The figure achieved is then divided by the total tail integration cell intensity.Ĭredit: Koncak / Materials The comet definition is determined by working out the percentage of DNA in the tail, dividing this by the integrated tail intensity and multiplying this by 100. In contrast, an alkaline Comet Assay is carried out at pH8 and a denaturing step is included so that both single and double-stranded breaks can be determined. In a Neutral Comet Assay, the DNA is kept as double strands so that the assay is used to detect double-stranded breaks. The underlying principle is likely that DNA breakage leads to a loss of supercoiling and results in the freedom to migrate towards the anode. The intensity, brightness and length of the comet tail relative to the head indicate the number of DNA breaks. The undamaged DNA stays where it is.Īs the damaged DNA travels through the agarose, it creates trails like comet tails – this is visualized with fluorescence microscopy. The damaged DNA, which has a negative charge, starts to move out of the nucleus towards the positively charged anode. The next stage is electrophoresis - a charge is applied to the cells by using an electrical field. This helps the cells to form nucleoids with the tightly coiled DNA in the nuclear matrix. This is often carried out on a microscope slide where the cell membrane is broken down (lysed) with the use of a detergent and high salt.

In the process, the single cells under investigation are placed in low melting point agarose which is a gel-like polysaccharide derived from seaweed. In the Comet Assay process, cell damage is determined by employing the theory that when a charge is applied to a cell, the amount of DNA that leaves the nucleus is an indication of the amount of DNA damage.

The Comet Assay is used in oncology to screen for genotoxicity (damage to genetic materials), gauge chemoprevention effectiveness, and determine the effectiveness of specific agents to halt progression to invasive cancer in the human body. The Comet Assay can be applied to both eukaryotic (cell with a membrane-bound nucleus with the DNA structured in chromosomes) and prokaryotic (a cell with neither a membrane-bound nucleus nor DNA structured in chromosomes) cells. In this case, damage is defined as single or double-stranded breaks in the DNA – such damage can be caused by chemicals or UV radiation. The DNA of an organism encodes the genetic instructions which govern the structure and functions of the constituent cells.ĭNA damage (tail intensity) detected by the Comet Assay, credit: Aviello et al. The Comet Assay, also known as single cell gel electrophoresis (SCRE), helps scientists to determine whether there has been deoxyribonucleic acid (DNA) damage to a single cell from apoptosis (cell death) or cytotoxicity (toxicity to cells) and the extent of this damage.

Comet definition