Manual Oligonucleotide Synthesis Using the Phosphoramidite Method

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Abstract

Oligodeoxyribonucleotides may be synthesized on a solid support, which allows for the elongation of the chain without intermediate purifications. The exocyclic amino groups on cytosine and the purines are protected as the alkali-labile benzoyl or iso-butyryl amides. The 3′-OH group of the first nucleoside is attached to the solid support by a spacer chain, and the synthesis procedes by coupling the 5′-OH group of the growing chain to the 3′-phosphorus of the monomer being added, which has its 5′-OH group protected by an acid-labile group. The phosphorus must also be protected to avoid side reactions. When the synthesis is completed, the chain must be cleaved from the support and the base, phosphorus, and terminal 5′-OH-protecting groups removed. The desired oligonucleotide is then separated from the mixture of shorter chains and modified chains (1).