Summary
A critical step in the chemical preparation of oligonucleotides is the chromatographic purification of the deprotected oligomers. In case of large quantities of reaction products, the oligonucleotides are first enriched on a QAE-Sephadex column at low pressure. The obtained fractions are then purified by multidimensional chromatography making use of three independent physical properties of the solutes: molecular size, ionic net charge and hydrophobicity. In the first dimension size exclusion chromatography (Sephadex G-15) is used. In the second dimension the high molecular weight fraction from the size exclusion chromatography is applied to a HPLC ion-exchange column (Partisil-10 SAX). Usually the last peak is collected and transferred to a HPLC reversed phase column (Nucleosil C18) where the components are separated according to their hydrophobicity in the third dimension. The efficiency of this multi-dimensional chromatographic procedure is demonstrated by the unequivocal fingerprints after radioactive labelling of the isolated oligonucleotides.
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Eckstein, H., Schott, H. The use of multidimensional chromatography for the isolation of synthetic oligodeoxyribonucleotides on a preparative scale. Chromatographia 19, 236–239 (1984). https://doi.org/10.1007/BF02687744
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DOI: https://doi.org/10.1007/BF02687744