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Public Library of Science, PLoS ONE, 10(6), p. e26222, 2011

DOI: 10.1371/journal.pone.0026222

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OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons

Journal article published in 2011 by Alexander Dennig, Amol V. Shivange, Jan Marienhagen ORCID, Ulrich Schwaneberg ORCID
This paper is made freely available by the publisher.
This paper is made freely available by the publisher.

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Abstract

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.