Purification of mitochondrial DNA from total cellular DNA of small tissue samples.
A method is presented for the isolation of highly purified mitochondrial (mt)DNA from a crude DNA extract, making use of the different mobilities of covalently closed circular mtDNA vs. endonuclease-digested nuclear DNA in agarose gels. The preparation is virtually free of any contaminating linear DNA, as judged from its electron microscopic appearance, and can be used for further procedures such as polymerase chain reaction (PCR). Since isolation of mitochondria is not a prerequisite for this method, it can be applied to tissue samples in the mg range. In principle, the method can be applied to every eukaryotic species, provided a molecular hybridization probe is available which permits the position of mtDNA to be located in an agarose gel. This probe can be a cDNA, a DNA fragment generated by PCR, or mtDNA itself, if only the approximate size of the genome is known.
Wiesner
R J
RJ
Department of Medicine, University of Chicago, IL 60637.
Swift
H
H
Zak
R
R
D000818:Animals; D015139:Blotting, Southern; D004247:DNA / Q000302:isolation & purification*; D015342:DNA Probes; D004272:DNA, Mitochondrial / Q000302:isolation & purification* / Q000648:ultrastructure; D015252:Deoxyribonucleases, Type II Site-Specific; D006801:Humans; D008854:Microscopy, Electron; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D051381:Rats
D015342:DNA Probes; D004272:DNA, Mitochondrial; D004247:DNA; C024408:endodeoxyribonuclease SacI; D015252:Deoxyribonucleases, Type II Site-Specific