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Site-Directed Mutagenesis Using Uracil-Containing Double-Stranded DNA Templates and DpnI Digestion

1999; Future Science Ltd; Volume: 27; Issue: 4 Linguagem: Inglês

10.2144/99274st03

1940-9818

Fusheng Li, Shan‐Lu Liu, James I. Mullins,

Bacteriophages and microbial interactions

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 uracilcontaining 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.