Comet

SINGLE CELL GEL ELECTROPHORESIS

This technique has also been called "Comet assay" after the shape of the images (comets) seen under the microscope. It is a microgel electrophoresis technique which allows to measure DNA damage cell by cell. The alkaline version, introduced by Singh et al. in 1988, detects DNA breakage, alkali labile sites, open repair sites and cross links. For this technique cells are mixed with agarose gel, which is spread onto a microscope slide. The cells are lysed with high salt concentrations and detergents. The remaining nuclear DNA is then denaturated in an alkali buffer and electrophoresed in the same buffer. The DNA fragments migrate out of the nucleus, towards the positive pole. After electrophoresis, the slides are stained with a fluorochrome, like ethidium bromide. An image analysis system is used to measure several damage parameters. The most useful are tail length (TL), measured from the center of the nucleus towards the end of the tail, the percentage of DNA in the tail (TD) and tail moment (TM = TL x TD)

To allow detection of oxidative DNA damage, the Fpg enzyme (Formamidopyrimidine DNA glycosylase), which detects mainly 8-hydroxydeoxyguanine and so called Fpg-sensitive sites, may be included (Hartwig et al., 1996).

Characteristics of COMET test:

Biomarker of exposure and/or effect: DNA damage is not a fixed mutation
Endpoint: DNA damage

Advantages/ Disadvantages

Advantages	Disadvantages
Cell/cell approach Applicable on many cell types No in vitro cultivation step required Possible estimation of global repair capacity after in vitro challenging experiments Cheap Fast Simplicity Give some indication on apoptosis	The detected DNA damage does not correspond to fixed mutations Need of internal reference to avoid experimental variation during the electrophoresis

Confounding factors:

controversial

References:

Singh NP, McCoy MT, Tice RR, Schneider EL. "A simple technique for quantitation of low levels of DNA damage in individual cells." Exp Cell Res. 1988 Mar;175(1):184-91.
Hartwig A, Dally H, Schlepegrell R. "Sensitive analysis of oxidative DNA damage in mammalian cells: use of the bacterial Fpg protein in combination with alkaline unwinding." Toxicol Lett. 1996 Nov;88(1-3):85-90.