Generation of epitope-tagged proteins by inverse PCR mutagenesis.

The polymerase chain reaction (PCR) has been extensively used for the mutation of DNA sequences (1–5,7,8). Many variations of the protocol have been implemented, but most involve a first step in which PCR is used to modify the molecule, and a second step in which the new sequence is subcloned into a vector. A new method, named inverse PCR mutagenesis (IPCRM) has been shown to be an efficient and rapid way to introduce point mutations into DNA sequences cloned in plasmid vectors without the requirement of a subcloning step (2,3,7,8). The technique consists of a PCR amplification of the vector using a set of two adjacent nonoverlapping oligonucleotides, one of which encodes the desired mutation. Since the PCR product can be longer than 10 kb, the use of Pfu DNA polymerase is recommended, not just because of its high fidelity but also because it generates blunt-ended PCR products, eliminating the need for end polishing. The PCR product is then ligated, treated with DpnI to digest methylated contaminating parental plasmid and subsequently introduced into competent cells through transformation. IPCRM is a suitable method to produce substitution and deletion mutations in sequences already cloned into a specific vector (3). We have extended the IPCRM method to permit insertion of epitope tags into a target protein of interest. Routinely, tagged proteins are generated by cloning the coding sequence into a multiple cloning site of a vector that already contains the epitope sequence (6). Changing the epitope or even its location with respect to the target protein requires either the use of new vectors or extensive cloning procedures. Using IPCRM, it is possible to insert short peptide sequences anywhere within the vector, requiring only a pair of primers. To illustrate the procedure, we have fused the FLAG epitope sequence