PROTOCOL

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.
(polive@bccrc.ca).

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 http://www.nature.com/natureprotocols

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.

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

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

 PROTOCOL
similar for mammalian cells using most of the published methods.
The most significant differences are the length of incubation in the
alkali and salt solution, and whether a detergent lysis step precedes
the alkaline denaturation step2. Here we include an overnight lysis
period for optimum sensitivity and reproducibility14, but there are
advantages to completing the protocol in a shorter time. As with
all techniques, paying rigorous attention to technical details will
improve assay reproducibility. For biomonitoring, there are impor-
tant advantages to standardizing comet assay protocols and analy-
sis methods. Recommendations for conducting the comet assay
for this purpose have evolved from several workshops and working
groups12,30−32. Considerations of various statistical approaches are
also available33,34.
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Results of various measurements of DNA damage by comet
assay are not often normally distributed. Typically, mean or
median values for 25−100 comets along with 75th-percentile val-
ues, shown as a box plot, can adequately describe most data sets.
Bivariate plots of percent DNA in the tail versus DNA content are
also useful for appreciating the role of cell cycle or ploidy in DNA
damage. When the goal is to measure heterogeneity in DNA dam-
age within a population, more comets are analyzed and methods
of data analysis must also be modified35. There continues to be dis-
cussion over which comet descriptor may best describe the results.
Percent DNA in the tail, tail length (at low damage levels only) and
tail moment, which is the percent DNA in the tail multiplied by the
distance between the means of the head and tail distributions, are
all useful measures.
Two variations on the comet protocol are described below. The
first one can be used to detect the combination of DNA single-
strand breaks, double-strand breaks and alkali-labile sites in the
DNA. Although a long lysis time is recommended to allow suf-
ficient time for denatured DNA to unwind from break points,
shorter lysis times are sufficient for screening purposes. Although
not described here, modifications to this method can also be used
to detect DNA cross-links and base damage30.
The second procedure is performed under neutral condi-
tions and detects only DNA double-strand breaks. This can be
confirmed by treating cells with hydrogen peroxide which pro-
a b
40 0 Gy 40 2 Gy 40 8 Gy
Mean tail moment
Tail moment
30 30 30
20 20 20
10 10 10
0 0 0 0
DNA content (arbitrary units)
c d
20 0 Gy 20 30 Gy 20 60 Gy
Mean tail moment
Tail moment
15 15 15
10 10 10
5 5 5
0 0 0 0
DNA content (arbitrary units)
24 | VOL.1 NO.1 | 2006 | NATURE PROTOCOLS
duces a 1,000-fold or more excess of single-strand versus double-
strand breaks, even at millimolar concentrations36. It should be
emphasized that simply performing the comet assay under non-
denaturing conditions does not ensure the measurement of DNA
double-strand breaks. In fact, the original Ostling and Johanson
method used neutral lysis, but the authors clearly thought that
they were detecting loss of DNA supercoiling caused by DNA sin-
gle-strand breaks1. Because about 2,000 or so breaks are adequate
to relax all supercoils, the method was limited to single-strand
break detection at lower damage levels. The version of the proce-
dure described here performed under neutral conditions can be
used to measure double-strand over a range of about 50−10,000
breaks per cell.
Limitations of the method
The ability to analyze individual cells is an advantage in terms of
identifying subpopulations that respond differently to a cytotoxic
treatment. There are, however, practical limitations to the num-
ber of cells and samples that can be analyzed. At best, 600 comets
per hour can be scored if analyzed individually, and on the order
of 50 slides per day can be scored using automated systems. As
indicated earlier, the recommended sample size of 50 comets may
not be adequate if there is significant heterogeneity in DNA dam-
age within a population.
A second limitation is the requirement for a viable single-cell
suspension. If samples contain predominantly necrotic or apop-
totic cells, accurate information on the presence of specific lesions
like strand breaks or base damage cannot be obtained. Tissue dis-
aggregation methods need to be developed to minimize any DNA
damage produced by the procedure. If performed sufficiently rap-
idly, mechanical dissociation methods (chopping and filtering)
can be effective37. But the possibility that there may be preferential
loss of heavily damaged cells during single cell preparation should
be considered.
The comet assay provides no information on DNA fragment size
since fragments are not separated during the short electrophoresis
period. Instead, as the number of DNA breaks increases, super-
coiled loops relax, more free ends are able to migrate, and therefore
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
alkali method that detects DNA single-strand breaks, double-strand breaks
104 breaks/cell
30 and alkali-labile lesions. (c,d) SiHa human cervical carcinoma cells were
examined using the neutral lysis method that detects double-strand breaks.
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
(Gray; middle), and after 8 or 60 Gy (right). Graphs in b and d show typical
dose-response curves (mean ± s.d.; n = 3). Note the 40-fold difference in
0 2 4 6 8
the slopes of the two dose-response curves, consistent with the difference
Dose (Gy)
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
2,500 breaks/cell versus comet tail moment indicate the response of individual cells in various
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,
under denaturing conditions replication forks appear as single-strand breaks,
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 20 40 60
S-phase cells retard DNA migration during electrophoresis.
Dose (Gy)

a larger fraction of the DNA moves away from the comet head.
Some information can be obtained by measuring the distribution
of DNA damage within the comet tail, but accurate fragment size
measurements will require use of a technique such as pulsed field
gel electrophoresis38.
Cells actively replicating their DNA behave differently during
gel electrophoresis, and this can be confused with a difference in
inherent sensitivity. Under alkaline conditions, replication forks
behave as single-strand breaks so that S-phase DNA migrates more
rapidly. Under neutral conditions, S-phase DNA operates as repli-
cation bubbles that retard migration during electrophoresis39. This
problem is readily apparent in Figure 1. But as the comet assay can
provide a measure of both DNA content and DNA damage, it is
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
possible to analyze damage in any phase of the cell cycle.
Interpretation of comet results is complicated by the fact that
there is no simple relationship between the amount of DNA
damage caused by a specific chemical and the biological impact
of that damage. Each drug can differ in terms of the number of
DNA breaks that are associated with a given biological effect36.
Chemicals that produce interstrand cross-links will block detec-
tion of single-strand breaks17,22. For this reason, analysis of the
effects of mixtures of drugs, or a drug with two different mecha-
nisms of producing DNA damage can be particularly problem-
atic. Comparing comet assay results with other measures of DNA
damage (e.g., micronuclei, mutations, adduct levels, chromosome
aberrations or cell killing) is necessary to interpret the biological
MATERIALS
REAGENTS
• Low-gelling-temperature agarose (Sigma; Type VII, cat. no. A-4018)
• Phosphate-buffered saline (Ca2+- and Mg2+-free)
• Proteinase K (EMD Chemicals; cat. no. 24568-2)
• 0.5 M Na2EDTA (pH 8.0): add 55.8 g Na2EDTA and 6.4 g NaOH to 270 ml
distilled water (stir for approximately 2 h and adjust pH to 8.0 with additional
NaOH)
• 5 M stock solution of NaOH: add NaOH slowly to ice-cold distilled water
! CAUTION NaOH is caustic.
• Fluorescent DNA stain: 10 µg/ml propidium iodide (Sigma; cat. no. P-4170);
other DNA stains (e.g., SYBR-green, YO-PRO-1, DAPI) may also be used,
but photostability should be assessed especially with UV-excitable dyes13
! CAUTION Propidium iodide is mutagenic; wear protective gloves.
• (Optional; Step 15A only) A1, alkaline lysis solution for single-strand break
detection: 1.2 M NaCl, 100 mM Na2EDTA, 0.1% sodium lauryl sarcosinate,
0.26 M NaOH (pH > 13); equilibrate at 4 °C. ▲ CRITICAL Prepare fresh on
day of experiment.
• (Optional; Step 15A only) A2, alkaline rinse and electrophoresis solution:
0.03 M NaOH, 2 mM Na2EDTA (pH ∼12.3)
• (Optional; Step 15B only) N1, neutral lysis solution for double-strand-break
detection: 2% sarkosyl, 0.5M Na2EDTA, 0.5 mg/ml proteinase K (pH 8.0);
equilibrate at 4 °C
PROCEDURE
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
PROTOCOL
relevance of the damage. It is important to appreciate that DNA
damage measured in the comet assay is not necessarily a result
of direct genotoxicity; mitochrondrial or membrane damage can
cause extensive DNA fragmentation via apoptosis or necrosis.
Many investigators are interested in examining the DNA repair
capacity of cells by measuring the decrease in damage as a func-
tion of time after exposure to a known genotoxic agent. If the
agent can be administered quickly (e.g., X-rays) or under condi-
tions that inhibit repair (4 °C), then the half-time for recovery
can be determined by placing the treated cells under conditions
that allow for repair (e.g., complete growth medium at 37 °C).
Repair processes, however, can often complicate measurement
of DNA damage40. For exposures over long times, DNA dam-
age is a measure of both induction and repair. Similarly, cis-
platin-induced cross-links require several hours to form so that
both amount of damage and efficiency of repair contribute to
the overall damage detected. For some agents (e.g., ultraviolet-C
radiation and N-methyl-N’-nitro-N-nitrosoguanidine), damage
measured using the comet assay can be a result of incision at sites
of base damage produced during nucleotide excision repair41. In
this situation, ‘damage’ becomes a measure of repair. Finally, repair
of some types of DNA damage can be very rapid making detection
in living organisms difficult. Ionizing radiation–induced strand
breaks and some types of base damage by reactive oxygen spe-
cies can be repaired with a half-time less than 30 min and as short
as 3 min.
• (Optional; Step 15B only) N2, Neutral rinse and electrophoresis solution:
90 mM Tris buffer, 90 mM boric acid, 2 mM Na2EDTA (pH 8.5)
EQUIPMENT
• Disposable plastic tubes (5 ml; Falcon; cat. no. 35-2054)
• Water baths for melting agarose at 100 °C and maintaining agarose at 40 °C
• Hemocytometer or electronic particle counter (e.g., Coulter counter) for
adjusting cell numbers
• Frosted-end microscope slides ▲ CRITICAL Better adhesion can be obtained
using fully frosted slides (Fisher Scientific; cat. no. 12-544-5CY), but slides
cannot be dried and stored after staining.
• Diamond-tipped pen for scoring slides
• Covered glass containers for slide lysis, staining and storage
• Large-bed horizontal gel electrophoresis chamber and power supply
• Epifluorescence microscope with 25× objective (long working distance
objective is best) and filter set for green-light excitation if using propidium
iodide to stain DNA (546 nm excitation from a 100 watt mercury bulb;
emission monitored using a 580 nm reflector and 590 bandpass filter)
• Charge-coupled device (CCD) camera (8-bit black-and-white camera is
adequate); high sensitivity and high pixel density are preferred
• Computer and comet analysis software (commercial systems or free software
available on the internet; see http://cometassay.com.)
NATURE PROTOCOLS | VOL.1 NO.1 | 2006 | 25
 PROTOCOL
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 http://www.nature.com/natureprotocols

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
bubbles.

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
chamber.
(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

 PROTOCOL
(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 http://www.nature.com/natureprotocols

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.

● TIMING
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.

? TROUBLESHOOTING
See Table 1.

TABLE 1 | Troubleshooting table.

PROBLEM POSSIBLE REASON SOLUTION
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

 PROTOCOL
TABLE 1 | Troubleshooting table (continued).
Step 6 Agarose gel falls off the slide.
Step 8 Untreated cells show comet tails.
Steps 15 (A) (iv) and 15 B (iv) Microscopic
appearance of comets differs at different ends
© 2006 Nature Publishing Group http://www.nature.com/natureprotocols
Slides are not adequately agarose- Pay special attention to Step 6.
precoated or humidity is high.
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).
Depth of electrophoresis buffer is variable Use a bubble level device to ensure that the
across the bed. electrophoresis chamber is level.

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).

ANTICIPATED RESULTS
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
software.
COMPETING INTERESTS STATEMENT The authors declare that they have no
competing financial interests.
Published online at http://www.natureprotocols.com
Reprints and permissions information is available online at http://npg.nature.
com/reprintsandpermissions
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