Chromatographic fractionation of nucleic acids using microcapsules made from plant cells

Abstract

The chromatographic fractionation of nucleic acids and oligodeoxyribonucleotides on a vesicular packing (VP) material has been investigated. As the VP material consists of microcapsules with a negatively charged ultrafiltration membrane, the experiments were focused on permeation chromatography in aqueous buffers of different ionic compositions. The adsorption of oligonucleotides and nucleic acids by the VP material occurring at pH values below 7 and especially in the presence of divalent cations, was negligible in neutral or weakly alkaline buffers. In such buffers the separation principle is based only on the permeation of polyanions into the stationary liquid within the vesicles. Polydisperse samples may be separated into an excluded peak fraction (at 40% of bed volume), fractions with intermediate elution volumes and a permeating peak fraction (at 95% of bed volume). The ratio between these fractions can be varied gradually by changing the salt concentration. Using defined oligonucleotides the maximum chain length for permeation into the whole stationary phase was smaller than the minimum chain length for complete exclusion. Both values were dependent on the salt concentration and pH. They could be varied gradually and reversibly over a wide range by altering the magnesium chloride concentration from 0 to 10 m M. In contrast to ion-exclusion effects known from gel permeation chromatography, in vesicle permeation chromatography there is no salt effect on the elution volume of the permeable fraction. Preparative applications for the fractionation of oligodeoxyribonucleotides and nucleic acids are presented.