Improvement of φ29 DNA polymerase amplification performance by fusion of DNA binding motifs

Proc Natl Acad Sci U S A. 2010 Sep 21;107(38):16506-11. doi: 10.1073/pnas.1011428107. Epub 2010 Sep 7.

Abstract

Bacteriophage ϕ29 DNA polymerase is a unique enzyme endowed with two distinctive properties, high processivity and faithful polymerization coupled to strand displacement, that have led to the development of protocols to achieve isothermal amplification of limiting amounts of both circular plasmids and genomic DNA. To enhance the amplification efficiency of ϕ29 DNA polymerase, we have constructed chimerical DNA polymerases by fusing DNA binding domains to the C terminus of the polymerase. The results show that the addition of Helix-hairpin-Helix [(HhH)(2)] domains increases DNA binding of the hybrid polymerases without hindering their replication rate. In addition, the chimerical DNA polymerases display an improved and faithful multiply primed DNA amplification proficiency on both circular plasmids and genomic DNA and are unique ϕ29 DNA polymerase variants with enhanced amplification performance. The reported chimerical DNA polymerases will contribute to make ϕ29 DNA polymerase-based amplification technologies one of the most powerful tools for genomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Bacillus Phages / enzymology*
  • Bacillus Phages / genetics
  • Base Sequence
  • Binding Sites / genetics
  • DNA Primers / genetics
  • DNA Replication
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Models, Molecular
  • Nucleic Acid Amplification Techniques / methods*
  • Plasmids / biosynthesis
  • Plasmids / genetics
  • Protein Conformation
  • Protein Engineering
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism

Substances

  • DNA Primers
  • Recombinant Fusion Proteins
  • DNA-Directed DNA Polymerase