This article describes a method for quantifying DNA damage in cancer cells induced by chemotherapeutic agents. The technique involves the alkaline comet assay, which visualizes DNA breaks through electrophoresis.
Chemotherapeutic agents induce single- and double-strand DNA breaks in cancer cells, causing DNA damage and cell death.
To quantify DNA damage, mix the chemotherapy-treated cancer cells with molten low-melting agarose. Pipette this mixture onto agarose-precoated glass slides. Allow the molten agarose to solidify, embedding the cells within the porous gel matrix.
Treat with a lysis buffer containing high concentrations of detergents and salts. Detergents solubilize cellular and nuclear membranes. Further, high salt strips off the histone proteins from DNA, leaving supercoiled DNA attached to the nuclear matrix.
Incubate in alkaline electrophoresis buffer. The alkaline environment causes DNA supercoils to unwind, exposing single-strand, double-strand DNA breaks.
Place the slides in electrophoresis buffer-containing gel tray. Initiate electrophoresis.
Being negatively charged, DNA is attracted towards the positive anode. The damaged DNA fragments migrate out from the nucleus and travel through the porous gel matrix, with longer-length fragments moving slower than shorter fragments. On the contrary, undamaged DNA does not migrate readily, staying within the nucleus.
Post-electrophoresis, stain the cells with fluorescent nucleic acid dye solution. The dye molecules bind to DNA.
Under a fluorescence microscope, single cells containing damaged DNA exhibit fluorescent comet structure. The comet ‘head' represents undamaged DNA, while the 'tail' comprises damaged DNA fragments.
The length and intensity of the comet 'tail' indicates the extent of DNA damage in individual chemotherapy-treated cancer cells.
Combine the cell suspension with 1% molten low melting point agarose, at a volume ratio of 1:10. Mix gently by pipetting up and down, and immediately pipette 30 microliters onto a slide. Use the side of the pipette tip to spread the agarose-cell mixture to ensure the formation of a thin layer. Place the slides flat at 4 degrees Celsius in the dark for 10 minutes. Immerse the slides in 4 degrees Celsius lysis solution, in the dark for one hour to overnight.
For the alkaline comet assay, gently remove slides from the lysis solution, drain excess liquid, and gently immerse slides in AES, in the dark at 4 degrees Celsius for one hour to allow DNA unwinding.
Add pre-chilled AES in the electrophoresis slide tray. Do not exceed 0.5 centimeters above the slides. Place the slides inside, and cover with a cap. Set the power supply voltage to 1 volt per centimeter, and run at 4 degrees Celsius for 30 minutes. When the electrophoresis is complete, drain excess AES from the slides.
Gently immerse the slides twice, in distilled water at room temperature, for five minutes each time. After that, gently immerse the slides in 70% ethanol at room temperature for five minutes.
Begin staining procedure by drying the slides in the dark at 37 degrees Celsius for 10 to 15 minutes. Next, place 50 to 100 microliters of green fluorescent nucleic acid staining solution onto the dried agarose of each slide, and stain for 15 minutes at room temperature in the dark. Rinse the slides briefly in distilled water, and dry completely at 37 degrees Celsius in the dark.
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