Codon based mutagenesis using trimer phosphoramidites

Abstract

A set of Trimer phosphoramidites was synthesized covering all 20 amino acid codons. These trimer phosphoramidites can be added during synthesis using standard DNA chemistry. A Reaction Factor (RF) was determined empirically for each trimer to compensate for differences in their relative rate of reaction during coupling. It is therefore possible to introduce an equimolar mix of all 20 amino acid codons, or subsets thereof, at any location within a sequence. By mutating a gene at the codon level rather than at individual bases, it is possible to avoid codon bias, frame-shift mutations and the introduction of stop codons, making Trimer phosphoramidites a highly efficient tool for the exploration of sequence space in proteins.