Researchers have investigated the sequence dependence on protein function for 4 decades. Early endeavors involved generation of a handful of point mutations followed by low-throughput assays for function; the ‘search space’ a protein scientist could feasibly explore was miniscule. The advent of next-generation sequencing (NGS) has presented protein scientists with the ability to economically observe entire populations of molecules before, during, and after a high-throughput screen or selection for function (HTS). However, the promise of NGS has been tempered by the lack of a robust and accessible method for the construction of high quality, user-defined mutational libraries. Commonly used mutagenesis methods such as error-prone PCR suffer from limited codon sampling and imprecise control over the number of mutations introduced. To solve this challenge, we invented a new mutagenesis method, called Nicking Mutagenesis, to allow user-defined, comprehensive mutagenesis libraries from routinely prepped plasmid dsDNA in a single day and single pot1. Nicking Mutagenesis can be used to produce comprehensive or single- or multi-site saturation mutagenesis libraries. During this talk I will present an overview of Nicking Mutagenesis and highlight demonstrations of its reproducibility and accessibility. I will also supply examples of protein engineering enabled by the method for enzymes and antibodies.

Nicking mutagenesis: User-defined, single-pot comprehensive mutagenesis

253rd National Meeting (2017)

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