Site-directed mutagenesis using uracil-containing double-stranded DNA templates and DpnI digestion.
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.
Li
F
F
University of Washington, Seattle, USA.
Liu
S L
SL
Mullins
J I
JI
D000225:Adenine / Q000378:metabolism; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019175:DNA Methylation; D004259:DNA-Directed DNA Polymerase; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D013698:Templates, Genetic; D014498:Uracil / Q000032:analysis*; D014764:Viral Proteins / Q000378:metabolism
D014764:Viral Proteins; C069983:gene 43 protein, Enterobacteria phage T4; D014498:Uracil; D004247:DNA; D004259:DNA-Directed DNA Polymerase; C020102:endodeoxyribonuclease DpnI; D015252:Deoxyribonucleases, Type II Site-Specific; D000225:Adenine