Plasmid-based one-pot saturation mutagenesis

Abstract

Deep mutational scanning is a foundational tool for addressing the functional consequences of large numbers of mutants, but a more efficient and accessible method for construction of user-defined mutagenesis libraries is needed. Here we present nicking mutagenesis, a robust, single-day, one-pot saturation mutagenesis method performed on routinely prepped plasmid dsDNA. The method can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries.

Figure 1

Comprehensive single-site nicking mutagenesis. Wild-type (WT) plasmid dsDNA containing a 7-bp BbvCI recognition site is nicked by Nt.BbvCI. Exonuclease III (ExoIII) degrades the nicked strand to generate ssDNA template, exonuclease I (ExoI) degrades insufficiently digested DNA (step (1)). Mutagenic oligos are then added at a 1:20 ratio with template, Phusion polymerase synthesizes mutant strands, and Taq DNA ligase seals nicks (step (2)). The reaction is column purified, and then WT template strand is nicked by Nb.BbvCI and digested by ExoIII (step (3)). A second primer is added, and the complementary mutant strand is synthesized to yield mutagenized dsDNA (step (4)).