THE SYNTHESIS OF BLOCKED TRIPLET-PHOSPHORAMIDITES AND THEIR USE IN MUTAGENESIS

A general approach for the synthesis of oligonucleotide-triplet phosph oramidites and the synthesis of four such blocks are described. A stra tegy was devised to minimize the number of dimer precursors needed for synthesis of a complete set of triplet-amidite blocks encoding all 20 amino acids. Whereas synthesis of 20 triplet-amidite blocks consistin g of codon sequences requires 16 dimer blocks, just seven dimer blocks are required to synthesize all required antisense sequences. The anti sense sequences are then converted to codons in template mediated repl ication. Using a mixture of four triplet-amidites and conventional aut omated solid-phase DNA synthesis, short (6mer) and medium length (30me r) oligonucleotide mixtures were synthesized and analyzed. The latter was replicated in vitro and used as a mutagenic cassette to produce fo ur mutants of Asp 221 in the enzyme thymidylate synthase. The method e stablishes the direction and utility for the production and use of tri plet-amidite blocks in DNA synthesis.