuclease activity involved in proof-reading, whereas the Taq polymerase is devoid of such activity. 

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