Comet assay

[1] It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing.

[2][3] The comet assay (single-cell gel electrophoresis) is a simple method for measuring deoxyribonucleic acid (DNA) strand breaks in eukaryotic cells.

Cells embedded in agarose on a microscope slide are lysed with detergent and high salt to form nucleoids containing supercoiled loops of DNA linked to the nuclear matrix.

A glass cover slip is held at an angle and the mono-suspension is applied to the point of contact between the coverslip and the slide.

All proteins, RNA, membranes and cytoplasmic and nucleoplasmic constituents are disrupted and diffuse into the agarose matrix.

In alkaline conditions the DNA double helix is denatured and the nucleoid becomes single stranded.

The concept underlying the SCGE assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus.

The individual strands of DNA lose their compact structure and relax, expanding out of the cavity into the agarose.

Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time.

It is sometimes stated[6][7] that alkaline conditions and complete denaturating of the DNA is necessary to detect single strand breaks.

These include genotoxicity testing, human biomonitoring and molecular epidemiology, ecogenotoxicology, as well as fundamental research in DNA damage and repair.

[15] For example, Swain and Rao, using the comet assay[16] reported marked increases in several types of DNA damages in rat brain neurons and astrocytes during aging, including single-strand breaks, double-strand breaks and modified bases (8-OHdG and uracil).