title: 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.
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Emily E
authors.identifier: http://orcid.org/0000-0003-0096-0768
authors.initials: EE
authors.lastname: Wrenbeck
authors.affiliation: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Justin R
authors.initials: JR
authors.lastname: Klesmith
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, USA.
authors.forename: James A
authors.initials: JA
authors.lastname: Stapleton
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
authors.forename: Adebola
authors.initials: A
authors.lastname: Adeniran
authors.affiliation: Department of Chemical and Biological Engineering, Northwestern University, Evanston, Illinois, USA.
authors.forename: Keith E J
authors.initials: KE
authors.lastname: Tyo
authors.affiliation: Department of Chemical Engineering and Materials Science, Michigan State University, East Lansing, Michigan, USA.|Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, Michigan, USA.
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Whitehead
mesh_terms: D000581:Amidohydrolases / Q000235:genetics; D004247:DNA / Q000235:genetics*; D053904:DNA Breaks, Single-Stranded; D004262:DNA Restriction Enzymes / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D004247:DNA; D004262:DNA Restriction Enzymes; D000581:Amidohydrolases; C019665:amidase; D001618:beta-Lactamases