Abstract:
Comet assay also known as single cell gel electrophoresis is used to detect DNA damage. DNA damage caused by double strand break, single strand break, alkali labile sites, oxidative base damage and DNA crosslinking with DNA or protein can be detected by Comet assay. It is a sensitive and rapid technique for quantifying and analysing any kind of DNA damage in individual cells. DNA damage detection at the level where an individual eukaryotic cell has high significance and has applications in the various fields of toxicology, pharmaceuticals genotoxicity testing, environmental and human bio-monitoring, diagnosis of genetic disorders.
Key words:
Single cell gel electrophoresis, Comet, DNA damage, Genotoxicity.
Introduction
In 1984, Ostling and Johansan developed the comet assay which is also called single cell gel electrophoresis. This assay was carried out under neutral conditions. Later this assay was modified by two groups of scientists. In 1988, the first group Singh and his co-workers used highly alkaline conditions for electrophoresis which enables supercoiled DNA to get relaxed and unwind. This modification enables the detection of low level of strand break with higher sensitivity. In 1990 the second group Olive and co-scientist performed this experiment under neutral and mild alkaline condition. This method is used for cells with varying sensitivity to drugs and radiations. The assay has been modified at various steps that include cell isolation, cell lysis, electrophoresis, staining to make it suitable for detecting various kinds of damage in different cells. The assay is now well established and simple, a rapid and sensitive technique to detect DNA damage qualitatively and quantitatively. Using lesion-specific antibodies or specific DNA repair enzymes lesions of DNA damage such as an oxidative DNA damage or DNA crosslinking may also be assessed. This assay has various applications such as a valuable tool in
fundamental DNA damage and repair studies, human bio-monitoring and genotoxicity testing.
The Comet assay technique has been accepted worldwide for detecting DNA damage and repair in prokaryotic and eukaryotic cells. Comet assay can detect low level of DNA damage (one break per 1010 Daltons of DNA and also require small numbers of cells (∼10 000) per sample. It has low cost estimation, flexible for proliferating as well as non-proliferating cells, ease of application and assay needed short time to complete a study.
Principle and procedure
Any eukaryote cell can be obtained as a single cell suspension. For example cells isolated from blood, cells from tissue biopsies that can be homogenized, buccal cells, whole blood and cultured cells can be used. As collecting single cell sample from blood or tissue is not always feasible, other sources of sample can be collected non-invasively for the test. DNA damage in various types of cell like epithelial cells as well as sperm cell can be detected by the assay. Individual cells are collected and then embedded in a thin agarose gel on a microscopic slide. In the next step all the cellular proteins have to be removed from cells by appropriate cell lysis
method. Once the cells are deproteinized, then the alkaline or neutral conditions have been provided to unwind the DNA.
After the unwinding, the DNA undergoes electrophoresis. Electrophoresis allows the broken DNA to migrate away from the nucleus. DNA specific fluorescent dye such as ethidium bromide or propidium iodide is used for staining. After staining is done the gel is read for amount of fluorescence in head and tail and length of tail. The extent of DNA migrated from the head of the comet is directly proportional to the amount of DNA damage present in the cell. In this test these resulting images resembles a comet so this method is also called comet test.
Figure 1: Cells that have DNA damage forms comet like structure. Comet tails contain damaged DNA while comet head
contain undamaged DNA. Image Courtesy: Comet Assay Principal: How to run a Comet Assay.
DNA is being negatively charged due to the electric current smaller DNA fragments are able to migrate through the gel faster than the larger unbroken /undamaged DNA fragments. Migration of damaged DNA occurs at a different rate. So Comet like structure is formed with damaged DNA fragments accumulating in the tail region. Therefore, the length of the tail is directly proportional to the extent of DNA damage.
A single cell gel electrophoresis also can be used for DNA repair assay. In this technique cells are embedded in the gel to repair before lysis. This idea recently adapted to study DNA repair kinetics in more detail like to study the regulation of BER proteins by post transcriptional modifications. But this can also be studied by comet technique. In this method fluorescent-labelled DNA probes are used. DNA probes will hybridize to the single stranded DNA in the comet tail.
Interpretation of results
When DNA is less damaged, the distance of DNA migration from the nuclear core is used to measure the extent of DNA. However this technique is not applied when the DNA damage is very high. Because at high level of DNA damage fluorescent staining intensity increases but not length. For this a scoring method based on visual inspection can be used as recommended by Collins. This method gives quantitative resolution which is sufficient for many purposes.
Another method introduced by Olive called ‘tail moment’ is used for comet evaluation. By this method both smallest detectable size of migrating DNA with the use of length of comet tail and the number of broken pieces of DNA with the visualisation of staining intensity of DNA in the tail can be detected. So method chosen depends on the investigator and the experiment design for the evaluation of DNA migration.
Conclusion
Even after lots modifications and years of the development the comet assay is still a simple technique. Certain limitations of the Comet assay are that a neugenic effects, and epigenetic mechanisms of DNA damage such as effects on cell-cycle checkpoints are not detected. Also it is versatile but labour-intensive assay. The other disadvantages of this method are as single-cell data which may be rate limiting, small cell sample leading to sample bias, technical variability and interpretation. However its advantages can outnumber the disadvantages and hence this technique has an importance in fields ranging from molecular epidemiology to genetic toxicology.
Figure 2: Comet tail moment measurement
Krina Patel
Department of Biochemistry and Biotechnology
St. Xavier’s College, Ahmedabad
References
Mahima Vajpayee, Ashutosh Kumar and Alok Dhawan, Chapter 1:The Comet Assay: A Versatile Tool for Assessing DNA Damage , in The Comet Assay in Toxicology, 2016, pp. 1-64 DOI: 10.1039/9781782622895-00001 https://pubs.rsc.org/en/content/chapterhtml/2016/bk9781782622871-00001?isbn=978-1-78262-287-1&sercode=bk
Sabine A. S. Langie,Amaya Azqueta and Andrew R. Collins(2015) https://www.frontiersin.org/articles/10.3389/fgene.2015.00266/full
Wenjuan, Michael A. McNutt and Wei-Guo Zhu(2009);Methods 48(1):46-53 DOI:10.1016/j.ymeth.2009.02.016 Source: PubMed