Nicking Mutagenesis: comprehensive single-site saturation mutagenesis

Abstract

The purpose of this method is to generate comprehensive single-site saturation mutagenesis libraries. The required input for Nicking Mutagenesis is double-stranded plasmid DNA, and any plasmid dsDNA can be used provided that it contains a 7-base pair BbvCI recognition site. The method works by first creating an ssDNA template using a site and strand specific nicking endonuclease (Nt.BbvCI) followed by exonuclease digestion of the nicked strand. After creation of a heteroduplex by thermal cycling template with mutagenic oligos, the parental template ssDNA is destroyed by employing the opposite strand nicking endonuclease (Nb.BbvCI) followed by exonuclease digestion. A schematic of the method is outlined in Figure 1. The protocol can be completed in a single day (with transformation) and libraries can be harvested the following day.

Plasmid-based one-pot saturation mutagenesis

“…The final optimized comprehensive nicking mutagenesis protocol is supplied in Supplementary Protocol 1 and at Protocol Exchange 21 . 1× CutSmart buffer (NEB) was used as an enzyme diluent when necessary.…”

Characterizing Protein-Protein Interactions Using Deep Sequencing Coupled to Yeast Surface Display

“…This protocol is exactly as described in Wrenbeck et al [16]. All reactions should be prepared on ice unless otherwise stated.…”

User-defined single pot mutagenesis using unamplified oligo pools

“…Preparation of mutagenesis libraries. Single and double-site saturation mutagenesis libraries were constructed using nicking mutagenesis as described in Wrenbeck et al 2016aWrenbeck et al , 2016b with the following changes. To conserve the 20:1 template to oligonucleotide ratio, the volume and concentration of oligos are determined using Supporting Note 1 for the AmiE and PYR1 libraries.…”

“…Directed mutagenesis is foundational for synthetic biology and protein engineering. Recent methods support the creation of large libraries of user-defined mutations in a single reaction (Firnberg and Ostermeier 2012;Wrenbeck et al 2016aWrenbeck et al , 2016bCozens and Pinheiro 2018). Such protocols rely on annealing a short oligonucleotide to a parental template, wherein the oligo encodes a mutation by template mismatch.…”