A rapid and efficient method for region- and strand-specific mutagenesis of cloned DNA.
The single-stranded viral DNA of an M13 phage recombinant containing the early promoter region of SV40 was hybridized with linear, double-stranded replicative form DNA of a related M13 phage containing a short deletion in the cloned SV40 sequence. The heteroduplexes formed between these DNA molecules contained a short, defined single-stranded region in an otherwise duplex molecule. These heteroduplexes were treated with sodium bisulphite to deaminate exposed unpaired cytosines to uracil residues. The single-stranded region was filled in with DNA polymerase I, which incorporates adenine opposite the mutated uracils, and the DNA then transfected into the M13 host JM103 . Viral DNA from the resultant plaques was used for the rapid dideoxy-DNA sequencing procedure; all of the plaques studied contained point mutations within the desired area. This method allows the very rapid and efficient generation of region-directed point mutants which can be quickly sequenced.
D001483:Base Sequence; D003001:Cloning, Molecular; D003090:Coliphages / Q000235:genetics*; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004277:DNA, Single-Stranded / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D009154:Mutation
D004277:DNA, Single-Stranded; D004247:DNA; D004262:DNA Restriction Enzymes