Olive P L, Banáth J P, Fjell C D
Medical Biophysics Dept., British Columbia Cancer Research Centre, Vancouver, Canada.
Cytometry. 1994 Aug 1;16(4):305-12. doi: 10.1002/cyto.990160404.
The alkaline comet assay is used to detect DNA single-strand breaks in individual cells embedded in agarose, lysed to denature DNA and remove proteins, and briefly exposed to an electric field to allow broken DNA to migrate. Total DNA fluorescence, measured by staining individual comets with propidium iodide, is reduced 30-40% by low doses of ionizing radiation, N-methyl-N-nitrosoguanidine (MNNG), etoposide, or hydrogen peroxide. Three possible explanations were examined: 1) these agents produce small fragments of DNA that are lost during lysis and electrophoresis, 2) the analysis of comet images is less efficient once the DNA is small enough to migrate in the electric field, and 3) DNA staining by propidium iodide is affected by changes in DNA structure caused by strand breaks and exposure to alkali. Our results indicate that the decrease in fluorescence after low doses is best explained by a change in ability of DNA, which has been denatured by alkali and subsequently renatured, to interact with fluorescent dyes. This change in fluorescence has the practical consequence of improving the ability of the alkaline comet assay to distinguish damaged from undamaged cells.
碱性彗星试验用于检测包埋在琼脂糖中的单个细胞内的DNA单链断裂，细胞经裂解使DNA变性并去除蛋白质，然后短暂暴露于电场中，使断裂的DNA迁移。用碘化丙啶对单个彗星进行染色来测量总DNA荧光，低剂量的电离辐射、N-甲基-N-亚硝基胍（MNNG）、依托泊苷或过氧化氢可使总DNA荧光降低30-40%。研究了三种可能的解释：1）这些试剂产生的DNA小片段在裂解和电泳过程中丢失；2）一旦DNA小到足以在电场中迁移，彗星图像分析的效率就会降低；3）碘化丙啶对DNA的染色受链断裂和碱处理导致的DNA结构变化的影响。我们的结果表明，低剂量后荧光的降低最好用已被碱变性并随后复性的DNA与荧光染料相互作用能力的变化来解释。这种荧光变化的实际结果是提高了碱性彗星试验区分受损细胞和未受损细胞的能力。
