Automated method to quantify formation and repair of DNA strand breaks

DNA strand breaks are among the lesions introduced by ionizing radiation but also represent intermediate steps in several DNA repair pathways. Therefore quantification of DNA strand breaks in living cells is an important endpoint in the assessment of genotoxicity. One of the available methods, the FADU assay, is thought to be very sensitive, yet is in its original, manually-operated version (Birnboim HC & Jevcak JJ, 1981, Cancer Res 41: 1889–1892) technically very demanding and requires large numbers of cells. In order to render this assay more convenient and reproducible, to increase throughput, and to reduce the number of cells needed we established an automated version based on the use of a commercial laboratory robot and additional modifications of the assay protocol (Moreno-Villanueva et al., 2009, BMC Biotechnol 23: 9: 39). Main advantages of the present protocol are the following: agitation-free handling of the samples placed in 96-well microtiter plate to avoid shearing of the DNA; Precise dispensing of solutions to the overlaid at a controlled rate without any mixing; perfect temperature control of the samples at all times; full protection from light at all times; reduction of the number of cells required by more than 100-fold; and finally automated performance of all the steps except cell preparation and DNA-damaging treatment. Several compounds which are toxic by different mechanisms have been tested using this new technology such as Methyl methanesulfonate (MMS), N-methyl-N′-nitroN-nitrosoguanidine (MNNG), 4-Nitroquinoline 1-oxide (4NQO), 7,12-Dimethyl-benz[a] Anthracene (DMBA), Camptothecin, Etoposide, Vinblastine, Rotenone, p-Nitrophenol and Paracetamol among others (Moreno-Villanueva et al., 2011, Altex 28: 295–303). The FADU assay basically measures DNA strand breaks. To assess DNA repair, disappearance of DNA strand breaks in living cells over time after a standard damaging procedure is monitored. The present FADU assay version requires as few as 6x106 cells for a standard repair assay and should greatly facilitate analyses of DNA damage and repair in a variety of settings. L4.2