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 critical step in this protocol is the control of the air suction (pressure and frequency) that depends on the size of the coalesced droplet. The discrepancy of hybridization affinity between the fully complementary sample DNA and the mismatched sample DNA is difficult to distinguish with the naked eye for a long DNA sequence fragment. The limitation of this detection technique is hence the fragment length of the sample DNA. For samples with long DNA sequences, the sizes or conformation of the growth AuNP are similar to those of varied DNA samples containing various mismatches. The mismatches of DNA samples to the probe appear in the middle segment in this protocol. With identical lengths and homomeric mismatches of sample DNA, the position of the mismatches could also affect the hybridization affinity and growth size of AuNP. Many illnesses are currently being traced to particular defective genes. The genome databases for particular illnesses are still expanding and improving. Once the DNA sequence variation of the specific DNA fragment is confirmed to be responsible for a specific disease, the specifically designed probe (with an appropriate length and mismatch position of the DNA) can be used to acquire the number of mismatches of sample DNA to the probe within the detectable concentration range on the basis already tested in this work.
The proposed pneumatic droplet manipulation platform is performed in an open environment. The evaporation of droplets increases the concentration of DNA samples and affects the accuracy of the colorimetric readout. To improve the accuracy of MNP analysis, a controlled environmental temperature and humidity are required. Laser micromachining was applied to fabricate a superhydrophobic surface in the presented platform. The superhydrophobicity of the PDMS membrane does not persist under many iterations of the droplet manipulation (&#62;20 times). The loss of superhydrophobicity decreases the mobility and maneuverability of this droplet manipulation platform. Once the droplet manipulation platform loses the superhydrophobicity on the surface, remodifying the superhydrophobicity of the PDMS membrane (repeat steps 2.6-2.8) is required. The simple fabrication of a more durable superhydrophobic surface is under future consideration. In this protocol, we used a solenoid valve and a simple controlling program (with data acquisition equipment) to control the air suction. The control of air suction can be utilized in other ways or with equipment including but not limited to use of a solenoid valve.
In this manuscript, we introduced the combination of a simple colorimetric method of DNA detection and a pneumatic droplet manipulation platform. Both techniques are unique and highly prospective; we found no alternative DNA detection method (that is both rapid and visual) to achieve the same results. Several droplet manipulation techniques on an open surface including optowetting,22 dielectrophoresis,23 electrowetting,24 vibration25 and thermos-capillary actuation26 have been reported. The drawbacks of all these droplet manipulation techniques were discussed in our previous work.20 The proposed pneumatic platform is more biocompatible than the aforementioned methods.
This technique to detect multi-nucleotide polymorphisms on a pneumatic droplet manipulation platform is straightforward for researchers in the biochemical field, which means that samples and reagents can be directly loaded and collected with pipettes. The integration of the presented platform is being undertaken with an automatic control system and a design of a user interface for improved and wide use in the future.

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 border="0" cellpadding="0" cellspacing="0" width="735">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:245px;">PDMS</td>
			<td style="width:204px;">Dow Corning</td>
			<td style="width:119px;">SYLGARD 184</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">benchtop engravers</td>
			<td style="width:204px;">&#160;Roland DG</td>
			<td style="width:119px;">EGX-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">laser cutting machine</td>
			<td style="width:204px;">Universal Laser Systems, Inc.</td>
			<td style="width:119px;">VLS 3.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">Oxygen plasma treatment system</td>
			<td style="width:204px;">Femto Science Inc. Korea</td>
			<td style="width:119px;">CUTE-MPR</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">solenoid valve</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td>home built</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">vacuum pump</td>
			<td>ULVAC KIKO, Inc.</td>
			<td style="width:119px;">DA-30D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">13-nm AuNP solution</td>
			<td style="width:204px;">TAN Bead Inc., Taiwan</td>
			<td style="width:119px;">NG-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">DNA (with 5 -end labeled thiol)</td>
			<td style="width:204px;">MDBio, Inc., Taiwan</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">phosphate buffered saline (PBS)</td>
			<td>UniRegion&#160;Bio-Tech,. Taiwan</td>
			<td>PBS001-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium dodecyl sulfate (SDS)</td>
			<td style="width:204px;">J. T. baker</td>
			<td style="width:119px;">4095-04</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Hydroxylamine solution (NH2OH)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td style="width:119px;">467804</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:245px;">Chloroauric acid (HAuCl4)</td>
			<td style="width:204px;">Sigma-Aldrich</td>
			<td>G4022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">sodium chloride (NaCl)</td>
			<td style="width:204px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:245px;">vortex mixer</td>
			<td style="width:204px;">Digisystem Laboratory Instruments Inc.</td>
			<td style="width:119px;">VM-2000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:245px;">centrifuge</td>
			<td style="width:204px;">Hermle Labortechnik GmbH.</td>
			<td style="width:119px;">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. Figure 6 shows the colors of the growth AuNP solutions for the DNA samples at varied concentration. For the perfectly matched DNA (CDNA), the hues of solutions vary from pink to indigo and then to transparent with increasing DNA concentration. For TMDNA, the color of solutions goes from pink to dark purple and then to indigo, and for SixMDNA, the color goes from pink to dark pink with increasing DNA concentration. The separate DNA samples possess varied hybridization affinities to the probe DNA that caused varied growth size of AuNP and varied color of solutions. The TMDNA sample, with three base-pair mutation from the probe, has less hybridization affinity than the CDNA sample, which caused a smaller growth size of the AuNP. As the results in Figure 6 show, there are distinctions of color between CDNA and TMDNA samples, especially at a large DNA concentration. Through many mismatches with the probe, SixMDNA sample has a weak hybridization affinity to the probe, which resulted in a few dsDNA conjugated to AuNP, even with the DNA concentration greater than 0.2 &#181;M. The growth size of AuNP is consequently small in this SixMDNA sample, causing the AuNP solution to change from pink to amaranth, which is not obviously measured with a naked eye. Based on the color images, the CDNA, TMDNA and SixMDNA are roughly differentiated for a DNA concentration from 0.11 to 0.50 &#181;M.
With the protocols and methods presented here, a simple and novel droplet-based manipulation platform for open-surface microfluidics was demonstrated. Using pneumatic suction as a force to generate the surface deformation of the PDMS membrane, the facile transport and manipulation of droplets can be achieved without interruption to the bio-sample, DNA in this case, in the droplets (see Figure 7A). The new and visual detection of MNP is demonstrated on this pneumatic open-surface droplet manipulation platform. As a result as shown in Figure 7B, the detection of a DNA mismatch is readily observable with the naked eye. In the original state, the AuNP probe droplet exhibited a red color. The maximum optical absorption of AuNP probes occurs at 520 nm, which is attributed to a surface-plasmon resonance (SPR). After hybridization with the various DNA sample droplets, the target-probe DNA duplex of AuNP with a fully complementary DNA (CDNA) sample droplet became transparent as the SPR feature of the increased AuNP disappeared. In contrast, AuNP hybridized with the three base-pair mismatched DNA (TMDNA) and six base-pair mismatched DNA (SixMDNA) were blue and purple respectively. With this approach, the proposed platform and method of DNA detection are combined and achieved in a simple way.
<img alt="Figure 1" src="/files/ftp_upload/54424/54424fig1.jpg" /> 
Figure 1. Principle of colorimetric detection of MNP. The thiol-modified single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) from the hybridization of the thiol-modified ssDNA on AuNP influenced the growth size and shape of the AuNP, and caused varied optical properties of the AuNP. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54424/54424fig2.jpg" /> 
Figure 2. Schematic diagram of the pneumatic droplet manipulation platform. With a pneumatic suction as a force to cause a deflection of the flexible PDMS-based superhydrophobic membrane, the droplet on the membrane becomes thereby activated. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54424/54424fig3.jpg" /> 
Figure 3. Fabrication of a pneumatic droplet manipulation platform. The fabrication included computer-numerical-control (CNC) micromachining, PDMS casting or replication techniques, and laser micromachining. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54424/54424fig4.jpg" /> 
Figure 4. Experimental setup of the pneumatic droplet manipulation platform. The vacuum pump generated an air suction to provide a diminished pressure in the air chamber and to cause the deflection of the PDMS membrane for droplet manipulation. The vacuum pump and the inlet of the air channel are connected through the solenoid valve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54424/54424fig5.jpg" /> 
Figure 5. Schematic illustration of the operation of this MNP detection technique. The droplets containing target DNA and the AuNP probes are loaded on the platform, mixed and hybridized. The droplet containing both NH2OH and HAuCl4 is loaded on the platform and mixed for AuNP growth. Finally, the target-probe DNA duplex of AuNP shifted the absorption maximum and altered the color. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/54424/54424fig6.jpg" /> 
Figure 6. Variation of color of various DNA samples with concentration. For the fully complementary DNA (CDNA), the hues of solutions vary from pink to indigo with increasing DNA concentration. For TMDNA, the color of solutions goes from pink to dark purple, and for SixMDNA, the color goes from pink to dark pink with increasing DNA concentration. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/54424/54424fig7.jpg" /> 
Figure 7. Images of droplet on the pneumatic platform. A) Serial images of droplet transport on the pneumatic platform with control by air suction. B) Images of the corresponding target-probe DNA duplex of AuNP on the proposed pneumatic droplet manipulation platform. The operating conditions of the driving frequency and the working pressure were 5 Hz and -80 kPa (gauge pressure), respectively. In the original state, the AuNP probe droplet exhibits a red color. The target-probe DNA duplex of AuNP in the presence of the CDNA was transparent. The AuNP probes hybridized with the TMDNA and SixMDNA were blue and purple respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54424/54424fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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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Nanyang Technological University

Research

JoVE Journal

Genetics

7.5K 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

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia coli, these transfer proteins make up a DNA transfer machinery known as the mating pair formation, or DNA transfer complex, which facilitates conjugative plasmid transfer. The objective of this paper is to provide a method that can be used to determine the role of a specific transfer protein that is involved in conjugation using a series of deletions and/or point mutations in combination with mating assays. The target gene is knocked out on the conjugative plasmid and is then provided in trans through the use of a small recovery plasmid harboring the target gene. Mutations affecting the target gene on the recovery plasmid can reveal information about functional aspects of the target protein that result in the alteration of mating efficiency of donor cells harboring the mutated gene. Alterations in mating efficiency provide insight into the role and importance of the particular transfer protein, or a region therein, in facilitating conjugative DNA transfer. Coupling this mating assay with detailed three-dimensional structural studies will provide a comprehensive understanding of the function of the conjugative transfer protein as well as provide a means for identifying and characterizing regions of protein-protein interaction.

Here, we present a protocol to knockout a gene of interest involved in plasmid conjugation and subsequently analyze the impact of its absence using mating assays. The function of the gene is further explored to a specific region of its sequence using deletion or point mutations.

The transfer of genetic material by bacterial conjugation is a process that takes place via complexes formed by specific transfer proteins. In Escherichia ...

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

Methodology for Accurate Detection of Mitochondrial DNA Methylation

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert unmethylated cytosine into uracil, in a single-stranded DNA context. Bisulfite sequencing can be targeted (using PCR) or performed on the whole genome and provides absolute quantification of cytosine methylation at the single base-resolution. Given the distinct nature of nuclear- and mitochondrial DNA, notably in the secondary structure, adaptions of bisulfite sequencing methods for investigating cytosine methylation in mtDNA should be made. Secondary and tertiary structure of mtDNA can indeed lead to bisulfite sequencing artifacts leading to false-positives due to incomplete denaturation poor access of bisulfite to single-stranded DNA. Here, we describe a protocol using an enzymatic digestion of DNA with BamHI coupled with bioinformatic analysis pipeline to allow accurate quantification of cytosine methylation levels in mtDNA. In addition, we provide guidelines for designing the bisulfite sequencing primers specific to mtDNA, in order to avoid targeting undesirable NUclear MiTochondrial segments (NUMTs) inserted into the nuclear genome.

Here, we present a protocol to allow accurate quantification of mitochondrial DNA (mtDNA) methylation. In this protocol, we describe an enzymatic digestion of DNA with BamHI coupled with a bioinformatic analysis pipeline which can be used to avoid overestimation of mtDNA methylation levels caused by the secondary structure of mtDNA.

Quantification of DNA methylation can be achieved using bisulfite sequencing, which takes advantage of the property of sodium bisulfite to convert ...

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 ...

Exploring the Effects of Spaceflight on Mouse Physiology using the Open Access NASA GeneLab Platform

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively and efficiently. Moreover, given the infrequency of these experiments it is imperative that their impacts be maximized. The rapid advancement of omics technologies offers an opportunity to dramatically increase the volume of data produced from precious spaceflight specimens. To capitalize on this, NASA has developed the GeneLab platform to provide unrestricted access to spaceflight omics data and encourage its widespread analysis. Rodents (both rats and mice) are common model organisms used by scientists to investigate space-related biological impacts. The enclosure that house rodents during spaceflight are called Rodent Habitats (formerly Animal Enclosure Modules), and are substantially different from standard vivarium cages in their dimensions, air flow, and access to water and food. In addition, due to environmental and atmospheric conditions on the International Space Station (ISS), animals are exposed to a higher CO2 concentration. We recently reported that mice in the Rodent Habitats experience large changes in their transcriptome irrespective of whether animals were on the ground or in space. Furthermore, these changes were consistent with a hypoxic response, potentially driven by higher CO2 concentrations. Here we describe how a typical rodent experiment is performed in space, how omics data from these experiments can be accessed through the GeneLab platform, and how to identify key factors in this data. Using this process, any individual can make critical discoveries that could change the design of future space missions and activities.

The NASA GeneLab platform provides unfettered access to precious omics data from biological spaceflight experiments. We describe how a typical mouse experiment is conducted in space and how data from such experiments can be accessed and analyzed.

Performing biological experiments in space requires special accommodations and procedures to ensure that these investigations are performed effectively ...

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

Manipulation of Gene Function in Mexican Cavefish

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, including eye degeneration, albinism, sleep loss, hyperphagia, and sensory processing. Species of cavefish from around the world display a convergent evolution of morphological and behavioral traits due to shared environmental pressures between different cave systems. Diverse cave species have been studied in the laboratory setting. The Mexican tetra, Astyanax mexicanus, with sighted and blind forms, has provided unique insights into biological and molecular processes underlying the evolution of complex traits and is well-poised as an emerging model system. While candidate genes regulating the evolution of diverse biological processes have been identified in A. mexicanus, the ability to validate a role for individual genes has been limited. The application of transgenesis and gene-editing technology has the potential to overcome this significant impediment and to investigate the mechanisms underlying the evolution of complex traits. Here, we describe a different methodology for manipulating gene expression in A. mexicanus. Approaches include the use of morpholinos, Tol2 transgenesis, and gene-editing systems, commonly used in zebrafish and other fish models, to manipulate gene function in A. mexicanus. These protocols include detailed descriptions of timed breeding procedures, the collection of fertilized eggs, injections, and the selection of genetically modified animals. These methodological approaches will allow for the investigation of the genetic and neural mechanisms underlying the evolution of diverse traits in A. mexicanus.&#160;

We describe approaches for the manipulation of genes in the evolutionary model system Astyanax mexicanus. Three different techniques are described: Tol2-mediated transgenesis, targeted manipulation of the genome using CRISPR/Cas9, and knockdown of expression using morpholinos. These tools should facilitate the direct investigation of genes underlying the variation between surface- and cave-dwelling forms.

Cave animals provide a compelling system for investigating the evolutionary mechanisms and genetic bases underlying changes in numerous complex traits, ...