A simple and reproducible method for directed evolution: combination of random mutation with dITP and DNA fragmentation with endonuclease V.

An alternative method to combine mutagenesis PCR with dITP and fragmentation by endonuclease V for directed evolution was developed. In comparison to the routine protocol for directed evolution, dITP was used as mutation reagent in the mutagenesis PCR. Subsequently, the incorporated dITP in the PCR products could represent as being the target of endonuclease V. Finally, the mutated dsDNA was fragmented by endonuclease V and then shuffled via assembly and reamplification as is usually done. In this study, the gene encoding kanamycin resistance has been used as reporter to verify the novel method for directed evolution. However, the mutation frequency could be easily adjusted by the amount of dITP used in the mutagenesis PCR reaction. Besides, this protocol yielded the mutation types with an obvious bias to transition substitutions as the normal error-prone PCR did. Conclusively, this novel method for directed evolution has been demonstrated to be efficient, reproducible, and easy to handle in actual practice. Using this protocol, we have successfully constructed a random mutation library for the gene encoding a serine alkaline protease.