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Anal Chem. 2011 Nov 15;83(22):8604-10. doi: 10.1021/ac202028g. Epub 2011 Oct 28.

High-throughput droplet digital PCR system for absolute quantitation of DNA copy number.

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1
Bio-Rad Laboratories, Inc., Pleasanton, California 94566, United States. ben_hindson@bio-rad.com

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.

PMID:
22035192
PMCID:
PMC3216358
DOI:
10.1021/ac202028g
[Indexed for MEDLINE]
Free PMC Article
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