OmniChange: simultaneous site saturation of up to five codons.

ABSTRACT

Multi-site-saturation mutagenesis allows altering of "localizable" properties such as activity and selectivity and enables the discovery of cooperative amino acid substitutions which are unlikely to be discovered by saturating single codons individually or iteratively. The herein presented method "OmniChange" does not require any DNA modifying enzyme (e.g., endonucleases or ligases), and diverse mutant libraries with up to five simultaneously saturated positions are generated in a robust and technically simple manner in four steps. The key feature of the OmniChange method is a highly efficient chemical cleavage of phosphorothiolated nucleotides by ethanol-iodine to generate 12-nucleotide-long 5' overhangs in double-stranded DNA. The generated vector and inserts can be hybridized in a one-pot assembly leading to fully functional mutagenic plasmids, and the employed E. coli host can easily ligate up to 10 DNA nicks without any further enzymatic treatment. OmniChange is furthermore a reliable and general tool for multi-DNA fragment assembly which is DNA sequence independent.