Ministry of Science and Technology of Taiwan provided financial support of this research under contracts MOST-103-2221-E-002 -097 -MY3.

1. Method to Detect MNP
Note: This section describes the procedure to detect the MNP based on the hybridization-mediated growth of gold nanoparticles.
<ol>
	<li>Prepare the probe DNA (5&#39;-thiol-GAGCTGGTGGCGTAGGCAAG-3&#39;) solution at a concentration 100 &#181;M.</li>
	<li>Prepare the probe DNA-modified AuNP (AuNP probe) particles.21 
	Note: The volume used here depends on the requirement of probe DNA-modified AuNP (AuNP probe) particles. It is hence dependent on the number of experiments to be conducted. We typically prepare excess AuNP probe particles as spares. The volume used in these steps is adjustable and flexible.
	<ol>
		<li>Add probe DNA (100 &#181;M, prepared in step 1.1) to a solution of gold nanoparticles (13 nm, 17 nM) at concentration 1 OD/ml.</li>
		<li>Bring the final concentrations of sodium dodecyl sulfate (NaC12H25SO4, SDS) and phosphate buffer (PBS) to 0.01% and 0.01 M, respectively.</li>
		<li>After 20 min, bring the concentration of sodium chloride (NaCl) to 0.05 M with a solution of NaCl (2 M) and PBS (0.01 M) while maintaining SDS at 0.01% and incubate for 20 min.</li>
		<li>Increase the NaCl concentration in increments of 0.1 M to a final concentration of 1 M over 20 min intervals.</li>
		<li>Incubate overnight with shaking on a vortex mixer at 23 &#176;C. The shaking speed does not affect the experiment as long as the DNA samples and AuNP probes become hybridized.</li>
		<li>Centrifuge the gold nanoparticles at 7,000 x g for 30 sec and remove the supernatant.</li>
	</ol>
	</li>
	<li>Add AuNP probe particles into deionized water (DI water) at a concentration of 25 nM (AuNP probe solution).</li>
	<li>Prepare target DNA samples (fully complementary: 5&#39;-CTTGCCTACGCCACCAGCTC-3&#39;; three base-pair mismatched: 5&#39;-CTTGCCTACTTTACCAGCTC-3&#39;; six base-pair mismatched: 5&#39;-CTTGCCTTTTTTTCCAGCTC-3&#39;) at concentrations of 0.06, 0.11, 0.17, 0.20, 0.30, 0.50 &#181;M, respectively.</li>
	<li>Add AuNP probe solution (6 &#181;l, 25 nM) into the DNA samples (350 &#181;l) with NaCl (14 &#181;M).</li>
	<li>Shake the mixture of DNA samples and AuNP probes with a vortex mixer at 23 &#176;C for 5 min for DNA hybridization.</li>
	<li>For AuNP growth, add NH2OH (6 &#181;l, 400 mM) and HAuCl4 (6 &#181;l, 25.4 mM) to the solution (the mixture of DNA samples and AuNP probes). The growth of AuNP takes approximately 30 sec for completion. The steady change of coloration maintains more than 1 hr. 
	CAUTION: Skin contact with HAuCl4 might produce severe toxic effects. Please consult the material safety data sheets (MSDS) before use. Please wear gloves and use a fume cupboard when using HAuCl4.</li>
</ol>
2. Fabrication of a Pneumatic Droplet Manipulation Platform
Note: The PDMS-based droplet manipulation platform comprises two components: a PDMS membrane (100 &#181;m) with a super-hydrophobic surface and an air-channel layer (5 mm). Without a MEMS process, the common machining processes were utilized to fabricate this device, which includes computer-numerical control (CNC) micromachining for making a mold, PDMS casting and replication for rapid prototyping of the microfluidic components, and laser micromachining for the fabrication of a super-hydrophobic surface (see Figure 3).
<ol>
	<li>Use the CNC machine equipped with a drill bit (0.5 mm) to produce PMMA-based master molds (see Figure 3) with microstructures. Use a feed speed of the drill bit 7 mm/sec and a rate of rotation 26,000 rpm. Use an air blower and DI water to remove the PMMA scrap and to clean the surface of the master molds.</li>
	<li>Prepare a mixture of the PDMS base and the curing agent in ratio 10:1. Degas the mixture inside a desiccator for 30 min, or until all air bubbles are removed.</li>
	<li>Pour the PDMS into the master mold and bake the PDMS for 3 hr at 60 &#176;C in the oven to obtain the inverse microstructures of the air chambers (PDMS membrane) and air channels (PDMS layer).</li>
	<li>Remove the PDMS layer from the master mold (by hand). Punch holes on the PDMS layer for air-suction inlets.</li>
	<li>Put the PDMS membrane, the PDMS layer and the glass substrate into the chamber of the oxygen-plasma treatment system. After oxygen-plasma treatment, bond the PDMS membrane, the PDMS layer and the glass substrate together on a hotplate (90 &#176;C) for 10 min.</li>
	<li>Put the composite chip into the laser-cutting machine (PDMS membrane side up). Import the .dwg file to define the superhydrophobic area (15 mm &#215; 12 mm). See Figure 3. Directly engrave the PDMS membrane with a CO2-laser machine to create the superhydrophobic area on the PDMS membrane (power 12 W, scanning velocity 106.68 mm/sec).</li>
	<li>Clean the superhydrophobic surface with DI water to wash away the carbonized residues on the surface.</li>
	<li>Connect the air-suction inlet and vacuum pump through a solenoid valve (see Figure 4). Connect the solenoid valve to the computer and control with a digital I/O USB module.</li>
</ol>
3. Operation for Detection of MNP on the Pneumatic Platform
Note: This section describes the operation to identify colorimetrically and rapidly the MNP on the pneumatic droplet manipulation platform (see Figure 5). All steps take place at 23 &#176;C (ambient temperature) and relative humidity 85%.
<ol>
	<li>Place the target DNA sample droplet (10 &#181;l, 0.5 &#181;M) on the superhydrophobic area of the pneumatic droplet manipulation platform.</li>
	<li>Place the AuNP probe droplet (10 &#181;l, 25 nM) on the superhydrophobic area of the pneumatic droplet manipulation platform.</li>
	<li>Control the air suction to cause the deflection of the PDMS membrane with a solenoid valve and a simple program (e.g., Labview). Provide air suction in each air chamber along the migration path of the droplets (containing target DNA and AuNP probes respectively) on the PDMS membrane such that the droplets collide with each other and coalesce. Use a working pressure of the vacuum pump of -80 kPa (gauge pressure).
	<ol>
		<li>Alternatively, control the air suction manually as long as the droplet is fully mixed. By hand, connect or disconnect the vacuum pump and inlets of air chamber with tubes in the early stages of testing.</li>
	</ol>
	</li>
	<li>Control the air suction to roll the coalesced droplet forward and backward to enhance the mixing efficiency for 3 min using the controlling system of the solenoid valve. The target DNA and AuNP probes become well mixed and hybridized within 3 min. Use a driving frequency and a working pressure of 5 Hz and -80 kPa (gauge pressure), respectively.</li>
	<li>Place a droplet containing NH2OH (2 &#181;l, 400 mM) and HAuCl4 (2 &#181;l, 25.4 mM) on the superhydrophobic area of the pneumatic droplet manipulation platform.</li>
	<li>Control the air suction (-80 kPa gauge pressure) to let the droplets collide with each other and coalesce. Then control the air suction (5 Hz and -80 kPa gauge pressure) to let the coalesced droplet roll forward and backward to enhance the mixing efficiency for 1 min.</li>
	<li>Measure and record the color of the coalesced droplet with the naked eye.</li>
</ol>

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The authors declare that they have no competing financial interests.

In this protocol, a simple colorimetric method to detect MNP can be implemented at concentrations ranging from 0.11-0.50 &#181;M in microcentrifuge tubes. Furthermore, the proposed MNP detection method is conducted on a pneumatic droplet manipulation platform that has a high potential for DNA screening and other bio-medical applications. In practice, the detectable range of the sample DNA concentration depends on the mixing efficiency of the operating platforms. To ensure that the coalesced droplet is fully mixed, the cr...

Single-nucleotide polymorphism (SNP), which is a single base-pair difference in a DNA sequence, is one of the most common genetic variations. Current studies report that SNPs are associated with disease risk, drug efficacy and side-effects of individuals by affecting gene function.1,2 Recent studies also revealed that two- or multi-point mutations (multi-nucleotide polymorphism) cause particular diseases and individual differences in the effects of disease.3,4 The detection of nucleotide polymorphism is therefore imperative in prescreening disease. Simple and efficient methods for the rapid detection of sequence-specific oligonucleotides were highly developed in the past two decades.1,5 Current approaches to identify DNA mutations typically involve procedures including probe immobilization, fluorescence labeling, gel electrophoresis, etc.,6,7 but those methods generally require a long analytical process, expensive equipment, well trained technicians, and significant consumption of samples and reagents.
A nanoparticle with a large ratio of surface area to volume and unique physicochemical properties is an ideal material as a highly sensitive and cheap detection platform for specific biomarkers. Gold nanoparticles (AuNP) are widely used for DNA detection because of their great ability to be modified with oligonucleotide probes.8-10 SNP detection techniques were also developed using AuNP.11-13 In this work we adopted a novel colorimetric approach to detect the multi-nucleotide polymorphism (MNP) through DNA hybridization-mediated growth of AuNP probes.14 This simple and rapid probing method is based on the theory that varied lengths of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) conjugated to AuNP influence the growth size and shape of the AuNP (see Figure 1).15 This method of DNA detection features a small consumption of reagents, a small assay duration (a few minutes), and a simple procedure without thermal control that is prospectively applicable for clinical diagnosis and domestic medical screening.
Several microfluidic systems to detect the DNA sequence have been developed;16 those microfluidic systems, evolved from traditional experimental protocols, required fewer pieces of large-scale equipment and simplified the experimental protocols so as to improve the sensitivity, detection limit and specificity of the DNA biosensor. DNA detection methods in the microfluidic systems still require, however, instruments of a subsequent process such as a PCR (polymerase chain reaction) machine for signal amplification and a fluorescence reader for a single readout to identify the heterogeneous SNP.17,18 Developing a simple platform without the subsequent processing to directly read out the results of multi-nucleotide polymorphism is highly desirable. Compared to well used, conventional, closed microfluidic systems, the open-surface microfluidic devices promisingly offer several advantages, such as a clear optical path, an easy way to access the sample, a direct environmental accessibility and no easily formed cavitation or interfacial obstruction in the channel.19 Our previous work introduced a simple pneumatic platform for open-surface droplet manipulation (see Figure 2).20 On this platform, droplets can be simultaneously transported and manipulated without interference from a driving energy using a suction force, which has a great potential in biological and chemical applications. This pneumatic platform was thus utilized to execute the manipulation of DNA samples for MNP detection in combination with the colorimetric approach using the concept of DNA hybridization-mediated growth of AuNP probes.
The protocol presented in this paper describes a simple visual detection of multi-nucleotide polymorphisms on the pneumatic droplet manipulation platform on an open surface. This work confirms that multi-nucleotide polymorphism is detectable with the naked eye; the proposed pneumatic platform is suitable for biological and chemical applications.

<table><tbody><tr><td>PDMS</td><td>Dow Corning</td><td>SYLGARD 184</td><td></td></tr><tr><td>benchtop engravers</td><td>Roland DG</td><td>EGX-400</td><td></td></tr><tr><td>laser cutting machine</td><td>Universal Laser Systems, Inc.</td><td>VLS 3.50</td><td></td></tr><tr><td>Oxygen plasma treatment system</td><td>Femto Science Inc. Korea</td><td>CUTE-MPR</td><td></td></tr><tr><td>solenoid valve</td><td></td><td></td><td>home built</td></tr><tr><td>vacuum pump</td><td>ULVAC KIKO, Inc.</td><td>DA-30D</td><td></td></tr><tr><td>13-nm AuNP solution</td><td>TAN Bead Inc., Taiwan</td><td>NG-13</td><td></td></tr><tr><td>DNA (with 5&#039;-end labeled thiol)</td><td>MDBio, Inc., Taiwan</td><td></td><td></td></tr><tr><td>phosphate buffered saline (PBS)</td><td>UniRegion&amp;nbsp;Bio-Tech,. Taiwan</td><td>PBS001-1L</td><td></td></tr><tr><td>sodium dodecyl sulfate (SDS)</td><td>J. T. baker</td><td>4095-04</td><td></td></tr><tr><td>Hydroxylamine solution (NH&lt;sub&gt;2&lt;/sub&gt;OH)</td><td>Sigma-Aldrich</td><td>467804</td><td></td></tr><tr><td>Chloroauric acid (HAuCl&lt;sub&gt;4&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>G4022</td><td></td></tr><tr><td>sodium chloride (NaCl)</td><td></td><td></td><td></td></tr><tr><td>vortex mixer</td><td>Digisystem Laboratory Instruments Inc.</td><td>VM-2000</td><td></td></tr><tr><td>centrifuge</td><td>Hermle Labortechnik GmbH.</td><td>Z 216 MK</td><td></td></tr></tbody></table>

the visual colorimetric detection of multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. This approach to colorimetric DNA detection was based on the hybridization-mediated growth of gold nanoparticle probes (AuNP probes). The growth size and configuration of the AuNP are dominated by the number of DNA samples hybridized with the probes. Based on the specific size- and shape-dependent optical properties of the nanoparticles, the number of mismatches in a sample DNA fragment to the probes is able to be discriminated. The tests were conducted via droplets containing reagents and DNA samples respectively, and were transported and mixed on the pneumatic platform with the controlled pneumatic suction of the flexible PDMS-based superhydrophobic membrane. Droplets can be delivered simultaneously and precisely on an open-surface on the proposed pneumatic platform that is highly biocompatible with no side effect of DNA samples inside the droplets. Combining the two proposed methods, the multi-nucleotide polymorphism can be detected at sight on the pneumatic droplet manipulation platform; no additional instrument is required. The procedure from installing the droplets on the platform to the final result takes less than 5 min, much less than with existing methods. Moreover, this combined MNP detection approach requires a sample volume of only 10 &#181;l in each operation, which is remarkably less than that of a macro system.

In this work, three DNA samples were tested using a simple and novel method of detection through the DNA hybridization-mediated growth of the AuNP probes. The sequences of probe DNA and DNA samples of three kinds, specifically, CDNA (fully complementary to probe DNA), TMDNA (three base-pair mismatched DNA), and SixMDNA (six base-pair mismatched DNA) are listed in Protocol step 1. The mismatches to the probe of the DNA samples tested here are both in the middle segments of the DNA samples....

Watch this Scientific Journal Video about The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform at JoVE.com

The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 &#176;C without the aid of advanced instruments.

A simple and visual method to detect multi-nucleotide polymorphism (MNP) was performed on a pneumatic droplet manipulation platform on an open surface. ...

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7.6K Views.  National Taiwan University. This work presents a simple and visual method to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform. With the proposed method, the entire experiment, including droplet manipulation and detection of multi-nucleotide polymorphisms, can be performed near 23 °C without the aid of advanced instruments.

Video: The Visual Colorimetric Detection of Multi-nucleotide Polymorphisms on a Pneumatic Droplet Manipulation Platform

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Bioengineering

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA sequence variation on a large scale. Specifically, detecting insertions and deletions (indels) at gene-edited sites generated by CRISPR guide RNA (gRNA)/Cas9-mediated non-homologous end-joining (NHEJ) is important for assessing the fidelity of the mutagenesis and the frequency of unintended changes. We describe here a protocol for digital-droplet PCR (ddPCR) that is well-suited for high-throughput NHEJ analysis. While this method does not produce data that identifies individual sequence variation, it provides a quantitative estimate of the sequence variation within a population. Additionally, with appropriate resources, this protocol can be implemented in a field-site laboratory setting more easily than next-generation or Sanger sequencing. ddPCR also has a faster turn-around time for results than either of those methods, which allows a more quick and complete analysis of genetic variation in wild populations during field trials of genetically-engineered organisms.

This protocol provides the steps from DNA extraction to experimental set-up for digital droplet PCR (ddPCR), including analysis for the identification and quantification of non-homologous end-joining (NHEJ) events at target sites following gRNA-induced Cas9 cleavage and DNA repair. Other uses of this method include applications such as polymorphism detection and gene-editing variant verification.

Recent advances in mosquito genomics and genetic engineering technologies have fostered a need for quick and efficient methods for detecting targeted DNA ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as multiplex assays, enzyme evolution, and molecular biology enhanced cell sorting require the detection of two or more colors of fluorescence. Standard multicolor detection systems that couple free space lasers to epifluorescence microscopes are bulky, expensive, and difficult to maintain. In this paper, we describe a scheme to perform multicolor detection by exciting discrete regions of a microfluidic channel with lasers coupled to optical fibers. Emitted light is collected by an optical fiber coupled to a single photodetector. Because the excitation occurs at different spatial locations, the identity of emitted light can be encoded as a temporal shift, eliminating the need for more complicated light filtering schemes. The system has been used to detect droplet populations containing four unique combinations of dyes and to detect sub-nanomolar concentrations of fluorescein.

Multicolor fluorescence detection in droplet microfluidics typically involves bulky and complex epifluorescence microscope-based detection systems. Here we describe a compact and modular multicolor detection scheme that utilizes an array of optical fibers to temporally encode multicolor data collected by a single photodetector.

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Methylation Specific Multiplex Droplet PCR using Polymer Droplet Generator Device for Hematological Diagnostics

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, along with a thermoplastic elastomer (TPE) microfluidic droplet generation device. Epigenetic markers are used for WBC&#160;subtyping which is of important prognostic value in different diseases. This is achieved through the quantification of DNA methylation patterns of specific CG-rich regions in the genome (CpG loci). In this paper, bisulfite-treated DNA from peripheral blood mononuclear cells (PBMCs) is encapsulated in droplets with mdPCR reagents including primers and hydrolysis fluorescent probes specific for CpG loci that correlate with WBC sub-populations. The multiplex approach allows for the interrogation of many CpG loci without the need for separate mdPCR reactions, enabling more accurate parametric determination of WBC sub-populations using epigenetic analysis of methylation sites. This precise quantification can be extended to different applications and highlights the benefits for clinical diagnosis and subsequent prognosis.

Epigenetic markers are used for white blood cell (WBC) subtyping through the quantification of DNA methylation patterns. This protocol presents a multiplex droplet polymerase chain reaction (mdPCR) method using a thermoplastic elastomer (TPE)-based microfluidic device for droplet generation allowing for precise and multiplex methylation-specific target quantification of WBC differential counts.

A multiplexed droplet PCR (mdPCR) workflow and detailed protocol for determining epigenetic-based white blood cell (WBC) differential count is described, ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

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 droplet-based 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&#160;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.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Biology

Digital Droplet Polymerase Chain Reaction for Indel Mutation Detection in Target Genes

Source: Carballar-Lejaraz&#250;, R., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/62607">Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations.</a> J. Vis. Exp. (2021).
This video demonstrates the role of digital droplet polymerase chain reaction in identifying indel mutation in a DNA sample. The high precision and sensitivity of ddPCR enable accurate and consistent detection of rare mutations present in a sample, even at low levels. It can identify and quantify indel mutations in different regions of the genome, providing valuable insights into the mechanisms of mutation and the functional consequences of these mutations.&#160;

Source: Carballar-Lejaraz&#250;, R., et al. Digital-Droplet PCR to Detect Indels Mutations in Genetically Modified Anopheline Mosquito Populations. J. ...

JoVE Encyclopedia of Experiments

Biological Techniques

PCR Techniques

Chip-in-a-Tube-Based Digital PCR for Quantification of Single Nucleotide Variants

Source: Kahyo, T., et al. <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/58199">Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format.</a> J. Vis. Exp. (2018).
This video demonstrates the utility of digital PCR in a chip-in-a-tube format for the detection of single nucleotide variations in DNA samples. A single PCR reaction is partitioned into chambers that act as independent PCR reactions, and the detection of fluorescence signals from the amplified targets in the chambers is used to compute the frequency of the variant allele in the sample.

Source: Kahyo, T., et al. Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the ...

Droplet Digital Polymerase Chain Reaction: A Method to Generate Nanodroplet PCR Reactions for Detecting Rare Tumor Mutations

Source: Bonner, E. R. et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/video/59721" target="_blank">Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids.</a> J. Vis. Exp. (2019)
In this video, we present a step-by-step procedure to generate emulsified nano-droplets to detect target nucleic acids by digital polymerase chain reaction or dPCR.

Source: Bonner, E. R. et al. Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids. J. Vis. Exp. (2019)
In this video, we ...