An efficient one-step site-directed and site-saturation mutagenesis protocol

We have developed a new primer design method based on the QuickChange site-directed mutagenesis protocol, which significantly improves the PCR amplification efficiency. This design method minimizes primer dimerization and ensures the priority of primer-template annealing over primer self-pairing during the PCR. 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. In comparison, all attempts failed when using complete-overlapping primer pairs as recommended in the standard QuickChange protocol. Our protocol was further extended to site-saturation mutagenesis by introducing…

• NS
  National Science Foundation
• SN
  Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung