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Method Article
Nicking Mutagenesis: comprehensive single-site saturation mutagenesis
Tim Whitehead
Whitehead Lab, Michigan State University
Corresponding Author
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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.
Keywords
saturation mutagenesis, comprehensive saturation mutagenesis, high-throughput mutagenesis, protein engineering, protein library
Figure 1
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- supplement0.xlsx
Table 1 Time required for nicking mutagenesis Estimated time required for comprehensive library construction using nicking mutagenesis. Steps follow numbering in Figure 1.
- supplement0.xlsx
Table 2 Sample library coverage statistics Nicking mutagenesis coverage statistics
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Subject Areas
Biochemistry
Genetics
Microbiology
Molecular Biology
Computational biology and bioinformatics
Associated Publications
Plasmid-based one-pot saturation mutagenesis
by Emily E Wrenbeck, Justin R Klesmith, James A Stapleton, +3
Nature Methods (19 October, 2016)
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This is a preprint. It has not completed peer review.
Method Article
Nicking Mutagenesis: comprehensive single-site saturation mutagenesis
Tim Whitehead
Whitehead Lab, Michigan State University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 06 Sep, 2016
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
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.
Figure 1
Associated Publications
Plasmid-based one-pot saturation mutagenesis
by Emily E Wrenbeck, Justin R Klesmith, James A Stapleton, +3
Nature Methods (19 October, 2016)