Mitochondrial DNA Structural Analysis

1 μg of isolated mtDNA was linearized with SacI, precipitated and dissolved in 10 mM Tris HCl pH 7.5. The DNA was hydrolyzed with 0.3 M NaOH at 55°C or digested with RNase H2 (New England Biolabs) at 37°C for 2 h. Samples were run on a 0.8% agarose alkaline gel (30 mM NaOH, 1 mM EDTA) at 25 V, 4°C for 20 h and blotted onto Hybond-N+ membrane (Amersham, GE Healthcare). Single-stranded probes were end-labelled with γ-³²P ATP using T4 polynucleotide kinase (Thermo Scientific) following the manufacturer’s protocol. Double-stranded probes were generated by labelling an approximately 500 bp PCR product with α-³²P dCTP using Prime-It II Random Primer Labeling kit (Agilent Technologies). Hybridization was for 16 h at 42°C for ssDNA probes and at 65°C for dsDNA probes. The membrane was exposed to a PhosphoImager screen and scanned in a Typhoon laser scanner (GE Healthcare). The radioactive intensity was quantified using ImageJ software and plotted on a distribution plot. The median size of alkali-treated products was determined from the distribution of the radioactivity intensity and related to the size marker that was run in parallel [7 (link)].