Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells

Andrew M Shaw; Payam A Gammage

doi:10.1007/978-1-0716-2922-2_3

Coupling Differential Centrifugation with Exonuclease Treatment and Size Exclusion Chromatography (DIFSEC) for Purification of mtDNA from Mammalian Cells

Methods Mol Biol. 2023:2615:31-40. doi: 10.1007/978-1-0716-2922-2_3.

Authors

Andrew M Shaw¹, Payam A Gammage^{2

3}

Affiliations

¹ CRUK Beatson Institute, Glasgow, UK.
² CRUK Beatson Institute, Glasgow, UK. payam.gammage@glasgow.ac.uk.
³ Institute of Cancer Sciences, University of Glasgow, Glasgow, UK. payam.gammage@glasgow.ac.uk.

PMID: 36807782
DOI: 10.1007/978-1-0716-2922-2_3

Abstract

Direct analysis of mtDNA using PCR-free methods is limited by the presence of persistent, contaminating nucleic acids originating from the nuclear genome, even following stringent mitochondrial isolations. Here we describe a method developed in our laboratory that couples existing, commercially available mtDNA isolation protocols with exonuclease treatment and size exclusion chromatography (DIFSEC). This protocol produces highly enriched mtDNA extracts from small-scale cell culture, with near-undetectable nuclear DNA contamination.

Keywords: DNA; Gel filtration; Purification; Size exclusion chromatography; mtDNA.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Animals
Centrifugation
Chromatography, Gel
DNA, Mitochondrial* / genetics
Mammals / genetics
Mitochondria* / genetics
Polymerase Chain Reaction

Substances

DNA, Mitochondrial