P3 site-directed mutagenesis: An efficient method based on primer pairs with 3'-overhangs.

ABSTRACT: Site-directed mutagenesis is a fundamental tool indispensable for protein and plasmid engineering. An important technological question is how to achieve the ideal efficiency of 100%. Based on complementary primer pairs, the QuickChange method has been widely used, but it requires significant improvements due to its low efficiency and frequent unwanted mutations. An alternative and innovative strategy is to utilize primer pairs with 3'-overhangs, but this approach has not been fully developed. As the first step toward reaching the efficiency of 100%, we have optimized this approach systematically (such as use of newly designed short primers, test of different Pfu DNA polymerases, and modification of PCR parameters) and evaluated the resulting method extensively with >100 mutations on 12 mam

SUBMITTER: Mousavi N 

PROVIDER: S-EPMC11910099 | biostudies-literature | 2025 Mar

REPOSITORIES: biostudies-literature