Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion. |
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Authors: | F Li S L Liu J I Mullins |
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Institution: | University of Washington, Seattle, USA. |
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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. |
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