Oligodeoxynucleotide-directed cleavage and repair of a single-stranded vector: A method of site-specific mutagenesis

Abstract

A simple and efficient site-specific mutagenesis method is described. First, a single-stranded (ss) circular vector is linearized at the site where the desired mulation will be introduced. To do this, an oligodeoxynucleotide complementary to the target region of the ss vector and containing a restriction enzyme recognition sequence is annealed to the circular ss vector, and the partial double-strand formed is subsequently cleaved with that enzyme. Then, another oligodeoxynucleotide spanning the nick and carrying the mutation is annealed to the linearized ss DNA template and the annealed mixture is used directly to transform Escherichia coli without prior enzymatic DNA synthesis in vitro. The procedure has been applied successfully to constructing insertion, deletion, and point mutations in both M13 phage vectors and plasmid vectors containing the f1 origin of replication.