High-throughput droplet digital PCR system for absolute quantitation of DNA copy number
Benjamin J Hindson, Kevin D Ness, Donald A Masquelier, Phillip Belgrader, Nicholas J Heredia, Anthony J Makarewicz, Isaac J Bright, Michael Y Lucero, Amy L Hiddessen, Tina C Legler, Tyler K Kitano, Michael R Hodel, Jonathan F Petersen, Paul W Wyatt, Erin R Steenblock, Pallavi H Shah, Luc J Bousse, Camille B Troup, Jeffrey C Mellen, Dean K Wittmann, Nicholas G Erndt, Thomas H Cauley, Ryan T Koehler, Austin P So, Simant Dube, Klint A Rose, Luz Montesclaros, Shenglong Wang, David P Stumbo, Shawn P Hodges, Steven Romine, Fred P Milanovich, Helen E White, John F Regan, George A Karlin-Neumann, Christopher M Hindson, Serge Saxonov, Bill W Colston, Benjamin J Hindson, Kevin D Ness, Donald A Masquelier, Phillip Belgrader, Nicholas J Heredia, Anthony J Makarewicz, Isaac J Bright, Michael Y Lucero, Amy L Hiddessen, Tina C Legler, Tyler K Kitano, Michael R Hodel, Jonathan F Petersen, Paul W Wyatt, Erin R Steenblock, Pallavi H Shah, Luc J Bousse, Camille B Troup, Jeffrey C Mellen, Dean K Wittmann, Nicholas G Erndt, Thomas H Cauley, Ryan T Koehler, Austin P So, Simant Dube, Klint A Rose, Luz Montesclaros, Shenglong Wang, David P Stumbo, Shawn P Hodges, Steven Romine, Fred P Milanovich, Helen E White, John F Regan, George A Karlin-Neumann, Christopher M Hindson, Serge Saxonov, Bill W Colston
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 ~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 precision 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 100,000-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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References
- Sykes P. J.; Neoh S. H.; Brisco M. J.; Hughes E.; Condon J.; Morley A. A. Biotechniques 1992, 13, 444–449.
- Vogelstein B.; Kinzler K. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9236–9241.
- McCaughan F.; Dear P. H. J. Pathol. 2010, 220, 297–306.
- Dube S.; Qin J.; Ramakrishnan R. PLoS One 2008, 3, e2876.
- Morrison T.; Hurley J.; Garcia J.; Yoder K.; Katz A.; Roberts D.; Cho J.; Kanigan T.; Ilyin S. E.; Horowitz D.; Dixon J. M.; Brenan C. J. Nucleic Acids Res. 2006, 34, e123.
- Warren L.; Bryder D.; Weissman I. L.; Quake S. R. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 17807–17812.
- Ottesen E. A.; Hong J. W.; Quake S. R.; Leadbetter J. R. Science 2006, 314, 1464–1467.
- Fan H. C.; Quake S. R. Anal. Chem. 2007, 79, 7576–7579.
- Dressman D.; Yan H.; Traverso G.; Kinzler K. W.; Vogelstein B. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 8817–8822.
- Diehl F.; Li M.; He Y.; Kinzler K. W.; Vogelstein B.; Dressman D. Nat. Methods 2006, 3, 551–559.
- Diehl F.; Diaz L. A. Jr. Curr. Opin. Oncol. 2007, 19, 36–42.
- Li M.; Diehl F.; Dressman D.; Vogelstein B.; Kinzler K. W. Nat. Methods 2006, 3, 95–97.
- Li M.; Chen W. D.; Papadopoulos N.; Goodman S. N.; Bjerregaard N. C.; Laurberg S.; Levin B.; Juhl H.; Arber N.; Moinova H.; Durkee K.; Schmidt K.; He Y.; Diehl F.; Velculescu V. E.; Zhou S.; Diaz L. A. Jr.; Kinzler K. W.; Markowitz S. D.; Vogelstein B.; Nat. Biotechnol. 2009, 27(9), 858−863.
- Beer N. R.; Hindson B. J.; Wheeler E. K.; Hall S. B.; Rose K. A.; Kennedy I. M.; Colston B. W. Anal. Chem. 2007, 79, 8471–8475.
- Beer N. R.; Wheeler E. K.; Lee-Houghton L.; Watkins N.; Nasarabadi S.; Hebert N.; Leung P.; Arnold D. W.; Bailey C. G.; Colston B. W. Anal. Chem. 2008, 80, 1854–1858.
- Kiss M. M.; Ortoleva-Donnelly L.; Beer N. R.; Warner J.; Bailey C. G.; Colston B. W.; Rothberg J. M.; Link D. R.; Leamon J. H. Anal. Chem. 2008, 80, 8975–8981.
- Weaver S.; Dube S.; Mir A.; Qin J.; Sun G.; Ramakrishnan R.; Jones R. C.; Livak K. J. Methods 2010, 50, 271–276.
- Gonzalez E.; Kulkarni H.; Bolivar H.; Mangano A.; Sanchez R.; Catano G.; Nibbs R. J.; Freedman B. I.; Quinones M. P.; Bamshad M. J.; Murthy K. K.; Rovin B. H.; Bradley W.; Clark R. A.; Anderson S. A.; O’Connell R J.; Agan B. K.; Ahuja S. S.; Bologna R.; Sen L.; Dolan M. J.; Ahuja S. K. Science 2005, 307, 1434–1440.
- Sudmant P. H.; Kitzman J. O.; Antonacci F.; Alkan C.; Malig M.; Tsalenko A.; Sampas N.; Bruhn L.; Shendure J.; Eichler E. E. Science 2010, 330, 641–646.
- Carter N. P. Nat. Genet. 2007, 39, S16–21.
- Schouten J. P.; McElgunn C. J.; Waaijer R.; Zwijnenburg D.; Diepvens F.; Pals G. Nucleic Acids Res. 2002, 30, e57.
- Aldhous M. C.; Abu Bakar S.; Prescott N. J.; Palla R.; Soo K.; Mansfield J. C.; Mathew C. G.; Satsangi J.; Armour J. A. Hum. Mol. Genet. 2010, 19, 4930–4938.
- Sherry S. T.; Ward M. H.; Kholodov M.; Baker J.; Phan L.; Smigielski E. M.; Sirotkin K. Nucleic Acids Res. 2001, 29, 308–311.
- Qin J.; Jones R. C.; Ramakrishnan R. Nucleic Acids Res. 2008, 36, e116.
- Alkan C.; Kidd J. M.; Marques-Bonet T.; Aksay G.; Antonacci F.; Hormozdiari F.; Kitzman J. O.; Baker C.; Malig M.; Mutlu O.; Sahinalp S. C.; Gibbs R. A.; Eichler E. E. Nat. Genet. 2009, 41, 1061–1067.
- Bartlett J. M.; Ibrahim M.; Jasani B.; Morgan J. M.; Ellis I.; Kay E.; Connolly Y.; Campbell F.; O’Grady A.; Barnett S.; Miller K. Amer. J. Clin. Pathol. 2009, 131, 106–111.
- Kauraniemi P.; Kuukasjarvi T.; Sauter G.; Kallioniemi A. Am. J. Pathol. 2003, 163, 1979–1984.
- Luoh S. W. Cancer Genet. Cytogenet. 2002, 136, 43–47.
- Benlloch S.; Paya A.; Alenda C.; Bessa X.; Andreu M.; Jover R.; Castells A.; Llor X.; Aranda F. I.; Massuti B. J. Mol. Diagn. 2006, 8, 540–543.
- Lo Y. M.; Corbetta N.; Chamberlain P. F.; Rai V.; Sargent I. L.; Redman C. W.; Wainscoat J. S. Lancet 1997, 350, 485–487.
- Wright C. F.; Burton H. Hum. Reprod. Update 2009, 15, 139–151.
- Pathak A. K.; Bhutani M.; Kumar S.; Mohan A.; Guleria R. Clin. Chem. 2006, 52, 1833–1842.
- Fan H. C.; Blumenfeld Y. J.; Chitkara U.; Hudgins L.; Quake S. R. Clin. Chem. 2010, 56, 1279–1286.
- Tong Y. K.; Jin S.; Chiu R. W.; Ding C.; Chan K. C.; Leung T. Y.; Yu L.; Lau T. K.; Lo Y. M. Clin. Chem. 2010, 56, 90–98.
- Fan H. C.; Blumenfeld Y. J.; Chitkara U.; Hudgins L.; Quake S. R. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 16266–16271.
- Hosono N.; Kubo M.; Tsuchiya Y.; Sato H.; Kitamoto T.; Saito S.; Ohnishi Y.; Nakamura Y. Hum. Mutat. 2008, 29, 182–189.
- Fan H. C.; Blumenfeld Y. J.; El-Sayed Y. Y.; Chueh J.; Quake S. R. Am. J. Obstet. Gynecol. 2009, 200 (543), e541–547.
Source: PubMed