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

By B. Hindson, K. Ness, D. Masquelier, P. Belgrader, Nick Heredia, A. Makarewicz, Isaac J. Bright, Mike Lucero, A. Hiddessen, T. Legler and 28 more. Analytical Chemistry, 2011

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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.

Subfields: Cancer Research, Molecular Biology, Pediatrics, Perinatology and Child Health

View Paper: https://www.semanticscholar.org/paper/044e4f0b0eb269e207e7c9d0342737519406c1ca

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Citations in the corpus

Clonal haematopoiesis harbouring AML-associated mutations is ubiquitous in healthy adults (2016, positive): “We validated 21 SNVs and 1 indel using the ddPCR probe assay (Bio-Rad) 42 .”
Environmental DNA surveillance for invertebrate species: advantages and technical limitations to detect invasive crayfish Procambarus clarkii in freshwater ponds. (2014, methodology): “Moreover, the DNA detection and quantification should be improved in the near future thanks to a new technology that has been commercialized recently, the digital PCR or dPCR (Vogelstein & Kinzler 1999; Hindson et al. 2011).”
Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets (2015, methodology): “Drop-seq will accelerate biological discovery by enabling routine transcriptional profiling at singlecell resolution.”
Integrated digital error suppression for improved detection of circulating tumor DNA (2016, positive): “…4b ; also see Reference standards for noninvasive tumor genotyping, above), we separately quantified 3 mutant alleles known to be present in the mixture: EGFR, KRAS, and BRAF on a Bio-Rad QX200 ddPCR instrument as previously described 46 , using reagents, primers and probes obtained from Bio-Rad.”
dPCR: A Technology Review (2018, methodology): “Microfluidics relies on microfabrication techniques adapted from microelectronics and its implementation relies either on fast prototyping by soft lithography in Polydimethylsiloxane (PDMS) [51], glass etching [52], or injection molding [53].”
Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA (2014, positive): “5 then read in an automated droplet flow cytometer (Figure 1) (14).”
The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020. (2020, background): “Restriction digestion to small linear DNA fragments may equalize template differences (36, 49, 64), and prevent underestimation of linked target molecules (18).”