Cut-and-paste-based cloning strategy for large gene site-directed mutagenesis

Abstract

ABSTRACT. Site-directed mutagenesis is an essential technique for investigating the mechanisms of gene regulation on a molecular level, as well as for exploring post-translational modifications and functional structure at the protein level. Polymerase chain reaction combining in vitro synthesis of oligonucleotide primers allows for site-directed mutation to be performed with ease. However, site-directed mutagenesis is difficult when larger plasmids are involved. Here, we present a novel method for generating large gene site-directed mutagenesis products based on a cut-and-paste-based cloning strategy. This method uses 4 primers, incorporating relevant mutations and restriction enzyme site sequences, to generate 2 DNA fragments by polymerase chain reaction. The fragments are then ligated into TA cloning vectors. Large genes containing mutations of interest were obtained by cutting and then pasting, and then inserting one fragment into another T-vector. We demonstrated the practicality of this method by creating a G59S mutation within the p150 Glued -encoding gene.

Site‐Directed Mutagenesis by Polymerase Chain Reaction

“…Wang et al [14] have described a method to generate site-directed mutagenesis in large genes. The method consists of two PCR products with four primers (containing mutations and restriction enzyme sites).…”