EDTA alkaline elution characteristics and measurement of DNA damage in unlabeled DNA using Hoechst 33258 fluorescence.
Hoechst 33258 fluorescence of single stranded DNA has been used to perform alkaline elution with unlabeled DNA. The high background fluorescence of "standard" elution solutions has prompted others to use EDTA but the elution characteristics of DNA in EDTA-containing solutions and the comparability of results with those using "standard" tetrapropyl ammonium hydroxide solutions have not previously been examined. We report here the elution characteristics of DNA in EDTA and the relevant parameters for the successful use of EDTA as an elution solution. An increase in elution pH to 12.4 is required but elution solutions of higher pH cause alkaline hydrolysis of undamaged DNA. Drug-treated DNA from which DNA-protein crosslinks have been removed can be completely removed from the filters at the end of the elution by a Pronase filter digestion. The simplest and most efficient removal of DNA-protein crosslinks is through the inclusion of proteinase-K in an SDS containing lysis solution. EDTA elution can measure interstrand crosslinks and single strand breaks as easily as is performed using radiolabeled DNA under "standard" elution conditions and requires only 1.5-2 x 10(6) cells per elution filter. DNA-protein crosslinking measurements were unsatisfactory, however, since even the Pronase digestion failed to completely remove protein-crosslinked DNA from the elution filters.
D006690:Bisbenzimidazole; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000302:isolation & purification*; D004492:Edetic Acid; D006863:Hydrogen-Ion Concentration; D011402:Pronase; D014462:Ultrafiltration
D004277:DNA, Single-Stranded; D004492:Edetic Acid; D011402:Pronase; D006690:Bisbenzimidazole