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comet assay

The comet assay can be applied to a broad spectrum of cells such as white blood, buccal, nasal, sperm, and exfoliated cells from urinary tract and lachrymal ducts, although technical variables should be carefully controlled both within and across laboratories. Biotechnol. Controlling variation in the comet assay. Collecting blood or tissues is not always feasible in all human subjects, and other sources of cells that can be collected non-invasively have been tested with the comet assay; for example, various types of epithelial cells (Rojas et al., 2014) as well as sperm (Cortés-Gutiérrez et al., 2014; Brunborg et al., 2015). 6:61. doi: 10.3389/fgene.2015.00061, Brunborg, G., Jackson, P., Shaposhnikov, S., Dahl, H., Azqueta, A., Collins, A. R., et al. 2007 Epub ahead of print. 724, 41–45. 8-oxoG was also preferentially repaired in the transcribed strand of the ATM gene which was revealed using a specific glycosylase in the Comet-FISH assay [58]. Cell. The test protocol involves embedding a small amount of cells in an agarose gel matrix on a microscope slide and lysing to remove the cell membrane and most of the cellular proteins. The simplest approach to DNA repair measurement is to treat cells with a DNA-damaging agent and then to incubate them to allow repair to proceed, measuring the amount of damage remaining at intervals.

Luke Simon, Douglas T. Carrell, in Encyclopedia of Reproduction (Second Edition), 2018.

Møller and Loft (2014) remind us that to keep the comet assay statistical analysis simple, appropriate study design and statistical power should be carefully considered when planning experiments. P.B. For calibration, hydrogen peroxide is usually used to provide a standardized level of DNA damage. Fairbairn DW, Olive PL, O婬l KL. At the molecular level, the formation of comet in the DNA of cells upon genotoxic insult can be visualized through the method of gel electrophoresis and indicates DNA strand breaks, as the damaged DNA migrates at a different rate than nondamaged DNA during electrophoresis. The comet assay, also known as the single-cell gel electrophoresis (SCGE) assay, measures DNA strand breaks in individual cells. Tests with cells from the gonads (testis and ovary) seem to be the most appropriate and a promising tool for demonstrating that a test compound reaches the gonads and is able to interact with the genetic material of germ cells.

Statistical analysis of comet assay results. The likely explanation for comet tail formation is that loops containing a break lose their supercoiling and become free to extend toward the anode (Tice et al., 2000; Collins, 2004). Res. No use, distribution or reproduction is permitted which does not comply with these terms. [106] is a method for measuring DNA strand breaks and can serve as an alternative to in vivo testing for DNA damage. These include genotoxicity testing, human biomonitoring and molecular epidemiology, ecogenotoxicology, as well as fundamental research in DNA damage and repair. Genet. [7][8][9][10] In alkaline conditions, however, additional DNA structures are detected as DNA damage: AP sites (abasic sites missing either a pyrimidine or purine nucleotide) and sites where excision repair is taking place.[11][12]. Briefly, cells placed in low-melting agarose on the surface of microscope slides are subjected to electrophoresis after the action of a damaging agent. Also including “omics” data will aid in unraveling the mode of action of genotoxic compounds (Slyskova et al., 2012, 2014; Santos et al., 2015)—though it is worth pointing out that several studies have shown that phenotypic measures of DNA repair do not necessarily correlate with genomic or transcriptomic data (Collins et al., 2012; Slyskova et al., 2012, 2014); the different approaches should be regarded as complementary. The aqueous salt disrupts proteins and their bonding patterns within the cell as well as disrupting the RNA content of the cell. Fluorescent, "DNA double-strand breaks measured in individual cells subjected to gel electrophoresis", "A simple technique for quantitation of low levels of DNA damage in individual cells",, Articles with dead external links from August 2017, Articles with permanently dead external links, Articles needing additional references from May 2014, All articles needing additional references, Creative Commons Attribution-ShareAlike License. Hughes CM, Lewis SEM, McKelvey-Martin V, Thompson W. Reproducibility of human sperm DNA measurements using a single cell gel electrophoresis assay.

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