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Comet assay

The Comet Assay, also called single cell gel electrophoresis (SCGE), is a sensitive and rapid technique for quantifying and analyzing DNA damage in individual cells. This is one of the techniques used in the area of cancer research for the evaluation of genotoxicity and effectiveness of chemoprevention. Swedish researchers stling & Johansson developed this technique in 1984. Singh, et al., later modified this technique, in 1988, as the Alkaline Comet Assay. The resulting image that is obtained resembles a "comet" with a distinct head and tail. The head is composed of intact DNA, while the tail consists of damaged (single-strand or double-strand breaks) or broken pieces of DNA. Individual cells are embedded in a thin agarose gel on a microscope slide. All cellular proteins are then removed from the cells by lysing. The DNA is allowed to unwind under alkaline/neutral conditions. Following the unwinding, the DNA undergoes electrophoresis, allowing the broken DNA fragments or damaged DNA to migrate away from the nucleus.

After staining with a DNA-specific fluorescent dye such as ethidium bromide or propidium iodide, the gel is read for amount of fluorescence in head and tail and length of tail. The extent of DNA liberated from the head of the comet is directly proportional to the amount of DNA damage. The Comet Assay can be used to detect DNA damage caused by double strand breaks, single strand breaks, alkali labile sites, oxidative base damage, and DNA cross-linking with DNA or protein. The Comet Assay is also used to monitor DNA repair by living cells.3

Comet assay
DNA fragments are released from nuclei using electrophoresis Isolated nuclei are mounted into electrophoretic gel after electrophoresis are stained with fluorescent dye. If DNA fragments are present a comet tail is present observed in the vicinity of the nuclei.

Comet assay

Possible results of a comet assay

Normal nucleus without fragments (DNA is not damaged mutagenicity excluded)

Two nuclei with DNA damage

Evaluation of a comet assay

Comet assay computer analysis

Lysis The slides are then immersed in a solution that cause the cells to lyse. The lysis solution often used in the comet assay consists of a highly concentrated aqueous salt (often, common table salt can be used) and a detergent (such as Triton X-100 or sarcosinate). The pH of the lysis solution can be adjusted (usually between neutral and alkaline pH) depending upon the type of damage the researcher is investigating. The aqueous salt disrupts proteins and their bonding patterns within the cell as well as disrupting the RNA content of the cell. The detergent dissolves the cellular membranes. Through the action of the lysis solution the cells are destroyed. All proteins, RNA, membranes and cytoplasmic and nucleoplasmic constituents are disrupted and diffuse into the agarose matrix. Only the DNA of the cell remains, and unravels to fill the cavity in the agarose that the whole cell formerly filled. This structure is called nucleoid (a general term for a structure in which DNA is concentrated).

Electrophoresis After lysis of the cells (typically 1 to 2 hours at 4C) the slides are washed in distilled water to remove all salts and immersed in a second solution - an electrophoresis solution. Again this solution can have its pH adjusted depending upon the type of damage that is being investigated. The slides are left for ~20 minutes in the electrophoresis solution prior to an electric field being applied. In alkaline conditions the DNA double helix is denatured and the nucleoid becomes single stranded. An electric field is applied (typically 1 V/cm) for ~20 minutes. The slides are then neutralised to pH 7, stained with a DNA-specific fluorescent stain and analysed using a microscope with an attached CCD (charge-coupled device - essentially a digital camera) that is connected to a computer with image analysis software. Electrophoresis refers to the migration of a charged molecule through a restrictive matrix , or gel, drawn by an electrical force. As the force drags the molecule through the gel, it encounters resistance from the strands of the gel, retarding its rate of migration.

In gel electrophoresis, larger molecules migrate more slowly than smaller ones, and so the distance of migration within a gel can be used to determine a molecule's size.

APPLICATIONS: Major applications of the Comet assay are in the following areas: 1.Genetic toxicology (DNA damage) In vivo & in vitro evaluation of genotoxic chemicals DNA damage: SSBs, DNA crosslinking, alkali labile sites DNA repair: Strand break repair Excision repair 2. Eco-toxicology: the assay has been used to monitor soil and aquatic toxicology 3. Nutrition 4. Bio-monitoring genotoxicity 5. Environmental biomonitoring Evaluation of genotoxic pollutants from hazardous waste sites 6. Hypoxia assessment 7. Human epidemiology For assessing levels of DNA damage in occupationally, clinically and environmentally exposed individuals or in evaluating the differences in DNA repair competency among control and exposed individuals. (a) Sperm bank (b) Blood bank (c) Monitoring Radio- and chemo- therapy in cancer patients 7. Miscellaneous

ADVANTAGES: 1. it is a non-invasive technique 2. it requires <10,000 cells and collection of data at the level of the individual cell, allowing for more robust types of statistical analyses 3. counting of 50-100 cells per individual / treatment group, through a computerised image analysis software gives a robust statistics 4. that virtually any eukaryotic cell population is amenable to analysis 5. its sensitivity (1 break in 1010 daltons) for detecting DNA damage and repair results obtained in a few hours compared to conventional cytogenetics techniques which take a few days 6. single strand breaks (SSBs) and alkali labile lesions (capable of being transformed into SSBs under alkaline conditions) in the DNA of individual cells can be assessed only few microlitres of blood (5-10 l), nasal & buccal mucosal cells, epithelial cells, male germ cells, fine needle biopsy, etc. required human studies.