KEYWORDS: Codon, gene library, randomization, synthesis, trinucleotide.
ABSTRACT: Over the past decades, the interest in efficient methods for site-directed mutagenesis in protein engineering has strongly grown. Oligonucleotide-directed mutagenesis is the favoured method for preparation of large peptide or protein libraries. Among a host of methods, the use of mixtures of pre-formed trinucleotide blocks representing codons for the 20 canonical amino acids stands out as allowing fully controlled partial (or total) randomization at any arbitrarily chosen codon positions of a given gene. Such trinucleotide synthons, however, are not easy to come by. Several strategies have been suggested making use of various 3'-O- and phosphate protecting groups. However, most of these methods suffer from one or more limitations, such as di- and mononucleotide side products, loss of protecting groups, bad coupling yields or false linked nucleotides. More recent work has successfully overcome these limitations. Herein we review the strategies for preparation of fully protected trinucleotide synthons. ...