Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion

Biotechniques. 1999 Oct;27(4):734-8. doi: 10.2144/99274st03.


DpnI can cleave fully methylated parental DNA while leaving hemi-methylated DNA intact. Based on this observation, we developed a rapid site-directed mutagenesis method using uracil-containing, double-stranded (ds)DNA templates and DpnI digestion. A 38% mutation efficiency was achieved by DpnI treatment of the mutagenic strand-extension reaction, and it increased to 70%-91% when uracil-containing dsDNA templates were used. This method compares favorably to the most efficient current methods, but is simpler and does not require the use of single-stranded templates or phage vectors.

Publication types

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

MeSH terms

  • Adenine / metabolism
  • DNA / chemistry*
  • DNA / metabolism
  • DNA Methylation
  • DNA-Directed DNA Polymerase*
  • Deoxyribonucleases, Type II Site-Specific / metabolism*
  • Escherichia coli / genetics
  • Genetic Vectors
  • Mutagenesis, Site-Directed*
  • Plasmids / genetics
  • Templates, Genetic
  • Uracil / analysis*
  • Viral Proteins / metabolism


  • Viral Proteins
  • gene 43 protein, Enterobacteria phage T4
  • Uracil
  • DNA
  • DNA-Directed DNA Polymerase
  • endodeoxyribonuclease DpnI
  • Deoxyribonucleases, Type II Site-Specific
  • Adenine