Measuring the Effects of Nucleoside Analogs on Mitochondrial DNA Content Using the Polymerase Chain Reaction

A sensitive method has been developed for analyzing mitochondrial (mt) DNA content in multiple species. Sequences specific for the mitochondrial cytochrome b gene (mtCytb) were selected as generic primers for use in the polymerase chain reaction (PCR), and were shown to amplify the expected 370-bp fragment in human, mouse, and rat. The specificity of the product was confirmed by hybridization analysis. Because of sequence diversity within the region defined by the primers, species-specific probes were synthesized to increase the sensitivity of detection. Because these mt sequences are not appreciably homologous to any nuclear chromosomal targets, total cellular DNA is a sufficient starting material for specific amplification. The relative amount of mtDNA from different tissues in mouse and rat assessed by PCR was in agreement with the histologic and biochemical characteristics of these tissues. The ability of the assay to measure previously described changes in mtDNA content in nucleoside analog-treated c...