Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis

Abstract

We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis. Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length. The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector. The oligonucleotides are hybridized and ligated to the M13 vector without any purification of the synthetic DNA segment. After cloning, about half of the progeny from such shot-gun ligations contained the predicted sequence demonstrating the efficacy of this method for gene synthesis and its potential for the extensive mutational analysis of genes.