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

• Main Authors: Alexander Dennig, Amol V Shivange, Jan Marienhagen, Ulrich Schwaneberg
• Format: Article
• Language: English
• Published: Public Library of Science (PLoS) 2011-01-01
• Series: PLoS ONE
• Online Access: https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22039444/pdf/?tool=EBI

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.