Islam M. El-Garawani and Sobhy E. Hassab El-Nabi
Keywords: DNA staining, safer dye, ethidium bromide, acridine orange and micronucleus test
Abstract: Electrophoresis is a common technique used in identification, quantification and purification of nucleic acid fragments. Some troubleshooting faced us in detection of apoptosis through DNA fragmentation assay. In some samples; electrophoretic pattern of extracted DNA has a flam shape nearly like sever necrotic fragmentation. The new method was evaluated in this study to standardize the apoptotic fragmentation detection. The direct incubation of extracted DNA samples which have a flam shape with ethidium bromide (EtBr) and acridine orange (AO) gave better results than the ordinary method of gel staining in apoptotic bands detection which appeared at 180 bp and its multiples on 1.8% agarose gel. Moreover, due to the hazardous effect of EtBr handling, acridine orange was tested in parallel on the same nucleic acid samples as an alternative fluorescent staining dye. Additionally, a novel micronucleus test can be accomplished in parallel with viability investigation by fluorescent dual AO/EtBr method. The obtained results showed obvious micronucleated and other nuclear abnormalities observations. In conclusion, although acridine orange is less sensitive than ethidium bromide staining in case of agarose electrophoresis, its safety, low cost and metachromatic properties should be considered and advisably, acridine orange can be used as an alternative nucleic acids fluorescent dye with increased sensitivity for apoptosis detection using direct incubation method. Acridine orange can also be integrated for micronucleus technique.
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