Rapid and efficient site-specific mutagenesis without phenotypic selection

Proc Natl Acad Sci U S A. 1985 Jan;82(2):488-92. doi: 10.1073/pnas.82.2.488.

Abstract

Several single-base substitution mutations have been introduced into the lacZ alpha gene in cloning vector M13mp2, at 40-60% efficiency, in a rapid procedure requiring only transfection of the unfractionated products of standard in vitro mutagenesis reactions. Two simple additional treatments of the DNA, before transfection, produce a site-specific mutation frequency approaching 100%. The approach is applicable to phenotypically silent mutations in addition to those that can be selected. The high efficiency, approximately equal to 10-fold greater than that observed using current methods without enrichment procedures, is obtained by using a DNA template containing several uracil residues in place of thymine. This template has normal coding potential for the in vitro reactions typical of site-directed mutagenesis protocols but is not biologically active upon transfection into a wild-type (i.e., ung+) Escherichia coli host cell. Expression of the desired change, present in the newly synthesized non-uracil-containing covalently closed circular complementary strand, is thus strongly favored. The procedure has been applied to mutations introduced via both oligonucleotides and error-prone polymerization. In addition to its utility in changing DNA sequences, this approach can potentially be used to examine the biological consequences of specific lesions placed at defined positions within a gene.

MeSH terms

  • Base Sequence
  • Coliphages / genetics
  • DNA Glycosylases*
  • DNA, Single-Stranded / analysis
  • DNA, Viral / analysis*
  • Escherichia coli / genetics
  • Mutation*
  • N-Glycosyl Hydrolases / metabolism
  • Phenotype
  • Transfection
  • Uracil / metabolism
  • Uracil-DNA Glycosidase

Substances

  • DNA, Single-Stranded
  • DNA, Viral
  • Uracil
  • DNA Glycosylases
  • N-Glycosyl Hydrolases
  • Uracil-DNA Glycosidase