A simple method for site-directed mutagenesis using the polymerase chain reaction

We have developed a general and simple method for directing specific sequence changes in a plasmid using primed amplification by the polymerase chain reaction (PCR). The method is based on the amplification of the entire plasmid using primers that include the desired changes. The method is rapid, simple in its execution, and requires only minute amounts of plasmid template DNA. It is significant that there are no special requirements for appropriately placed restriction sites in the sequence to be manipulated. In our system the yield of transformants was high and the fraction of them harboring plasmids with only the desired change was consistently about 80%. The generality of the method should make it useful for the direct alteration of most cloned genes. The only limitation may be the total length of the plasmid to be manipulated. During the study we found that the Taq DNA polymerase used for PCR adds on a single extra base (usually an A) at the end of a large fraction of the newly synthesized chains. These had to be removed by the Klenow fragment of DNA polymerase to insure restoration of the gene sequence.