Single-molecule PCR using water-in-oil emulsion

Nakano, Michihiko; Komatsu, Jun; Matsuura, Shun-ichi; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

doi:10.1016/s0168-1656(03)00023-3

Cited by 192 publications

(141 citation statements)

References 15 publications

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“…The contents of individual containers should if possible be independently controllable so that distinct reactions can take place in separate containers. An emulsion in which one liquid forms droplets in another immiscible liquid (water-droplet-in-oil or oil-droplet-in-water) is a simple and attractive way of creating such containers (Lu et al 1998;Nakano et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Scalable attoliter monodisperse droplet formation using multiphase nano-microfluidics

Shui

Berg

Eijkel

2011

Microfluid Nanofluid

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We demonstrate a robust method to produce monodisperse femtoliter to attoliter droplets by using a nano-microfluidic device. Two immiscible liquids are forced through a nanochannel where a steady nanoscopic liquid filament forms, thinning close to the nanochannel exit to a microchannel due to the capillary focusing. When the nanoscopic filament enters the microchannel, monodisperse droplets are formed by capillary instability. In a certain range of physical parameters and geometrical configurations, the droplet size is only determined by the nanochannel height and independent of liquid flow rates and ratios, surfactants, and continuous phase viscosity. By using nanochannels with a height of 100-900 nm, 0.4-3.5 lm diameter droplets (volume down to 30 aL) have been produced. The generated droplets are stable for at least weeks.

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Section: Introductionmentioning

confidence: 99%

Scalable attoliter monodisperse droplet formation using multiphase nano-microfluidics

Shui

Berg

Eijkel

2011

Microfluid Nanofluid

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“…Technologic advances have facilitated the development of methods for highly sensitive and quantitative detection of ctDNA, including microfluidic dropletbased digital PCR (dPCR) (3,4 ) and optimized nextgeneration sequencing (NGS) strategies (5)(6)(7). Building on previous bulk dPCR strategies (8 ), droplet-based dPCR uses millions of water-in-oil droplets for parallel amplification of millions of individual DNA fragments (9,10 ) leading to highly sensitive mutation detection (11)(12)(13)(14). This method is truly quantitative and highly precise, allowing finely tuned monitoring of ctDNA levels (3,15 ), paving the way for early cancer recurrence detection, identification of resistant subclones as well as early and cost-effective evaluation of treatment efficacy (16 -20 ).…”

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confidence: 99%

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

et al. 2016

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BACKGROUND Circulating tumor DNA (ctDNA) has emerged as a good candidate for tracking tumor dynamics in different cancer types, potentially avoiding repeated tumor biopsies. Many different genes can be mutated within a tumor, complicating procedures for tumor monitoring, even with highly sensitive next-generation sequencing (NGS) strategies. Droplet-based digital PCR (dPCR) is a highly sensitive and quantitative procedure, allowing detection of very low amounts of circulating tumor genetic material, but can be limited in the total number of target loci monitored. METHODS We analyzed hypermethylation of 3 genes, by use of droplet-based dPCR in different stages of colorectal cancer (CRC), to identify universal markers for tumor follow-up. RESULTS Hypermethylation of WIF1 (WNT inhibitory factor 1) and NPY (neuropeptide Y) genes was significantly higher in tumor tissue compared to normal tissue, independently of tumor stage. All tumor tissues appeared positive for one of the 2 markers. Methylated ctDNA (MetctDNA) was detected in 80% of metastatic CRC and 45% of localized CRC. For samples with detectable mutations in ctDNA, MetctDNA and mutant ctDNA (MutctDNA) fractions were correlated. During follow-up of different stage CRC patients, MetctDNA changes allowed monitoring of tumor evolution. CONCLUSIONS These results indicate that MetctDNA could be used as a universal surrogate marker for tumor follow-up in CRC patients, and monitoring MetctDNA by droplet-based dPCR could avoid the need for monitoring mutations.

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“…In addition, preliminary studies seem to suggest that ddPCR is 130 robust against many of the factors that can negatively influence 131 conventional PCR (Dingle et al, 2013), because the DNA template, 132 when confined, is sequestered from cross-reacting DNA templates 133 and inhibitory moieties (Nakano et al, 2003).…”

mentioning

confidence: 99%

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

Yang

Paparini

Monis

et al. 2014

International Journal for Parasitology

166

127

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Single-molecule PCR using water-in-oil emulsion

Cited by 192 publications

References 15 publications

Scalable attoliter monodisperse droplet formation using multiphase nano-microfluidics

Scalable attoliter monodisperse droplet formation using multiphase nano-microfluidics

A Study of Hypermethylated Circulating Tumor DNA as a Universal Colorectal Cancer Biomarker

Comparison of next-generation droplet digital PCR (ddPCR) with quantitative PCR (qPCR) for enumeration of Cryptosporidium oocysts in faecal samples

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