Differentiation of apurinic/apyrimidinic sites and single-strand breaks in DNA by formamide- and alkaline-sucrose gradient sedimentation |
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| Authors: | J Ross M S Tang |
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| Affiliation: | 1. Institute of Pharmaceutical Chemistry, Philipps-University Marburg, 35037 Marburg, Germany;2. Center for Anatomy & Cell Biology, Medical University of Vienna, 1090 Vienna, Austria;3. Chemistry Department and A.N. Belozersky Institute of Physico-Chemical Biology, M.V. Lomonosov Moscow State University, 119991 Moscow, Russia;4. Skolkovo Institute of Science and Technology, 3 Nobel street, Innovation Center “Skolkovo”, 143026 Skolkovo, Russia;1. Department of Cellular Biochemistry, University Medical Center Goettingen, Humboldtallee 23, D-37073 Goettingen, Germany;2. Research Group Structure and Function of Molecular Machines, Max Planck Institute for Multidisciplinary Sciences, Am Fassberg 11, D-37077 Goettingen, Germany;3. Cluster of Excellence Multiscale Bioimaging: from Molecular Machines to Networks of Excitable Cells (MBExC), University of Goettingen, D-37075 Goettingen, Germany;1. Department of Genetics, University of Georgia, Athens, GA 30605, USA;2. Department of Microbiology, University of Georgia, Athens, GA 30605, USA |
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| Abstract: | The excision repair of DNA damaged by physical or chemical agents may produce either apurinic/apyrimidinic (AP) sites or single-strand breaks (SSB) in the DNA. Alkaline-sucrose gradient sedimentation and alkaline elution, techniques generally used for the study of DNA repair which depend upon high pH to denature the DNA, cannot differentiate between these possibilities. A simple method for the quantitative measurement of SSB in DNA which leaves any AP sites intact is presented. This method relies upon the separation by size of the fragments resulting from the denaturation of the DNA under neutral conditions by sedimentation through gradients of sucrose in formamide. By combining the use of both formamide- and alkaline-sucrose sedimentation methods, we can quantify both AP sites and SSB in DNA. |
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