Modifications of Alkaline Microgel Electrophoresis for Sensitive Detection of DNA Damage

Abstract

The alkaline microgel electrophoresis technique was modified to achieve a substantial increase in sensitivity for the detection of radiation-induced DNA damage in human lymphocytes. This increased sensitivity was achieved through: (1) the addition of free radical scavengers to the electrophoresis solution to reduce DNA damage generated during alkaline unwinding and electrophoresis; (2) the modification of the electrophoresis unit to achieve a more uniform electric field; (3) the use of YOYO-1, a DNA dye, producing fluorescence 500-fold more intense than ethidium bromide; and (4) the introduction of an image analysis system for the quantitation of DNA migration. In addition to increasing sensitivity, these modifications have increased the speed with which observations can be quantified, and improved reproducibility from experiment to experiment. In human lymphocytes, these modifications have resulted in an increased sensitivity of several fold, allowing the detection of DNA damage in the range of 50 mGy. This increased sensitivity for the detection of DNA damage should extend the utility of this technique.

Comet Assay measurements: a perspective

“…4). Other dyes used are SYBR® green/gold (Tice et al 1998), AO, YOYO dye (Singh et al 1994) and propidium iodide. In addition, non-fluorescent staining, such as silver stain, has also been used by some workers (Kizilian et al 1999;Garcia et al 2007).…”

Measurements using the alkaline comet assay predict bladder cancer cell radiosensitivity

“…The alkaline comet assay (ACA) is a highly sensitive method for the assessment of SSBs and alkali labile sites (ALSs), and can readily detect levels of damage induced by clinically relevant doses of radiation (Singh et al, 1994;Singh, 1996). In a previous study of just three bladder cancer cell lines, an inverse correlation was obtained between clonogenic survival and mean tail moment (TM) for comet formation, suggesting that ACA could potentially be used to predict the radio response of the single cell lines (McKelvey-Martin et al, 1998).…”

HIV-1 Tat increases cell survival in response to cisplatin by stimulating Rad51 gene expression

“…The colonogenic potential of the cells was evaluated at 27 days after treatment by counting the number of colonies after staining with methylene blue (1% in ethanol). (e) Cisplatin-induced DNA damage in PC12 and PC12-Tat cells was tested by COMET assay (Green et al, 1994;Singh et al, 1994). Briefly, PC12 and PC12-Tat cells at various times after treatment with cisplatin (3 mg/ml) were spread on a slide precoated with 1% agarose type I-A.…”

“…To elucidate the underlying mechanism involved in Tat stimulation of DNA repair and/or Tat-mediated resistance against DNA damage, we examined the levels of Rad51 and Ku70, key proteins involved in homologous recombination, and nonhomologous end-joining, respectively (Singh et al, 1994;Featherstone and Jackson, 1999;Aguilera, 2001;Venkitaraman, 2002). Protein extracts from PC12 and PC12-Tat cells were prepared and analysed by Western blot after 24 and 48 h cisplatin treatment.…”

“…Next, we employed single-cell gel electrophoresis (SCGE), also known as COMET assay (Green et al, 1994;Singh et al, 1994), to assess DNA repair by homologous recombination in the Tat-producing cells. In this assay, propidium iodide stained DNA of cells with undamaged DNA appears as well-rounded fluorescent circles, while the nuclei of cells with damaged/ unrepaired DNA appear as comets with a highly fluorescent head and a tail.…”