2. Comet assay electrophoresis in principle

they were used only for a limited spectrum of cell types. The assay mostly used during the last three decades was first published by the Swedes Östling and Johanson in 1984 [1]; other versions followed, from N.P. Singh and R. Tice in 1988 [2], and Peggy Olive and co-workers [3]. These protocols are in principle quite similar but vary in their use of neutral or alkaline electrophoresis conditions and also in some other steps of the procedures, such as lysis cell conditions. The methods were initially given different names: microelectrophoresis [1], single cell gel electrophoresis [2], and Comet Assay [3]. The latter name—which we will mostly use in this chapter—stems from the comet-like image seen in micrographs of damaged cellular DNA

In the comet assay, cellular DNA embedded in thin layers of agarose (researchers choose different concentrations) are electrophoresed while placed in a horizontal electrophoresis tank. At this stage the cells are dead and devoid of most of their proteins—whether they originate from a live organism: human blood or a specific organ, an animal, a fish or a sea star, or a plant (there is in fact hardly no limitation)—or from cells or organ cultures treated with radiation, or

The comet assay has often been used simply to show the formation or presence of DNA damage in cells in a relative scale, without any reference to a standardized output or a calibration. However, in various applications, there is a need for standardization and calibration of comet assay data. Examples are when results from different experiments, cell types, and laboratories are to be compared, or when different human cohorts are combined in international collaborative studies. In the EU COST action hCOMET, the main aims are to create a unified database of comet assay data relating to human health and disease that will be the base for pooled analysis on DNA damage and repair in humans; the ultimate purpose is to investigate the significance of comet assay results as prognostic markers of disease. Such comparisons

Attempts have indeed been made to establish standardized protocols, and/or identifying the experimental steps that are most significant in determining the sensitivity and reproducibility of the comet assay. Validation studies and ring trials of various types have been described [4–9], involving, for example, distribution of treated cells to different laboratories followed by local comet assay analysis, or local treatment, and analysis of cells with chemical mutagens according to specific protocols. In spite of these attempts, no key factor explaining the observed rather large inter-laboratory differences has yet been identified [8]. It is concluded that there is hence still a

The separation of DNA fragments according to their size and structure—either single- or double-stranded—is in all versions of the comet assay based on electrophoresis at low-voltage (across the tank approximately 25 V) and short duration (20–30 mins). Cell isolation and manipulation, including long-time storage through freezing, are of uttermost importance for the final output measured by the assay; this concerns, in particular, the background level of DNA damage in "untreated" cells. We shall not deal with such issues here, but rather focus on

technical aspects of the electrophoresis, including some of its physics and chemistry.

do not make sense if the sensitivity of the assays used is very different.

as it stretches out after electrophoresis.

64 Electrophoresis - Life Sciences Practical Applications

need for further validation of the assay [5].

chemicals in vitro.

Analysis of charged molecules by means of an electric potential (or electric field) is a technique that has been known, understood and applied in experimental biology and biochemistry for decades. Electrophoresis separates charged molecules according to their mobility. The driving force is the electric potential; the mobility depends on the charge, size and shape of the molecules, and the movement is inhibited by viscous forces (depending on the medium including its pore size). Cellular DNA (in a diploid human cell) has a total size of approximately 2 \*10<sup>12</sup> Da, which in an intact cell is organized in 23 pairs of chromosomes. Under alkaline conditions, DNA from a lysed cell exists in stretches of single helixes, which may be of all sizes, either linear and free or more or less entangled with each other. When these negatively charged molecules are broken—either into discrete fragments or into a distribution of differently sized molecules—they are separated in size when subjected to an electric potential in a medium through which they have some freedom to move.

The charge per unit length of DNA is determined by its base composition, which is quite constant on a large scale. The movement (velocity per time) may be adjusted via the electric potential (V/cm) and the viscosity (related, primarily, to the agarose concentration), so that the separation of a distribution of differently sized DNA molecules may be optimized. This optimization may vary depending on the specific application of the assay. For most purposes, however, molecular sizes expected to be measurable in the comet assay would be in the range 2\*10<sup>8</sup> –10<sup>10</sup> Da (i.e., from a few hundred to ten thousand breaks per cell). The DNA distribution after electrophoresis is determined with fluorescent micrographic imaging. Using various algorithms, characteristic parameters have been worked out to characterize the population of differently sized molecules, to give the medium, the average and the distribution of the molecular weight or size (denoted scoring).
