The comet assay – from toy to tool
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1
Ulm University, Human Genetics, Germany
The comet assay is nowadays the most common method for measuring DNA damage and repair in single cells. It is based on the microelectrophoretic study published by Ostling and Johanson (1984) and was developed by Singh and coworkers (1988) to a versatile technique for quantitation of low levels of DNA damage in individual cells. This alkaline version still is the basis for the triumphant success of the comet assay in basic research into mechanisms of DNA damage and DNA repair, genotoxicity testing, ecotoxicology and human biomonitoring. Important technical improvements (e.g., the use of precoated slides, introduction of image analysis, high throughput methods, automated scoring systems) made the assay more robust and more efficient. Modifications of the standard protocol provide more specific information on the type and biological significance of the damage studied. The introduction of lesion-specific endonucleases allowed the characterization of oxidative base damage, alkylation damage and UV-induced pyrimidine dimers. The combination with fluorescence in situ hybridization (FISH) made it possible to identify DNA of particular chromosome regions and measure effects of damage and repair in particular genes.
The comet assay is being increasingly used in genotoxicity testing. In particular, the in vivo comet assay has become a component of some genotoxicity test strategies and generally accepted test protocols have evolved over the years. A large international collaborative trial sponsored by the Japanese Center for the Validation of Alternative Methods (JaCVAM) was recently completed and an OECD test guideline was approved.
The comet assay is widely used in human biomonitoring to measure DNA damage as a marker of exposure to genotoxic agents or to investigate genoprotective effects. However, there are still problems in comparing results from different laboratories and there is need for reducing inter-laboratory variation and identification of standard conditions for the assay protocol, the study design and the statistical analysis. The recently launched ComNet project aims to validate the comet assay as a reliable biomonitoring tool.
Unfortunately, the comet assay has sometimes been used without the necessary knowledge about the principles underlying the method and the kind of information it provides. Such knowledge gaps may lead to misconceptions regarding the use of the assay and the interpretation of results.
This presentation will briefly discuss the developments and applications of the comet assay, its advantages and limitations and the requirements for appropriate test performance.
Keywords:
History,
developments,
applications,
performance,
misconceptions
Conference:
ICAW 2015 - 11th International Comet Assay Workshop, Antwerpen, Belgium, 1 Sep - 4 Sep, 2015.
Presentation Type:
Oral Presentation
Topic:
Origin and history of the Comet Assay
Citation:
Speit
G
(2015). The comet assay – from toy to tool.
Front. Genet.
Conference Abstract:
ICAW 2015 - 11th International Comet Assay Workshop.
doi: 10.3389/conf.fgene.2015.01.00052
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Received:
23 Apr 2015;
Published Online:
23 Jun 2015.
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Correspondence:
Prof. Guenter Speit, Ulm University, Human Genetics, Ulm, Germany, guenter.speit@uni-ulm.de