A genetic enrichment for mutations constructed by oligodeoxynucleotide-directed mutagenesis

Abstract

A genetic enrichment procedure for mutations constructed by oligodeoxynucleotide(oligo)-directed mutagenesis of DNA cloned in M 13mp vectors is described. The procedure uses an M13 vector that contains the cloned target DNA and amber (am) mutations within the phage genes I and II. This vector cannot replicate in a suppressor-free ( sup o) bacterial strain. A gapped heteroduplex is formed by annealing portions of a complementary (−)strand containing wild-type copies of genes I and II to the am-containing template (+)strand. The oligo is annealed to the single-stranded (ss) region and the remaining gaps and nicks are repaired enzymatically to form a closed circular heteroduplex structure. By transfecting the DNA into a sup o host we promote the propagation of heteroduplexes with the oligo-containing (−)strand since only this construction contains the wild-type copies of genes I and II. This procedure eliminates the need for any physical separation of the covalently closed circular DNA that contains the oligo from the ss template. Using this technique we have constructed 17 point mutations with mutation frequencies ranging from 2–20% for single base changes and from 0.3–9% for multiple base changes. In addition, we found that the mutation frequencies were affected by the state of DNA methylation in the (+) and (−)strands.