Li F, Liu SL, Mullins JI (1999). Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion. BioTechniques, 27(4), 734-8. (pubmed)
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.