High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number
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- 作者：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
- 刊名：Analytical Chemistry
- 出版年：2011
- 出版时间：November 15, 2011
- 年：2011
- 卷：83
- 期：22
- 页码：8604-8610
- 全文大小：916K
- 年卷期：v.83,no.22(November 15, 2011)
- ISSN：1520-6882
文摘
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鈥?00-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.