Sensitive Assay for Mitochondrial DNA

Background: The mitochondrial DNA polymerase g is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activ-ity of DNA polymerase g may lead to mitochondrial DNA depletion. We describe a sensitive and robust clinical assay for this enzyme. Methods: The assay was performed on mitochondria isolated from skeletal muscle biopsies. High-molecular weight polynucleotide reaction products were captured on ion-exchange paper, examined qualitatively by auto-radiography, and quantified by scintillation counting. Results: Kinetic analysis of DNA polymerase g by this method showed a Km for dTTP of 1.43 mmol/L and a Ki for azidothymidine triphosphate of 0.861 mmol/L. The assay was linear from 0.1 to 2 mg of mitochondria