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 modification...

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Autores principales:	Alexander Dennig, Amol V Shivange, Jan Marienhagen, Ulrich Schwaneberg
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/5e3e714e6b2648028332b834bf2f1569
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spelling	oai:doaj.org-article:5e3e714e6b2648028332b834bf2f15692021-11-18T07:36:09ZOmniChange: the sequence independent method for simultaneous site-saturation of five codons.1932-620310.1371/journal.pone.0026222https://doaj.org/article/5e3e714e6b2648028332b834bf2f15692011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22039444/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Focused 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.Alexander DennigAmol V ShivangeJan MarienhagenUlrich SchwanebergPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 10, p e26222 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Alexander Dennig Amol V Shivange Jan Marienhagen Ulrich Schwaneberg OmniChange: the sequence independent method for simultaneous site-saturation of five codons.
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	article
author	Alexander Dennig Amol V Shivange Jan Marienhagen Ulrich Schwaneberg
author_facet	Alexander Dennig Amol V Shivange Jan Marienhagen Ulrich Schwaneberg
author_sort	Alexander Dennig
title	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_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_sort	omnichange: the sequence independent method for simultaneous site-saturation of five codons.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/5e3e714e6b2648028332b834bf2f1569
work_keys_str_mv	AT alexanderdennig omnichangethesequenceindependentmethodforsimultaneoussitesaturationoffivecodons AT amolvshivange omnichangethesequenceindependentmethodforsimultaneoussitesaturationoffivecodons AT janmarienhagen omnichangethesequenceindependentmethodforsimultaneoussitesaturationoffivecodons AT ulrichschwaneberg omnichangethesequenceindependentmethodforsimultaneoussitesaturationoffivecodons
_version_	1718423224089313280

OmniChange: the sequence independent method for simultaneous site-saturation of five codons.

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