Droplet-Based Microfluidic Systems for High-Throughput Single DNA Molecule Isothermal Amplification and Analysis

The amplification and digital quantification of single DNA molecules are important in biomedicine and diagnostics. Beyond quantifying DNA molecules in a sample, the ability to express proteins from the amplified DNA would open even broader applications in synthetic biology, directed evolution, and proteomics. Herein, a microfluidic approach is reported for the production of condensed DNA nanoparticles that can serve as efficient templates for in vitro protein synthesis. Using phi29 DNA polymerase and a multiple displacement amplification reaction, single DNA molecules were converted into DNA nanoparticles containing up to about 104 clonal gene copies of the starting template. DNA nanoparticle formation was triggered by accumulation of inorganic pyrophosphate (produced during DNA synthesis) and magnesium ions from the buffer. Transcription–translation reactions performed in vitro showed that individual DNA nanoparticles can serve as efficient templates for protein synthesis in vitro.

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“…Previous reports found a similar degree of amplification when phi29 reactions were performed in bulk8 and in droplets 10…”

supporting

confidence: 67%

DNA Nanoparticles for Improved Protein Synthesis In Vitro

et al. 2016

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“…We transferred the droplets from experiment (b), performed in Teflon tubing, to silica tubing (fused silica tubing with a polyimide coating -150µm ID, 360µm OD, from IDEX health and science) using a linear junction (IDEX health and science). The internal silica tubing wall was coated with a commercial hydrophobic surface-coating agent (Aquapel®, PPG industry) (Mazutis et al, 2009) to ensure droplet stability. The silica tubing containing the droplets was directly mounted on a magnetic base extracted from standard SPINE sample loops, ready for transfer to the X-ray setup (figure 3).…”

Section: Xrd Characterizationmentioning

confidence: 99%

Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction

Gerard¹,

Ferry²,

Vuillard³

et al. 2017

Acta Cryst Sect F

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“…Mazutis et al described a droplet-based microfluidic system for single molecule amplification by RCA [161]. Quantification was done by analyzing an intercalating fluorochrome.…”

Section: Lateral Flow Strip Through Dual Immunoreactionsmentioning

confidence: 99%

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

Tröger¹,

Niemann²,

Gärtig³

et al. 2015

J Nanomed Nanotechnol

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Nucleic acid amplification technologies (NAATs) offer the most sensitive tests in the clinical laboratory. These techniques are used as a powerful tool for screening and diagnosis of infectious diseases. Isothermal methods, as an alternative to polymerase chain reaction (PCR), require no thermocycling machine and can mostly be performed with reduced time, high throughput, and accurate and reliable results.However, current molecular diagnostic approaches generally need manual analysis by qualified and experienced personal which is a highly complex, time-consuming and labor-intensive task. Thus, the demand for simpler, miniaturized systems and assays for pathogen detection is steadily increasing. Microfluidic platforms and lab-on-a-chip devices have many advantages such as small sample volume, portability and rapid detection time and enable point-of-care diagnosis.In this article, we review several isothermal amplification methods and their implementation in microsystems in relation to quantification of nucleic acids. miniaturized systems can be diverse. The majority of publications are focussed on clinical diagnostics including foodborne organisms for environmental monitoring [10][11][12]. In case of an infectious disease, cultivation and phenotypic characterization are still the standard methods of microbiologists which need days up to weeks to obtain a diagnostic result. Hence, scientists started to make use of nucleic acid amplification methods to accelerate the analysis. The most widespread and well known technique for this purpose is the polymerase chain reaction (PCR) [10,13], which exploits the activity of the DNA polymerase. The method is based on a thermal cycling process consisting of repeated cycles of heating and cooling steps allowing for DNA melting, sequence specific primer binding and enzymatic amplification of the target nucleic acid. The quantitative assessment of target copies is possible by using a real-time PCR approach, where a concentration dependent signal (e.g. fluorescence) increases over time [14]. The latter enables the user to easily assess the pathogen load of patient samples [15][16][17][18]. Since the first example of a PCR on a miniaturized system in 1994 [19,20], numerous devices were presented, which integrate not just the amplification, but also sample preparation and detection steps [9,[21][22][23][24][25][26][27][28]. Just a few of those microsystems have reached the market so far, but some have shown a fascinating potential to replace the classical laboratory-oriented state-of-the art [29][30][31][32][33]. setups has been shown to obtain a similar result [34]. A smaller heat capacity allows for rapid changes in temperature beneficial for the PCR time as well as a higher parallelism of multiple genetic samples [34].However, a major drawback of the integration of PCR to microsystems is the necessity of sophisticated instrumentation. The reaction requires a thermal cycling instrumentation, space and considerable expertise [35]. Therefore, isothermal reactions for the ta...

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Droplet-Based Microfluidic Systems for High-Throughput Single DNA Molecule Isothermal Amplification and Analysis

Cited by 239 publications

References 32 publications

DNA Nanoparticles for Improved Protein Synthesis In Vitro

DNA Nanoparticles for Improved Protein Synthesis In Vitro

Crystallization via tubing microfluidics permits both in situ and ex situ X-ray diffraction

Isothermal Amplification and Quantification of Nucleic Acids and its Use in Microsystems

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