High-Throughput Droplet Digital PCR System for Absolute Quantitation of DNA Copy Number

作者：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

来源：Analytical Chemistry, 2011, 83(22): 8604-8610.

DOI：10.1021/ac202028g

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.

• 出版日期2011-11-15