Modification of the Overlap Extension Method for Extensive Mutagenesis on the Same Template

Polymerase chain reaction (PCR) is a powerful and efficient method allowing enzymatic amplification of small quantities of DNA. This technology has also been widely used as a quick and efficient alternative for introducing mutations into specific DNA sequences. Various general methods for site-directed mutagenesis using the PCR have been described recently (1 –5 ). In the overlap extension method two pairs of primers are used to generate two DNA fragments with overlapping ends containing the desired mutation. These two DNA products are combined and subsequently annealed. Finally, the resulting hybrids are amplified by PCR to generate homogeneous products containing the mutation. This method has proved to be simpler, faster, and much more efficient than traditional methods for site-directed mutagenesis (2 ). However, because synthesis of two complementary primers is required for each mutation, the overlap extension is not the method of choice for extensive mutagenesis of a target sequence.