TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons.

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4'-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides.