Comet Assay - Background

Background

The concept underlying the SCGE assay is that undamaged DNA retains a highly organized association with matrix proteins in the nucleus. When damaged, this organization is disrupted. The individual strands of DNA lose their compact structure and relax, expanding out of the cavity into the agarose. When the electric field is applied the DNA, which has an overall negative charge, is drawn towards the positively charged anode. Undamaged DNA strands are too large and do not leave the cavity, whereas the smaller the fragments, the farther they are free to move in a given period of time. Therefore, the amount of DNA that leaves the cavity is a measure of the amount of DNA damage in the cell.

The image analysis measures the overall intensity of the fluorescence for the whole nucleoid and the fluorescence of the migrated DNA and compares the two signals. The stronger the signal from the migrated DNA the more damage there is present. The overall structure resembles a comet (hence "comet assay") with a circular head corresponding to the undamaged DNA that remains in the cavity and a tail of damaged DNA. The brighter and longer the tail, the higher the level of damage.

The comet assay is a versatile technique for detecting damage and with adjustments to the protocol can be used to quantify the presence of a wide variety of DNA altering lesions (damage). The damage usually detected are single strand breaks and double strand breaks. It is sometimes stated that alkaline conditions and complete denaturating of the DNA is necessary to detect single strand breaks. However this is not true, both single- and double strand breaks are also detected in neutral conditions. In alkaline conditions, however, additional DNA structures are detected as DNA damage: AP sites (abasic sites missing either a pyrimidine or purine nucleotide) and sites where excision repair is taking place.

The comet assay is an extremely sensitive DNA damage assay. This sensitivity needs to be handled carefully as it is also vulnerable to physical changes which can affect the reproducibility of results. Essentially, anything that can cause DNA damage or denaturation except the factor(s) being researched is to be avoided. The most common form of the assay is the alkaline version although there is as yet no definitive alkaline assay protocol. Due to its simple and inexpensive setup, it can be used in conditions where more complex assays are not available.

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