39] Chromatographic characterization of oligonucleotides on DEAE paper

Abstract

Publisher Summary This chapter discusses the chromatographic characterization of oligonucleotides and nucleotides on Diethylaminoethyl (DEAE) paper. Chromatography on DEAE paper is a useful technique for the partial resolution of complex mixtures of oligonucleotides. The overall nucleotide composition of an oligonucleotide fraction can be obtained by enzymatic degradation followed by two-dimensional chromatography on DEAE paper. One-dimensional chromatography of oligonucleotide separates complex oligonucleotide mixtures according to chain length. This technique is useful in checking the fractions obtained from column chromatography of oligonucleotides as the homogeneity of the eluted fractions may be visually inspected on the DEAE chromatogram under an ultraviolet lamp. Two-dimensional chromatography increases the resolution of oligonucleotide mixtures, partially separating by composition and by chain length. The technique results in a concentration of oligonucleotides rich in guanine residues near the point of application and those rich in cytosine traveling the greatest distance. This chromatography is useful for further resolution of oligonucleotide fractions resulting from the partial resolution of complex oligonucleotide mixtures according to chain length. The separation of nucleotides, undigested oligonucleotides, nucleosides, and inorganic phosphate can be accomplished by two-dimensional chromatography on DEAE paper. The preparation, degradation, and chromatography of unlabeled oligonucleotides are accomplished by similar methods.