“The comet assay is performed to detect the amount of double-stranded or single-stranded DNA damaged in a eukaryotic cell.”
We know everything about DNA. It is a foundation for our life, therefore if it damages, it creates a big problem for once’s health. There are so many intrinsic and extrinsic factors that damage our DNA.
For instance, the process of uncontrolled apoptosis and UV light exposure are two common intrinsic and extrinsic factors, respectively, which damage DNA.
DNA should remain intact during cell division to package properly on chromosomes. Otherwise, it can cause several types of cancer and other problems.
By performing a comet assay, also known as single-cell gel electrophoresis, the amount of DNA damage can be determined.
In the present article, we will learn what is comet assay and how it works. We will also look after some of the applications of this technique in molecular genetics.
What is a comet assay?
Simply put, it is a method to measure the amount of nucleic acid damaged.
During 1984, Ostling and Johnsson (Swedish scientists) had originally developed a technique of comet assay, however, major modifications in it were done by Singh et al, in 1988 by using alkaline treatment in the main protocol.
Principle:
The comet assay function on the principle of electrophoresis in which charged particles migrates under the influence of current.
As the cell migrates under the current in a gel, a comet-like structure is formed with intact DNA in a head and a tail of damaged DNA which looks like an actual comet.
To perform a comet assay, cells are first treated with a lysis buffer to digest and remove all the protein portion of a cell.
In the very next step, DNA unwinding is performed under alkaline or neutral conditions. During the unwinding, the damaged single-stranded or double-stranded DNA releases or relaxed. However, the intact DNA remains tightly wrapped.
Next, the cells are run under low melting point agarose gel. The agarose power is extracted from seaweed and widely used in DNA gel electrophoresis. The DNA can migrate from the matrix of gel pores.
Although, unlike conventional gel electrophoresis, the comet gel is performed on a glass slide. The slide is employed to run under the electrical current for some time and results and analyzed.
The concept…
DNA is highly compacted and wrapped structure. When it damages the higher level of DNA organization disrupted and releases the DNA fragment. When we run DNA in a gel, it trying to migrate towards the positive pole. If DNA damage occurs, single-stranded and double-stranded damaged DNA migrated and creates a tail-like structure.
The amount of DNA damage is directly proportional to the length of a tail.
If you are interested to read articles on DNA packaging you can read these two articles:
Protocol:
Collect 1 to 2 ml blood sample or cultured cells under aseptic conditions and transfer it to the falcon tube. Ass 2 to 3 ml of lymphocyte separation media to the sample and centrifuge it at 1500rpm for 20 to 30 minutes.
The buffy coat of the mononuclear cells is separated and transfer to another tube.
The cells or eukaryotic cells are first encapsulated in a low-melting-point gel at 37°C. Prepare 0.5% LMPA and 0.75% NMPA in a PBS and heat it in the microwave to melt. (both gels are separately melted and added on a slide one after another).
Note: care must be taken while pouring a gel to avoid bubble formation.
Aspire 200μl of the melted gel and pour it on a microscopic glass slide and covered with a coverslip.
Soon after that, the gel allows to cool at a 4°C temperature and a coverslip is removed. The matrix of gel encapsulates cells in its carbohydrate matrix.
Once the slide is prepared, after that the slide is treated with the lysis solution. As we discussed the lysis solution digest the protein portion of a cell. Therefore, a nucleoid of supercoiled DNA remains linked to the nuclear matrix.
Usually, detergents like Triton X-100 or any other salt solution is used for it at higher pH.
Here the detergents also digest the RNA as well. Lysis occurs at 4°C for at least 2 hours. In the next step, the slide is washed with distilled water to remove the traces of chemicals other contaminants and placed in the electrophoresis buffer.
An electrophoresis buffer is a high pH alkaline solution which denatures DNA double helix. Rest the slide in a buffer for at least 15 minutes to denature.
Run electrophoresis fro 20 minutes and neutralize slide in pH 7.0 solution.
In the last step, stain the slide with fluorescence dye and observe under a microscope.
Results and interpretation:
To analyze a gel, the gel is stained with fluorescent dye like EtBr (Ethidium bromide) or propidium iodide. Notably, EtBr is used widely. It can intercalate between the DNA molecules and emit fluorescence.
The slide is analyzed under a fluorescent microscopy system that detects the amount of fluorescence emitted from the head part and tail part of a cell.
The amount of fluorescence emitted from the tail is directly proportional to the damaged DNA.
While running the gel, the intact and supercoiled nuclear DNA migrates very slowly and remains nearer to where it started migration but that is not the case with the damaged DNA.
The damaged DNA fragments of ssDNA and dsDNA migrate faster and create a tail-like appearance.
The present assay not only detects the damaged DNA but also measures the quantity of it in each cell.
Applications:
It detects and measures single-strand and double-strand break.
It is widely used in genotoxicity testing and studies, drug evaluation, epidemiological studies, toxicological studies, nutritional studies, genetic disorder studies and DNA repair studies.
It is widely used in male infertility screening via sperm cell fragmentation study.
The assay is also powerful enough to detect Ap sites, excision damage, alkali labile site and other breaks associated with the DNA.
Sperm DNA fragmentation study…
One of the most fascinative uses of the comet assay nowadays is to detect male infertility. Sperm DNA damage is directly proportional to the degree of infertility therefore it is an important tool used for the in-vitro fertilization process.
Before doing the artificial fertilization, the degree of DNA damage from a sample is detected to know whether the sample can be used for fertilization or not.
Although some minor modification in the native comet assay is needed to get success in the process.
Conclusion:
Conclusively, we can say a comet assay is an excellent technique to detect DNA damage at the cellular level. It is commonly used in cancer research viz to detect, monitor and detect the success rate of therapy used to cure cancer.
Sources:
Nandhakumar S, Parasuraman S, Shanmugam MM, Rao KR, Chand P, Bhat BV. Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay). J Pharmacol Pharmacother. 2011;2(2):107-111. doi:10.4103/0976-500X.81903
