A RAPID AND EFFICIENT METHOD FOR SITE-DIRECTED MUTAGENESIS USING ONE-STEP OVERLAP EXTENSION PCR

A rapid method is described to efficiently perform site-directed mutag enesis based on overlap extension polymerase chain reaction (OE-PCR). Two template DNA molecules in different orientations relative to only one universal primer were amplified in parallel. By choosing a high di lution of mutagenic primers it was possible to run an overlap extensio n PCR in only one reaction without purification of intermediate produc ts. This method which we have named one-step overlap extension PCR (OO E-PCR) can in principle be applied to every DNA fragment which can be cloned into a multiple cloning site of any common cloning vector.