Microgel Electrophoresis of DNA from Individual Cells
Rydberg and Johanson (1978) quantitated DNA damage using a novel approach that involved embedding cells in agarose, lysing them and denaturing their DNA with NaOH, then neutralizing the samples and staining the nucleoid thus formed with acridine orange. The ratio of red to green fluorescence from samples was used as a measure of DNA damage. Cells with less DNA damage showed higher ratios of green to red fluorescence and cells with more DNA damage showed lower ratios in photometric measurement. The technique is based on two assumptions:(1) a double-stranded DNA with more breaks denatures faster to single-stranded DNA in mild alkaline conditions and (2) acridine orange molecules in relatively high concentrations make a polymer along the length of single-stranded DNA. Polymer of the dye with single-stranded DNA yields a red fluorescence. Acridine molecules in low concentration intercalate in native double-stranded DNA and emit green fluorescence. In another important development, Ostling and Johanson (1984) exposed murine lymphoma cells to gamma rays and showed that lysed cells when electrophoresed in agarose on microscope slides in neutral conditions displayed a dose-dependent increase in DNA migration. These authors also used acridine orange, and claimed the sensitivity of their technique to detect DNA damage was as low as 50 rads.
KeywordsCover Glass Human Lymphocyte Acridine Orange Repair Time Representative Cell
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