Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants.
Deletion loop mutagenesis is a new, general method for site-directed mutagenesis that allows point mutations to the introduced within a sequence of DNA defined by a previously isolated deletion mutant. Wild type and deletion mutant DNA are cloned into a bacterial plasmid and each is cleaved with a different single cut restriction enzyme. Heteroduplexes are formed between the two DNAs to produce circular molecules containing a nick in each strand and a single-stranded deletion loop. The deletion loops are mutagenised using sodium bisulphite and the DNA transfected directly into a uracil repair deficient strain of Escherichia coli. Up to half of the resultant clones contain DNA produced by replication of the wild-type length strand and bear mutations exclusively within the target area. An example is given in which a deletion mutant lacking 21 nucleotides from the region coding for SV40 large-T was used. Eight of the possible nine target cytosine residues were mutagenised. The method described is specific, efficient and simple.
D001483:Base Sequence; D002872:Chromosome Deletion; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D009154:Mutation; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D013447:Sulfites / Q000494:pharmacology; D014162:Transfection
D004269:DNA, Bacterial; D013447:Sulfites; D004262:DNA Restriction Enzymes