volume 13 Number 5 1985 Nucleic Acids Research A simple and efficient method for chemical mutagenesis of DNA

A simple and e f f ic ient procedure for the generation of random GC to AT transit ion mutations 1n a specific DNA segment 1s described. A restr ic t ion fragment 1s Inserted 1n each orientation Into an H13 vector, single-stranded vir ion ONA from each recombinant phage 1s treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restr ict ion fragment 1s obtained. This methoxylamine-derivatized DNA segment 1s then joined with l inearized M13 RF DNA, competent E. col l Is transfected, and mutations are d i rect ly Ident i f ied by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 5OT In a 56-base pair segment of the signal codons of the TEH B-lactamase