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Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides.

Kind, Barbara and Wolf, Christine and Engel, Kerstin and Rapp, Alexander and Cardoso, M. Cristina and Lee-Kirsch, Min Ae (2018):
Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides.
In: Methods in molecular biology (Clifton, N.J.), 1672, pp. 311-318. ISSN 1940-6029,
[Article]

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.

Item Type: Article
Erschienen: 2018
Creators: Kind, Barbara and Wolf, Christine and Engel, Kerstin and Rapp, Alexander and Cardoso, M. Cristina and Lee-Kirsch, Min Ae
Title: Single Cell Gel Electrophoresis for the Detection of Genomic Ribonucleotides.
Language: English
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.

Journal or Publication Title: Methods in molecular biology (Clifton, N.J.)
Journal volume: 1672
Divisions: 10 Department of Biology
10 Department of Biology > Cell Biology and Epigenetics
Date Deposited: 18 Sep 2018 12:14
Identification Number: pmid:29043632
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