Ultrasensitive detection of DNA damage by the combination of the comet and TUNEL assays.

Abstract

The assessment of cellular DNA damage is crucial in many areas of biology including immunology, developmental biology, aging, cancer, and environmental science. A variety of experimental techniques including alkaline sucrose gradient centrifugation, alkaline elution, nucleoid sedimentation, viscoelastic measurements of DNA, alkaline unwinding, and gel electrophoresis (, , , ) have been developed to detect DNA damage in large cell populations. However, these methods can only detect the average amount of DNA damage over millions of cells. These approaches do not reveal the heterogeneity of DNA damage within a sample; some cells may experience extensive damage whereas others may display no damage at all. Moreover, one might want to analyze the amount of DNA damage in variety of cell subpopulations within a sample. In more specialized applications (e.g., a specific target cell undergoing immunologic recognition and destruction) the extent of DNA damage in a particular cell may be required. Furthermore, there are some clinical experiments where the amount of sample is severely limited (e.g., biopsy material) and therefore large-scale assays are impossible. Thus, there are many biological circumstances that require the use of small cell samples. To extract DNA damage information from these samples, techniques that rely upon the evaluation of DNA damage at the level of single cells is required. The single cell gel electrophoresis (SCGE) or “comet” assay is the most widely applied method for the detection of DNA damage in single cells. In this approach damaged DNA in individual cells is electrophoresed away from a nucleus into an agarose gel followed by staining.