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Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.

ABSTRACT: DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase) is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world. In this study, the gene fragments of the family A DNA polymerases were amplified by PCR from the DNAs from microorganisms within environmental soil samples, using a primer set for the two conserved regions. The corresponding region of the pol gene for Taq polymerase was substituted with the amplified gene fragments, and various chimeric DNA polymerases were prepared. Based on the properties of these chimeric enzymes and their sequences, two residues, E742 and A743, in Taq polymerase were found to be critical for its elongation ability. Taq polymerases with mutations at 742 and 743 actually showed higher DNA affinity and faster primer extension ability. These factors also affected the PCR performance of the DNA polymerase, and improved PCR results were observed with the mutant Taq polymerase.

SUBMITTER: Yamagami T

PROVIDER: S-EPMC4153296 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.

Yamagami Takeshi T Ishino Sonoko S Kawarabayasi Yutaka Y Ishino Yoshizumi Y

Frontiers in microbiology 20140903

DNA polymerases are widely used for DNA manipulation in vitro, including DNA cloning, sequencing, DNA labeling, mutagenesis, and other experiments. Thermostable DNA polymerases are especially useful and became quite valuable after the development of PCR technology. A DNA polymerase from Thermus aquaticus (Taq polymerase) is the most famous DNA polymerase as a PCR enzyme, and has been widely used all over the world. In this study, the gene fragments of the family A DNA polymerases were amplified ...[more]

PMID: 25232352

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Project description:PurposeTo investigate the potential for a suture tape-reinforcement technique to prevent elongation with repetitive use of a human dermal (HD) allograft traditionally used for superior capsule reconstruction in a biomechanical model.MethodsUsing 8 scapulae and humeri Sawbones models, the standard superior capsule reconstruction was performed using 8 HD allografts. Four grafts were tested in the native state, whereas 4 were tested using reinforcement. Allografts were reinforced using suture tape in a running 360° fashion around the allograft borders, maintaining 5 mm of space from the edges. Allografts were measured pre- and postdynamic testing for length, width, and thickness. All specimens were affixed to a materials testing machine that allowed for allograft orientation in a longitudinal plane throughout testing. Specimens were preloaded to 10 N and then cyclically loaded to 100 N at a rate of 15 mm/s for 30 cycles.ResultsAfter dynamic, cyclic loading, suture tape-reinforced allografts experienced a significantly smaller percent change in anterior length (6.36% vs 14.50%, P = .013), posterior length (6.00% vs 13.68%, P = .002), medial width (5.80% vs 21.05%, P = .001), lateral width (5.45% vs 19.29%, P < .001), medial thickness (4.38% vs 17.93%, P = .005), central thickness (7.03% vs 16.11%, P = .026), and lateral thickness (4.55% vs 20.80%, P < .001). Linear stiffness values obtained for suture tape -reinforced allografts were significantly greater than those for native allografts when measured at cycles 1 (21.18 ± 1.03 N/mm vs 17.69 ± 1.91 N/mm, P = .02), 15 (29.90 ± 1.45 vs 24.93 ± 2.79, P = .03), and 30 (32.13 ± 1.98 N/mm vs 25.72 ± 3.01 N/mm, P = .01) of dynamic testing.ConclusionsThe suture tape-reinforcement technique described decreased HD allograft elongation, maintained graft thickness, and improved linear stiffness values following uniplanar cyclic loading in a biomechanical Sawbones model.Clinical relevanceSuture tape reinforcement of the HD allograft could serve as one strategy to overcome the issue of allograft elongation with time following superior capsule reconstruction.

Dataset Information

Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.

Publications

Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.

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