Comet Assay

The Comet Assay, or Single Cell Gel Electrophoresis (SCGE) is an electrophoresis technique used to asses the damage to genomic DNA, applicable to a range of cell types.

Contents

An Overview of the Comet assay steps
An Overview of the Comet assay steps

Overview

To assess the extent of DNA damage, cells are exposed to a ‘treatment’ of interest, such as a novel pharmaceutical or an environmental contaminant. These cells are then embedded in agarose on a glass microscope slide to immobilise them and undergo a number of further treatment steps.

The cells are lysed in a lysis buffer to remove the cell membrane. In the most common form of the assay, the Alkaline Comet Assay, the cells are then placed in an alkaline solution, to undergo alkaline unwinding of the DNA and conversion of alkali labile site to double stranded DNA breaks.

The microscope slides are then placed in am electrophoresis chamber and electrophoresed in alkaline conditions to separate the damaged DNA. This separation causes the distinct “Comet” Shape in which damaged DNA forms a tail to the cells nucleus which resembles a comet.

Finally, the slides are neutralised, then stained with a DNA stain and imaged using fluorescent microscopy to quantify the tail regions.

Comet Assay results
Cells that have DNA damage show a distinct comet shape when analysed

Applications

The Comet assay method has a wide range of applications related to assessing DNA damage and DNA repair.

As an OECD approved test for genotoxicity the assay is widely used in the testing of new pharmaceuticals to ensure there are no damaging effects to DNA integrity. Many pharmaceutical companies and contract research organisations conduct Comet Assays on a frequent basis for this reason. The Comet assay provides a quantitative measure of DNA damage in both cultures and in vivo cells.

The Comet assay is also widely used in the study of DNA damage repair mechanisms. By using a known genotoxic agent to damage DNA, and then allowing cells to recover, the extend of DNA damage repair can be quantified. Researchers use this method to study changes in genetic or proteomic profiles that may affect the repair of DNA.

There are further applications for the Comet assay in fields such as Bio-monitoring for ecological contaminants, occupational exposure to chemicals and diagnostic quantification of DNA damage due to disease. For a full overview of techniques and applications you can find papers via google scholar here.

Important Considerations

When determining the suitability of equipment for use in the Comet Assay there are several factors that need to be considered. The most important of these is to prevent background DNA damage.

Background Damage can be caused by exposure to light (UV damage) or exposure to heat, so both these factors should be controlled during the assay.

Cleaver Scientific’s Comet Assay equipment is specifically designed to limit background DNA damage by reducing exposure to light and heat.

Electrophoresis tanks

Cleaver Scientific electrophoresis tanks for the Comet Assay are constructed from opaque acrylic, so no light can penetrate the tank during electrophoresis. The tanks come equipped with connections for a recirculating water chiller to keep the tank at a constant temperature during electrophoresis and eliminate joule heating effects. These tanks can be used with ethylene glycol, allowing cooling to 4°C in the electrophoresis buffer.

Cell embedding

The Comet Assay chilling plate provides a convenient way to keep cells cool while embedding them in agarose. The simple cool block based aluminium base provides space for 25 slides to stay cool while the cell embedded agarose sets.

Slide Processing

Our unique COMRAC slide system allows processing of 25 slides simultaneously inside our opaque acrylic staining dishes. These can be placed directly in a fridge during the treatment and staining processes to prevent light and heat exposure. Using the COMRAC system can save up to 80& of processing time compared with traditional Coplin jar methods.