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

• Published in: Molecular biotechnology Vol. 61; no. 12; pp. 938 - 944
• Main Authors: Lu, Shuhong, Zhang, Xuesong, Chen, Kaiying, Xie, Bingbin, Shan, Dapeng, Shen, Yulong, Li, Zhuo
• Format: Journal Article
• Language: English
• Published: New York Springer US 01.12.2019; Springer Nature B.V
• Subjects: Amplification; Binding; Biochemistry; Biological Techniques; Biotechnology; Cell Biology; Chemistry; Chemistry and Materials Science; Denaturation; Deoxyribonucleic acid; DNA; DNA polymerase; DNA Primers - chemistry; DNA-Binding Proteins - genetics; DNA-Binding Proteins - metabolism; DNA-directed DNA polymerase; DNA-Directed DNA Polymerase - metabolism; E coli; Escherichia coli; Escherichia coli - enzymology; Escherichia coli - genetics; Exodeoxyribonucleases - chemistry; Exodeoxyribonucleases - genetics; Exodeoxyribonucleases - metabolism; Exonuclease; Hot Temperature; Human Genetics; Mutation; Nuclease; Original Paper; Polymerase chain reaction; Polymerase Chain Reaction - methods; Protein Science; Room temperature; Temperature; Polymerase chain reaction; Hot start; Exonuclease III
• ISSN: 1073-6085; 1559-0305
• DOI: 10.1007/s12033-019-00216-z

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.