Comet assay

The single cell gel electrophoresis assay (SCGE, also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term 'comet' refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet. The single cell gel electrophoresis assay (SCGE, also known as comet assay) is an uncomplicated and sensitive technique for the detection of DNA damage at the level of the individual eukaryotic cell. It was first developed by Östling & Johansson in 1984 and later modified by Singh et al. in 1988. It has since increased in popularity as a standard technique for evaluation of DNA damage/repair, biomonitoring and genotoxicity testing. It involves the encapsulation of cells in a low-melting-point agarose suspension, lysis of the cells in neutral or alkaline (pH>13) conditions, and electrophoresis of the suspended lysed cells. The term 'comet' refers to the pattern of DNA migration through the electrophoresis gel, which often resembles a comet. 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. Electrophoresis at high pH results in structures resembling comets, observed by fluorescence microscopy; the intensity of the comet tail relative to the head reflects the number of DNA breaks. The likely basis for this is that loops containing a break lose their supercoiling and become free to extend toward the anode. This is followed by visual analysis with staining of DNA and calculating fluorescence to determine the extent of DNA damage. This can be performed by manual scoring or automatically by imaging software. A sample of cells, either derived from an in vitro cell culture or from an in vivo test subject is dispersed into individual cells and suspended in molten low-melting-point agarose at 37 °C. This mono-suspension is cast on a microscope slide. A glass cover slip is held at an angle and the mono-suspension applied to the point of contact between the coverslip and the slide. As the coverslip is lowered onto the slide the molten agarose spreads to form a thin layer. The agarose is gelled at 4 °C and the coverslip removed. The agarose forms a matrix of carbohydrate fibres that encapsulate the cells, anchoring them in place. The agarose is considered to be osmotic-neutral, therefore solutions can penetrate the gel and affect the cells without cells shifting position. In an in vitro study the cells would be exposed to a test agent – typically UV light, ionising radiation, or a genotoxic chemical – to induce DNA damage in the encapsulated cells. For calibration, hydrogen peroxide is usually used to provide a standardized level of DNA damage. 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). After lysis of the cells (typically 1 to 2 hours at 4 °C) 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.