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

Aurelio Reyes, Takehiro Yasukawa, Tricia J. Cluett, Ian J. Holt

Research output: Chapter in Book/Report/Conference proceedingChapter

11 Citations (Scopus)

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.

Original languageEnglish
Title of host publicationMitochondrial DNA
Subtitle of host publicationMethos and Protocols
Pages15-35
Number of pages21
Volume554
DOIs
Publication statusPublished - 2009
Externally publishedYes

Publication series

NameMethods in molecular biology (Clifton, N.J.)
PublisherHumana Press
ISSN (Print)1064-3745

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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