RAPID SOLID-PHASE PHOSPHOTRIESTER SYNTHESIS OF DNA FRAGMENTS ON CONTROLLED PORE GLASS AND APPLICATION TO THE PREPARATION OF A GENE FOR SOMATOMEDIN C

Abstract

A solid-phase method for micro-scale synthesis of oligodeoxyribonucleotides is described. The method involves use of controlled pore glass as support and phosphotriester chemistry for chain assembly. The 9-phenylxanthen-9-yl (pixyl) group was used for 5' hydroxyl protection and the phthaloyl group was used for protection of the ne position of deoxyadenosine residues. A new, more alkali-stable linkage between oligonucleotide and support was used, based on the sequential reac-— tion of tolylene-2,6-diisocyanate with a protected nucleoside and then support. Using the improved chemical methods all 23 oligonucleotides required for assembly of a gene for Somatomedin C (insulin-like growth factor I) were synthesised. The oligonucleotides were 5' phosphorylated and ligated in one step to form the complete DNA duplex of 230 base pairs. The duplex was cloned into a bacteriophage M13 vector and clones sequenced by the 'dideoxy' method.