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

Fusheng Li | Zendy; ShanLu Liu | Zendy; James I. Mullins | Zendy

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

Author(s) -

Fusheng Li,

ShanLu Liu,

James I. Mullins

Publication year - 1999

Publication title -

biotechniques

Language(s) - English

Resource type - Journals

SCImago Journal Rank - 0.617

H-Index - 131

eISSN - 1940-9818

pISSN - 0736-6205

DOI - 10.2144/99274st03

Subject(s) - mutagenesis , dna , biology , template , uracil , site directed mutagenesis , genetics , mutation , microbiology and biotechnology , gene , mutant , nanotechnology , materials science

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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