Large-scale synthesis, purification, and analysis of oligodeoxynucleotide phosphorothioates.

Abstract

Synthesis of oligonucleotides has been carried out on 1-, 2-, and 5-mmol scales using an appropriately modified automated, commercially available DNA synthesizer. The reaction cycles were optimized to obtain efficient coupling (> or = 97%). The synthesized oligonucleotide was purified by preparative reversed-phase liquid chromatography, followed by detritylation and desalting to obtain the oligonucleotides in the Na+ form. The purified oligonucleotides were characterized by 31P NMR, mass spectrometry, capillary gel electrophoresis, and ion-exchange high-performance liquid chromatography. By using these protocols, a 25-mer oligodeoxyribonucleotide can be synthesized on a 1-, 2-, or 5-mmole scale to obtain approximately 2.4, 4.8, or 12 g of purified product.