A simple and efficient method for chemical mutagenesis of DNA

Abstract

A simple and efficient procedure for the generation of random GC to AT transition mutations in a specific DNA seynent is described. A restriction fragment is inserted in each orientation into an M13 vector, single-stranded virion DNA from each recombinant phage is treated with methoxylamine, and, after reannealing of the mutagenized strands, a double-stranded restriction fragnent is obtained. This methoxylmine-derivatized DNA segment is then joined wlth linearized M13 RF DNA, competent E. coli is transfected. and mutations are directly identified by sequencing of the phage DNA. Using this technique, single and double nucleotide substitutions were generated at a frequency greater than 50% in a 56-base pair segment of the signal codons of the TEM β-lactamase. © 1985 IRL Press Limited.