Comet assay: A brief history
- First Online:
- Cite this article as:
- Piperakis, S.M. Cell Biol Toxicol (2009) 25: 1. doi:10.1007/s10565-008-9081-y
- 248 Views
In the last two decades, new methodologies that are able to assess DNA damage have been developed.
In 1976 Cook et al. published a paper investigating the nuclear structure based on the lysis of cells with nonionic detergent and high-molarity sodium chloride. This treatment removes membranes, cytoplasm, and nucleoplasm, and disrupts nucleosomes, almost all histones being solubilized by the high salt. What is left is the nucleoid, consisting of a nuclear matrix or scaffold composed of RNA and proteins, together with the DNA, which is negative supercoiled as a consequence of the turns made by the double helix around the histones of the nucleosome. The survival of the supercoils implies that free rotation of the DNA is not possible. Cook et al. proposed a model with the DNA attached at intervals to the matrix so that it is effectively arranged as a series of loops, rather than as a linear molecule. When the negative supercoiling was unwound by adding intercalating agent (i.e. ethidium bromide), the loops expanded out from the nucleoid core to form a “halo”. Similar effects is seen when radiation is used to relax the loops, one single-strand break is sufficient to relax the supercoiling in that loop.
The earliest attempt to quantify DNA strand breaks directly was made by Rydberg and Johanson in 1978 with cells embedded in agarose on slides and lysed under mild alkaline conditions.
In 1984 Ostling and Johanson, based on the approach described above, developed the comet assay, also called single-cell gel electrophoresis (SCGE).
This was an assay in which the lysis and electrophoresis were performed under neutral conditions. The staining of the DNA was done with acridine orange. The image obtained looked like a “comet” with a distinct head, comprising intact DNA and a tail, consisting of damaged or broken pieces of DNA. As a consequence the name “comet assay” was given. The amount of the DNA liberated from the head of the comet depends on the dose of mutagen used. However, in this procedure, only double-strand breaks could be analyzed.
The first group performed electrophoresis under highly alkaline conditions (pH > 13). This enables the DNA supercoils to get relaxed and unwind and makes possible the detection of alkali labile sites and single-strand breaks in DNA during electrophoresis. This method measures low level of strand breaks with high sensitivity.
The second group conducted electrophoresis under neutral or mild alkaline conditions to detect single strand breaks. This method was optimized to detect a subpopulation of cells with varying sensitivity to drugs or radiation.
The version of the comet assay developed by Singh et al. was found to be up to two orders of magnitude more sensitive.
The alkaline comet assay became very popular in the 1990s and today probably is one of the most used assays for the assessment of DNA damage repair. The popularity of the method is illustrated by the total number of entries in the PubMed database when using “comet assay” as search term (3575 entries by March 2008).
The simplest types of DNA damage detected by comet assay are double-strand breaks (DSBs). DSBs result in DNA fragments and can be detected by merely subjecting them to electrophoretic mobility at the neutral pH. Single-strand breaks (SSBs) do not produce DNA fragments unless the two strands of the DNA are separated/denatured. This is accomplished by unwinding the DNA at pH 12.1. It is also possible that single-strand breaks can relax the DNA and hence can also be detected with the comet assay at a neutral pH. Other types of DNA damage broadly termed alkali labile sites (ALS) are expressed when the DNA is treated with alkali at pH greater than pH 13. Breaks can also be introduced at the sites of DNA base modifications by treating the DNA with lesion-specific glycosylases/endonucleases and the fragments thus produced can also be detected by the comet assay. Therefore, by controlling the conditions that produce nicks at the sites of specific DNA lesions the comet assay can be used to detect various classes of DNA damage. While breaks increase DNA migration, DNA binding and crosslinks can retard DNA migrations and can also be detected by the comet assay. Therefore increased migration in the comet assay can be attributed to strand breaks, alkali labile sites and incomplete excision repair sites, while decreased DNA migration could be attributed to crosslinks DNA–DNA or DNA–protein interactions.
Cell lines are suitable.
It has been applied in a wide variety of eukaryotic cells in any organ, including plants and many prokaryotic cells, with successful results.
It detects damage at the single-cell level.
Highly sensitive (50–15,000 breaks/cell).
Results are obtained on the same day.
Damage can be detected in cycling as well as in noncycling cells.
It is fast, simple and inexpensive.
Fresh or frozen samples are suitable.
Sample size is very small (from 10,000 to 50,000 cells).
Human studies: The comet assay is ideal for human investigations since it does not require prelabelling with radioactivity or other harmful procedures. It has been used in biomonitoring including occupational exposure to genotoxic chemicals or radiation. In nutrition studies the effects of diet on DNA damage repair was assessed. The comet assay could also be used to aid diagnosis (xeroderma pigmentosum, Nijmegen breakage syndrome). Finally the technique could be used to assess the background levels of DNA damage in individuals.
Ecological monitoring: The comet assay in combination with suitable organisms (mussels, earthworms, small rodents) can be used as biosensors for measuring contamination of the environment with genotoxins.
Genotoxicity testing: The comet assay is now been used as a standard test to assess the safety of novel chemicals or pharmaceuticals.
Estimation of DNA repair: The technique can be used in measuring repair capacity at the cellular level. Alternatively the repair capacity of cells extract can be assessed in an in vitro assay.
This present special issue titled: the comet assay, its uses and recent developments is comprised of seven articles addressing various topics on the technique.
Anderson et al. in their communication give an overview of the use of comet assay utilising sperm or testicular cells in reproductive toxicology. This includes consideration of damage assessed by protocol modification, cryopreservation versus the use of fresh sperm, viability and statistics.
Collins et al. analyse their aim to develop a simple comet-based in vitro assay for nucleotide excision repair (NER), similar to that already in use for base excision repair (BER), and then to apply these in vitro assays to lymphocyte samples collected on several occasions from healthy subjects to gain an impression of the degree of intra- and inter-individual variability.
Dhawan et al. provide an overview of in vivo as well as in vitro studies assessing the DNA damage from bacteria to man with the comet assay demonstrating the versatility of the technique. In particular they examine the use of the comet assay in studies with bacteria, lower (fungi, algae) and higher plants, lower animals, invertebrates, mussels, earthworms, drosophila, fishes, amphibians, birds, rodents, and finally humans.
Garaj-Vrhovac and her group used the comet assay to study the effects of microwave radiation on exposed population. In particular with the use of alkaline comet assay they studied the effects of microwave radiation emitted by radar equipment and antenna system service on daily exposed workers.
Kumaravel et al., in their article, provide an overview of evolution of measurements of DNA damage using the comet assay and summarise and critically analyse the advantages and disadvantages of different approaches currently being adopted while using this assay.
Marcos et al. attempted to enhance the ability of the comet assay technique to detect excision repair sites this could be done by the inclusion of repair inhibitors, DNA synthesis inhibitors or chain terminators. In this sense they used hydroxyurea and cytosine arabinoside for detecting lesions produced by the alkylating agents ethyl methanesulfonate and methyl methanesulfonate in three different cell systems.
Finally Piperakis et al. assessed with the comet assay basal DNA damage and induced DNA damage and repair in populations of different ages. For this they used three distinct populations of 5- to 10-, 40- to 50- and 60- to 70-years old. Their results show increased DNA damage and declined DNA repair with age.
We hope that this special issue will be well received by the Cell Biology and Toxicology Journal’s readers. Personally, I would like to thank the Editor-in-chief of the periodical, Prof. John Masters, also Naomi Portnoy, Fabio de Castro, Ruben Kramers, Amit Dixit and all the staff at Springer in the Netherlands for encouragement and assistance during the preparation of this issue.