Mixed oligonucleotides for random mutagenesis: best way of making them

Abstract

The generation of proteins, especially enzymes, with pre-deliberated, novel properties is a big challenge in the field of protein engineering. This aim, over the years was critically facilitated by newly emerging methods of combinatorial and evolutionary techniques, such as combinatorial gene synthesis followed by functional screening of many structural variants generated in parallel (library). Libraries can be generated by a large number of available methods. Therein the use of mixtures of pre-formed trinucleotide blocks representing codons for the 20 canonical amino acids for oligonucleotide synthesis stands out as allowing fully controlled partial (or total) randomization individually at any number of arbitrarily chosen codon positions of a given gene. This has created substantial demand of fully protected trinucleotide synthons of good reactivity in standard oligonucleotide synthesis. We here review methods for the preparation of oligonucleotide mixtures with a strong focus on codon-specific trinucleotide blocks.