A highly efficient procedure for site-specific mutagenesis of full-length plasmids using Vent DNA polymerase

Genome Res. 1995 Nov;5(4):404-7. doi: 10.1101/gr.5.4.404.


Careful titration of Vent polymerase activity allows efficient amplification of full-length plasmids (12 kb). The high processivity and fidelity of this enzyme made oligonucleotide-directed site-specific mutagenesis of plasmids a straight-forward process. Using only two primers, a mutagenic and a complementary, single-base mutants of recombinant plasmids were obtained consistently with > 90% efficiency from a single round of PCR. This procedure also made site-specific deletion, insertion, and several bases mutagenesis facile and efficient.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Base Sequence
  • Cloning, Molecular
  • DNA Primers
  • DNA-Directed DNA Polymerase / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Plasmids / genetics*
  • Point Mutation
  • Polymerase Chain Reaction
  • Respirovirus / genetics


  • DNA Primers
  • Tli polymerase
  • DNA-Directed DNA Polymerase