Sensitive assay for mitochondrial DNA polymerase gamma

Background: The mitochondrial DNA polymerase gamma is the principal polymer ase required for mitochondrial DNA replication. Primary or secondary defici encies in the activity of DNA polymerase gamma may lead to mitochondrial DN A depletion. We describe a sensitive and robust clinical assay for this enz yme. Methods: The assay was performed on mitochondria isolated from skeletal mus cle biopsies. High-molecular weight polynucleotide reaction products were c aptured on ion-exchange paper, examined qualitatively by autoradiography, a nd quantified by scintillation counting. Results: Kinetic analysis of DNA polymerase gamma by this method showed a K -m for dTTP of 1.43 mu mol/L, and a K-i for azidothymidine triphosphate of 0.861 mu mol/L. The assay was linear from 0.1 to 2 mu g of mitochondrial pr otein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 mi n). The linear dynamic range was three orders of magnitude; 30-30 000 units . Imprecision (CV) was 6.4% within day and 12% between days. Application of this assay to a mixed population of 38 patients referred for evaluation of mitochondrial disease revealed a distribution with a range of 0-2506 U/mu g, reflecting extensive biologic variation among patients with neuromuscula r disease. Conclusion: This assay provides a useful adjunct to current laboratory meth ods for the evaluation of patients with suspected mitochondrial DNA depleti on syndromes. (C) 1999 American Association for Clinical Chemistry.