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Analytical Chemistry 2011, Vol. 83 (22) :8604 -8610 doi:10.1021/ac202028g <<-上一篇 下一篇 ->>
High-Throughput Droplet Digital PCR System for Absolute Quantitation of;DNA Copy Number
Hindson, Benjamin J.;Ness, Kevin D.;Masquelier, Donald A.;Belgrader, Phillip;Heredia, Nicholas J.;Makarewicz, Anthony J.;Bright, Isaac J.;Lucero, Michael Y.;Hiddessen, Amy L.;Legler, Tina C.;Kitano, Tyler K.;Hodel, Michael R.;Petersen, Jonathan F.;Wyatt, Paul W.;Steenblock, Erin R.;Shah, Pallavi H.;Bousse, Luc J.;Troup, Camille B.;Mellen, Jeffrey C.;Wittmann, Dean K.;Erndt, Nicholas G.;Cauley, Thomas H.;Koehler, Ryan T.;So, Austin P.;Dube, Simant;Rose, Klint A.;Montesclaros, Luz;Wang, Shenglong;Stumbo, David P.;Hodges, Shawn P.;Romine, Steven;Milanovich, Fred P.;White, Helen E.;Regan, John F.;Karlin-Neumann, George A.;Hindson, Christopher M.;Saxonov, Serge;Colston, Bill W.
[Hindson, Benjamin J.; Ness, Kevin D.; Masquelier, Donald A.; Belgrader, Phillip; Heredia, Nicholas J.; Makarewicz, Anthony J.; Bright, Isaac J.; Lucero, Michael Y.; Hiddessen, Amy L.; Legler, Tina C.; Kitano, Tyler K.; Hodel, Michael R.; Petersen, Jonathan F.; Wyatt, Paul W.; Steenblock, Erin R.; Shah, Pallavi H.; Bousse, Luc J.; Troup, Camille B.; Mellen, Jeffrey C.; Wittmann, Dean K.; Erndt, Nicholas G.; Cauley, Thomas H.; Koehler, Ryan T.; So, Austin P.; Dube, Simant; Rose, Klint A.; Montesclaros, Luz; Wang, Shenglong; Stumbo, David P.; Hodges, Shawn P.; Romine, Steven; Milanovich, Fred P.; Regan, John F.; Karlin-Neumann, George A.; Hindson, Christopher M.; Saxonov, Serge; Colston, Bill W.] Biorad Labs Inc, Pleasanton, CA 94566 USA.;[White, Helen E.] Salisbury Dist Hosp, Natl Genet Reference Lab, Salisbury SP2 8BJ, Wilts, England.
Abstract: Digital PCR enables the absolute quantitation of nucleic acids in a sample. The lack of scalable and practical technologies for digital PCR implementation has hampered the widespread adoption of this inherently powerful technique. Here we describe a high-throughput droplet digital PCR (ddPCR) system that enables processing of similar to 2 million PCR reactions using conventional TaqMan assays with a 96-well plate workflow. Three applications demonstrate that the massive partitioning afforded by our ddPCR system provides orders of magnitude more p:recision and sensitivity than real-time PCR. First, we show the accurate measurement of germline copy number variation. Second, for rare alleles, we show sensitive detection of mutant DNA in a 100000-fold excess of wildtype background. Third, we demonstrate absolute quantitation of circulating fetal and maternal DNA from cell-free plasma. We anticipate this ddPCR system will allow researchers to explore complex genetic landscapes, discover and validate new disease associations, and define a new era of molecular diagnostics.
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