The Single Cell Gel Electrophoresis Assay, commonly known as the Comet Assay, is a relatively inexpensive method to qualitatively and quantitatively detect genetic damage, specifically DNA strand breaks at the cellular level. This assay is widely used in research, genotoxicity investigations, and as a pre-screening assay in the pharmaceutical industry.
Genetic damage occurs all the time due to aging and other factors. Therefore, as in most scientific tests, a control must be used for the purposes of comparison. If the number of strand breaks and the length of those strands can be measured, then a conclusion can be drawn that the agent did or did not create additional damage to the cell, as compared to the amount of damage in the control cells.
To fully understand the results of this assay, we must first try to understand the changes each cell undergoes during the process.
|First, animals/cells are exposed to a suspected damaging agent. Then, sample cells from the animal are suspended in agarose on a slide. Next, the cells are lysed, removing their nuclear membranes and proteins. After this lysing procedure, the sample is soaked in either a neutral buffer or an alkaline buffer where the DNA mass “loosens”, allowing the broken DNA strands to unwind as the nucleic mass beings to fray.Then the slides are bathed in an electrophoresis tank where an electrostatic field pulls the damaged DNA strands in a single direction, resulting in the cell taking on a comet-like appearance.
Then, the slide is dried, causing the DNA mass to settle or “flatten”. This settling of the DNA mass should be taken into account during the analysis of the comet as the image is taken from directly above and the 2-dimensional representation of the DNA mass will appear larger than the original cell.
Finally, the samples are stained with a fluorescence dye, making the DNA visible under a microscope objective using proper illumination.
Through the use of CCD and computer technology, the cells are imaged and stored electronically for analysis. The primary assumption made in imaging the comets with a CCD camera is :
Anything that remains after the camera background image has been extracted digitally from the video image is a part of a comet.
The human eye can usually distinguish about 25 gray levels (and the response is logarithmic), whereas an optimized video imaging system can resolve more than 200 gray levels (8x). Thus, the electronic image processing approach can use information not readily visible to the eye. This increased sensitivity provides the capability to maximize the acquisition of fluorescence from a cell to improve confidence in the metrics.
To quantify the amount of genetic damage, the cells are analyzed via image processing algorithms. The most common method is based on the intensity profiles of the cell image. The intensity of each pixel in the image is directly proportional to the amount of DNA at that location in the cell. Therefore, the area under the profile generated from the summation of intensity values in each column is directly proportional to the amount of DNA at that x-coordinate of the cell. Below, the intensity profile is shown in yellow.
Further, the head and the tail of the comet can be determined using the same methodology. The assumption is made that the highest peak of the intensity profile is the center of the head. Mirroring the head about that point and subtracting the resulting head profile from the original intensity profile produces the tail profile. The area under the tail profile is directly proportional to the amount of DNA in the tail. In the following images, the purple head profile and the aqua tail profile have been overlaid.
When quantifying DNA damage, many different measurements and equations are used. Among the most common are Total Length, Tail Length, Olive Moment, and Tail Area.