Analysis of mitochondrial DNA by two-dimensional agarose gel electrophoresis.

TitleAnalysis of mitochondrial DNA by two-dimensional agarose gel electrophoresis.
Publication TypeJournal Article
Year of Publication2009
AuthorsReyes, A, Yasukawa, T, Cluett, TJ, Holt, IJ
JournalMethods Mol Biol
Volume554
Pagination15-35
Date Published2009
ISSN1064-3745
KeywordsAnimals, Blotting, Southern, Cells, Cultured, DNA Replication, DNA Restriction Enzymes, DNA, Mitochondrial, Electrophoresis, Agar Gel, Electrophoresis, Gel, Two-Dimensional, Mice, Mitochondria, Liver
Abstract

In higher vertebrates, the DNA of mitochondria takes the form of circular molecules of approximately 16 kbp. These circles are arranged in multigenomic nucleoprotein complexes or nucleoids. It is envisaged that nucleoid superstructure makes a critical contribution to the twin processes of replication and segregation of mtDNA. Replication intermediates can be isolated from cells or solid tissues and separated on agarose gels in two dimensions to reveal a wealth of data on mechanisms of DNA replication. Using this technique we have demonstrated that many molecules of replicating mtDNA have extensive regions of RNA: DNA hybrid in higher vertebrates. More recently, we have extracted mitochondrial nucleoprotein and analyzed it by the same method to derive information on the distribution of DNA-binding proteins on mitochondrial DNA. Here we describe the procedures used to isolate intact mitochondrial replication intermediates from liver and cultured cells of higher vertebrates and the process of separating DNA fragments on neutral two-dimensional agarose gels.

DOI10.1007/978-1-59745-521-3_2
Alternate JournalMethods Mol. Biol.
Citation Key10.1007/978-1-59745-521-3_2
PubMed ID19513665
Grant ListMC_U105663140 / / Medical Research Council / United Kingdom