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The comet assay: a method to measure DNA damage

in individual cells
Peggy L Olive & Judit P Banáth

British Columbia Cancer Research Center, 675 W. 10th Avenue, Vancouver, British Columbia V5Z 1L3, Canada. Correspondence should be addressed to P.L.O.

Published online 27 June 2006; doi:10.1038/nprot.2006.5

We present a procedure for the comet assay, a gel electrophoresis–based method that can be used to measure DNA damage in
individual eukaryotic cells. It is versatile, relatively simple to perform and sensitive. Although most investigations make use of
© 2006 Nature Publishing Group

its ability to measure DNA single-strand breaks, modifications to the method allow detection of DNA double-strand breaks, cross-
links, base damage and apoptotic nuclei. The limit of sensitivity is approximately 50 strand breaks per diploid mammalian cell.
DNA damage and its repair in single-cell suspensions prepared from yeast, protozoa, plants, invertebrates and mammals can also
be studied using this assay. Originally developed to measure variation in DNA damage and repair capacity within a population of
mammalian cells, applications of the comet assay now range from human and sentinel animal biomonitoring (e.g., DNA damage in
earthworms crawling through toxic waste sites) to measurement of DNA damage in specific genomic sequences. This protocol can
be completed in fewer than 24 h.

Development of the method Applications of the comet assay
The concept of microgel electrophoresis was first introduced Since the initial development of the comet assay, efforts have been
in 1984 by Ostling and Johanson1 as a method to measure DNA made to improve assay sensitivity and reliability, extend applica-
single-strand breaks that caused relaxation of DNA supercoils. A tions to the analysis of various types of DNA damage in various cell
modified version was published by Singh and colleagues in 1988, types7–9, increase sample-handling capacity10,11, and standardize
which used alkaline conditions2. The idea was to combine DNA gel the protocols and analysis12. Efforts to optimize agarose concen-
electrophoresis with fluorescence microscopy to visualize migra- tration, lysis buffers and DNA stains were undertaken by several
tion of DNA strands from individual agarose-embedded cells. If groups13,14. A version of the method done in neutral conditions was
the negatively charged DNA contained breaks, DNA supercoils introduced that allowed detection of DNA double-strand breaks
were relaxed and broken ends were able to migrate toward the independent of the presence of single-strand breaks15. Base damage
anode during a brief electrophoresis. If the DNA was undamaged, could be identified by incubating lysed cells with base damage–spe-
the lack of free ends and large size of the fragments prevented cific endonucleases before electrophoresis16. Interstrand cross-links
migration. Determination of the relative amount of DNA that could be identified by the failure to detect single-strand breaks that
migrated provided a simple way to measure the number of DNA were known to be present17. The extensive DNA fragmentation that
breaks in an individual cell. Although equally sensitive methods occurs in apoptotic cells made these cells simple to detect18.
for the detection of DNA single-strand breaks were introduced The ability to measure heterogeneity in response to DNA-dam-
in the mid 1970s3,4, three facts (in addition to the obvious appeal aging agents was first tested on cells exposed to the cancer chemo-
of ‘seeing’ the damaged DNA) made this method attractive. First, therapeutic drug, bleomycin19. A wide range in appearances of
only about a thousand cells were required. Second, the cells did the comets indicated that some nuclei contained large numbers of
not need to be tagged with a radioisotope, thus allowing measure- strand breaks whereas others were undamaged. The importance of
ment of damage in any nucleated cell. Perhaps most important, the heterogeneity in DNA damage in explaining resistance to cancer
method could be used to measure variations in response to DNA treatment was subsequently demonstrated in cells from animal
damaging agents between cells of the same exposed population. tumor models and clinical biopsies20–22. The potential for predict-
In 1990, a modification of the original method of Ostling and ing tumour response to specific treatments that cause DNA dam-
Johanson was introduced and named the “comet assay” after the age has now been demonstrated for several agents23−25.
appearance of the DNA from an individual cell5. The comet head Much of the interest in this method comes from its potential
containing the high-molecular-weight DNA and the comet tail applications in human biomonitoring and in ecological assess-
containing the leading ends of migrating fragments were mea- ment of sentinel organisms exposed to environmental contami-
sured in real time from digitized images using software developed nants9,26. New applications include magnetic-bead identification
for this purpose6. Tail moment, a measure of both amount of DNA of cell-surface markers present on cells embedded in agarose27
in the tail and distribution of DNA in the tail, became a common and fluorescence in situ hybridization to detect sequence-specific
descriptor along with tail length and percentage of DNA in the tail. effects in damaged DNA28,29.
The comet assay interest group offers a useful introduction to
this area, helpful information on various protocols and image Variations in the method for mammalian cells
analysis systems, and a forum for discussion of issues related to Although several protocols exist for preparing slides, lysing cells, per-
this method ( forming electrophoresis and staining slides, results are remarkably

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 23

similar for mammalian cells using most of the published methods. duces a 1,000-fold or more excess of single-strand versus double-
The most significant differences are the length of incubation in the strand breaks, even at millimolar concentrations36. It should be
alkali and salt solution, and whether a detergent lysis step precedes emphasized that simply performing the comet assay under non-
the alkaline denaturation step2. Here we include an overnight lysis denaturing conditions does not ensure the measurement of DNA
period for optimum sensitivity and reproducibility14, but there are double-strand breaks. In fact, the original Ostling and Johanson
advantages to completing the protocol in a shorter time. As with method used neutral lysis, but the authors clearly thought that
all techniques, paying rigorous attention to technical details will they were detecting loss of DNA supercoiling caused by DNA sin-
improve assay reproducibility. For biomonitoring, there are impor- gle-strand breaks1. Because about 2,000 or so breaks are adequate
tant advantages to standardizing comet assay protocols and analy- to relax all supercoils, the method was limited to single-strand
sis methods. Recommendations for conducting the comet assay break detection at lower damage levels. The version of the proce-
for this purpose have evolved from several workshops and working dure described here performed under neutral conditions can be
groups12,30−32. Considerations of various statistical approaches are used to measure double-strand over a range of about 50−10,000
also available33,34. breaks per cell.
© 2006 Nature Publishing Group

Results of various measurements of DNA damage by comet

assay are not often normally distributed. Typically, mean or Limitations of the method
median values for 25−100 comets along with 75th-percentile val- The ability to analyze individual cells is an advantage in terms of
ues, shown as a box plot, can adequately describe most data sets. identifying subpopulations that respond differently to a cytotoxic
Bivariate plots of percent DNA in the tail versus DNA content are treatment. There are, however, practical limitations to the num-
also useful for appreciating the role of cell cycle or ploidy in DNA ber of cells and samples that can be analyzed. At best, 600 comets
damage. When the goal is to measure heterogeneity in DNA dam- per hour can be scored if analyzed individually, and on the order
age within a population, more comets are analyzed and methods of 50 slides per day can be scored using automated systems. As
of data analysis must also be modified35. There continues to be dis- indicated earlier, the recommended sample size of 50 comets may
cussion over which comet descriptor may best describe the results. not be adequate if there is significant heterogeneity in DNA dam-
Percent DNA in the tail, tail length (at low damage levels only) and age within a population.
tail moment, which is the percent DNA in the tail multiplied by the A second limitation is the requirement for a viable single-cell
distance between the means of the head and tail distributions, are suspension. If samples contain predominantly necrotic or apop-
all useful measures. totic cells, accurate information on the presence of specific lesions
Two variations on the comet protocol are described below. The like strand breaks or base damage cannot be obtained. Tissue dis-
first one can be used to detect the combination of DNA single- aggregation methods need to be developed to minimize any DNA
strand breaks, double-strand breaks and alkali-labile sites in the damage produced by the procedure. If performed sufficiently rap-
DNA. Although a long lysis time is recommended to allow suf- idly, mechanical dissociation methods (chopping and filtering)
ficient time for denatured DNA to unwind from break points, can be effective37. But the possibility that there may be preferential
shorter lysis times are sufficient for screening purposes. Although loss of heavily damaged cells during single cell preparation should
not described here, modifications to this method can also be used be considered.
to detect DNA cross-links and base damage30. The comet assay provides no information on DNA fragment size
The second procedure is performed under neutral condi- since fragments are not separated during the short electrophoresis
tions and detects only DNA double-strand breaks. This can be period. Instead, as the number of DNA breaks increases, super-
confirmed by treating cells with hydrogen peroxide which pro- coiled loops relax, more free ends are able to migrate, and therefore

a b Figure 1 | Typical dose response relationships for human cells exposed

Alkali method to ionizing radiation using the two methods described in this protocol.
(a,b) WIL2NS human lymphoblast cells were examined using the overnight
40 0 Gy 40 2 Gy 40 8 Gy
alkali method that detects DNA single-strand breaks, double-strand breaks
Mean tail moment

104 breaks/cell
30 and alkali-labile lesions. (c,d) SiHa human cervical carcinoma cells were
Tail moment

30 30 30
examined using the neutral lysis method that detects double-strand breaks.
20 20 20 Single cell suspensions were exposed to X-rays on ice to inhibit strand break
10 rejoining. Shown in a and c are results from controls (left), after 2 or 30 Gy
10 10 10
(Gray; middle), and after 8 or 60 Gy (right). Graphs in b and d show typical
0 0 0 0 dose-response curves (mean ± s.d.; n = 3). Note the 40-fold difference in
0 2 4 6 8
DNA content (arbitrary units) the slopes of the two dose-response curves, consistent with the difference
Dose (Gy)
c d Neutral method
in induction of single-strand breaks versus double-strand breaks by ionizing
radiation. The appearance of the microscopic images of representative
comets for the three doses are shown, and bivariate plots of DNA content
20 0 Gy 20 30 Gy 20 60 Gy
Mean tail moment

2,500 breaks/cell versus comet tail moment indicate the response of individual cells in various
Tail moment

15 15 15 10 phases of the cell cycle (a,c). Although S-phase DNA contains the same
number of radiation-induced single- and double-strand breaks per Dalton,
10 10 10
under denaturing conditions replication forks appear as single-strand breaks,
5 5 5
and thus S-phase DNA migrates faster than DNA from cells in G1 or G2 phase.
The opposite occurs using the neutral method because replication bubbles in
0 0 0 0
0 20 40 60
S-phase cells retard DNA migration during electrophoresis.
DNA content (arbitrary units) Dose (Gy)

24 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

a larger fraction of the DNA moves away from the comet head. relevance of the damage. It is important to appreciate that DNA
Some information can be obtained by measuring the distribution damage measured in the comet assay is not necessarily a result
of DNA damage within the comet tail, but accurate fragment size of direct genotoxicity; mitochrondrial or membrane damage can
measurements will require use of a technique such as pulsed field cause extensive DNA fragmentation via apoptosis or necrosis.
gel electrophoresis38. Many investigators are interested in examining the DNA repair
Cells actively replicating their DNA behave differently during capacity of cells by measuring the decrease in damage as a func-
gel electrophoresis, and this can be confused with a difference in tion of time after exposure to a known genotoxic agent. If the
inherent sensitivity. Under alkaline conditions, replication forks agent can be administered quickly (e.g., X-rays) or under condi-
behave as single-strand breaks so that S-phase DNA migrates more tions that inhibit repair (4 °C), then the half-time for recovery
rapidly. Under neutral conditions, S-phase DNA operates as repli- can be determined by placing the treated cells under conditions
cation bubbles that retard migration during electrophoresis39. This that allow for repair (e.g., complete growth medium at 37 °C).
problem is readily apparent in Figure 1. But as the comet assay can Repair processes, however, can often complicate measurement
provide a measure of both DNA content and DNA damage, it is of DNA damage40. For exposures over long times, DNA dam-
© 2006 Nature Publishing Group

possible to analyze damage in any phase of the cell cycle. age is a measure of both induction and repair. Similarly, cis-
Interpretation of comet results is complicated by the fact that platin-induced cross-links require several hours to form so that
there is no simple relationship between the amount of DNA both amount of damage and efficiency of repair contribute to
damage caused by a specific chemical and the biological impact the overall damage detected. For some agents (e.g., ultraviolet-C
of that damage. Each drug can differ in terms of the number of radiation and N-methyl-N’-nitro-N-nitrosoguanidine), damage
DNA breaks that are associated with a given biological effect36. measured using the comet assay can be a result of incision at sites
Chemicals that produce interstrand cross-links will block detec- of base damage produced during nucleotide excision repair41. In
tion of single-strand breaks17,22. For this reason, analysis of the this situation, ‘damage’ becomes a measure of repair. Finally, repair
effects of mixtures of drugs, or a drug with two different mecha- of some types of DNA damage can be very rapid making detection
nisms of producing DNA damage can be particularly problem- in living organisms difficult. Ionizing radiation–induced strand
atic. Comparing comet assay results with other measures of DNA breaks and some types of base damage by reactive oxygen spe-
damage (e.g., micronuclei, mutations, adduct levels, chromosome cies can be repaired with a half-time less than 30 min and as short
aberrations or cell killing) is necessary to interpret the biological as 3 min.

REAGENTS • (Optional; Step 15B only) N2, Neutral rinse and electrophoresis solution:
• Low-gelling-temperature agarose (Sigma; Type VII, cat. no. A-4018) 90 mM Tris buffer, 90 mM boric acid, 2 mM Na2EDTA (pH 8.5)
• Phosphate-buffered saline (Ca2+- and Mg2+-free) EQUIPMENT
• Proteinase K (EMD Chemicals; cat. no. 24568-2) • Disposable plastic tubes (5 ml; Falcon; cat. no. 35-2054)
• 0.5 M Na2EDTA (pH 8.0): add 55.8 g Na2EDTA and 6.4 g NaOH to 270 ml • Water baths for melting agarose at 100 °C and maintaining agarose at 40 °C
distilled water (stir for approximately 2 h and adjust pH to 8.0 with additional • Hemocytometer or electronic particle counter (e.g., Coulter counter) for
NaOH) adjusting cell numbers
• 5 M stock solution of NaOH: add NaOH slowly to ice-cold distilled water • Frosted-end microscope slides ▲ CRITICAL Better adhesion can be obtained
! CAUTION NaOH is caustic. using fully frosted slides (Fisher Scientific; cat. no. 12-544-5CY), but slides
• Fluorescent DNA stain: 10 µg/ml propidium iodide (Sigma; cat. no. P-4170); cannot be dried and stored after staining.
other DNA stains (e.g., SYBR-green, YO-PRO-1, DAPI) may also be used, • Diamond-tipped pen for scoring slides
but photostability should be assessed especially with UV-excitable dyes13 • Covered glass containers for slide lysis, staining and storage
! CAUTION Propidium iodide is mutagenic; wear protective gloves. • Large-bed horizontal gel electrophoresis chamber and power supply
• (Optional; Step 15A only) A1, alkaline lysis solution for single-strand break • Epifluorescence microscope with 25× objective (long working distance
detection: 1.2 M NaCl, 100 mM Na2EDTA, 0.1% sodium lauryl sarcosinate, objective is best) and filter set for green-light excitation if using propidium
0.26 M NaOH (pH > 13); equilibrate at 4 °C. ▲ CRITICAL Prepare fresh on iodide to stain DNA (546 nm excitation from a 100 watt mercury bulb;
day of experiment. emission monitored using a 580 nm reflector and 590 bandpass filter)
• (Optional; Step 15A only) A2, alkaline rinse and electrophoresis solution: • Charge-coupled device (CCD) camera (8-bit black-and-white camera is
0.03 M NaOH, 2 mM Na2EDTA (pH ∼12.3) adequate); high sensitivity and high pixel density are preferred
• (Optional; Step 15B only) N1, neutral lysis solution for double-strand-break • Computer and comet analysis software (commercial systems or free software
detection: 2% sarkosyl, 0.5M Na2EDTA, 0.5 mg/ml proteinase K (pH 8.0); available on the internet; see
equilibrate at 4 °C

Agarose preparation
1| Equilibrate two water baths: one at 40 °C and one at ∼100 °C.

2| Prepare 1% low-gelling-temperature agarose by mixing powdered agarose with distilled water in a glass beaker or bottle.

3| Place bottle in the 100 °C water bath for several minutes. Alternatively, carefully microwave bottle at low power for short
intervals (avoid vigorous boiling of the agarose and ensure that all agarose is dissolved). ? TROUBLESHOOTING

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 25

4| Place bottle with agarose into a 40 °C water bath. If the cell sample contains red blood cells or hemoglobin, add 2%
DMSO to the agarose and/or alkaline lysis solution to avoid damage by iron-catalyzed reactive oxygen species, which can cause
strand breaks.

Slide precoating
5| To improve agarose adhesion, score the edges of dust-free frosted-end microscope slides using a diamond-tipped pen.

6| Prepare agarose-precoated slides by dipping the slides into molten 1% agarose and wiping one side clean. It is best to
work in a low-humidity environment to ensure agarose adhesion. ? TROUBLESHOOTING

7| Allow agarose to air-dry to a thin film. Slides can be prepared ahead of time and stored with desiccant.

Sample preparation
© 2006 Nature Publishing Group

8| Prepare a single-cell suspension using enzyme disaggregation or mechanical dissociation. Never use fixed cells because
the DNA will not migrate. Keep cells in ice-cold medium or phosphate-buffered saline to minimize cell aggregation and inhibit
DNA repair. Always include a sample of untreated cells to confirm that background damage is low. A positive control (e.g., cells
exposed to 20 µM freshly prepared hydrogen peroxide or 8 Gray (Gy) X-rays on ice for the alkaline version, and 75 Gy for the
neutral version) is useful when first setting up the method. ? TROUBLESHOOTING

9| Using a hemocytometer or particle counter, adjust cell density to about 2 × 104 cells/ml in phosphate-buffered saline
lacking divalent cations.

10| Label slide on frosted end using a pencil, not a pen.

11| Pipet 0.4 ml of cells into a 5 ml plastic disposable tube.

12| Add 1.2 ml 1% low-gelling-temperature agarose at 40 °C.

13| Mix and rapidly pipet 1.2 ml of cell suspension onto the agarose-covered surface of a pre-coated slide; avoid producing

14| Allow agarose to gel for about 2 min. Be consistent with the time and temperature used for gelling, and ensure that
agarose is fully set before submerging in lysis solution.

Lysis and electrophoresis

15| Lysis and electrophoresis can be performed in alkaline (option A) or neutral (option B) conditions. Use option A to detect
the combination of DNA single-strand breaks, double-strand breaks and alkali-labile sites in the DNA, and option B to detect
only DNA double-strand breaks.
(A) Alkaline lysis and electrophoresis.
(i) After agarose has gelled, submerge slides in a covered dish containing A1 lysis solution. Handle slides gently, e.g., hold
slides horizontally and lower into solutions, do not pour solutions over slides or move containers containing slides.
(ii) Lyse samples overnight (18−20 h) at 4 °C in the dark. If time is limited, a 1-h lysis procedure can also be used, but this
will be at the expense of some decrease in sensitivity (up to twofold) and reproducibility14.
(iii) After 1-h or overnight lysis, carefully remove slides and submerge in room temperature (18−25 °C) A2 rinse solution for
20 min. Repeat two times to ensure removal of salt and detergent. Take care not to allow DNA to renature even briefly
(i.e., by lowering pH below 12.3) until after electrophoresis, as this will result in DNA tangling and reduced migration.
(iv) After these three rinses, submerge slides in fresh A2 solution in an electrophoresis chamber. The chamber should be
filled with a consistent volume of buffer that is about 1–2 mm above the top of the agarose. Ensure that the chamber is
level using a bubble leveling device. ? TROUBLESHOOTING
(v) Conduct electrophoresis in solution A2 for 25 min at a voltage of 0.6 V/cm. The current should be about 40 mA if using
20 V. The distance in centimeters is measured between the negative and positive electrodes in the electrophoresis
(B) Neutral lysis and electrophoresis.
(i) After agarose has gelled, gently submerge slides in a covered dish containing N1 lysis solution at 4 °C, and avoid moving
the dish containing the slides.

26 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

(ii) Place dish in an incubator at 37 °C overnight (18−20 h) in the dark. Although greater sensitivity is achieved using 50 °C
lysis, the lower temperature reduces heat-labile damage42.
(iii) After overnight lysis, remove slides and submerge in room temperature N2 rinse buffer for 30 min at room temperature.
Repeat two more times.
(iv) Submerge slides in fresh N2 solution in an electrophoresis chamber that has been leveled and filled with a measured
volume of buffer about 1−2 mm above the top surface of the agarose. ? TROUBLESHOOTING
(v) Conduct electrophoresis in solution N2 for 25 min at 0.6 V/cm. Current is typically 7 mA when using 20 V.

Slide staining
16| Remove slides from electrophoresis chamber and rinse and neutralize in 400 ml of distilled water.

17| Place slides in staining solution containing 2.5 µg/ml of propidium iodide in distilled water for 20 min. Alternatively, pipet
100 µl of a 10 µg/ml stock solution of propidium iodide directly onto the slide and incubate for 20 min.
© 2006 Nature Publishing Group

18| Rinse slides with 400 ml distilled water to remove excess stain.
■ PAUSE POINT Slides can be stored in humidified, light tight boxes in the cold room (at 4 °C). If storing for more than 2−3 d,
dip slides in ethanol and dry them. Dried slides can be stored for several months, but before analysis, rehydrate by adding a
thin layer of agarose to reduce background fluorescence, and restain with propidium iodide. If the intensity of the comet image
appears to decrease while viewing with the fluorescence microscope, try using an antifade solution.

Slide analysis
19| Analyze cells by examining at least 50 comet images from each slide. Avoid analyzing doublets or comets at slide edges.
If two or more populations are present, or if heterogeneity in DNA is high, more images should be scored (up to 1,000 can be
scored from a slide). When information on DNA content is required, ensure that the image is not ‘saturated’, that is, that the
intensity of fluorescence in any part of the digitized comet image does not exceed the maximum of the digital range (e.g., if
256 channels are available, data should be collected between 0 and 254). This can be accomplished most easily by color-coding
the comet image to define specific intensity ranges and then adjusting the light intensity to avoid the color range assigned
to channels above 254. Technical methods for extending the dynamic range have also been developed by some comet assay
software companies.

20| Using image analysis software, analyze individual comet images for several features including total intensity (DNA
content), tail length, percent DNA in tail and tail moment. Alternatively, visual scoring can be used especially when the
population is homogenous and noncycling, large differences are expected and adequate controls have been included (e.g., use
of coded slides and clearly defined ranges of response). The use of five damage classes introduced by Collins43 has been widely
adopted. Triplicate repeat experiments are recommended.

21| Apply appropriate statistical analysis using means or medians depending on population distributions. Error bars typically
represent the between-experiment variability, not the within-slide variability for a specific parameter.

Slide preparation, cell-sample preparation, agarose embedding for 10 samples: 1 h.
Alkaline or neutral lysis: typically 18–20 h, but as short as 1 h.
Rinse after lysis: 1 h.
Electrophoresis under alkaline or neutral conditions: 25 min.
DNA staining: 20 min.
Comet image capture and analysis: 1 h for approximately 600 images.

See Table 1.

TABLE 1 | Troubleshooting table.

Step 3 Results are not reproducible. Agarose concentration is variable. Ensure that agarose is fully dissolved and
the concentration has not been altered
(e.g., by evaporation).

NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 27

TABLE 1 | Troubleshooting table (continued).
Step 6 Agarose gel falls off the slide. Slides are not adequately agarose- Pay special attention to Step 6.
precoated or humidity is high.
Step 8 Untreated cells show comet tails. DNA in the cells is damaged. Evaluate cell viability. If an enzyme
disaggregation method is used, try reducing
exposure time or enzyme concentration.
Cells are in S phase (if alkali lysis is used). Determine whether damaged cells are
in S phase by analysis of DNA content
(fluorescence intensity of an individual
comet is proportional to DNA content).
Steps 15 (A) (iv) and 15 B (iv) Microscopic Depth of electrophoresis buffer is variable Use a bubble level device to ensure that the
appearance of comets differs at different ends across the bed. electrophoresis chamber is level.
© 2006 Nature Publishing Group

of the same slide or for slides from different

parts of the electrophoresis chamber.
Step 15 Agarose gel falls off the slide. Slides are not handled gently during lysis Pay special attention to 15 (A)(i) and
and electrophoresis. 15 (B)(i).

The comet assay is sensitive to damage above about 50 breaks per diploid mammalian cell, and will lose sensitivity above
about 10,000 breaks per cell. Linear dose response relationships should be observed over that range for cells exposed to
X-rays and examined using the alkali lysis method (Fig. 1). DNA content (total image fluorescence) should remain relatively
constant as the number of DNA breaks increases (i.e., with increasing dose). But changes in chromatin structure after alkali
denaturation and renaturation can affect dye binding after low amounts of damage44. Using an imaging system, it should be
possible to identify cells in different phases of the cell cycle based on fluorescence intensity of individual comets. Note that
the appearance of comets with tails in the alkaline method could be indicative of the presence of replication forks in S-phase
cells as well as cells containing treatment-induced single-strand breaks.
Double-strand breaks occur much less frequently than single-strand breaks, but they are essential precursors to chromosome
aberrations. Cells prepared using the neutral lysis method develop a ‘halo’ of DNA loops that gives the comets a slightly
different appearance compared to comets prepared using alkali method. Comets from untreated cells appear more elongated so
that more DNA is assigned to the ‘tail’ region in the untreated cells (note the higher tail moment for the “0 Gy” cells analyzed
using the neutral method in Fig. 1). As indicated earlier, the increase in DNA measured after low doses of a DNA damaging
agent can be a result of relaxation of DNA supercoils caused by single-strand breaks, not an indication of double-strand break
induction36. A rapid increase in damage at low doses followed by a much slower but linear increase over the higher-dose range
(true double-strand breaks) is therefore anticipated, although the longer lysis time recommended here should be adequate to
relax DNA supercoils.

ACKNOWLEDGMENTS We thank R. Durand for developing the first comet analysis DNA damage and repair in tumor and normal cells measured using the
software. “comet” assay. Radiat. Res. 122, 86−94 (1990).
7. Fairbairn, D.W., Olive, P.L. & O’Neill, K.L. The comet assay: a comprehensive
COMPETING INTERESTS STATEMENT The authors declare that they have no review. Mutat. Res. 339, 37−59 (1995).
competing financial interests. 8. Salagovic, J., Gilles, J., Verschaeve, L. & Kalina, I. The comet assay for the
detection of genotoxic damage in the earthworms: a promising tool for
Published online at assessing the biological hazards of polluted sites. Folia Biol. (Praha.) 42,
Reprints and permissions information is available online at http://npg.nature. 17−21 (1996).
com/reprintsandpermissions 9. Moller, P. Genotoxicity of environmental agents assessed by the alkaline
comet assay. Basic Clin. Pharmacol. Toxicol. 96 (Suppl. 1), 1−42 (2005).
1. Ostling, O. & Johanson, K.J. Microelectrophoretic study of radiation-induced 10. Kiskinis, E., Suter, W. & Hartmann, A. High-throughput Comet assay using
DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 96-well plates. Mutagenesis 17, 37−43 (2002).
123, 291−298 (1984). 11. Frieauff, W., Hartmann, A. & Suter, W. Automatic analysis of slides processed
2. Singh, N.P., McCoy, M.T., Tice, R.R. & Schneider, E.L. A simple technique for in the Comet assay. Mutagenesis 16, 133−137 (2001).
quantitation of low levels of DNA damage in individual cells. Exp. Cell Res. 12. Tice, R.R. et al. Single cell gel/comet assay: guidelines for in vitro and in vivo
175, 184−191 (1988). genetic toxicology testing. Environ. Mol. Mutagen. 35, 206−221 (2000).
3. Rydberg, B. The rate of strand separation in alkali of DNA of irradiated 13. Bauch, T., Bocker, W., Mallek, U., Muller, W.U. & Streffer, C. Optimization and
mammalian cells. Radiat. Res. 61, 274−287 (1975). standardization of the “comet assay” for analyzing the repair of DNA damage
4. Kohn, K.W. & Grimek-Ewig, R.A. Alkaline elution analysis, a new approach in cells. Strahlenther. Onkol. 175, 333−340 (1999).
to the study of DNA single-strand interruptions in cells. Cancer Res. 33, 14. Banath, J.P., Kim, A. & Olive, P.L. Overnight lysis improves the efficiency
1849−1853 (1973). of detection of DNA damage in the alkaline comet assay. Radiat. Res. 155,
5. Olive, P.L. Cell proliferation as a requirement for development of the contact 564−571 (2001).
effect in Chinese hamster V79 spheroids. Radiat. Res. 117, 79−92 (1989). 15. Olive, P.L., Wlodek, D. & Banáth, J.P. DNA double-strand breaks measured
6. Olive, P.L., Banáth, J.P. & Durand, R.E. Heterogeneity in radiation-induced in individual cells subjected to gel electrophoresis. Cancer Res. 51,

28 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS

4671−4676 (1991). the detection of DNA damage. Methods Mol. Biol. 291, 85−95 (2005).
16. Collins, A.R., Duthie, S.J. & Dobson, V.L. Direct enzymic detection of 31. Hartmann, A. et al. Recommendations for conducting the in vivo alkaline
endogenous oxidative base damage in human lymphocyte DNA. Carcinogen. Comet assay. 4th International Comet Assay Workshop. Mutagenesis 18,
14, 1733−1735 (1993). 45−51 (2003).
17. Merk, O. & Speit, G. Detection of crosslinks with the comet assay in 32. Albertini, R.J. et al. IPCS guidelines for the monitoring of genotoxic effects
relationship to genotoxicity and cytotoxicity. Environ. Mol. Mutagen. 33, of carcinogens in humans. International Programme on Chemical Safety.
167−172 (1999). Mutat. Res. 463, 111−172 (2000).
18. Olive, P.L., Frazer, G. & Banáth, J.P. Radiation-induced apoptosis measured 33. Duez, P., Dehon, G., Kumps, A. & Dubois, J. Statistics of the Comet assay:
in TK6 human B lymphoblast cells using the comet assay. Radiat. Res. 136, a key to discriminate between genotoxic effects. Mutagenesis 18, 159−166
130−136 (1993). (2003).
19. Ostling, O. & Johanson, K.J. Bleomycin, in contrast to gamma irradiation, 34. Verde, P.E. et al. Application of public-domain statistical analysis software
induces extreme variation of DNA strand breakage from cell to cell. Int. J. for evaluation and comparison of comet assay data. Mutat. Res. 604, 71−82
Radiat. Biol. Relat. Stud. Phys. Chem. Med. 52, 683−691 (1987). (2006).
20. Olive, P.L., Johnston, P.J., Banáth, J.P. & Durand, R.E. The comet assay: A 35. Olive, P.L. & Durand, R.E. Heterogeneity in DNA damage using the comet
new method to examine heterogeneity associated with solid tumors. Nat. assay. Cytometry A 66, 1−8 (2005).
Med. 4, 103−105 (1998). 36. Olive, P.L. & Johnston, P.J. DNA damage from oxidants: influence of lesion
21. Olive, P.L., Durand, R.E., LeRiche, J.C., Olivotto, I.A. & Jackson, S.M. Gel complexity and chromatin organization. Oncol. Res. 9, 287−294 (1997).
© 2006 Nature Publishing Group

electrophoresis of individual cells to quantify hypoxic fraction in human 37. Sasaki, Y.F. et al. The comet assay with multiple mouse organs: comparison of
breast cancers. Cancer Res. 53, 733−736 (1993). comet assay results and carcinogenicity with 208 chemicals selected from the
22. Olive, P.L. & Banáth, J.P. Multicell spheroid response to drugs predicted with IARC monographs and U.S. NTP Carcinogenicity Database. Crit. Rev. Toxicol.
the comet assay. Cancer Res. 57, 5528−5533 (1997). 30, 629−799 (2000).
23. Almeida, G.M., Duarte, T.L., Steward, W.P. & Jones, G.D. Detection of 38. Cedervall, B. et al. Methods for the quantification of DNA double-strand
oxaliplatin-induced DNA crosslinks in vitro and in cancer patients using the breaks determined from the distribution of DNA fragment sizes measured by
alkaline comet assay. DNA Repair (Amst.) 5, 219−225 (2006). pulsed-field gel electrophoresis. Radiat. Res. 143, 8−16 (1995).
24. Olive, P.L. et al. The comet assay in clinical practice. Acta Oncol. 38, 839−844 39. Olive, P.L. DNA damage and repair in individual cells: applications of the
(1999). comet assay in radiobiology. Int. J. Radiat. Biol. 75, 395−405 (1999).
25. Dorie, M.J. et al. DNA damage measured by the comet assay in head and neck 40. Speit, G. & Hartmann, A. The contribution of excision repair to the DNA
cancer patients treated with tirapazamine. Neoplasia. 1, 461−467 (1999). effects seen in the alkaline single cell gel test (comet assay). Mutagenesis
26. Speit, G. & Hartmann, A. The comet assay: a sensitive genotoxicity test for 10, 555−559 (1995).
the detection of DNA damage. Methods Mol. Biol. 291, 85−95 (2005). 41. Alapetite, C., Wachter, T., Sage, E. & Moustacchi, E. Use of the alkaline comet
27. Strauss, G.H., Peters, W.P. & Everson, R.B. Measuring DNA damage in assay to detect DNA repair deficiencies in human fibroblasts exposed to UVC,
individual cells of heterogeneous mixtures: a novel application of an UVB, UVA and γ rays. Int. J. Radiat. Biol. 69, 359−369 (1996).
immunological typing technique. Mutat. Res. 304, 211−216 (1994). 42. Rydberg, B. Radiation-induced heat-labile sites that convert into DNA
28. Santos, S.J., Singh, N.P. & Natarajan, A.T. Fluorescence in situ hybridization double-strand breaks. Radiat. Res. 153, 805−812 (2000).
with comets. Exp. Cell Res. 232, 407−411 (1997). 43. Collins, A.R. The comet assay for DNA damage and repair: principles,
29. Rapp, A., Hausmann, M. & Greulich, K.O. The comet-FISH technique: a tool applications, and limitations. Mol. Biotechnol. 26, 249−261 (2004).
for detection of specific DNA damage and repair. Methods Mol. Biol. 291, 44. Olive, P.L., Banáth, J.P. & Fjell, C.D. DNA strand breakage and DNA structure
107−119 (2005). influence staining with propidium iodide using the alkaline comet assay.
30. Speit, G. & Hartmann, A. The comet assay: a sensitive genotoxicity test for Cytometry 16, 305−312 (1994).

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