Method of site-directed mutagenesis using long primer-unique site elimination and exonuclease III.

troducing undesired mutations during the preparation of the vector, can negatively affect the frequency of gene targeting in ES cells (13). In summary, the method described here offers an attractive alternative for the preparation of replacement vectors for gene targeting. This protocol can be used to perform site-directed mutagenesis in long genomic sequences cloned into plasmids with the highest degree of fidelity of DNA replication. This method provides also an approach for correcting mutations on long PCR products.