In Vitro Mammalian Cells COMET Assay

Theoretical Background / Context of COMET Assay for DNA Damage

• -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.

In Vitro Comet Assay DNA Damage - 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 DNA Comet assay protocol  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 denaturation of DNA (with disruption of hydrogen bonds between the double-stranded DNA) and 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.