Molecular evolution of a defined DNA sequence with accumulation of mutations in a single round by a dual approach to random chemical mutagenesis (DuARCheM).

Directed evolution has paved the way to a new era of protein and nucleic acid molecules with improved and enhanced properties. The utmost important component of directed evolution is random mutations in a defined DNA sequence. The utility of random chemical mutagenesis in directed evolution studies is dwindling due to the inherent flaws with whole-organism mutagenesis and the in vitro approach. Here, we report a novel Dual Approach to Random Chemical Mutagenesis (DuARCheM) to introduce random mutations in a defined DNA fragment. DuARCheM involves in vivo chemical mutagenesis and in vitro genetic manipulations. The resulting library revealed an accumulation of mutations in its members. These results imply that the parent mutation is carried in the further generations within the same library. This method might help to change random chemical mutagenesis because the combination of in vivo and in vitro approaches mimics the amplification and mutation that is performed by PCR-based mutagenesis, and at the same time the mutations are confined to the desired gene. Moreover, the mutagen pressure is greater in chemical mutagenesis than in a Taq-polymerase-based error-prone system. Concomitant amplification and mutation in the DuARCheM method leads to a better spectrum of mutants because the plasmid construct is exponentially amplified in the presence of mutagen pressure, unlike in the in vitro chemical mutagenesis system in which the template molecule does not replicate. This work is able to nullify all the disadvantages that are associated with random chemical mutagenesis, and could make random chemical mutagenesis an indispensable tool in directed evolution studies.