The simplest and most broadly applicable protocol is currently the QuickChange™ Site-Directed Mutagenesis System (QCM) developed by Stratagene (La Jolla, CA). Numerous mutagenesis methods have been developed based upon the PCR ( 1 – 4). It is also widely used for studying protein structure–function relationships. Site-directed mutagenesis (SDM) is an invaluable tool to modify DNA sequences in molecular biological studies and genetic engineering. Received JRevised JAccepted JINTRODUCTION This method should be useful to facilitate the preparation of high-quality site saturation libraries. Our data indicated no specific sequence selection during library construction, with the randomized positions resulting in average occurrence of each base in each position. Our protocol was further extended to site-saturation mutagenesis by introducing randomized codons. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange™ protocol. Several different multiple mutations (up to 7 bases) were successfully performed with this partial overlapping primer design in a variety of vectors ranging from 4 to 12 kb in length. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. We have developed a new primer design method based on the QuickChange™ site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency.
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