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The magic of the comet assay: Why does DNA move like a comet in electrophoresis?
In the world of science, detecting DNA damage has never been easy. However, the single-cell gelatin electrophoresis assay (also known as the comet assay) provides a simple and sensitive method to detect DNA damage in individual eukaryotic cells. This technique was first developed by Östling and Johansson in 1984 and then improved by Singh et al. in 1988. It has since become one of the standard techniques for DNA damage/repair, environmental monitoring, and mutagenicity testing.
The comet assay gets its name from the migration pattern of DNA in an electrophoretic gel, which often resembles a comet.
The basic protocol for this assay involves encapsulating cells in a suspension of low melting point agarose, then lysing the cells under neutral or alkaline (pH > 13) conditions, and electrophoresing the suspended lysed cells. During this process, the cell's DNA structure is examined, forming unique "comet" patterns that reflect the extent of DNA damage.
How it works
The basic principle of the comet assay is that undamaged DNA retains a highly ordered association with matrix proteins in the cell nucleus, and this structure will be disrupted when the DNA is damaged. The damaged DNA strands lose their compact structure and relax, beginning to expand into the agarose. When an electric field is applied, the negatively charged DNA is attracted to the positively charged anode, thus creating a "comet" shape.
The greater the degree of damage, the longer and brighter the DNA tails will be, as more DNA fragments are able to be released from the cell into the agarose.
Steps of the comet assay
Cellular Encapsulation
First, researchers need to obtain cells from in vitro cell culture or in vivo samples, then disperse these cells into single cells and suspend them in low-melting point agarose melted at 37°C. This single-cell suspension is then spread onto a microscope glass slide and forms a thin layer of agarose as it cools. The neutral osmotic pressure of agarose allows reagents to penetrate without affecting the position of cells.
Cracking
Next, the slides are immersed in a solution that causes the cells to lyse. This solution usually contains highly concentrated salt and detergent. Salt water can destroy the protein structure inside cells and dissolve the cell membrane. In this way, only the DNA remains intact and fills the space previously occupied by the cell, forming a so-called karyotype structure.
Electrophoresis
Once the cells are lysed, the slides are then washed to remove excess salts and immersed in the electrophoresis solution. During electrophoresis, the applied electric field causes damaged DNA to move toward the anode. In this way, the degree of damage directly affects the migration range of the DNA and thus the shape of the "comet".
This technology has extremely high sensitivity for DNA damage, making it a broad detection tool.
Application Areas
The comet assay is commonly used in the fields of genotoxicity testing, human biomonitoring, molecular epidemiology and eco-genetic toxicology. Recent studies have shown that during aging, multiple DNA damage types, such as single-strand breaks and double-strand breaks, can be detected using the comet assay. In the diagnosis of male infertility, researchers can also use the comet assay to assess the degree of DNA fragmentation in sperm cells.
Overall, the comet assay provides an effective and flexible technique to detect DNA damage in cells, which can play a unique role in both basic research and applied science. As this technology continues to advance, will there be more innovative ways to detect and repair DNA damage in the future, allowing human health to reach new milestones?
