DNA polymerase is widely used for manipulating DNA in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. thermostable DNA polymerase is very useful and can be quite worthwhile after the development of PCR technology. A DNA polymerases from Thermus aquaticus (Taq polymerase) is the most well-known DNA polymerase as PCR enzyme, and has been widely used around the world. In this study, the gene fragment of family A DNA polymerase was amplified by PCR from the DNA of microorganisms in environmental soil samples, using a set of primers for two conserved regions.
The corresponding region of the pol gene for Taq polymerase gene fragment was replaced with reinforced, and a variety of chimeric DNA polymerases prepared. Based on these properties chimeric enzymes and their sequence, two residues, E742 and A743, in the Taq polymerase was found to be important for the ability of elongation. Taq polymerase with mutations in the 742 and 743 actually showed a higher affinity DNA and primer extension capabilities faster. These factors also affect the performance of PCR on DNA polymerase, and the PCR yield improvement was observed with Taq polymerase mutants. Fast-cycling PCR formulations, protocols, and instruments have been developed to address the need to improve throughput and shorter turn-around times for PCR-based tests.
Although the run time can be cut by up to 50% shorter cycle times have been correlated with a lower detection sensitivity and increased variability. To solve this problem, we apply the self compartmentalized Replication (CSR) to develop faster-cycling mutant Taq DNA polymerase. After five rounds of selection using a shorter PCR extension times, individual mutations identified in the fastest-cycling clones randomly combined using ligation-based multi-site mutagenesis.
The best performing combination mutants showed 35- 90-fold higher affinity (lower Kd) for excellent template and (2-fold) increase in the rate of extension moderate compared to wild-type Taq. Further characterization revealed that mutations selected CSR provides increased resistance to inhibitors, and in particular, enable direct amplification of up to 65% whole blood. We discuss the contribution of individual mutations for fast-cycling and blood-resistant phenotype.
Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.
The increase in the gap repair cloning in yeast: vector treatment gap with Taq DNA polymerase Avoid self-ligation of the vector.
Gap repair a fast and efficient method for the assembly of recombinant DNA molecules in Saccharomyces cerevisiae. This method produces a circular DNA molecule by homologous recombination between two or more linear DNA fragments, one of which is usually the vector carrying the replicative sequence and a selective marker.
This technique avoids laborious and expensive in vitro purification and DNA ligation. DNA repair machinery can also close and ligate linear vector by a mechanism other than homologous recombination, resulting in an empty vector. The frequency of adverse events can be reduced by removing the 5′-phosphate group using a phosphatase, which is the standard method used for in vitro ligation.
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). This product is supplied with 10x PCR reaction buffer, containing MgCl2, which produces a final Mg2+ concentration of 1.5 mM. Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends.Product Includes:Taq DNA Polymerase10x PCR Buffer with Mg2+5x Magic Enhancer10mM dNTP Mix
Description: Taq DNA Polymerase is a highly thermostable DNA Polymerase that catalyzes the 5’-3’ synthesis of DNA. This polymerase has 5’-3’ exonuclease activities, lacks 3’-5’ exonuclease activity, and produces 3’-dA-tailed amplicons. PCR products made with Taq can be used with TA cloning vectors.
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/μl)
Description: Intact Genomics Taq DNA Polymerase 2x Premix with Dye is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends. Intact Genomics Taq DNA Polymerase 2x Premix with Dye is ready to use, containing Taq DNA Polymerase, dNTPs, MgCl2, and stabilizers. It has been optimized for routine PCR applications.Product Includes:Taq DNA Polymerase 2x Premix with Dye5x Magic Enhancer
Description: Intact Genomics Taq DNA Polymerase 2x Premix with Dye is a thermostable DNA polymerase that possesses a 5´→3´ polymerase activity (1, 2) and a 5´ flap endonuclease activity (3, 4). Ideal for primary extension reaction with DNA fragments having dA overhang on 3’ ends. Intact Genomics Taq DNA Polymerase 2x Premix with Dye is ready to use, containing Taq DNA Polymerase, dNTPs, MgCl2, and stabilizers. It has been optimized for routine PCR applications.Product Includes:Taq DNA Polymerase 2x Premix with Dye5x Magic Enhancer
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/ul)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/ul)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/ul)
Description: The enzyme is supplemented with two inert gel tracking dyes (red and yellow),allowing direct loading of PCR products to agarose gels (1.0 U/ul)
However, phosphatase treatment is less effective for gap repair cloning rather than in vitro ligation, probably due to the ability of S. cerevisiae DNA repair machinery to efficiently repair the lost phosphate groups to allow religation. We have developed a more efficient method to prevent vector religation, the vector fragment by treatment with Taq DNA polymerase and dATP. It prevents the procedure recircularization vector almost completely, to facilitate the screening of recombinant clones correctly.
Jordan
