A combined alkaline unwinding/Southern blotting assay for measuring low levels of cellular DNA breakage within specific genomic regions.
A method is described which combines alkaline induced DNA unwinding and Southern blotting to measure DNA damage occurring in specific genomic regions. Damage induced by gamma-rays at levels as low as 2 Gy was measured in bulk DNA and in a one megabase region surrounding the actively transcribed oncogene, c-myc, as well as in the inactive alpha-satellite DNA of chromosome 17. Although the unwinding kinetics for bulk DNA were consistent with random strand breakage throughout the genome as a whole, measurements at specific loci indicated that the region encompassing c-myc was at least 2-fold more susceptible to damage than either the bulk of the genome or the alpha-satellite region. The results of this study indicate that the combined alkaline unwinding/Southern blotting assay is a sensitive method for the detection of DNA damage within specific chromatin regions, at biologically relevant doses.
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Department of Pharmacology, Virginia Commonwealth University/Medical College of Virginia, Richmond 23298.
Gewirtz
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Povirk
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D015139:Blotting, Southern; D004247:DNA / Q000528:radiation effects*; D004249:DNA Damage; D004276:DNA, Satellite; D005720:Gamma Rays; D016259:Genes, myc / Q000528:radiation effects*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D014407:Tumor Cells, Cultured
D004276:DNA, Satellite; D004247:DNA