An improved protocol for efficient one-step PCR site-directed mutagenesis with partial-overlapping primers

Described here is an improved protocol of the reported method for one-step PCR site-directed mutagenesis with partial-overlapping primers [1]. The improvements include the new design of partial-overlapping primers, and optimized PCR amplification at the annealing temperature of 60°C, instead of recommended 52°C. Under such conditions, the non-specific PCR products could be remarkably reduced or even completely eliminated. Sufficient amount of specific PCR products were obtained, which allowed not only to omit the gel purification step from the reported protocol and making the procedures automated, but also to markedly reduce the cost of mutagenesis experiments.