Simple chromatographic systems permitting both DNA purification and separation of 2'-deoxyribonucleoside 3'-monophosphates as substrates for 32P-postlabelling studies.
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.
Polverelli
M
M
DRFMC/SESAM/LAN, Centre d'Etudes Nucléaires de Grenoble, France.
Berger
M
M
Odin
F
F
Cadet
J
J
D000818:Animals; D002021:Buffers; D002417:Cattle; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000302:isolation & purification*; D003854:Deoxyribonucleotides / Q000302:isolation & purification*; D007553:Isotope Labeling; D010761:Phosphorus Radioisotopes; D012639:Seeds / Q000737:chemistry
D002021:Buffers; D003854:Deoxyribonucleotides; D010761:Phosphorus Radioisotopes; D004247:DNA