Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site.
Enhancement of Polymerase Activity of the Large Fragment in DNA Polymerase I from Geobacillus stearothermophilus by Site-Directed Mutagenesis at the Active Site.
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通过活性位点定点诱变增强嗜热脂肪地芽孢杆菌 DNA 聚合酶 I 大片段的聚合酶活性
DOI：
10.1155/2016/2906484
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发表时间：
2016
影响因子：
--
通讯作者：
Li S
中科院分区：
文献类型：
--
作者：
Ma Y;Zhang B;Wang M;Ou Y;Wang J;Li S
嗜热脂肪土芽孢杆菌GIM1.543的DNA聚合酶Ⅰ（Bst DNA聚合酶）大片段具有较高的热稳定性和聚合酶活性，但不具有5′-3′核酸外切酶活性。Bst DNA聚合酶应用于等温多重自匹配启动扩增（IMSA），可高选择性地扩增目的序列，广泛应用于人类流行病的快速检测。但商业化的Bst DNA聚合酶的详细信息未公开，且受专利保护较好，这使得商业化试剂盒价格偏高。在本研究中，野生型Bst DNA聚合酶（WT）和用于提高DNA聚合效率的置换突变在E.杆菌Bst DNA聚合酶活性位点上的4个保守残基（Gly 310、Arg 412、Lys 416和Asp 540）的定点取代影响dNTPs的聚合效率。用丙氨酸或亮氨酸取代Gly 310残基和用谷氨酸取代Asp 540残基可提高聚合酶活性的效率。利用所有聚合效率较高的突变株，以相对较短的时间完成EV 71相关手足口病（HFMD）的IMSA快速检测，适用于农村和欠发达地区的基层诊断。
The large fragment of DNA polymerase I from Geobacillus stearothermophilus GIM1.543 (Bst DNA polymerase) with 5′-3′ DNA polymerase activity while in absence of 5′-3′ exonuclease activity possesses high thermal stability and polymerase activity. Bst DNA polymerase was employed in isothermal multiple self-matching initiated amplification (IMSA) which amplified the interest sequence with high selectivity and was widely applied in the rapid detection of human epidemic diseases. However, the detailed information of commercial Bst DNA polymerase is unpublished and well protected by patents, which makes the high price of commercial kits. In this study, wild-type Bst DNA polymerase (WT) and substitution mutations for improving the efficiency of DNA polymerization were expressed and purified in E. coli. Site-directed substitutions of four conserved residues (Gly310, Arg412, Lys416, and Asp540) in the activity site of Bst DNA polymerase influenced efficiency of polymerizing dNTPs. The substitution of residue Gly310 by alanine or leucine and residue Asp540 by glutamic acid increased the efficiency of polymerase activity. All mutants with higher polymerizing efficiency were employed to complete the rapid detection of EV71-associated hand, foot, and mouth disease (HFMD) by IMSA approach with relatively shorter period which is suitable for the primary diagnostics setting in rural and underdeveloped areas.
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中国的手足口病：关键社区规模和空间疫苗接种策略
影响因子：
4.6
作者：
Van Boeckel TP;Takahashi S;Liao Q;Xing W;Lai S;Hsiao V;Liu F;Zheng Y;Chang Z;Yuan C;Metcalf CJ;Yu H;Grenfell BT
通讯作者：
Grenfell BT
DOI：
10.4103/1755-6783.92879
发表时间：
2012-01-01
期刊：
ANNALS OF TROPICAL MEDICINE AND PUBLIC HEALTH
影响因子：
--
作者：
Dias, Edwin;Dias, Meena
通讯作者：
Dias, Meena
影响因子：
3.7
作者：
Chen, Ping;Liu, Zhongfa;Liu, Shujun;Xie, Zhiliang;Aimiuwu, Josephine;Pang, Jiuxia;Klisovic, Rebecca;Blum, William;Grever, Michael R.;Marcucci, Guido;Chan, Kenneth K.
通讯作者：
Chan, Kenneth K.
影响因子：
4.8
作者：
Xu, Y;Potapova, O;Joyce, CM
通讯作者：
Joyce, CM
基于混合染料的无标记灵敏双荧光用于核酸等温多重自匹配启动扩增的产品检测
影响因子：
7.4
作者：
Ding, Xiong;Wu, Wenshuai;Mu, Ying
通讯作者：
Mu, Ying