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OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons
Focused mutant library generation methods have been developed to improve mainly “localizable” enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we...
Autores principales: | , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2011
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198389/ https://www.ncbi.nlm.nih.gov/pubmed/22039444 http://dx.doi.org/10.1371/journal.pone.0026222 |
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author | Dennig, Alexander Shivange, Amol V. Marienhagen, Jan Schwaneberg, Ulrich |
author_facet | Dennig, Alexander Shivange, Amol V. Marienhagen, Jan Schwaneberg, Ulrich |
author_sort | Dennig, Alexander |
collection | PubMed |
description | Focused mutant library generation methods have been developed to improve mainly “localizable” enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day. |
format | Online Article Text |
id | pubmed-3198389 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2011 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-31983892011-10-28 OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons Dennig, Alexander Shivange, Amol V. Marienhagen, Jan Schwaneberg, Ulrich PLoS One Research Article Focused mutant library generation methods have been developed to improve mainly “localizable” enzyme properties such as activity and selectivity. Current multi-site saturation methods are restricted by the gene sequence, require subsequent PCR steps and/or additional enzymatic modifications. Here we report, a multiple site saturation mutagenesis method, OmniChange, which simultaneously and efficiently saturates five independent codons. As proof of principle, five chemically cleaved DNA fragments, each carrying one NNK-degenerated codon, were generated and assembled to full gene length in a one-pot-reaction without additional PCR-amplification or use of restriction enzymes or ligases. Sequencing revealed the presence of up to 27 different codons at individual positions, corresponding to 84.4% of the theoretical diversity offered by NNK-degeneration. OmniChange is absolutely sequence independent, does not require a minimal distance between mutated codons and can be accomplished within a day. Public Library of Science 2011-10-19 /pmc/articles/PMC3198389/ /pubmed/22039444 http://dx.doi.org/10.1371/journal.pone.0026222 Text en Dennig et al. http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Dennig, Alexander Shivange, Amol V. Marienhagen, Jan Schwaneberg, Ulrich OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title | OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title_full | OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title_fullStr | OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title_full_unstemmed | OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title_short | OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons |
title_sort | omnichange: the sequence independent method for simultaneous site-saturation of five codons |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3198389/ https://www.ncbi.nlm.nih.gov/pubmed/22039444 http://dx.doi.org/10.1371/journal.pone.0026222 |
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