A method for site-specific mutagenesis which can be used with any gene in any vector has been devised. This method has been named "phoenix mutagenesis." Upon selection of the mutation site, the plasmid bearing the gene in question is cleaved with one or two restriction enzymes that generate fragments with random staggered ends. The enzymes are chosen to maximize the protrusions while minimizing the size of the fragment to be mutated. Ligation of the fragments reconstitutes the original plasmid. Mutations are obtained by allowing mutant fragments, added in 10-fold molar excess, to compete with their wild-type counterparts for a place in the reassembled vector. In a test system with the restriction enzyme PflMI, which cleaves an 8-kb vector bearing the prothymosin α gene into four fragments, the efficiency of reassembly exceeded that of sealing linearized molecules. The recovery of mutated molecules was 20-50%. With BsaI, which also cleaves the plasmid into four fragments, both the efficiency of reassembly and the production of mutants were reduced. To expedite the use of phoenix mutagenesis, a simple technique for generating mutant fragments in tandem polymerase chain reactions is presented. In the first step, a mutated subfragment is synthesized from the region of the mutation to one terminus; in the second step, the complete fragment is made with the aid of an oligomer targeted to the other terminus and one strand of the product of the first reaction. The complementary strand is present, but does not interfere with the amplification process. © 1993 Academic Press, Inc.
