Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.
Mutant Taq DNA polymerases with improved elongation ability as a useful reagent for genetic engineering.
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DOI：
10.3389/fmicb.2014.00461
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发表时间：
2014
影响因子：
5.2
通讯作者：
Ishino Y
中科院分区：
文献类型：
--
作者：
Yamagami T;Ishino S;Kawarabayasi Y;Ishino Y
DNA聚合酶广泛用于体外DNA操作，包括DNA克隆、测序、DNA标记、诱变和其他实验。热稳定性 DNA 聚合酶特别有用，并且在 PCR 技术发展后变得非常有价值。来自水生栖热菌的 DNA 聚合酶（Taq 聚合酶）是最著名的 PCR 酶 DNA 聚合酶，已在世界各地广泛使用。在这项研究中，使用两个保守区域的引物组，通过 PCR 从环境土壤样品中微生物的 DNA 中扩增 A 家族 DNA 聚合酶的基因片段。用扩增的基因片段替换Taq聚合酶的pol基因的相应区域，并制备各种嵌合DNA聚合酶。根据这些嵌合酶的特性及其序列，发现 Taq 聚合酶中的两个残基 E742 和 A743 对于其延伸能力至关重要。在 742 和 743 处突变的 Taq 聚合酶实际上表现出更高的 DNA 亲和力和更快的引物延伸能力。这些因素也影响 DNA 聚合酶的 PCR 性能，并且使用突变型 Taq 聚合酶观察到 PCR 结果有所改善。
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.
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影响因子：
46.9
作者：
Ghadessy, FJ;Ramsay, N;Holliger, P
通讯作者：
Holliger, P
影响因子：
14.9
作者：
BRAITHWAITE, DK;ITO, J
通讯作者：
ITO, J
影响因子：
1.1
作者：
Matsukawa, Hiroaki;Yamagami, Takeshi;Ishino, Yoshizumi
通讯作者：
Ishino, Yoshizumi
影响因子：
3.2
作者：
Cann, IKO;Ishino, S;Ishino, Y
通讯作者：
Ishino, Y
DOI：
10.1073/pnas.91.6.2216
发表时间：
1994-03-15
影响因子：
11.1
作者：
BARNES, WM
通讯作者：
BARNES, WM