title: OmniChange: the sequence independent method for simultaneous site-saturation of five codons.
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.
authors: Lehrstuhl für Biotechnologie, RWTH Aachen University, Aachen, Germany.
authors: Alexander
authors: A
authors: Dennig
authors: Amol V
authors: AV
authors: Shivange
authors: Jan
authors: J
authors: Marienhagen
authors: Ulrich
authors: U
authors: Schwaneberg
mesh_terms: D001483:Base Sequence; D003062:Codon; D016296:Mutagenesis; D016133:Polymerase Chain Reaction
chemical_list: D003062:Codon