COMBINATION OF DMT-MONONUCLEOTIDE AND FMOC-TRINUCLEOTIDE PHOSPHORAMIDITES IN OLIGONUCLEOTIDE SYNTHESIS AFFORDS AN AUTOMATABLE CODON-LEVEL MUTAGENESIS METHOD

Background: Synthetic DNA has been used to introduce variability into protein-coding regions, In protocols that produce a few mutations per gene, the sampling of amino-acid sequence space is limited by the bias imposed by the genetic code. It has long been apparent that the incor poration of trinucleotides in the synthetic regime would circumvent th is problem and significantly enhance the usefulness of the technique. Results: A new method is described for the creation of codon-level deg enerate oligodeoxyribonucleotides that combines conventional dimethoxy trityl (DMT) mononucleoside phosphoramidite chemistry with 9-fluorenyl methoxycarbonyl (Fmoc) trinucleotide phosphoramidites (whose synthesis is reported in the paper), The substoichiometric use of these Fmoc-tr inucleotides in an automatable, solid-phase synthesis procedure afford ed DNA fragments comprising the wild-type sequence and a controllable distribution of mutants within two- and three-codon stretches of DNA, within the multiple cloning site of the conventional cloning vector pU C19. Conclusions: DMT and Fmoc are compatible protecting groups in con ventional oligonucleotide synthesis methods, resulting in controllable levels of codon-based mutagenesis.