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Biotechniques. 1999 Oct;27(4):734-8.

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

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1
University of Washington, Seattle, USA.

Abstract

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.

PMID:
10524315
DOI:
10.2144/99274st03
[Indexed for MEDLINE]
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