DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. While this counts for only a small part of the human genome's approximately 6 billion bases (3 billion base pairs), unrepaired lesions to critical genes can impede a cell's ability to carry out its function.
2. DNA damage and cancer
When DNA is damaged and DNA repair is not sufficient, and the cell cannot function properly, it increases the likelihood of cancer.
3. Technique for measuring DNA damage
The comet assay is used universally for the detection of DNA damage.
Comet assay kits and slides can detect DNA / RNA damage and modification.
It is a useful screening tool for various types of DNA damage.
The alkaline SCG assay can be effectively used to evaluate the in vivo genotoxicity of chemicals in multiple organs, providing for a good assessment of potential carcinogenicity.
4. Comet assay
Different versions of Comet Assay:
Comet assay kits and slides can detect DNA / RNA damage and modification.
It is a useful screening tool for various types of DNA damage.
4. Comet assay
The comet assay is also known as single-cell gel electrophoresis (SCGE) assay. This simple assay has many scientific applications. It is a common technique for measurement of DNA damage in individual cells. Under an electrophoretic field, damaged cellular DNA (containing fragments and strand breaks) is separated from intact DNA, yielding a classic “comet tail” shape under the fluorescent microscope. Extent of DNA damage is usually visually estimated by comet tail measurement; however, image analysis software is also available for measuring various parameters.
Different versions of Comet Assay:
http://www.cometassay.com/index_files/Page290.htm
The comet assay is sufficiently sensitivite for detecting genotoxicity
http://www.hindawi.com/journals/jna/2010/541050/
- Alkaline Comet Assay
- Neutral Comet Assay
- Comet assay at pH 12.1
- Comet assay with DNA repair enzymes
- Acellular Comet Assay
- Comet-FISH Assay
- Bromodeoxyuridine Labeling to Detect Replicating DNA
- Detection of incomplete DNA excision repair sites
- Halo assay or non-electrophoretic assay
The comet assay is sufficiently sensitivite for detecting genotoxicity
http://www.hindawi.com/journals/jna/2010/541050/
6. Commercial assay kits
The DNA damage is quantified by measuring the displacement between the genetic material of the nucleus ('comet head') and the resulting 'tail'. Tail Moment and Tail DNA% are the two most commonly parameters to analyze comet assay results. At least 50 -100 cells should be analyzed per sample. The Tail Moment has been suggested to be an appropriate index of induced DNA damage in considering both the migration of the genetic material as well as the relative amount of DNA in the tail.
Tail DNA% = 100 x Tail DNA Intensity/Cell DNA Intensity
Tail Moment can be measured using one of the following methods:
(a) Olive Tail Moment = Tail DNA% x Tail Moment Length*
(b) Extent Tail Moment = Tail DNA% x Length of Tail (see diagram)
*Tail Moment Length is measured from the center of the head to the center of the tail (see diagram)7. Comet analysis software programs
A number of comet analysis software programs are commercially available, such as:
1. LACAAS from Loats Associates, Inc. http://www.loats.com/comet.html and
2. Comet Assay IV from Perceptive Instruments http://www.scorecomets.com/
3. WOLFRAM Comet Assay Analysis http://library.wolfram.com/examples/cometassay/
8. Comet Assay IV scoring system
Comet Assay IV is the world's fastest, most consistent comet assay scoring system. Scoring with Comet Assay IV is a uniquely effortless process. Just click on each comet to get your results.
Comet Assay IVTM is the latest in a highly successful series of comet scoring systems from Perceptive Instruments. Comet Assay IV is the professional choice for those seeking accuracy, reproducibility and GLP compliance.
Comet Assay IV has the ability to score comets from a live video. This makes Comet Assay IV the fastest, easiest and most accurate way to score comets to date.
Comet Assay IV is supplied with a FireWire video camera delivering 3x the resolution of previous models. This makes it possible to use lower power microscope objectives, offering a larger field of view and greater depth of field. This combination greatly improves scoring speed, with more comets per field and less time spent at the controls of the microscope.
Getting started with Comet Assay IV is as simple as installing the software and connecting the camera.This can be done in a few minutes either by yourself or your IT department. Once running, Comet Assay IV displays a live video image onscreen that mirrors what you see by looking down the microscope's eyepiece. Any refocusing or stage movements performed at the microscope level are shown live on screen without any lag or delay.
9. Cell Biolabs comet assay kit
The OxiSelect™ Comet Assay Kits provide a fast, convenient way to screen for general DNA damage, regardless of the source or nature of the damage.
The OxiSelect™ Comet Assay Control Cells set contains both positive and negative control cells for detection by fluorescence microscopy.
The OxiSelect™ Comet Assay Control Cells are provided as a set; the set containing vials of healthy, untreated cells and DNA damaged, Etoposide-treated cells. These cells are intended for use as controls in the Cell Biolabs’ OxiSelect™ Comet Assay under alkaline electrophoresis conditions.
10. Cell Biolabs comet assay procedure
Cell Biolabs’ OxiSelect™ Comet Assay Control Cells are designed for use in a single cell gel electrophoresis assay (SCGE) for simple evaluation of cellular DNA damage (under alkaline conditions).
- First, individual cells are mixed with molten agarose before application to the OxiSelect™ Comet Slide.
- These embedded cells are then treated with a lysis buffer and alkaline solution, which relaxes and denatures the DNA.
- Finally, the samples are electrophoresed in a horizontal chamber to separate intact DNA from damaged fragments.
- Following alkaline electrophoresis, the samples are dried, stained with a DNA dye, and visualized by epifluorescence microscopy.
- Under these conditions, the damaged DNA (containing cleavage and strand breaks) will migrate further than intact DNA and produce a “comet tail” shape.
- For a complete protocol, refer to Cell Biolabs’ OxiSelect™ Comet Assay Kit insert (STA-350).
11. Comet assay special interest group
12. References
- Ostling, O., and Johanson, K. J. (1984). Micro gel electrophoretic study of radiation induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun. 123, 291–298.
- Singh, N. P., McCoy, M. T., Tice, R. R., and Schneider, E. L. (1988). A simple technique for quantification of low levels of DNA damage in individual cells. Exp. Cell. Res. 175, 184–191.
- Olive, P. L., Banath, J. P., and Durand, R. E. (1990a). Heterogeneity in radiation induced DNA damage and repair in tumor and normal cells using the "Comet" assay. Radiat. Res. 122, 86–94.
- De Boeck, M., Touil, N., De Visscher, G., Vande, P. A., and Kirsch-Volders, M. (2000). Validation and implementation of an internal standard in Comet assay. Mutat. Res. 469, 181–197.
- Yu F Sasakia, Keiko Fujikawaa, Kumiko Ishidaa, et al. The alkaline single cell gel electrophoresis assay with mouse multiple organs: results with 30 aromatic amines evaluated by the IARC and U.S. NTP. http://www.sciencedirect.com/science/article/pii/S1383571899000066
Study Questions
What is the comet assay?
How is the comet assay performed?
What is the comet assay good for?
Is the comet assay sensitive for detecting genotoxicity?
What is the comet assay good for?
Is the comet assay sensitive for detecting genotoxicity?
0 comments:
Post a Comment