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.

中文翻译：

---

一种使用大肠杆菌中核酸酶缺陷型ExoIII的替代热启动PCR方法。

热启动聚合酶链反应（Hot Start PCR）旨在通过在室温下阻断DNA聚合酶的延伸直至达到所需温度来减少脱靶扩增。在这项研究中，我们研究了一种热启动PCR的新方法，该方法使用经过修饰的大肠杆菌核酸外切酶III（EcoExoIIIM）取代了DNA结合袋和催化中心中的残基。结果表明，通过添加EcoExoIIIM可以显着提高PCR扩增的产量和特异性。我们假设在室温下引物的非特异性结合可以通过EcoExoIIIM结合引发的模板来防止，该模板随后在第一个PCR循环之前通过热变性从DNA中释放出来。通过这种机制，可以通过减少室温下的脱靶延伸来增强PCR。