Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission.
The present study describes the development and characterization of a novel technique, the alkaline-halo assay, for the assessment of DNA single strand breakage in mammalian cells. This technique allows the measurement of DNA lesions at the single cell level and presents the additional advantages of being rapid, sensitive, virtually costless and environmentally friendly, because it does not require the use of isotopes. The alkaline halo assay involves a series of sequential steps in which the cells are first treated, then embedded in melted agarose and spread onto microscope slides that are incubated for 2 min at ice-bath temperature to allow complete geling. The slides are then incubated for 20 min in a high salt alkaline lysis solution, for an additional 15 min in a hypotonic alkaline solution and, finally, for 10 min in ethidium bromide. Under these conditions, single-stranded DNA fragments spread radially from the nuclear cage and generate a fluorescent image that resembles a halo concentric to the nucleus remnants. The area of the halos increased at increasing levels of DNA fragmentation and this process was associated with a progressive reduction of areas of the nuclear remnants. These events were conveniently monitored with a fluorescence microscope and quantified by image processing analysis. The sensitivity of the alkaline-halo assay, which is based on the osmotically driven radial diffusion of single-stranded DNA fragments through agarose pores, is remarkably similar to that of the widely used alkaline elution and comet assays.
Sestili
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Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Università di Urbino, Italy. sestili@fis.uniurb.it
Cantoni
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D000818:Animals; D002467:Cell Nucleus / Q000737:chemistry / Q000648:ultrastructure; D004249:DNA Damage; D004277:DNA, Single-Stranded / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004058:Diffusion; D004586:Electrophoresis / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D008322:Mammals; D009691:Nucleic Acid Denaturation; D009994:Osmolar Concentration; D012685:Sepharose; D020298:U937 Cells; D020122:tert-Butylhydroperoxide / Q000494:pharmacology
D004277:DNA, Single-Stranded; D007202:Indicators and Reagents; D012685:Sepharose; D020122:tert-Butylhydroperoxide