Abstract
Single cell gel electrophoresis or comet assay enables the quantification of DNA damage such as single-strand or double-strand breaks on a single cell level. Here, we describe a variant of this method for the detection of ribonucleotides embedded in genomic DNA. Briefly, cells are embedded in agarose on a microscopic slide, lysed under high salt and alkaline conditions and then subjected to in situ treatment with E. coli RNase HII which nicks 5′ to a ribonucleotide within the context of a DNA duplex thereby converting genomic ribonucleotides into strand breaks. After unwinding of genomic DNA using a highly alkaline buffer, electrophoresis under mild alkaline conditions is performed resulting in formation of comets due to migration of fragmented DNA toward the anode. Following SYBR Gold staining comets can be visualized by fluorescence microscopy. In this setting, the length and the intensity of comets formed reflect the level of genomic ribonucleotides present in a given cell.
Barbara Kind and Christine Wolf are co-first authors.
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Acknowledgment
This work was supported by grants by the Deutsche Forschungsgemeinschaft (KFO 249; LE 1074/4-1 and LE 1074/4-2 to M.L.-K.; KI 1956/2-1 to B.K.) and the Bundesministerium für Forschung und Bildung (grants 02S8355 and 02NUK017D to M.C.C.; grant 02NUK036D to A.R.). B.K. is a recipient of a Maria Reiche fellowship of TU Dresden.
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Kind, B., Wolf, C., Engel, K., Rapp, A., Cristina Cardoso, M., Lee-Kirsch, M.A. (2018). Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides. In: Muzi-Falconi, M., Brown, G. (eds) Genome Instability. Methods in Molecular Biology, vol 1672. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7306-4_21
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DOI: https://doi.org/10.1007/978-1-4939-7306-4_21
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