Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme

Gene. 1988;62(1):135-9. doi: 10.1016/0378-1119(88)90587-2.


Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.

Publication types

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

MeSH terms

  • Bacterial Proteins / metabolism*
  • Base Sequence
  • Coliphages / genetics
  • DNA Polymerase III / metabolism*
  • DNA, Recombinant
  • DNA, Viral / biosynthesis
  • DNA, Viral / genetics
  • DNA-Directed DNA Polymerase / metabolism*
  • Escherichia coli / enzymology
  • Escherichia coli / genetics*
  • Genetic Techniques*
  • Molecular Sequence Data
  • Mutation*


  • Bacterial Proteins
  • DNA, Recombinant
  • DNA, Viral
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase