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Molecular Biotechnology

, Volume 61, Issue 12, pp 938–944 | Cite as

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

  • Shuhong Lu
  • Xuesong Zhang
  • Kaiying Chen
  • Bingbin Xie
  • Dapeng Shan
  • Yulong Shen
  • Zhuo LiEmail author
Original paper
  • 27 Downloads

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.

Keywords

Polymerase chain reaction Hot start Exonuclease III 

Notes

Acknowledgements

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.

Supplementary material

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Supplementary material 1 (TIFF 959 kb)
12033_2019_216_MOESM2_ESM.tif (202 kb)
Supplementary material 2 (TIFF 201 kb)
12033_2019_216_MOESM3_ESM.docx (16 kb)
Supplementary material 3 (DOCX 16 kb)

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.College of Ocean and Earth SciencesXiamen UniversityXiamenChina
  2. 2.Third Institute of Oceanography, Ministry of Natural Resources of ChinaXiamenChina
  3. 3.State Key Laboratory of Microbial TechnologyShandong UniversityQingdaoChina

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