The gapped duplex DNA approach to oligonucleotide-directed mutation construction

TLDR

The study presents a simple, efficient method to introduce predetermined mutations into filamentous phage M13 genomes. The method uses gapped duplex DNA intermediates, where a synthetic oligonucleotide with partial homology is annealed and incorporated into the shorter strand via enzymatic gap filling, enabling selection of phage carrying the desired mutation in mismatch‑repair deficient E. coli.

Abstract

A simple and efficient method is described to introduce structurally pre-determined mutations into recombinant genomes of filamentous phage M13. The method rests on gapped duplex DNA (gdDNA) molecules of the phage M13 genome as the key intermediate. In this gdDNA, the (+) and the (shorter) (−)strand carry different genetic markers in such a way, that a rigorous selection can be applied for phage carrying the markers of the (−)strand. For introduction of the mutation, a synthetic oligonucleotide with partial homology to a target site within the single stranded DNA region is annealed to the gdDNA. The oligonucleotide subsequently becomes part of the (−)strand by enzymatic DNA gap filling and sealing. This physical linkage is preserved at the genetic level after transfection of a recipient E.coli strain deficient in DNA mismatch correction, so that the synthetic marker can be selected from the phage progeny independent from its potential phenotype. It is demonstrated that by this method mutants can be constructed with marker yields in excess of 70%.