Comet assay:Quantifying DNA damage and repair at single cell level
Comet image courtesy of Marie Vasquez, Helex 3 Inc.
What is a Comet assay?
No, it’s not a technique in astronomy whereby the solar system and beyond is scanned to assess the risk of the human race following the fate of the dinosaurs! For those that do "Single Cell Gel (SCG) electrophoresis", Comet is a highly sensitive and efficient means of assessing and quantifying DNA damage and repair at the level of the individual cell, and is applicable to any eukaryotic cell. Types of DNA damage that occur include single-/double-strand breaks, DNA-drug crosslinking, DNA-protein or DNA-DNA crosslinking and oxidative DNA base damage. The name Comet comes about since the samples look like a comet having a head and a tail extending to different lengths relative to the amount of DNA integrity or damage.
The Comet Technique
The Comet technique is based on applying gel electrophoresis to the stained nucleic acid sample(s) and then performing fluorescence microscopy analysis of the migrated product(s).
The DNA is stained with a fluorescent dye
Gel Electrophoresis is performed
Product is subsequently imaged under an epifluorescence microscope.
Analysis of DNA damage
The resulting structure is reminiscent of a comet, consisting of the nucleus head, and tail of damaged DNA. During electrophoresis, fractured DNA fragments migrate away from the nucleus and the extent of DNA damage can be quantified by measuring the relative % DNA between head and tail. Importantly, the exact class of DNA damage can be examined by careful control over cell preparation conditions. For example, the extent of alkali treatment (pH control) that is used to convert a DNA damage site into a DNA fragment (essential for Comet detection) can discriminate between different types of strand breaks. Sites of DNA base modifications can be converted to DNA fragments by introduction of enzymes that act specifically on such lesions. Furthermore, DNA crosslinking and binding can also be detected by the Comet assay, by determining the extent to which these interactions retard migration of DNA. Since its "impact", Comet has become a core technique within fields or study such as toxicology, apoptosis, DNA repair, aging, cell cycle analysis and free radical biology.
Comet slides are examined under a fluorescent microscope, often by widefield epifluorescence microscopy. Widefield epifluorescence microscopy has developed into a universally accessible technique for study of fluorescently labelled cells and tissues. Over the last few decades, this drive has been accelerated by improvements in fluorescent probes, labelling chemistry, optical instrumentation (such as filters and objectives) and detector technology.
The widefield technique involves flood-illumination of the field of view by a wavelength or small wavelength range (often though use of an excitation filter and arc lamp). The stoke-shifted fluorescent emission transmits though the dichroic, that was initially used to reflect the shorter wavelength excitation light onto the sample, gets optically filtered once again by an emission filter (often called barrier filter), and focused onto the camera detector.
Komet: Providing a Solution for Comet Experiments
Komet is Andor’s software solution for image acquisition through to analysis and assessment of DNA damage. Komet 7 and Komet 7-GLP are the latest versions of the original Komet software first released in 1992 by Kinetic Imaging. Komet 7 and Komet 7-GLP embrace new OECD requirements building on the experience developed over the last 25+ years. Komet 7 maintains backward compatibility for hundreds of existing users, while also enabling new possibilities using the latest computer systems (including Windows 10 Support), cameras (via virtual camera functionality) and new light source developments. Komet remains the most highly referenced comet assay analysis solution available for research.