COMET ASSAY INTEREST GROUP
Frequently Asked Questions
Contributed by Prof. Andrew Collins and Dr. Gunnar Brunborg.
1. Do you need to use alkaline conditions to see single strand breaks?
Answer: No. Other methods, such as alkaline unwinding, depend on the high pH to unravel the DNA molecules, starting at DNA breaks; the rate of unravelling depends on the frequency of breaks, and is assessed as the proportion of single-stranded DNA present after a certain unwinding time. But the comet assay is different. The original comet paper (Ístling and Johanson, 1984; BBRC 123, 291-298) used pH 10 (effectively neutral - certainly not high enough to unwind DNA), and reported the effect of low doses of ionising radiation. The dose range giving detectable damage was almost the same as that reported for the alkaline version by Singh et al. (1988; Exp. Cell Res. 175, 184-191). The most likely explanation is in terms of supercoiling. The DNA in the nucleoids (the structures remaining after lysis with Triton X-100 and 2.5 M salt) is supercoiled in a negative sense. Imagine the DNA as a series of supercoiled loops, associated at their bases to the nuclear matrix (a scaffold of protein and RNA which supports the DNA). Comets from undamaged cells have tightly packed, supercoiled DNA and no tail. Damage that leads to DNA breaks relaxes the supercoiling in the loops with breaks; these loops relax, and are pulled into a tail under electrophoresis. A single-strand break is sufficient to relax supercoiling. So the neutral and the alkaline comet assays detect both single- and double-strand breaks, and it is impossible to distinguish between them.
2. Does the neutral comet assay only detect double strand breaks?
Answer: No. This is the same issue as Question 1 (q.v.) [There is a special version of the neutral assay, with prolonged protease digestion at high temperature, which does apparently only detect double strand breaks. It is not clear how this works, but the digestion conditions probably destroy the nuclear matrix and loop attachments. Perhaps double stranded fragments then migrate in a conventional way.] It is possible to produce tails without breaks (single or double) in neutral comets. Relaxation of torsional tension with the intercalating dye ethidium bromide gave tails while higher concentrations of ethidium bromide caused the tails to diminish as positive supercoils built up; no such effect was seen in alkaline comets (Belyaev et al., 1999, BBA 1428).
3. How can I prevent gels from falling off the slides?
Answer: Assuming you are using the recommended method of precoating slides with agarose: Don't go back to using frosted glass slides, or expensive commercial coated slides. Gels falling off is an occupational hazard, that happens to everyone at some time. Usually whatever you do to cure the problem has no effect, and then one day the problem goes away. This is frustrating; it is impossible to give a sure solution. Some claim that the slides should be dried on the open bench, rather than at high temperature. Others find that if the laboratory atmosphere is humid, the gels do not stick as well as in a dry atmosphere. Another observation is that leaving the slides in lysis solution for more than a few hours makes the gels fall off - but only if a the cell suspension contains a trace of medium with serum. You could consider using plastic film (Gelbond) instead of glass slides. The film costs more than slides, but gels never fall off - providing you use the hydrophilic and not the hydrophobic surface.
4. Can the comet assay be used to detect damage in mitochondrial DNA?
Answer: No. Mitochondrial DNA molecules are small (about 17 kb) and are - of course - not attached to the nuclear matrix. They have been visualised by fluorescent in situ hybridisation with 'padlock' probes (Shaposhnikov et al., Mutagenesis, in press) and appear clustered around the nucleus of embedded cells, but as soon as lysis starts, the mtDNA begins to disperse, and by the time electrophoresis begins virtually no mtDNA molecules are left.
5. Can the comet assay be used to detect apoptotic cells? Are 'hedgehog' comets a sign of apoptosis?
Answer: 'Hedgehog' comets can be produced by treating cells with 0.1 M H2O2 for 5 min on ice (no chance for induction of apoptotic processes) and immediately processing for the comet assay. If the treated cells are incubated for half an hour, 'hedgehog' comets are no longer seen - the DNA damage has been repaired by the cell. Therefore hedgehog comets do not necessarily indicate apoptosis. In fully developed apoptosis, fragmented DNA is so small (down to nucleosome-sized pieces) that it would disappear completely by diffusion. But hedgehogs might be a sign of apoptosis in certain cases, indicating limited fragmentation (i.e. the earliest stages of apoptosis). [Comet 'ghosts' - faint images with just a small percentage of normal DNA stain - are sometimes seen. Perhaps these are aopoptotic cells?] In summary, hedgehogs may be a good indicator of apoptosis if you already are sure that apoptosis is in progress - but in that case you won't need hedgehogs to tell you.
6. Does it matter if there are non-viable cells among those used in the comet assay? What % is acceptable?
Answer: 'Viability' is usually measured with the trypan blue exclusion test. This is actually not a test of viability, but of membrane integrity. The membrane can be made leaky simply by scraping cells off the culture surface, but it soon gets repaired. In an experiment with HeLa cells, harvested by scraping or by trypsinisation, the scraped cells showed more than 80% trypan blue positive, whereas the trypsinised cells were 90% negative. But both gave the same low background damage scores. Furthermore, the scraped and trypsinised cells were replated in fresh medium and cultured for 24 h. After that time, the number of attached, living cells was the same, whether they had been trypsinised or scraped. Really 'unhappy' cells, such as cells grown under sub-optimal conditions, will show high background levels of breaks. So - if in an experiment untreated, control cells give a low background level of DNA breaks, that is a good test of viability. Don't rely on trypan blue. If the density of comets in the gel is less than expected from the number of cells inoculated, this may be because there were dead cells, which disappear as the heavily fragmented DNA disperses.
7. What is the best image analysis parameter to use?
Answer: % DNA in tail. This shows a good linearity with dose of damage over a reasonable range. Tail length tends to reach a maximum at a low level of damage. Please do not use tail moment (product of tail length and relative tail content of DNA). This is the least informative parameter. It has no generally accepted units. If you read in a paper that comets have a certain tail moment, it tells you nothing about the appearance of the comets, whereas with % tail DNA you can immediately visualise them. It is often important to know the sort of level of damage being reported - for instance, to be sure that the assay is not at saturation level. [Tail moment could be useful, if everyone used the same units, and calibrated their analysis system. Tail moment has dimensions - length x relative DNA content. So the appropriate units would be Ám.%, or Ám.tail fraction. Meanwhile, just use % DNA in tail!].
8. Do I need to buy image analysis software?
Answer: No (although, depending on the questions being asked, image analysis may be preferred. In the case of genetic toxicology testing, it has been recommended to use image analysis - see Hartmann et al., Mutagenesis 18, 45-51, 2003). . As an alternative, you can use the visual scoring approach, assigning each comet to a class according to how developed the tail is. Conventionally, 5 classes are distinguished, from 0 (no visible tail) to 4 (almost all DNA in tail). [It turns out that the human eye and brain are good at categorising visual appearance - of various kinds - in up to 5 grades, but can't cope well with more distinctions than that.] A visual score is computed (for 100 comets) by giving each comet the value corresponding to its class, so that the total ranges between 0 and 400 arbitrary units. The correspondence between visual score and image analysis results (% tail DNA) has been determined. There is an excellent correlation between visual score and % tail DNA for different samples analysed by both methods. Visual scoring is fast. Or you can download free image analysis programmes. If you decide to try such a programme, make sure that the counting rate is relatively high. It should not take more than 10 min to score 50 comets, preferably less. The cost of counting time should not be forgotten.
9. How important is pH?
Answer: The comet assay detects strand breaks and not all damage is in the form of strand breaks. Some other types of damage can be converted to strand breaks with the use of lesion-specific endonucleases. Some types of damage, such as base or phosphate alkylations, will decompose to form strand breaks in a process dependent on pH and time of alkaline treatment. These are called 'alkali-labile sites' or ALS. (Intermediates in base excision repair, the base-less sugars left by glycosylase action, are also ALS.) The most commonly used concentration of NaOH in the comet assay, 0.3M, gives a pH of around 13, which is enough to convert many types of ALS into strand breaks. 0.03M NaOH (pH around 12.1) is much less efficient. Thus it can be said, as an approximation, that 0.03M NaOH will detect mainly strand breaks, while 0.3M will also detect ALS. Not all base damage is alkali-labile, and not all ALS are equally alkali-labile. Regarding optimal pH and incubation time, we do not know the whole story, and research remains to be done. It is likely that different types of damage will have different time and pH optima.
10. How important is voltage?
Answer: It is voltage that determines movement of DNA in the gel. Strictly speaking, it is the voltage gradient over the gel that determines movement of DNA. The voltage gradient depends on resistance, and so will be less in the chambers of the tank with (low resistance) buffer, and more in the part of the tank with gels covered in a thin layer of buffer, where the resistance is higher. Increasing the layer of buffer above the gels will increase the current by reducing the overall resistance - and will also decrease the voltage gradient. It is complicated! As a rough guide, most of us would aim at a voltage gradient of about 1V/cm over the part of the tank where the slides are placed, with a layer of 1-2 mm of buffer above the slides.
11. How important is current?
Answer: Current can be important, since many power supplies have a cut-off of around 400 or 500 mA. Current is usually adjusted to be below the cut-off, by adjusting the volume of buffer in the tank and therefore the depth of buffer over the gels.
12. How important is lysis time?
Answer: One hour is generally regarded as the minimum required. It is also a convenient time in which to go to have lunch! Overnight is OK, unless you have a problem with gel attachment, in which case the extended lysis can cause losses.
13. How do I measure other kinds of damage, in addition to strand breaks?
Answer: After lysis of cells in the gel, incubate with a repair enzyme that will convert particular base lesions to DNA breaks. Compare the breaks seen with the enzyme with the breaks seen on incubation for the same time with buffer but no enzyme. Enzymes: Endonuclease III converts oxidised pyrimidines to breaks; formamidopyrimidine DNA glycosylase (FPG) converts 8-oxoguanine and some other oxidised purines to breaks; AlkA converts alkylated bases to AP (apurinic/apyrimidinic) sites; T4 endonuclease V converts cyclobutane pyrimidine dimers to breaks. (Unfortunately, as yet, there is no enzyme that recognises bulky adducts. UvrABC is the bacterial enzyme complex that is potentially useful in this respect, but so far it has not been possible to use it on nucleoid DNA in gels.) Some of these enzymes have an associated AP lyase or endonuclease activity that carries out the actual strand breakage at the AP site left by a glycosylase. In other cases, it is left to the high pH to convert the AP site to a break.
14. Can the comet assay be used to measure DNA repair?
Answer: Yes. There are several approaches. The simplest is to treat cells with damaging agent and measure the damage remaining (as strand breaks or enzyme-sensitive sites) at intervals during incubation. Thus you can measure the kinetics of cellular repair after very low ('physiological') doses of DNA damage. Another method is the in vitro repair assay - a biochemical approach which measures the repair capacity. A substrate is prepared, of cells treated with an agent delivering an excess of damage of the relevant sort for the enzyme of interest. An extract is prepared from the cells whose repair capacity is to be investigated, and this extract is incubated with the substrate, for 10 or 20 min. Breaks accumulate as the incision events take place. Rates of repair of oxidised bases vary between individuals. Also, inhibitors of DNA synthesis (hydroxyurea plus cytosine arabinoside or aphidicolin) may be used to block the resynthesis step of nucleotide excision repair, causing breaks to accumulate; these give a measure of repair capacity, since incision is the rate-limiting step. Some hints: Preincubate cells for 15-30 min before giving damage; do not incubate for longer than 20-30 min after treatment (as breaks do not accumulate linearly after that); beware of the rapid rejoining of breaks when inhibitors are removed (so scraping may be better than trypsinisation to harvest the cells).
15. Why do I sometimes see images resembling halos, rather than comets, even when there is sufficient damage to produce tails?
Answer: We wish we knew. Any suggestions?