Measuring mtDNA turnover, synthesis, and supercoiling via selective bromodeoxyuridine incorporation

Abstract

An often-overlooked aspect of mitochondrial biology is the mitochondrial DNA (mtDNA). The multi-copy mtDNA is highly dynamic, with changes in supercoiling, synthesis rates, and turnover rates that are tightly associated with mitochondrial and cellular functions. To better understand the state of mtDNA, here we describe a protocol that selectively incorporates bromodeoxyuridine into mtDNA for subsequent measurement via an adapted Southern blot followed by immunoblotting (a.k.a. Southwestern blot). This basic protocol can be applied with slight modifications for the measurement of mtDNA synthesis, turnover or supercoiling to understand mtDNA changes.