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Single Cell Gel Electrophoresis

This document provides information about performing the comet assay technique for examining DNA damage at the single cell level. It introduces the comet assay and notes its simplicity, sensitivity, versatility, speed and low cost. Contact information is provided for two individuals, Ms. Rachel Kwok and Dr. G. Don Jones, who can provide further information and assistance with the comet assay in the Biocentre laboratory. A brief overview of the protocol is also given, explaining how cells embedded in agarose are lysed and subjected to electrophoresis, resulting in comet-shaped structures that can then be analyzed using image software to assess DNA strand break damage. Material costs for a single comet assay experiment are estimated to be approximately £20.

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113 views3 pages

Single Cell Gel Electrophoresis

This document provides information about performing the comet assay technique for examining DNA damage at the single cell level. It introduces the comet assay and notes its simplicity, sensitivity, versatility, speed and low cost. Contact information is provided for two individuals, Ms. Rachel Kwok and Dr. G. Don Jones, who can provide further information and assistance with the comet assay in the Biocentre laboratory. A brief overview of the protocol is also given, explaining how cells embedded in agarose are lysed and subjected to electrophoresis, resulting in comet-shaped structures that can then be analyzed using image software to assess DNA strand break damage. Material costs for a single comet assay experiment are estimated to be approximately £20.

Uploaded by

sivaram888
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We take content rights seriously. If you suspect this is your content, claim it here.
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CSMM

Single Cell Gell


Electrophoresis
(Comet Assay)

For information and enquiries, please contact:

Ms Rachel Kwok
Laboratory 105
Biocentre
Tel: 0116 223 1842
Email: rwlk3@le.ac.uk

Or, if Ms Kwok is unavailable:

Dr G Don Jones
Biocentre
Telephone: 0116 223 1841
Email: gdj2@le.ac.uk
The Comet Assay

Single cell gel (SCG) electrophoresis or ‘Comet assay’ is a rapid and very sensitive
fluorescent microscopy-based method to examine DNA damage and repair at the level of
individual cells. Furthermore, the assay is cheap, has low material requirements and can
use virtually any eukaryotic cell. Since the introduction of the alkaline Comet assay in
1988, a number of advancements have greatly increased the flexibility and utility of this
technique for detecting various forms of DNA damage (e.g. single- and double-strand
breaks, oxidative DNA base damage, and DNA-DNA/DNA-protein/DNA-drug crosslinking).

The assay is attractive because of its simplicity, sensitivity, versatility, speed and
economy, and the number of publications it leads to rises each year with applications in
genotoxicity testing, human biomonitoring and molecular epidemiology,
ecogenotoxicology, as well as fundamental research in DNA damage and repair. Comet
assay not only provides an estimate of how much damage is present in cells, but what
form it takes. Although it is essentially a method for measuring DNA breaks, the
introduction of lesion-specific endonucleases allows detection of, for example, ultraviolet
(UV)-induced pyrimidine dimers, oxidized bases and alkylation damage.

The protocol is very straightforward. Cells embedded in agarose on a microscope slide are
lysed with detergent and high salt to form nucleoides containing supercoiled loops of DNA
linked to the nuclear matrix. Electrophoresis at high pH results in structures resembling
comets, observed by fluorescence microscopy (see Figures A-D); the intensity of the comet
tail relative to the head reflects the number of DNA breaks. The basis for this is that
loops containing a break lose their supercoiling and become free to extend toward the
anode. Cells containing greater levels of DNA strand break damage generate comets with
more intense tails (i.e. Figures C & D). Densitometric and geometric parameters of the
comets are determined using image analysis software and the extent of DNA strand break
damage is assessed.

As a rough guide, material costs for a single straight forward Comet Assay experience is
~£20 per experiment and we prefer to work in collaboration.

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