Skip to main content
Article
A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA
Gene (1992)
  • Rudolf Jung, United States Department of Agriculture
  • M. Paul Scott, United States Department of Agriculture
  • Luiz O. Oliveira, United States Department of Agriculture
  • Niels C. Nielsen, United States Department of Agriculture
Abstract
A method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded DNA without the necessity for phenotypic selection is described. Plasmids denatured with alkali and purified by adsorption to and elution from nitrocellulose have single-stranded regions where primers can hybridize and serve as templates for a T7 DNA polymerase-catalyzed synthesis of complementary mutant DNA strands. When this procedure was carried out such that the original nonmutant strand contained uracil [method of Kunkel, Proc. Natl. Acad. Sci. USA 82(1985)488–492], mutation frequencies of between 30% and 40% were obtained. The technique has been used to generate mutant genes in plasmids of a wide variety of sizes. The largest plasmid manipulated and successfully mutagenized was 22 kb. The method is rapid and efficient and is not dependent upon either f1 phage vectors or the presence of restriction sites in the vicinity of the sequence targeted for mutation.
Keywords
  • Nucleotide sequence,
  • uracil N-glycosylase,
  • alkali-denatured DNA,
  • nitrocellulose,
  • T7 DNA polymerase,
  • large plasmids
Publication Date
November 2, 1992
DOI
10.1016/0378-1119(92)90157-K
Publisher Statement
Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted.
Citation Information
Rudolf Jung, M. Paul Scott, Luiz O. Oliveira and Niels C. Nielsen. "A simple and efficient method for the oligodeoxyribonucleotide-directed mutagenesis of double-stranded plasmid DNA" Gene Vol. 121 Iss. 1 (1992) p. 17 - 24
Available at: http://works.bepress.com/paul-scott/48/