Method of isolating pure mitochondrial DNA

A method for preparing circular double stranded mitochondrial DNA (mtDNA) substantially free of linear genomic DNA (gDNA) comprising the steps of:

• a) providing a solution comprising circular, double stranded mitochondrial DNA (mtDNA) and linear genomic DNA (gDNA) purified from a mammalian cell having linear genomic DNA of length greater than a billion base pairs and circular double stranded mtDNA of length less than about 30,000 base pairs;
• b) incubating the solution with an amount of an Exonuclease selected from the group consisting of Hind Exonuclease V, T5 exonuclease, and Exonuclease V for a time and at a concentration effective to cleave linear genomic DNA and obtain circular, double stranded mtDNA;
• c) incubating said circular double stranded mtDNA with an amount of carboxylated solid phase reversible immobilization magnetic beads effective to bind said circular double stranded mtDNA;
• d) washing said beads with ethanol;
• e) eluting said circular double stranded mtDNA from said beads,
• wherein said method is free of ultra-centrifugation and provides circular double stranded mtDNA from mammalian cells that is at least 90% free of linear genomic DNA;
• and
• f) evaluating the purity of the circular double stranded mtDNA by amplifying the genomic and mt DNA using genomic and mitochondrial specific primers.

A method for preparing circular double stranded mitochondrial DNA substantially free of linear genomic DNA (gDNA) comprising the steps of:

• a) providing a cellular lysate by lysing mammalian cells having about 6 billion base pairs of linear genomic DNA and about 16,000 base pairs of circular double stranded mt DNA and free of protein and RNA contaminants;
• b) precipitating cellular debris and proteins out of said lysate and obtaining a solution comprising purified circular double stranded mitochondrial DNA (mtDNA) and linear genomic DNA (gDNA);
• c) incubating said solution with an amount of an Exonuclease, selected from the group consisting of Hind Exonuclease V, T5 exonuclease, and Exonuclease V for a time and at a temperature effective to cleave linear genomic DNA and obtain circular double stranded mtDNA;
• d) incubating said circular double stranded mtDNA with an amount of carboxylated solid phase reversible immobilization magnetic beads effective to bind said circular double stranded mtDNA;
• e) washing said beads with ethanol,
• f) eluting said mtDNA from said beads, and
• g) evaluating the purity of the mtDNA by amplifying the linear genomic and circular double stranded mt DNA using genomic and mitochondrial specific primers,
• wherein said method is free of ultra-centrifugation and provides mtDNA from the mammalian cells that is at least 90% free of linear gDNA.

A method for preparing circular double stranded DNA substantially free of linear DNA comprising the steps of:

• a) providing a lysate from mammalian cells having about 6 billion base pairs of linear DNA and about 16,000 base pairs of circular, double-stranded DNA that is free of protein and RNA contaminants;
• b) incubating said lysate with an amount of an Exonuclease, selected from the group consisting of Hind Exonuclease V, T5 exonuclease, and Exonuclease V for a time and at temperature effective to cleave linear DNA and obtain circular double stranded DNA;
• c) incubating said circular double stranded DNA with an amount of carboxylated solid phase reversible immobilization magnetic beads effective to bind said circular double stranded DNA;
• d) washing said beads with ethanol, and
• e) eluting said circular double stranded DNA from said beads,
• wherein said method is free of ultra-centrifugation and provides circular double stranded DNA from mammalian cells that is at least 80% free of linear DNA.