CANLILARDA “TEK HÜCRE JEL ELEKTROFOREZ” YÖNTEMİ İLE DNA HASAR ANALİZİ (TEKNİK NOT): COMET ANALİZ YÖNTEMİ

Yıl 2010, Cilt: 14 Sayı: 2, 77 - 89, 21.02.2014

Murat Dikilitaş , Abdurrahim Koçyiğit

Öz

Comet analiz yöntemi olarak da bilinen tek hücre jel elektroforez yöntemi son yıllarda genişleyen uygulama alanı, güvenilirliği ve uygulaması kolay olması bakımından kimyasal ve
fiziksel etmenlerin canlılar üzerinde yol açtığı genotoksik ve sitotoksik etkilerin bir göstergesi olan DNA hasar seviyelerinin ölçülmesini sağlayan önemli bir metotdur. Bu metot, izole edilen DNA içeren tek hücrelerin DNA’larının lam üzerinde hazırlanan agar jel içerisinde elektroforetik ortamda yürütülmesi ve hasar seviyesine göre göç eden farklı yük ve molekül ağırlıklarına sahip DNA parçalarının DNA spesifik floresan boya ile boyandıktan sonra, floresan mikroskop altında değerlendirilmesi esasına dayanmaktadır. Metot, oksidatif stres, toksik ağır metaller, kimyasal ajanlar, ilaçlar ve ultraviole gibi çeşitli genotoksik ajanların DNA sarmalları üzerinde oluşturduğu tek veya çift zincir kırıklarını doğru, hassas, hızlı, ucuz ve az bir örnek hacmi kullanarak ölçen bir yöntem olup, tüm canlı hücreleri üzerinde yapılan çalışmalarda yaygın olarak kullanılmaktadır.

Anahtar Kelimeler

Alkali Tek Hücre Jel Elektroforezi, Comet Analizi, SCGE, DNA Hasarı, DNA Tamiri.

ANALYSIS OF DNA DAMAGE IN ORGANIZMS VIA “SINGLE CELL GEL ELECTROPHORESIS” (TECHNICAL NOTE): COMET ASSAY METHOD

Öz

Single cell gel electrophoresis known also as the comet assay is an important method which enables to measure DNA damage and the evaluation of mechanisms of genotoxic and cytotoxic
effects of physical and chemical agents on organisms. The method is reliable, easily applicable and has been widely used in recent years. This method depends on the isolated DNA of single cells running in agarose gel on a microscopy slide in an electrophoretic condition and the migration of damaged DNA fragments with different polarity and molecular weights and the evaluation of DNA fragments following the use of DNA specific fluorescence dyes under a microscopy. The assay method determines the single or double-strand breaks caused by the
agents such as oxidative stress, chemical agents, drugs, heavy metal toxicity and ultraviolet lights with fast, sensitive, reliable and cheap way as well as requiring the little amount of cell samples. The method is commonly applied in all living cells. 

Anahtar Kelimeler

The Alkaline Single Cell Gel Electrophoresis, Comet Assay, SCGE, DNA Damage, DNA Repair

Kaynakça

• Abas, Y., Touil, N., Kirsch-Volders, M., Angenon, G., Jacobs, M. ve Famelaer, I.D.H. 2007. Evaluation of UV damage at DNA level in Nicotiana plumbaginifolia protoplasts using single-cell gel electrophoresis. Plant Cell Tissue Organ Culture, 91: 145-154.
• Achary, V. M. M., Jena, S., Panda, K. K. ve Panda, B. B. 2008. Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicology and Environmental Safety, 70: 300–310.
• Angelis, K.J., Dusinska, M. ve Collins, A.R. 1999. Single cell gel electrophoresis: detection of DNA damage at different levels of sensitivity. Electrophoresis, 20:2133-2138.
• Bhanoori, M., Venkateswerlu, G., 1998. The alkaline single cell gel electrophoresis: a new test for assessing DNA single strand breaks in Neurospora crassa. Mutation Research, 405:29-34.
• De Pinto, M.C., Francis, D. ve De Gara, L. 1999. The redox state of the ascorbate- dehydroascorbate pair as a specific sensor of cell division in tobacco BY-2 cells. Protoplasma, 209: 90-97.
• Dhawan, A., Bajpayee, M. ve Parmar, D. 2009. Comet assay: a reliable tool for the assessment of DNA damage in different models. Cell Biolology Toxicology, 25:5–32.
• Dikilitas, M., Kocyigit, A. ve Yigit, F. 2009. A molecular-based fast method to determine the extent of DNA damages in higher plants and fungi. African Journal of Biotechnology, 8 (14): 3118-3127.
• Dikilitas, M. 2003. Effect of salinity & its interactions with Verticillium albo-atrum on the disease development in tomato (Lycopersicon esculentum Mill.) and lucerne (Medicago sativa & M. media) plants. Ph.D. Thesis, University of Wales, Swansea
• Ecobichon, D.J. 2001. Toxic effects of pesticides. In: Klaassen CD(ed.) Casarett and Doull’s toxicology. The basic science of poisons. McGraw-Hill, New York, pp 763-810.
• Gichner, T. ve Plewa, M.J. 1998. Induction of somatic DNA damage as measured by single cell gel electrophoresis and point mutation in leaves of tobacco plants. Mutation Research, 401:143-152.
• Gichner, T., Patkova, Z., Szakova, J., Demnerova, K. 2004. Cadmium induces DNA damage in tobacco roots, but no DNA damage, somatic mutations or homologous recombinations in tobacco leaves. Mutation Research, 559:49-57.
• Gichner, T., Mukherjee, A. ve Veleminsky, J. 2006. DNA staining with the fluorochromes EtBr, DAPI and YOYO-1 in the comet assay with tobacco plants after treatment with ethyl methanesulphonate, hyperthermia and DNase- I. Mutation Research, 605:17-21.
• Gichner, T., Znidar, I. ve Szakova, J. 2008a. Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants. Mutation Research, 652:186-190.
• Gichner, T., Patkova, Z., Szakova, J., Znidar, I. ve Mukherjee, A. 2008b. DNA damage in potato plants induced by cadmium, ethyl methanesulfonate and γ-rays. Environmental and Experimental Botany, 62: 113-119.
• Gichner, T., Znidar, I., Wagner, E.D. ve Plewa, M.J. 2009. The use of higher plants in the comet assay. In: The Comet Assay in Toxicology Edited by Alok Dhawan and Diana Anderson. Royal Society of Chemistry, p: 98- 119.
• Hahn, A. ve Hock, B. 1999. Assessment of DNA damage in filamentous fungi by single cell gel electrophoresis, comet assay. Environmental Toxicology and Chemistry, 18 (7): 1411-1424.
• Kocyigit, A., Keles, H., Selek, S., Guzel, S., Celik, H. ve Erel, O. 2005. Increased DNA damage and oxidative stress in patients with Cutaneous leishmaniasis. Mutation Research, 585:71-78.
• Konca, K., Lankoff, A., Banasik, A., Lisowska, H., Kuszewski, T., Gozdz S, Koza, Z. ve Wojcik, A. 2003. A cross-platform public domain PC image-analysis program for the comet assay. Mutation Research 534: 15-20.
• Lin, A., Zhang, X., Chen, M., Cao, Q. 2007. Oxidative stress and DNA damages induced by cadmium accumulation. Journal of Environmental Science, 19:596-602.
• Lin, A., Zhang, X., Zhu, Y.G. ve Zhao, F.J. 2008. Arsenate-induced toxicity: effects on antioxidative enzymes and DNA damage inVicia faba. Environmental Toxicology and Chemistry 27 (2): 413-419.
• Mancini, A., Buschini, A., Restivo, F.M., Rossi, C. ve Poli, P. 2006. Oxidative stress as DNA damage in different transgenic tobacco plants. Plant Science 170:845-852.
• Miloshev, G., Mihaylov, I. ve Anachkova, B. 2002. Application of the single cell gel electrophoresis on yeast cells. Mutation Research, 513: 69-74.
• Mohan Murali Achary, V., Jena, S., Panda, K.K. ve Panda, B.B. 2008. Aluminium induced oxidative stress and DNA damage in root cells of Allium cepa L. Ecotoxicology Environmental Safety, 70: 300-310.
• Östling, O., Johanson, K.J. 1984. Microelectrophoretic study of radiation- induced DNA damages in individual mammalian cells. Biochem. Biophys. Res. Commun, 123:291-298.
• Piperakis, S.M., Petrakou, E., Tsilimigaki, S., Sagnou, M., Monogiudis, E., Haniotakis, G., Karkaseli, H. ve Sarikaki, E. 2003. Biomonitoring with the comet assay of Greek greenhouse workers exposed to pesticides. Environmental Molecular Mutagenesis, 41:104-110.
• Rank, J., Syberg, K., ve Jensen, K. 2009. Comet assay on tetraploid yeast cells. Mutation Research 673: 53-58.
• Singh, N.P., McCoy, M.T., Tice, R.R. ve Schneider, E.L. 1988. A simple technique for quantitation of low levels of DNA damage in individual cells. Experimental Cell Research, 175:184-91.
• Sriussadaporn, C., Yamamoto, K., Fukushi, K. ve Simazaki, D. 2003. Comparison of DNA damage detected by plant comet assay in roadside and non-roadside environments. Mutation Research, 541:31-44.
• Stavreva, D.A. ve Gichner, T. 2002. DNA damage induced by hydrogen peroxide in cultured tobacco cells is dependent on the cell growth stage. Mutation Research, 514:147-152.
• Tatli, M.M., Minnet, C., Kocyigit, A., Karadag, A. 2008. Phototherapy increases DNA damage in lymphocytes of hyperbbilirubinemic nonates. Mutation Research, 654 (1): 93-95.
• Tice, R.R., Agurell, E., Anderson, D., Burlinson, B., Hartmann, A., Kobayashi, H. 2000. The single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environmental Molecular Mutagenesis, 35:206-21.
• Trevigen Inc. 2000. TREVIGEN TM Instructions for Comet Assay TM .
• Ündeğer, Ü. ve Başaran, N. 2005. Effects of pesticides on human peripheral lymphocytes in vitro: induction of DNA damage. Archives of Toxicology 79: 169-176.
• Vajpayee, P., Dhawan, A. ve Shanker, R. 2006. Evaluation of the alkaline comet assay conducted with the wetlands plant Bacopa monnieri L. as a model for ecogenotoxicity assessment. Environmental and Molecular Mutagenesis 47: 483-489.
• WHO 1986. Environmental Health Criteria 64. Carbamate pesticides: a general introduction. World Health Organisation, Geneva.
• WHO 1988. The WHO recommended classification of pesticides by hazard and guidelines to the classification 1988-1989. World Health Organisation, Geneva.
• www.sigmaaldrich.com. 2010. Use of trypan blue stain and the hemocytometer to determine total cell counts and viable cell number.