Improving the mutagenesis efficiency of the Kunkel method by codon optimization and annealing temperature adjustment

The Kunkel method is a widely used site-directed mutagenesis strategy that introduces point mutations by annealing mutation-containing oligonucleotides to single-stranded uracil-containing DNA (dU-ssDNA) templates. The method is fast and inexpensive and has been routinely employed to generate point mutations and multi-site mutations. However, its efficiency for point mutations is highly variable. In this work, codons in both DNA templates and mutagenic oligonucleotides were optimized to lower the GC percentage (GC%) of the complementary regions, and the oligonucleotide length was also extended to reduce the GC difference between upstream and downstream regions. These modifications largely increased the mutation efficiency of single-site mutagenesis. In addition, a multi-stage cooling programme was developed in the annealing step specifically for multi-site mutagenesis, which increased the simultaneous mutation efficiency. The modifications will help in generating antibody libraries by effectively randomizing multiple CDRs simultaneously.