一种使用大肠杆菌无核酸酶 ExoIII 的热启动 PCR 替代方法。

An Alternative Hot Start PCR Method Using a Nuclease-Deficient ExoIII from Escherichia coli.

机构信息

College of Ocean and Earth Sciences, Xiamen University, Xiamen, 361102, Fujian, China.

Third Institute of Oceanography, Ministry of Natural Resources of China, Xiamen, 361005, Fujian, China.

出版信息

Mol Biotechnol. 2019 Dec;61(12):938-944. doi: 10.1007/s12033-019-00216-z.

DOI:10.1007/s12033-019-00216-z

PMID:31641996

Abstract

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.

摘要

热启动聚合酶链反应（Hot Start PCR）旨在通过在室温下阻止 DNA 聚合酶延伸来减少非特异性扩增，直到达到所需温度。在这项研究中，我们研究了一种新的热启动 PCR 方法，该方法通过取代 DNA 结合口袋和催化中心中的残基来使用改良的大肠杆菌核酸外切酶 III（EcoExoIIIM）。结果表明，添加 EcoExoIIIM 可显著促进 PCR 扩增产量和特异性。我们假设 EcoExoIIIM 通过与引物标记的模板结合来防止引物在室温下的非特异性结合，然后在第一个 PCR 循环之前通过热变性将其从 DNA 上释放出来。通过这种机制，通过减少室温下的非特异性延伸来增强 PCR。