Clinical Chemistry
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Clinical Chemistry 45: 1725-1733, 1999;
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(Clinical Chemistry. 1999;45:1725-1733.)
© 1999 American Association for Clinical Chemistry, Inc.

Articles

Sensitive Assay for Mitochondrial DNA Polymerase {gamma}

Robert K. Naviaux1,a, David Markusic2, Bruce A. Barshop3, William L. Nyhan3 and Richard H. Haas2,3

The Mitochondrial and Metabolic Disease Center, University of California, San Diego, School of Medicine, Departments of
1 Medicine,
2 Neurosciences, and
3 Pediatrics, 200 West Arbor Dr., San Diego, CA 92103-8467.
a Author for correspondence. Fax 619-543-7868; e-mail naviaux{at}ucsd.edu

Background: The mitochondrial DNA polymerase {gamma} is the principal polymerase required for mitochondrial DNA replication. Primary or secondary deficiencies in the activity of DNA polymerase {gamma} 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 autoradiography, and quantified by scintillation counting.

Results: Kinetic analysis of DNA polymerase {gamma} by this method showed a Km for dTTP of 1.43 µmol/L and a Ki for azidothymidine triphosphate of 0.861 µmol/L. The assay was linear from 0.1 to 2 µg of mitochondrial protein. The detection limit was 30 units (30 fmol dTMP incorporated in 30 min). 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/µg, reflecting extensive biologic variation among patients with neuromuscular disease.

Conclusion: This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.




The following articles in journals at HighWire Press have cited this article:


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 Annals of Clinical & Laboratory ScienceHome page
E. Fosslien
Mitochondrial Medicine - Molecular Pathology of Defective Oxidative Phosphorylation
Ann. Clin. Lab. Sci., January 1, 2001; 31(1): 25 - 67.
[Abstract] [Full Text] [PDF]


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