Rapid site-specific mutagenesis in plasmids.
A quick and simple method for introducing site-specific mutations into plasmids is described. The procedure involves restriction-enzyme digestion of the plasmid to give a linear fragment. A second preparation of the same plasmid is digested with other restriction enzymes to remove the targeted mutational region to give a gapped fragment. The linear fragment and the gapped fragment are mixed, then denatured and annealed in the presence of a short, synthetic oligodeoxynucleotide corresponding to the targeted region and containing the desired mutation. The mix is then transformed directly into cells where host enzymes fill single-stranded gaps to make a complete double-stranded, mutant plasmid.
Foss
K
K
Department of Bacteriology, University of Wisconsin, Madison 53706.
McClain
W H
WH
D001483:Base Sequence; D004262:DNA Restriction Enzymes; D005818:Genetic Engineering / Q000379:methods; D009154:Mutation; D009693:Nucleic Acid Hybridization; D009838:Oligodeoxyribonucleotides; D010957:Plasmids
D009838:Oligodeoxyribonucleotides; D004262:DNA Restriction Enzymes