Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device

Dube, Simant; Qin, Jian; Ramakrishnan, Ramesh

doi:10.1371/journal.pone.0002876

Cited by 266 publications

(285 citation statements)

References 17 publications

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“…dPCR measurement is well documented as being based on the Poisson distribution (20,41 ) which, for a given mean, has a constant variance. Although this may be true for the measurement of a given pure DNA extract, inhibitory substances copurified or introduced during the extraction may or may not affect the overall measurement variability (31 ).…”

Section: Error: Variancementioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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BACKGROUND Digital PCR (dPCR) is an increasingly popular manifestation of PCR that offers a number of unique advantages when applied to preclinical research, particularly when used to detect rare mutations and in the precise quantification of nucleic acids. As is common with many new research methods, the application of dPCR to potential clinical scenarios is also being increasingly described. CONTENT This review addresses some of the factors that need to be considered in the application of dPCR. Compared to real-time quantitative PCR (qPCR), dPCR clearly has the potential to offer more sensitive and considerably more reproducible clinical methods that could lend themselves to diagnostic, prognostic, and predictive tests. But for this to be realized the technology will need to be further developed to reduce cost and simplify application. Concomitantly the preclinical research will need be reported with a comprehensive understanding of the associated errors. dPCR benefits from a far more predictable variance than qPCR but is as susceptible to upstream errors associated with factors like sampling and extraction. dPCR can also suffer systematic bias, particularly leading to underestimation, and internal positive controls are likely to be as important for dPCR as they are for qPCR, especially when reporting the absence of a sequence. SUMMARY In this review we highlight some of the considerations that may be needed when applying dPCR and discuss sources of error. The factors discussed here aim to assist in the translation of dPCR to diagnostic, predictive, or prognostic applications.

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Section: Error: Variancementioning

confidence: 99%

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

2015

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“…The use of multiple independent chambers facilitates the detection of rare copies of a mutated allele in a limited amount of patient sample. The digital array has previously been used for a variety of different applications, including absolute quantification (26 ), mutation detection (28,29 ), and studies of variation in copy number (30,31 ).…”

Section: Mutations In the Krasmentioning

confidence: 99%

Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer

et al. 2012

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“…The results show good performance in droplet ratio quantification across two logs of dynamic range (R 2 = 0.984; Figure 3B). The input analyte concentration is easily calculated from the fraction of positive or negative droplets 38 .…”

Section: Representative Resultsmentioning

confidence: 99%

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Morinishi

Blainey

2015

JoVE

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Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of dropletbased digital assays.We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology. Video LinkThe video component of this article can be found at

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Mathematical Analysis of Copy Number Variation in a DNA Sample Using Digital PCR on a Nanofluidic Device

Cited by 266 publications

References 17 publications

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Considerations for Digital PCR as an Accurate Molecular Diagnostic Tool

Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

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