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.
Publication types
-
Research Support, U.S. Gov't, P.H.S.
-
Technical Report
MeSH terms
-
Adenine / metabolism
-
DNA / chemistry*
-
DNA / metabolism
-
DNA Methylation
-
DNA-Directed DNA Polymerase*
-
Deoxyribonucleases, Type II Site-Specific / metabolism*
-
Escherichia coli / genetics
-
Genetic Vectors
-
Mutagenesis, Site-Directed*
-
Plasmids / genetics
-
Templates, Genetic
-
Uracil / analysis*
-
Viral Proteins / metabolism
Substances
-
Viral Proteins
-
gene 43 protein, Enterobacteria phage T4
-
Uracil
-
DNA
-
DNA-Directed DNA Polymerase
-
endodeoxyribonuclease DpnI
-
Deoxyribonucleases, Type II Site-Specific
-
Adenine