Evaluation of a Droplet Digital Polymerase Chain Reaction Format for DNA Copy Number Quantification

By Leonardo B. Pinheiro, V. Coleman, Christopher M. Hindson, J. Herrmann, B. Hindson, Somanath Bhat, K. Emslie. Analytical Chemistry, 2011

1,001 citations recorded by the source corpus.

Droplet digital polymerase chain reaction (ddPCR) is a new technology that was recently commercialized to enable the precise quantification of target nucleic acids in a sample. ddPCR measures absolute quantities by counting nucleic acid molecules encapsulated in discrete, volumetrically defined, water-in-oil droplet partitions. This novel ddPCR format offers a simple workflow capable of generating highly stable partitioning of DNA molecules. In this study, we assessed key performance parameters of the ddPCR system. A linear ddPCR response to DNA concentration was obtained from 0.16% through to 99.6% saturation in a 20,000 droplet assay corresponding to more than 4 orders of magnitude of target DNA copy number per ddPCR. Analysis of simplex and duplex assays targeting two distinct loci in the Lambda DNA genome using the ddPCR platform agreed, within their expanded uncertainties, with values obtained using a lower density microfluidic chamber based digital PCR (cdPCR). A relative expanded uncertainty under 5% was achieved for copy number concentration using ddPCR. This level of uncertainty is much lower than values typically observed for quantification of specific DNA target sequences using currently commercially available real-time and digital cdPCR technologies.

Subfields: Biomedical Engineering, Information Systems and Management, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/3a2d352bde75f73b9de1bc14926bbe9366fed4a2

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In Biomedical Engineering

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Citations in the corpus

• dPCR: A Technology Review (2018, background): “The precision of dPCR is limited by the uncertainty of the measurement due to: (1) specimen sampling, whose effect is prevalent for low target concentrations and can only be minimized by using technical replicates; (2) its statistical nature whose effect on precision can be reduced by increasing the”
• Noninvasive Identification and Monitoring of Cancer Mutations by Targeted Deep Sequencing of Plasma DNA (2012, methodology): “Enhanced detection down to 1 mutant allele in 10,000 or more wild-type alleles can be obtained using a variety of methods, such as peptide nucleic acid and primer extension (“PPEM”) (38), ligation followed by quantitative PCR (“LigAmp”) (39), bead-based digital PCR in emulsions (“BEAMing”) (2, 3), m”