Simple chromatographic systems permitting both DNA purification and separation of 2'-deoxyribonucleoside...

The 32P-postlabelling method has recently been applied to the measurement of oxidative DNA damage. The assay requires the isolation of 2'-deoxyribonucleoside 3'-monophosphates subsequent to the extraction of DNA followed by its enzymatic digestion. As an alternative to the use of toxic and oxidizing solvents such as phenol, a simple purification method is proposed, based mainly on size-exclusion chromatography carried out either with ready-made columns (NAP-10, SEC-2000) or, more conveniently, with stainless-steel laboratory-packed columns (Fractogel HW 65 F). This method was applied to the purification of the DNA extracted from seeds of Lactuca sativa. After enzymatic digestion of DNA, the 2'-deoxyribonucleoside 3'-monophosphates may be further separated in less than 30 min by high-performance liquid chromatography on a Hypersil octadecylsilylsilica gel column in the ion-suppression mode by using either ammonium formate (0.05 M, pH 6.5) or sodium succinate (0.02 M, pH 6.0). The use of these eluent systems is compatible with straightforward 32P-labelling of the 2'-deoxyribonucleoside 3'-monophosphates without any concentration and desalting steps.