NDLI: Assaying DNA damage in hippocampal neurons using the comet assay.

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Year: 2012 Issue: 70

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Assaying DNA damage in hippocampal neurons using the comet assay.

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Assaying DNA damage in hippocampal neurons using the comet assay.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Nowsheen, Somaira Xia, Fen Yang, Eddy S.
Description	Author Affiliation: Nowsheen S ( Department of Radiation Oncology, University of Alabama-Birmingham.)
Abstract	A number of drugs target the DNA repair pathways and induce cell kill by creating DNA damage. Thus, processes to directly measure DNA damage have been extensively evaluated. Traditional methods are time consuming, expensive, resource intensive and require replicating cells. In contrast, the comet assay, a single cell gel electrophoresis assay, is a faster, non-invasive, inexpensive, direct and sensitive measure of DNA damage and repair. All forms of DNA damage as well as DNA repair can be visualized at the single cell level using this powerful technique. The principle underlying the comet assay is that intact DNA is highly ordered whereas DNA damage disrupts this organization. The damaged DNA seeps into the agarose matrix and when subjected to an electric field, the negatively charged DNA migrates towards the cathode which is positively charged. The large undamaged DNA strands are not able to migrate far from the nucleus. DNA damage creates smaller DNA fragments which travel farther than the intact DNA. Comet Assay, an image analysis software, measures and compares the overall fluorescent intensity of the DNA in the nucleus with DNA that has migrated out of the nucleus. Fluorescent signal from the migrated DNA is proportional to DNA damage. Longer brighter DNA tail signifies increased DNA damage. Some of the parameters that are measured are tail moment which is a measure of both the amount of DNA and distribution of DNA in the tail, tail length and percentage of DNA in the tail. This assay allows to measure DNA repair as well since resolution of DNA damage signifies repair has taken place. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell (1,2). Cells treated with any DNA damaging agents, such as etoposide, may be used as a positive control. Thus the comet assay is a quick and effective procedure to measure DNA damage.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/50049
Journal	Journal of Visualized Experiments
Issue Number	70
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-12-19
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Comet Assay Dna Damage Hippocampus Physiology Neurons Chemistry Genetics Cytology Research Support, Non-u.s. Gov't Video-audio Media
Content Type	Text
Resource Type	Article

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