A marker-coupled method for site-directed mutagenesis.

A marker-coupled method for site-directed mutagenesis (SDM) has been developed. In this method, target DNA is first cloned into a plasmid vector which carries an inactivated tetracycline-resistance (TcR)-encoding tet gene. Using this cloned plasmid as template, polymerase chain reaction (PCR) is performed with a mutagenic primer and a marker primer. The mutagenic primer contains the desired mutations to be introduced into the target DNA, and the marker primer contains a mutation for restoring the activity of the inactivated tet gene. The PCR product is annealed with a gapped duplex plasmid template, extended and ligated in vitro. The resulting uni-strand-mutated plasmid is converted into the gapped duplex form, transformed into Escherichia coli JM109 and spread on yeast extract/tryptone culture medium + Tc plates. The TcR colonies grown on these plates all carry active tet genes. Due to the 'tight coupling' between the marker primer and the mutagenic primer formed in the PCR product, these TcR colonies should also carry the mutagenic primer, e.g., the desired mutations in the target DNA. In fact, practically all of the TcR colonies have been found to be the desired mutants in the present experiments. Therefore, this method provides a very efficient approach for SDM.