Polymerase Chain Reaction-Mediated Mutagenesis in Sequences Resistant to Homogeneous Amplification

For many biologists, the polymerase chain reaction (PCR) has become an indispensable tool serving many diverse applications, from simple DNA amplification to complex diagnostics. In basic research, a very important application has been the use of PCR in the introduction of site-specific mutations into target DNA (1 ,2 ). A simple and commonly used example of this approach is the two-step “megaprimer” method Fig. 1 ), in which the mutant oligomer is first incorporated into DNA in one direction, then this DNA itself is used as a megaprimer to complete the mutant sequence in the other direction (3). Over the years, such an approach has been used in many laboratories with considerable success. In the authors’ experiments, this approach has been used for over a decade to introduce many changes into the ribosomal genes of yeast cells (4 –6 ). Although some modifications were made some modifications in the amplification conditions (7 ), to improve the efficiency of the megaprimer method, the basic strategy, as originally described, has proven to be effective and reliable in most instances.

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