Written and maintained by CASRAI Editorial Board
Last updated
Short answer
The comet assay, also called single-cell gel electrophoresis, measures DNA damage in individual cells. Cells are embedded in agarose, lysed, and electrophoresed; damaged DNA migrates out of the nucleoid and forms a comet-shaped tail whose intensity reflects how many breaks are present. The standard alkaline version detects strand breaks and alkali-labile sites, and enzyme-modified versions extend it to specific lesions.
Definition and scope
The assay was first described by Östling and Johanson in 1984 using a neutral version, applied to strand-break rejoining in gamma-irradiated mouse lymphoma cells. Singh and colleagues introduced the first alkaline version in 1988, using X-irradiated human lymphocytes. The nucleoid principle it relies on goes back to Cook and colleagues in 1976.
The Minimum Information for Reporting on the Comet Assay (MIRCA) recommendations describe the alkaline comet assay as a technically simple, sensitive assay for DNA damage and for DNA repair activity. It does not count specific lesions directly, which matters when you read a result. It has been used in cell culture, animal studies, Drosophila, and human nutrition, occupational and clinical studies.
How it works in practice
Lysis strips histones from the cell and leaves a nucleoid, in which DNA remains attached to the nuclear matrix as supercoiled loops. Strand breaks relax that supercoiling, so looped DNA can migrate during electrophoresis and forms the tail of the comet. The more breaks, the more DNA in the tail. Under alkaline conditions, alkali-labile sites are converted into breaks, so the standard alkaline assay reports strand breaks plus alkali-labile sites together.
For background on the separation principle, see our guides on gel electrophoresis and agarose gel electrophoresis. The comet assay applies electrophoresis to whole nucleoids in a gel, rather than to extracted DNA fragments.
Alkaline, neutral and enzyme-modified versions
Alkaline versus neutral
MIRCA notes that the neutral version, run at neutral pH, is rarely used, and its reporting recommendations cover the alkaline protocol. The two are not interchangeable: the alkaline version also picks up alkali-labile sites.
Enzyme-modified comet assay
Lesion-specific enzymes cut DNA at particular damaged bases, turning them into measurable breaks. The main enzymes and their targets are:
- FPG and OGG1 for oxidized purines
- Endonuclease III for oxidized pyrimidines
- T4 endonuclease V for UV dimers
- Uracil-DNA glycosylase (UDG) for uracil
The result is reported as net enzyme-sensitive sites: the value for enzyme-treated samples minus the value for the same samples incubated in enzyme buffer alone. MIRCA lists enzyme source, concentration, incubation duration and temperature as essential to report, because enzymes from different manufacturers can differ in activity and specificity.
Scoring descriptors
MIRCA names four descriptors in common use: % DNA in tail, tail length, tail moment and a visual score. It states that these are different scales and are not directly comparable, so a paper reporting tail moment cannot simply be set beside one reporting % DNA in tail. An optional calibration, using an ionizing-radiation curve, expresses damage as lesions per 109 nucleotides; MIRCA calls this desirable. No fixed number of cells is mandated, but the number of comets per gel and the number of gels scored must be reported.
Controls and what a methods section must report
MIRCA separates two kinds of control. Experimental negative and positive controls are treated as experimental groups, as in OECD Test Guideline 489, and positive controls are effectively essential in cell culture; human biomonitoring studies have none. Assay controls are different: cryopreserved aliquots of one cell batch exposed to an agent such as ionizing radiation, hydrogen peroxide or methyl methanesulfonate (or, for lesion-specific work, Ro19-8022 plus light, or potassium bromate). MIRCA classes reporting damage levels in assay controls, and the assay variation, as essential, because they support comparison between experiments and laboratories.
The checklist below condenses the MIRCA items with its essential (E) and desirable (D) grades:
| Area | Essential (E) | Desirable (D) |
|---|---|---|
| Samples | Cell type; transfer temperature and duration before processing; storage and thawing | – |
| Gel and lysis | Final agarose concentration; lysis buffer composition | Lysis duration and temperature |
| Alkaline treatment and electrophoresis | Alkaline treatment composition, duration and temperature; electrophoresis buffer, voltage per cm, duration and temperature | Neutralization |
| Staining and imaging | Dye type | Dye concentration; magnification; representative images |
| Scoring | Primary descriptor; comets per gel and gels scored; mean or median and the independent unit; image-analysis software name, manufacturer and version | – |
| Statistics | Methods suited to the design | – |
This is the kind of checklist a reporting guideline exists to provide: the assay is technically simple, so unreported differences in lysis or electrophoresis conditions can make results hard to compare across laboratories.
Common misunderstandings
- It counts specific lesions. The standard alkaline assay detects strand breaks and alkali-labile sites; named lesions need the enzyme-modified version.
- Any tail metric will do. Descriptors are on different scales and are not directly comparable.
- A bigger tail always means the treatment broke DNA. Nonspecific nuclease activity, which varies by tissue, and test compounds that break DNA or interfere with the assay (vitamin C is one example in the literature) can both distort results. Undamaged control nucleoids are needed to detect the nuclease problem.
- It replaces a viability readout. It answers a different question; cytotoxicity is assessed with other methods such as the one in our MTT assay guide.
FAQ
What is the comet assay used to measure?
DNA damage in individual cells and, with suitable adaptations, DNA repair activity. The standard alkaline version detects strand breaks and alkali-labile sites.
Is the comet assay the same as single-cell gel electrophoresis?
Yes. Both names describe the same method.
Why is it called a comet assay?
Breaks relax the supercoiled DNA loops of the nucleoid so DNA migrates during electrophoresis, producing a bright head with a trailing tail. Tail intensity depends on the frequency of breaks.
Can it identify oxidative DNA damage?
Not in the standard version. Adding FPG or OGG1 reveals oxidized purines, and endonuclease III oxidized pyrimidines, reported as net enzyme-sensitive sites.
Sources
- Møller P, Azqueta A, Boutet-Robinet E, et al. Minimum Information for Reporting on the Comet Assay (MIRCA). Nature Protocols 2020;15(12):3817-3826. doi:10.1038/s41596-020-0398-1. https://pmc.ncbi.nlm.nih.gov/articles/PMC7688437/. Date checked: 2026-10-29.
- Azqueta A, et al. Comet assay to measure DNA repair: approach and applications. Frontiers in Genetics 2014;5:288. doi:10.3389/fgene.2014.00288. https://pmc.ncbi.nlm.nih.gov/articles/PMC4142706/. Date checked: 2026-10-29.
