Large-Scale Synthesis, Purification, and Analysis of Oligodeoxynucleotide Phosphorothioates

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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 (≥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.