Oligonucleotide Synthesis Using the Manual Phosphotriester Method

Abstract

During the past few years, synthetic DNA, used as a primer in DNA polymerization, in site-directed mutagenesis or as a probe in gene selection, has assumed a central role in recombinant DNA technology (1). This was made possible by the development of methods for efficient solid phase chemical synthesis. Deoxyribonucleotides are ideally suited to solid-phase synthesis since their relative chemical uniformity allows for application of oligodeoxyribonucleotide (oligonucleotide) purification techniques, which are largely dependent on chain length. Hence, the potential disadvantage of omitting purification after each coupling step is minimized. This contrasts with peptide synthesis in which purification of the final product is more difficult, thereby placing greater demands on coupling efficiency. In practice, coupling efficiencies of at least 95% are now attainable in oligonucleotide synthesis because of the availability of highly reactive mononucleotides and specially developed coupling catalysts. In optimized systems, this allows for synthesis of oligomers greater than 50 bases in length.