Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates.
An approach for generating structures capable of directing full-length complementary-strand synthesis for double-stranded plasmid DNA is described. The structures are formed following heat denaturation and cooling of linearized plasmid DNA molecules in the presence of what is referred to as a "closing" oligonucleotide. Consisting of a sequence complementary to the free ends of one of the two plasmid strands, the closing oligonucleotide functions as an agent for recircularization of a DNA strand and generation of a primer-circular template structure suitable for polymerase-dependent full-length complementary-strand synthesis and ligation into a covalently closed heteroduplex molecule. When combined with a mutagenic oligonucleotide and uracil-substituted DNA templates, this approach allows site-directed mutagenesis to be performed directly on double-stranded DNA with a mutant formation efficiency of about 50%, a level amenable to rapid screening by DNA sequencing.
Slilaty
S N
SN
Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec.
Fung
M
M
Shen
S H
SH
Lebel
S
S
D001483:Base Sequence; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D004269:DNA, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D015183:Restriction Mapping; D013698:Templates, Genetic; D014169:Transformation, Bacterial; D001616:beta-Galactosidase / Q000235:genetics
D004269:DNA, Bacterial; D009841:Oligonucleotides; D004247:DNA; D004259:DNA-Directed DNA Polymerase; D001616:beta-Galactosidase