SORS: A Universal One-Round PCR-Based Method for Site-Directed Mutagenesis

We have developed a novel protocol for site-directed mutagenesis of double-stranded DNA. The procedure, termed SORS (named because it undergoes the sequential procedure of segmentation-overhang creating PCR-reannealing-splicing) mutagenesis, is exemplified by a substitution, a deletion, and an insertion of nucleotide(s) in target genes. The template DNA is PCR-amplified into two separate segments divided at the prospective mutation site, and each segment is amplified in two parallel PCRs using primers introducing the mutation. The primers are designed to be able to create protruding bases upon pooling, denaturing, and reannealing the two parallel reactions. The protruding bases at the prospective junction of the two segments are mutually complementary; therefore, the two segments can be re-spliced together to generate the mutated gene. Compared to previously published protocols, this procedure is rapid, restriction-independent and ensures higher success rate and lower potential to produce second-site mutations.