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.
MeSH terms
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Amidohydrolases / genetics
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DNA / genetics*
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DNA Breaks, Single-Stranded
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DNA Restriction Enzymes / genetics
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Escherichia coli / enzymology
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Escherichia coli / genetics
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Gene Library
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Genes, Bacterial
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Mutagenesis, Site-Directed / methods*
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Mutation
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Plasmids / genetics*
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Pseudomonas aeruginosa / enzymology
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Pseudomonas aeruginosa / genetics
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Sequence Analysis, DNA
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beta-Lactamases / genetics
Substances
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DNA
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DNA Restriction Enzymes
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Amidohydrolases
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amidase
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beta-Lactamases