omnichange the sequence independent method for simultaneous site-saturation of five codonsomnichange序列独立同时site-saturation五个基码的方法.pdfVIP

OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons Alexander Dennig., Amol V. Shivange., Jan Marienhagen, Ulrich Schwaneberg* ¨ Lehrstuhl fur Biotechnologie, RWTH Aachen University, Aachen, Germany 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. Citation: Dennig A, Shivange AV, Marienhagen J, Schwaneberg U (2011) OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons. PLoS ONE 6(10): e26222. doi:10.1371/journal.pone.0026222 Editor: Martin G. Marinus, University of Massachusetts Medical School, United States of America Received July 21, 2011; Accepted September 22, 2011; Published October 19, 2011 Copyright: 2011 Dennig et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted