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Determination of the ribonucleotide content of mtDNA using alkaline gels
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0001-5704-3093
Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics.ORCID iD: 0000-0002-8607-7564
2023 (English)In: Mitochondrial DNA: methods and protocols / [ed] Thomas J. Nicholls; Jay P. Uhler; Maria Falkenberg, New York: Humana Press, 2023, Vol. 2615, p. 293-314Chapter in book (Refereed)
Abstract [en]

Impaired mitochondrial DNA (mtDNA) maintenance, due to, e.g., defects in the replication machinery or an insufficient dNTP supply, underlies a number of mitochondrial disorders. The normal process of mtDNA replication leads to the incorporation of multiple single ribonucleotides (rNMPs) per mtDNA molecule. Given that embedded rNMPs alter the stability and properties of the DNA, they may have consequences for mtDNA maintenance and thereby for mitochondrial disease. They also serve as a readout of the intramitochondrial NTP/dNTP ratios. In this chapter, we describe a method for the determination of mtDNA rNMP content using alkaline gel electrophoresis and Southern blotting. This procedure is suited for the analysis of mtDNA in total genomic DNA preparations as well as in purified form. Moreover, it can be performed using equipment found in most biomedical laboratories, allows the simultaneous analysis of 10-20 samples depending on the gel system employed, and can be modified for the analysis of other mtDNA modifications.

Place, publisher, year, edition, pages
New York: Humana Press, 2023. Vol. 2615, p. 293-314
Series
Methods in Molecular Biology, ISSN 1064-3745, E-ISSN 1940-6029 ; 2615
Keywords [en]
Alkaline gels, Alkaline hydrolysis, Denaturing gels, Ribonucleotides, rNMPs, Southern blot
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:umu:diva-205504DOI: 10.1007/978-1-0716-2922-2_21PubMedID: 36807800Scopus ID: 2-s2.0-85148677775ISBN: 9781071629215 (print)ISBN: 9781071629222 (electronic)OAI: oai:DiVA.org:umu-205504DiVA, id: diva2:1743462
Available from: 2023-03-15 Created: 2023-03-15 Last updated: 2023-07-11Bibliographically approved

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Gorospe, Choco MichaelRepolês, Bruno MarçalWanrooij, Paulina H.

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Gorospe, Choco MichaelRepolês, Bruno MarçalWanrooij, Paulina H.
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