Comet Assay Virtual Lab Simulation | PraxiLabs

In Vitro Mammalian Cells COMET Assay

Biology | Toxicology | Biochemistry | Pharmacology

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General Aim

The single-cell gel electrophoresis assay or the Comet assay is a versatile, simple, sensitive, and rapid tool that aims at detecting primary DNA damage at an early stage in a 96-well tissue culture plate after exposure to the geometric concentration of different nanoparticles.


In Vitro Mammalian Cells COMET Assay. Single Cell Gel Electrophoresis (SCGE) Assay

Learning Objectives (ILO’s)

  • Successfully handle the required instruments and consumables needed in the experiment.

  • Check the confluence and count cells under the microscope.

  • Dilute the cells to a specific count suitable for seeding in the 96-well plate.

  • Calculate the concentration of tested chemicals and prepare the calculated doses in the cell culture medium.

  • Aspirate the old medium and add the new medium containing the tested chemicals in the appropriate wells.

  • Follow the protocol step by step till the end of the experiment to achieve results.

  • Read the results of cells exposed to different doses of tested chemical(s) using the fluorescent microscope and COMET software.

Theoretical Background/Context

In genetics, genotoxicity describes the property of chemical agents that damages the genetic information within a cell causing mutations, which may lead to cancer. While genotoxicity is often confused with mutagenicity, all mutagens are genotoxic, whereas not all genotoxic substances are mutagenic. 

The alteration can have direct or indirect effects on the DNA: the induction of mutations mistimed event activation, and direct DNA damage leading to mutations. The permanent, heritable changes can affect either somatic cells of the organism or germ cells to be passed on to future generations. Cells prevent the expression of the genotoxic mutation by either DNA repair or apoptosis; however, the damage may not always be fixed leading to mutagenesis.

Principle of Work

The duplex DNA is organized in the nucleus around the histone proteins, which help in its supercoiling and packaging. Interaction of the DNA with genotoxicants may cause DNA strand breaks. The Comet assay is based on the principle that strand breaks, reduce the size of the large duplex DNA molecule, and under high pH, the relaxed strands are pulled out during electrophoresis. 

The high salt concentration in the lysis step of the assay (pH 10), removes the cell membranes, histone proteins, cytoplasm, and nucleoplasm, and disrupts the nucleosomes, leaving behind the nucleoid, consisting of the negatively supercoiled DNA. The breaks present in the DNA cause local relaxation of supercoils and loops of DNA are then free. A high pH (>13) at the unwinding step facilitates the denaturation of DNA (with disruption of hydrogen bonds between the double-stranded DNA) and the expression of alkali labile sites as frank breaks.

During electrophoresis, this damaged DNA is pulled out toward the anode, thus forming the distinct “Comet,” with a head (intact DNA) and a tail (damaged DNA), visualized after fluorescent staining.


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