Alkaline Single Cell Gel Electrophoresis: A Sensitive Technique for Quantitative Estimation of DNA Damage in Individual Cancer Cells Following Chemotherapy
筆記録
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.
