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Simultaneous mapping and quantitation of ribonucleotides in human mitochondrial DNA

Journal article
Authors Katrin Kreisel
Martin K.M. Engqvist
Anders R Clausen
Published in Journal of Visualized Experiments
Volume 2017
ISSN 1940087X
Publication year 2017
Published at Institute of Biomedicine, Department of Medical Biochemistry and Cell Biology
Language en
Links dx.doi.org/10.3791/56551
https://www.jove.com/video/56551/si...
Keywords 5´-End-seq, DNA damage, DNA replication, Human mitochondrial DNA, HydEn-seq, Issue 129, Molecular biology, Next-generation sequencing, Quantitation and mapping of ribonucleotides in DNA
Subject categories Cell and Molecular Biology, Medical Genetics

Abstract

© 2017. Established approaches to estimate the number of ribonucleotides present in a genome are limited to the quantitation of incorporated ribonucleotides using short synthetic DNA fragments or plasmids as templates and then extrapolating the results to the whole genome. Alternatively, the number of ribonucleotides present in a genome may be estimated using alkaline gels or Southern blots. More recent in vivo approaches employ Next-generation sequencing allowing genome-wide mapping of ribonucleotides, providing the position and identity of embedded ribonucleotides. However, they do not allow quantitation of the number of ribonucleotides which are incorporated into a genome. Here we describe how to simultaneously map and quantitate the number of ribonucleotides which are incorporated into human mitochondrial DNA in vivo by Next-generation sequencing. We use highly intact DNA and introduce sequence specific double strand breaks by digesting it with an endonuclease, subsequently hydrolyzing incorporated ribonucleotides with alkali. The generated ends are ligated with adapters and these ends are sequenced on a Next-generation sequencing machine. The absolute number of ribonucleotides can be calculated as the number of reads outside the recognition site per average number of reads at the recognition site for the sequence specific endonuclease. This protocol may also be utilized to map and quantitate free nicks in DNA and allows adaption to map other DNA lesions that can be processed to 5´-OH ends or 5´-phosphate ends. Furthermore, this method can be applied to any organism, given that a suitable reference genome is available. This protocol therefore provides an important tool to study DNA replication, 5´-end processing, DNA damage, and DNA repair.

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