An Alternative Hot Start PCR Method Using a Nuclease-Deficient ExoIII from Escherichia coli
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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.
KeywordsPolymerase chain reaction Hot start Exonuclease III
This work was supported by grants from the National Natural Science Foundation of China to Z.L. (31500027), and the Startup Funding of TIO to Z.L.
- 28.Zhong, Y., Huang, L., Zhang, Z., Xiong, Y., Sun, L., & Weng, J. (2016). Enhancing the specificity of polymerase chain reaction by graphene oxide through surface modification: zwitterionic polymer is superior to other polymers with different charges. International Journal of Nanomedicine, 11, 5989–6002.CrossRefGoogle Scholar