Multiple Site-Directed and Saturation Mutagenesis by the Patch Cloning Method
Constructing protein-coding genes with desired mutations is a basic step for protein engineering. Herein, we describe a multiple site-directed and saturation mutagenesis method, termed MUPAC. This method has been used to introduce multiple site-directed mutations in the green fluorescent protein gene and in the moloney murine leukemia virus reverse transcriptase gene. Moreover, this method was also successfully used to introduce randomized codons at five desired positions in the green fluorescent protein gene, and for simple DNA assembly for cloning.
Key wordsSite-directed mutagenesis Saturation mutagenesis Cloning Protein engineering
This work was supported by the Funding Program for Next Generation World-Leading Researchers [LR011 to H.M.] and Grant-in-Aid for Scientific Research (A) [15H02006 to H.M.] from Japan Society for the Promotion of Science (JSPS).