Determination of the Ribonucleotide Content of mtDNA Using Alkaline Gels.
Alkaline gels
Alkaline hydrolysis
Denaturing gels
Ribonucleotides
Southern blot
rNMPs
Journal
Methods in molecular biology (Clifton, N.J.)
ISSN: 1940-6029
Titre abrégé: Methods Mol Biol
Pays: United States
ID NLM: 9214969
Informations de publication
Date de publication:
2023
2023
Historique:
entrez:
22
2
2023
pubmed:
23
2
2023
medline:
25
2
2023
Statut:
ppublish
Résumé
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.
Identifiants
pubmed: 36807800
doi: 10.1007/978-1-0716-2922-2_21
doi:
Substances chimiques
DNA, Mitochondrial
0
Ribonucleotides
0
Nucleotides
0
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
293-314Informations de copyright
© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.
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