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- Title
OmniChange: The Sequence Independent Method for Simultaneous Site-Saturation of Five Codons.
- Authors
Dennig, Alexander; Shivange, Amol V.; Marienhagen, Jan; Schwaneberg, Ulrich
- 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.
- Subjects
GENETIC code; ENZYMES; GENETIC mutation; POLYMERASE chain reaction; NUCLEOTIDE sequence
- Publication
PLoS ONE, 2011, Vol 6, Issue 10, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0026222