Comet Assay for DNA Damage Detection in Cells
Transcrição
Exposure to harmful agents induces single- and double-stranded breaks in the cellular DNA, which lead to DNA damage.
To detect DNA damage, begin with a single-cell suspension of chemical-treated cells in liquefied, low-melting point agarose. Load the suspension onto a glass slide, and allow the agarose to solidify — enabling the immobilization of cells within the porous gel matrix.
Treat the cells with lysis buffer. The detergents in the buffer solubilize the cellular and nuclear membranes, while the salts remove the histones from the DNA, forming a nucleoid body containing DNA attached to the nuclear matrix.
Incubate the slides in an alkaline electrophoresis buffer. This causes the DNA to unwind and generate single-stranded fragments. Place the slide in an electrophoresis apparatus, and connect it to the power supply. This allows the migration of negatively charged DNA toward the positive anode.
The undamaged DNA remains restricted within the nucleoid body. In contrast, the smaller, damaged DNA fragments begin moving toward the anode. This differential movement forms a comet-like structure, in which the undamaged DNA forms the head, while the damaged DNA forms the comet's tail.
Incubate the slide in a neutralization buffer to allow the single-stranded DNA fragments to renature. Next, treat the slide with a fluorescent dye that stains DNA. Visualize the slide under a fluorescence microscope.
The intensity and length of the tail correlate with the degree of cellular DNA damage.
