Development and Use of a Novel Random Mutagenesis Method: In Situ Error-Prone PCR (is-epPCR).

Directed evolution methods are increasingly needed to improve gene and protein properties. Error-prone PCR is the most efficient method to introduce random mutations by reducing the fidelity of the DNA polymerase. However, a highly efficient process is required for constructing and screening a diverse mutagenesis library since a large pool of transformants is needed to generate a desired mutant. We developed a method called in situ error-prone PCR (is-epPCR) to improve the efficiency of constructing a mutation library for directed evolution. This method offers the following advantages: (1) closed-circular PCR products can be directly transformed into competent E. coli cells and easily selected by using an alternative antibiotic; (2) a mutant library can be created and screened by one-step error-prone amplification of a variable DNA region in an expression plasmid; and (3) accumulation of desired mutations in one sequence can be obtained by multiple rounds of is-epPCR. Is-epPCR offers a novel, convenient, and efficient approach for improving genes and proteins through directed evolution.