High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number
| Autor: | Nicholas G. Erndt, Christopher Hindson, Shenglong Wang, Isaac J. Bright, Dean K. Wittmann, Pallavi Shah, Tyler K. Kitano, Serge Saxonov, Jonathan Petersen, Fred P. Milanovich, Steven Romine, Luz Montesclaros, Phillip Belgrader, Paul Wyatt, John F. Regan, Thomas H. Cauley, Kevin D. Ness, Michael R. Hodel, Luc J. Bousse, Tina C. Legler, Donald A. Masquelier, George Karlin-Neumann, Ryan T. Koehler, Benjamin J. Hindson, Amy L. Hiddessen, Helen E. White, David P. Stumbo, Camille B. Troup, Simant Dube, Michael Y. Lucero, Shawn Hodges, Austin P. So, Erin R. Steenblock, Nicholas J. Heredia, Anthony J. Makarewicz, Bill W. Colston, Jeffrey Clark Mellen, Klint Rose |
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| Jazyk: | angličtina |
| Rok vydání: | 2011 |
| Předmět: |
Nucleic acid quantitation
Gene Dosage Computational biology Gene dosage Polymerase Chain Reaction Article Analytical Chemistry law.invention 03 medical and health sciences chemistry.chemical_compound 0302 clinical medicine law TaqMan Humans Digital polymerase chain reaction Copy-number variation Polymerase chain reaction 030304 developmental biology 0303 health sciences High-Throughput Nucleotide Sequencing DNA Molecular biology Orders of magnitude (mass) chemistry 030220 oncology & carcinogenesis |
| Zdroj: | Analytical Chemistry |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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