To independently determine the impact of circular structure on methylation analysis of mtDNA, intracellular DNA was extracted using phenol-chloroform from a mouse neuroblastoma cell line Neuro-2a (N2a) and then digested with two restriction endonucleases, SacI and NcoI. These endonucleases target adjacent sites in the mouse mtDNA (Fig. 2A). The gene discussed is ADCY10; the disease is neuroblastoma.