Diagnostic assays for defects in mitochondrial DNA replication and transcription in yeast and human cells

Abstract

Publisher Summary This chapter describes several simple assays employed for the determination of relative mitochondrial DNA (mtDNA) copy number (as a measure of mtDNA replication and stability) and steady-state levels of mitochondrial transcripts (as a measure of an intact mitochondrial transcription apparatus) in human and yeast cells. These diagnostic assays allow potential defects in mtDNA replication and transcription to be identified in a time- and labor-efficient manner. Once a defect of this type is postulated based on the preliminary assays described, the use of more intensive in vitro and in organello methods are discussed addressing the molecular nature of the defect more precisely. The chapter also outlines assays in human and yeast cells; the basic methodology should be applicable to any mitochondrial system where genome sequence information is available. The assays described in the chapter provide an initial assessment of whether mtDNA replication or mitochondrial transcription is occurring normally in human or yeast cells.