Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions.
Mitochondrial DNA (mtDNA) mutations are a common cause of primary mitochondrial disorders, and have also been implicated in a broad collection of conditions, including aging, neurodegeneration, and cancer. Prevalent among these pathogenic variants are mtDNA deletions, which show a strong bias for the loss of sequence in the major arc between, but not including, the heavy and light strand origins of replication. Because individual mtDNA deletions can accumulate focally, occur with multiple mixed breakpoints, and in the presence of normal mtDNA sequences, methods that detect broad-spectrum mutations with enhanced sensitivity and limited costs have both research and clinical applications. In this study, we evaluated semi-quantitative and digital PCR-based methods of mtDNA deletion detection using double-stranded reference templates or biological samples. Our aim was to describe key experimental assay parameters that will enable the analysis of low levels or small differences in mtDNA deletion load during disease progression, with limited false-positive detection. We determined that the digital PCR method significantly improved mtDNA deletion detection sensitivity through absolute quantitation, improved precision and reduced assay standard error.
University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute, Pittsburgh, PA, 15261, United States.
Frances R
FR
Belmonte
University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute, Pittsburgh, PA, 15261, United States.
James L
JL
Martin
Departments of Pediatrics and Medicine, McMaster University Medical Center, Hamilton, ON, L8N 3Z5, Canada.
Kristin
K
Frescura
Department of Comparative Biomedical Sciences, Royal Veterinary College, London, UK.
Joana
J
Damas
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto, Porto, Portugal.
Filipe
F
Pereira
Departments of Pediatrics and Medicine, McMaster University Medical Center, Hamilton, ON, L8N 3Z5, Canada.
Mark A
MA
Tarnopolsky
University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute, Pittsburgh, PA, 15261, United States.
Brett A
BA
Kaufman
D002460:Cell Line; D004272:DNA, Mitochondrial / Q000235:genetics*; D006801:Humans; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017384:Sequence Deletion
D004272:DNA, Mitochondrial