Megaprimer Method for Polymerase Chain Reaction-Mediated Generation of Specific Mutations in DNA

During the past decade, a number of methods using polymerase chain reaction (PCR) for the generation of specific mutations in any nucleotide sequence have been described, these methods include such drawbacks as the possibility of generating undesired secondary mutations, because of the low fidelity of some of the thermostable DNA polymerases, the need for four or more specific oligonucleotide primers, and the use of sophisticated, individually optimized protocols. Finally, the selection of correctly mutated clones may also prove to be laborious (1 –3 ). In contrast, the megaprimer principle described here is costefficient, fast, reliable, and convenient.