Sensitive assay for mitochondrial DNA polymerase gamma.
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 K(m) for dTTP of 1.43 micromol/L and a K(i) for azidothymidine triphosphate of 0.861 micromol/L. The assay was linear from 0.1 to 2 microgram 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/microgram, reflecting extensive biologic variation among patients with neuromuscular disease.


CONCLUSIONS
This assay provides a useful adjunct to current laboratory methods for the evaluation of patients with suspected mitochondrial DNA depletion syndromes.
Naviaux
R K
RK
The Mitochondrial and Metabolic Disease Center, University of California, San Diego, USA. naviaux@ucsd.edu
Markusic
D
D
Barshop
B A
BA
Nyhan
W L
WL
Haas
R H
RH
D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002852:Chromatography, Ion Exchange; D002854:Chromatography, Paper; D000074002:DNA Polymerase gamma; D004272:DNA, Mitochondrial / Q000032:analysis*; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D006801:Humans; D008929:Mitochondria, Heart / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D004272:DNA, Mitochondrial; D000074002:DNA Polymerase gamma; D004259:DNA-Directed DNA Polymerase