Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions
Identifieur interne : 000387 ( Main/Exploration ); précédent : 000386; suivant : 000388Digital PCR methods improve detection sensitivity and measurement precision of low abundance mtDNA deletions
Auteurs : Frances R. Belmonte [États-Unis] ; James L. Martin [États-Unis] ; Kristin Frescura [Canada] ; Joana Damas [Royaume-Uni] ; Filipe Pereira [Portugal] ; Mark A. Tarnopolsky [Canada] ; Brett A. Kaufman [États-Unis]Source :
- Scientific Reports [ 2045-2322 ] ; 2016.
Abstract
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.
Url:
DOI: 10.1038/srep25186
PubMed: 27122135
PubMed Central: 4848546
Affiliations:
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Belmonte</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute</institution>, Pittsburgh, PA, 15261,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Martin, James L" sort="Martin, James L" uniqKey="Martin J" first="James L." last="Martin">James L. Martin</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute</institution>, Pittsburgh, PA, 15261,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Frescura, Kristin" sort="Frescura, Kristin" uniqKey="Frescura K" first="Kristin" last="Frescura">Kristin Frescura</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Departments of Pediatrics and Medicine, McMaster University Medical Center</institution>, Hamilton, ON, L8N 3Z5,<country>Canada</country></nlm:aff><country xml:lang="fr">Canada</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Damas, Joana" sort="Damas, Joana" uniqKey="Damas J" first="Joana" last="Damas">Joana Damas</name><affiliation wicri:level="1"><nlm:aff id="a3"><institution>Department of Comparative Biomedical Sciences, Royal Veterinary College</institution>, London,<country>UK</country></nlm:aff><country xml:lang="fr">Royaume-Uni</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Pereira, Filipe" sort="Pereira, Filipe" uniqKey="Pereira F" first="Filipe" last="Pereira">Filipe Pereira</name><affiliation wicri:level="1"><nlm:aff id="a4"><institution>Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), University of Porto</institution>, Porto,<country>Portugal</country></nlm:aff><country xml:lang="fr">Portugal</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Tarnopolsky, Mark A" sort="Tarnopolsky, Mark A" uniqKey="Tarnopolsky M" first="Mark A." last="Tarnopolsky">Mark A. Tarnopolsky</name><affiliation wicri:level="1"><nlm:aff id="a2"><institution>Departments of Pediatrics and Medicine, McMaster University Medical Center</institution>, Hamilton, ON, L8N 3Z5,<country>Canada</country></nlm:aff><country xml:lang="fr">Canada</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author><author><name sortKey="Kaufman, Brett A" sort="Kaufman, Brett A" uniqKey="Kaufman B" first="Brett A." last="Kaufman">Brett A. Kaufman</name><affiliation wicri:level="1"><nlm:aff id="a1"><institution>University of Pittsburgh School of Medicine, Division of Cardiology, Center for Metabolism and Mitochondrial Medicine and Vascular Medicine Institute</institution>, Pittsburgh, PA, 15261,<country>United States</country></nlm:aff><country xml:lang="fr">États-Unis</country><wicri:regionArea># see nlm:aff country strict</wicri:regionArea></affiliation></author></analytic><series><title level="j">Scientific Reports</title><idno type="eISSN">2045-2322</idno><imprint><date when="2016">2016</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>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.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Schon, E A" uniqKey="Schon E">E. a. Schon</name></author><author><name sortKey="Dimauro, S" uniqKey="Dimauro S">S. DiMauro</name></author><author><name sortKey="Hirano, M" uniqKey="Hirano M">M. Hirano</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Barthelemy, C" uniqKey="Barthelemy C">C. Barthélémy</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Villarroya, J" uniqKey="Villarroya J">J. Villarroya</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Taylor, R W" uniqKey="Taylor R">R. W. Taylor</name></author><author><name sortKey="Turnbull, D M" uniqKey="Turnbull D">D. M. 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DiMauro</name></author><author><name sortKey="Moraes, C T" uniqKey="Moraes C">C. T. Moraes</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Chan, S S L" uniqKey="Chan S">S. S. L. Chan</name></author><author><name sortKey="Copeland, W C" uniqKey="Copeland W">W. C. Copeland</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Nesbitt, V" uniqKey="Nesbitt V">V. Nesbitt</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Greaves, L C" uniqKey="Greaves L">L. C. Greaves</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Tang, Y" uniqKey="Tang Y">Y. Tang</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Kolesar, J E" uniqKey="Kolesar J">J. E. Kolesar</name></author><author><name sortKey="Wang, C Y" uniqKey="Wang C">C. Y. Wang</name></author><author><name sortKey="Taguchi, Y V" uniqKey="Taguchi Y">Y. V. Taguchi</name></author><author><name sortKey="Chou, S H" uniqKey="Chou S">S. H. Chou</name></author><author><name sortKey="Kaufman, B A" uniqKey="Kaufman B">B. A. Kaufman</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Grady, J P" uniqKey="Grady J">J. P. Grady</name></author></analytic></biblStruct></listBibl></div1></back></TEI><affiliations><list><country><li>Canada</li><li>Portugal</li><li>Royaume-Uni</li><li>États-Unis</li></country></list><tree><country name="États-Unis"><noRegion><name sortKey="Belmonte, Frances R" sort="Belmonte, Frances R" uniqKey="Belmonte F" first="Frances R." last="Belmonte">Frances R. Belmonte</name></noRegion><name sortKey="Kaufman, Brett A" sort="Kaufman, Brett A" uniqKey="Kaufman B" first="Brett A." last="Kaufman">Brett A. Kaufman</name><name sortKey="Martin, James L" sort="Martin, James L" uniqKey="Martin J" first="James L." last="Martin">James L. Martin</name></country><country name="Canada"><noRegion><name sortKey="Frescura, Kristin" sort="Frescura, Kristin" uniqKey="Frescura K" first="Kristin" last="Frescura">Kristin Frescura</name></noRegion><name sortKey="Tarnopolsky, Mark A" sort="Tarnopolsky, Mark A" uniqKey="Tarnopolsky M" first="Mark A." last="Tarnopolsky">Mark A. Tarnopolsky</name></country><country name="Royaume-Uni"><noRegion><name sortKey="Damas, Joana" sort="Damas, Joana" uniqKey="Damas J" first="Joana" last="Damas">Joana Damas</name></noRegion></country><country name="Portugal"><noRegion><name sortKey="Pereira, Filipe" sort="Pereira, Filipe" uniqKey="Pereira F" first="Filipe" last="Pereira">Filipe Pereira</name></noRegion></country></tree></affiliations></record>Pour manipuler ce document sous Unix (Dilib)
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