Megaprimer PCR

A large variety of procedures of site-directed mutagenesis based on polymerase chain reaction (PCR) have been developed over the last decade. Among them, the “megaprimer” method, originally reported in 1990 (1 ), and its subsequent updates (8 ,10 ) still retain their popularity because they combine simplicity and versatility. Our most recent search in the PUBMED, using “megaprimer” as the keyword, generated 24 publications, many of which were improvements on the original theme. This is an impressive number, considering that “megaprimer” is essentially a specialized technique. In this chapter, we provide an updated protocol incorporating the variations and improvements of the basic technique published over the past decade. These include: a combination of magapriming and overlap extension, improvement of yield, use of single-stranded DNA, spiking with a proofreading polymerase (e.g., Pfu) to avoid unwanted mutations arising from nontemplated insertions by Taq polymerase, and the inclusion of various kinds of mutations, including multiple, nonadjacent ones (2 -12 ,18 ,19 ,26 -32 ).