Plasmid-based one-pot saturation mutagenesis

• Published in: Nature methods Vol. 13; no. 11; pp. 928 - 930
• Main Authors: Wrenbeck, Emily E, Klesmith, Justin R, Stapleton, James A, Adeniran, Adebola, Tyo, Keith E J, Whitehead, Timothy A
• Format: Journal Article
• Language: English
• Published: New York Nature Publishing Group US 01-11-2016; Nature Publishing Group
• Subjects: 631/1647/1511; 631/1647/1513; 631/337; 631/61/338/552; Amidohydrolases - genetics; beta-Lactamases - genetics; Bioinformatics; Biological Microscopy; Biological Techniques; Biomedical Engineering/Biotechnology; brief-communication; DNA - genetics; DNA Breaks, Single-Stranded; DNA Restriction Enzymes - genetics; Escherichia coli - enzymology; Escherichia coli - genetics; Gene Library; Genes, Bacterial; Genetic aspects; Life Sciences; Methods; Mutagenesis; Mutagenesis, Site-Directed - methods; Mutation; Plasmids; Plasmids - genetics; Proteomics; Pseudomonas aeruginosa - enzymology; Pseudomonas aeruginosa - genetics; Sequence Analysis, DNA; Thermal cycling; United States; United States > US; East Lansing Michigan; Michigan
• ISSN: 1548-7091; 1548-7105
• DOI: 10.1038/nmeth.4029

Nicking mutagenesis is a streamlined protocol for plasmid-based single-pot comprehensive saturation mutagenesis.
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.