Measuring DNA repair with the comet assay
There are two main approaches to assessing the ability of cells to repair DNA damage; the cellular repair
assay and the in vitro assay. In the first of these, cells are treated with an appropriate genotoxic agent
to induce damage and incubated; the comet assay is used to measure the damage remaining at each time-point.
In the simplest case, rejoining of strand breaks can be followed after treating cells with ionising radiation
or with H2O2. Typically, such repair is rapid, and samples need to be taken at intervals of a few minutes
in order to estimate accurately either the initial rate of removal or the t1/2 of the lesions (both valid
measures of repair activity). Monitoring the repair of other lesions, such as oxidised bases (by base
excision repair, BER), or UV-induced pyrimidine dimers (by nucleotide excision repair, NER), requires the
use of enzymes that recognise the lesions and convert them to strand breaks.
Formamidopyrimidine DNA glycosylase (FPG) recognises 8-oxoguanine and other purine oxidation products; endonuclease III deals
with oxidised pyrimidines; and T4 endonuclease V is able to incise at sites of pyrimidine dimers.
Digestion with these enzymes is carried out after the initial lysis step. The excision repair pathways
act more slowly than strand break rejoining (Collins & Horvathova, 2001), and samples should be taken over
a period of a few hours.
Figure 1. The in vitro DNA repair assay, applied to the measurement of base excision repair in
The in vitro DNA repair assay (Figure 1) (Gaiv„o et al., 2009) is a more biochemical approach. It measures
the ability of repair enzymes in a cell extract to detect and make breaks at the site of specific lesions
in a DNA substrate. The extract is made by suspending cells at high density in a buffer, snap-freezing the
suspension in liquid nitrogen and thawing (this disrupts the structure of the cell), adding detergent to
complete the release of soluble components, and centrifuging to remove cell debris.
The substrate consists of cells embedded in agarose and lysed - as in the standard comet assay - to leave the DNA in the form of
nucleoids. These cells were previously treated with a specific DNA-damaging agent, and this is what defines
the kind of repair activity that is assessed. Thus, the substrate cells can be irradiated with UV(C) in
order to measure NER; or treated with the photosensitiser Ro 19-8022 and visible light to measure the
activity of 8-oxoguanine DNA glycosylase (OGG). The repair reaction is carried out by adding extract to the
substrate DNA in the gel, covering with a cover slip, and incubating for a fixed time. T4 endonuclease V or
FPG is included as a positive control alongside the extracts. DNA breaks produced by glycosylase, endonuclease
and/or lyase activities in the extract are measured by completing the comet assay as usual. Figure 2
illustrates the importance of cell concentration in the in vitro assay, and the dependence of the reaction on
Figure 2. Extract prepared from different numbers of lymphocytes: repair activities at different
incubation times. (Data from Amaya Azqueta.)
The two approaches examine different aspects of repair. The cellular assay looks at the overall process,
removal of lesions, while the in vitro assay focuses on the intial, rate-limiting steps. In both, the
end-result is comets, and the preferred parameter for representing DNA damage is % tail DNA, or relative
tail intensity, conveniently measured with Perceptive Instruments Comet Assay IV. After scoring 50 or 100
comets per sample and time-point, the median % tail DNA is calculated.
The cellular repair assay is probably most useful for testing variant cell types for altered repair functions,
and examining factors that inhibit or stimulate DNA repair. For example, we demonstrated an acceleration of
strand break rejoining and BER of 8-oxoguanine on incubating cells with the carotenoid β-cryptoxanthin (Lorenzo
et al., 2009).
The in vitro assay was first developed for use in human biomonitoring (Collins et al., 2001). Figure 3 shows
the variation in BER rates (on substrate containing 8-oxoguanine) found with extracts from different individuals.
It is convenient, when collecting large numbers of samples in an epidemiological study, to prepare the extracts
and store them frozen, analysing them together in batches at a later date. As an example, BER was shown by
Vodicka et al. (2004) to be apparently induced by styrene in the environment of the workplace; the repair
rate was highest in the subjects with the highest level of exposure. In the context of nutritional exposure,
we found an enhancement of BER of 8-oxoguanine in lymphocytes from subjects following supplementation with
kiwifruit (Collins et al., 2003).
Figure 3. Repair activities in lymphocyte extracts from different subjects, compared with positive
(FPG) and negative (buffer) controls. Substrate contained 8-oxoguanine. Incubation time: 30 min.
(Data from Amaya Azqueta.)
Details of all experimental procedures can be found in pdf form at the website of the EC project NewGeneris
(www.newgeneris.org) together with a downloadable set of training videos. A general introduction to the comet
assay can also be found in Collins (2004).
Note: Measuring repair activity in multiple samples from human trials is simplified and speeded up by setting
12 mini-gels on a microscope slide, instead of the usual one or two. The slide is clamped together with a
silicone rubber gasket in a special chamber so that gels can be incubated with different extracts, or reagents.
In principle, 240 gels can be run in a standard electrophoresis tank taking 20 slides. Scoring using Comet
Assay IV is simplified by having comets in a small area of gel, and by not having to change slides so often.
Comet assay R & D; the Collins group:
The research group of Andrew Collins has played a leading role in the development and application of the comet
assay over a period of almost two decades, first at the Rowett Research Institute, Aberdeen, and now at the
University of Oslo. We modified the assay to detect specific lesions using repair enzymes; applied the assay
in human nutritional intervention studies; combined fluorescent in situ hybridisation with the comet assay
to monitor damage and repair of specific genes; developed in vitro repair assays for BER and NER; and most
recently led an EC project, COMICS, developing high throughput comet assay methods. Our main objective for
the near future is to validate the comet assay as a biomonitoring tool, and to establish whether comet assay
scores of DNA damage or repair capacity in human lymphocytes can be regarded as biomarkers related to cancer
Collins, A.R. (2004) The comet assay for DNA damage and repair, Molec. Biotech. 26, 249-261.
Collins, A.R., Dusinska, M., Horvathova, E., Munro, E., Savio, M. and Stetina, R. (2001) Inter-individual
differences in DNA base excision repair activity measured in vitro with the comet assay. Mutagenesis, 16, 297-301
Collins, A.R., Harrington, V., Drew, J. and Melvin, R. (2003) Nutritional modulation of DNA repair in a
human intervention study. Carcinogenesis, 24, 511-515.
Collins, A.R. and Horvathova, E. (2001) Oxidative DNA damage, antioxidants and DNA repair; applications of
the comet assay. Biochem. Soc. Trans., 29, part 2, 337-342
Gaiv„o, I., Piasek, A., Brevik, A., Shaposhnikov, S. and Collins, A.R. (2009) Comet assay-based methods
for measuring DNA repair in vitro; estimates of inter- and intra-individual variation. Cell Biology and
Toxicology, 25, 45-52
Lorenzo,Y., Azqueta,A., Luna,L., Bonilla,F., Dominguez,G., Collins,A.R (2009) The carotenoid
Ŗ-cryptoxanthin stimulates the repair of DNA oxidation damage in addition to acting as an antioxidant in
human cells. Carcinogenesis, 30, 308-314
Vodicka, P., Tuimala,J., Stetina,R., Kumar,R., Manini,P., et al. (2004) Cytogenetic markers, DNA
single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers.
Envir. Health Perspect., 112, 867-871