Three steps in the primer design for the OmniChange method on the examples of positions E31 and T77.

<p>(A) Selection of targeted codons for NNK saturation (dark-grey highlighted letters). (B) Twelve nucleotides downstream of a targeted codon are selected as phosphorothiolated nucleotides for subsequent overhang generation by chemical cleavage into multiple small fragments. Remaining overhangs enable efficient hybridization of generated DNA fragments (light-grey highlighted letters). (C) Design of each oligonucleotide 3′end for successful PCR amplification of DNA fragments. Arrows indicate Phusion DNA Polymerase amplifcation direction. Italic letters are the phosphorothiolated nucleotides on the 5′ends of every primer used in the OmniChange method.