Chapter 10 Chromatography of Synthetic and Natural Oligo-Nucleotides

Abstract

Publisher Summary This chapter presents a study on the chromatography of synthetic and natural oligonucleotides. The chapter describes the principles and limitations of the chromatographic methods that are used currently for the purification of oligonucleotides resulting from synthesis or from partial hydrolysates of DNA in analytical as well as preparative amounts. The choice of the various separation principles depends on both the amount of the nucleotides that have to be separated and the origin of the sample. The usually small amount of the desired oligonucleotide, prepared by using automatic synthesizers, can be separated on an analytical reversed-phase column. A general procedure is proposed in which different modes of chromatography are combined. In the first step, the tritylated or deprotected oligonucleotides are separated from the low molecular weight by-products with Sephadex G-15 or G-25. Alternatively, the reaction mixture is prechromatographed on an ion-exchange column, to enrich the desired oligonucleotides. By application of both ion-exchange and reversed-phase high-performance liquid chromatography (RP–HPLC) mixtures of sequence-isomeric pyrimidine oligonucleotides obtained from partial hydrolysates of depurinated DNA are separated up to the pentamers. Sequence isomers that are not separated by ion-exchange chromatography are separated by reversed-phase chromatography and vice versa.