Abstract:
The separation of common and modified deoxyribonucleosides derived from DNA hydrolyzates was examined under different chromatographic conditions on silica-based octadecyl (C18) columns, involving hydrophobic interactions with the matrix. A novel method for the analysis of the DNA composition is described. It involves the removal of RNA contaminants and enzymatic hydrolysis of DNA, first to deoxyribonucleoside monophosphates and then dephosphorylation of the latter to deoxyribonucleosides. Hydrolysis conditions were sought to avoid deamination of dA and dC residues to dI and dU contaminants, respectively. Elution of these contaminants and the artifacts (ribonucleosides derived from RNA) is described in relation to the elution of deoxyribonucleosides. Chromatographic separation of the hydrolyzate derived from a 15-micrograms sample of DNA under selected separation conditions and on one high-performance liquid chromatographic column is achieved in 18 min at room temperature. Detection of modified components (and contaminants) present in minute amounts is enhanced with the use of a diode-array detector. The power of this technique lies in its ability to characterize and quantitate accurately the amount of modified species present in the DNA structure (less than 2% of all the other residues). Examples of the composition analysis of DNA derived from a prokaryote (Escherichia coli B) and a eukaryote (salmon sperm) are described. Details of quantitation (calibration graphs) of different nucleosides are furnished for peak-area integration by commercially available software, and spectral properties of the nucleoside in the elution buffer are described for quantitation by other means. Application of the composition analysis is shown here for probing the DNA conformation in solution by chemical means, while using chloroacetaldehyde as the modifying agent.