Name: a droplet-based microfluidic approach and microsphere-pcr amplification for single-stranded dna amplicons
Uploaded: 2018-11-14T13:45:00
Description: Watch this Scientific Journal Video about A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons Video at JoVE.com

This study is supported by a project entitled &#34;Cooperative Research Program for Agriculture Science &#38; Technology Development (Project No. PJ0011642)&#34; funded by the Rural Development Administration, Republic of Korea. This research was also partly supported by a grant (NRF-2017R1A2B4012253) of the Basic Science Research Program through the National Research Foundation (NRF) funded by the Ministry of Science, ICT &#38; Future Planning, Republic of Korea. This research was also supported by a grant (N0000717) of the Education program for Creative and Industrial Convergence funded by the Ministry of Trade, Industry and Energy, Republic of Korea.

1. Fabrication of a PDMS Microfluidic Platform
<ol>
	<li>Prepare 20 mL of liquid PDMS prepolymer by mixing base polymer and catalyst in a volume ratio of 10:1. Pour 10 mL of the liquid PDMS onto a prepared SU-8 mold on a silicon wafer for the upper part of the microfluidic network. For the bottom flat part, pour the same volume of liquid PDMS on the silicon wafer without a mold structure. 
	Note: The microfluidic network is designed in a CAD program and then converted into a photomask in order to fabricate a maste...

<ol>
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Contaminants of dsDNA are a major issue in ssDNA amplification. It remains difficult to minimize dsDNA amplification in conventional asymmetric PCR amplification15. In addition, although technical improvements for generating ssDNA have enabled us to increase the efficiency of sample throughput, ssDNA isolation is still problematic due to its high costs and incomplete purification yields.
Asymmetric PCR is one of the most challenging methods used when working with ssDNA....

Single-stranded DNA (ssDNA) has been extensively considered as a molecular recognition element (MRE) due to its intrinsic properties for DNA-DNA hybridization1,2. The development of ssDNA synthetic systems can lead to biological applications such as DNA microarrays3, oligotherapeutics, diagnostics, and integrated molecular sensing based on complementary interactions4,5.
To date, micrometer-scale polymer particles have been successfully demonstrated using microfluidic devices. Several microfluidic techniques have been proven to be powerful for producing highly homogenous microspheres on continuous flow in the microchannel environment6,7.
In the study of Lee et al.8, a droplet-based microfluidic platform for the microfluidic synthesis of copolymerizable oligo-microsphere and ssDNA amplification was reported. The microfluidic platform consists of two PDMS (polydimethylsiloxane) layers: an upper part with a microfluidic channel network for generating microsphere and a bottom flat part. These consist of three kinds of PDMS fluidic channels: 1) a flow focusing channel for droplet generation, 2) a serpentine channel for mixing two solutions, and 3) a sequential polymerization channel for microsphere solidification. Once two immiscible flows are introduced into a single PDMS fluidic channel, the flows can be forced through the narrow orifice structure. The flow behaviors such as channel geometry, flow-rate, and viscosity affect the size and morphology of the microsphere. Therefore, the main liquid stream can be divided into microscale monospheres9,10.
Here, a detailed microsphere-PCR protocol is provided for the amplification of ssDNA. First, a droplet-based microfluidic device design process is described. Then, the way in which polyacrylamide microspheres can be functionalized with random DNA template in a complementary manner is explained. Finally, a microsphere-PCR protocol for amplifying ssDNA is shown.

<table>
	<tbody>
		<tr>
			<td>liquid polydimethylsiloxane, PDMS</td>
			<td>Dow Corning Inc.</td>
			<td>Sylgard 184</td>
			<td>Components of chip</td>
		</tr>
		<tr>
			<td>40% Acrylamide:bis solution (19:1)</td>
			<td>Bio-rad</td>
			<td>1610140</td>
			<td>Components of Copolymerizable oligo-microsphere</td>
		</tr>
		<tr>
			<td>Ammonium persulfate, APS</td>
			<td>Sigma Aldrich</td>
			<td>A3678</td>
			<td>Hardener of acrylamide:bis solution</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine, TEMED</td>
			<td>Sigma Aldrich</td>
			<td>T9281</td>
			<td>Catalyst of ammonium persulfate</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma Aldrich</td>
			<td>M5904</td>
			<td>Table 1. Solution III. Component of microsphere reagents</td>
		</tr>
		<tr>
			<td>Cy3 labeled complementary oligonucleotide probes</td>
			<td>Bioneer</td>
			<td>synthesized</td>
			<td>Table 3. Sequence information&#160;</td>
		</tr>
		<tr>
			<td>ssDNA acrydite labeled probe</td>
			<td>Bioneer</td>
			<td>synthesized</td>
			<td>Table 1. Solution I. Component of microsphere reagents</td>
		</tr>
		<tr>
			<td>Tris</td>
			<td>Biosesang</td>
			<td>&#160;T1016</td>
			<td>Components of TE buffer, pH buffer solution</td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma Aldrich</td>
			<td>EDS</td>
			<td>Components of TE buffer, removal of ion (Ca2+)</td>
		</tr>
		<tr>
			<td>Ex taq</td>
			<td>Takara</td>
			<td>RR001A</td>
			<td>ssDNA amplification</td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Carl Zeiss</td>
			<td>LSM 510</td>
			<td>Identifying oligonucleotides expossure of microsphere surface</td>
		</tr>
		<tr>
			<td>Light Microscope</td>
			<td>Nikon Instruments Inc.</td>
			<td>eclipse 80i</td>
			<td>Caculating number of microspheres</td>
		</tr>
		<tr>
			<td>T100 Thermal Cycler</td>
			<td>Bio-rad</td>
			<td>1861096</td>
			<td>ssDNA amplification</td>
		</tr>
		<tr>
			<td>Hand-held Corona Treater</td>
			<td>Electro-Technic</td>
			<td>BD-20AC Laboratory Corona Treater</td>
			<td>Hydrophilic surface treatment</td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>As one</td>
			<td>HI-1000</td>
			<td>heating plate for curing of liquid PDMS</td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>kd Scientific</td>
			<td>78-1100</td>
			<td>Uniform flow of Solution I and Solution II</td>
		</tr>
		<tr>
			<td>Compressor</td>
			<td>Kohands</td>
			<td>KC-250A</td>
			<td>Flow control of Solution III</td>
		</tr>
		<tr>
			<td>Bright-Line Hemacytometer</td>
			<td>Sigma Aldrich</td>
			<td>Z359629</td>
			<td>Caculating number of microspheres</td>
		</tr>
	</tbody>
</table>

a droplet-based microfluidic approach and microsphere-pcr amplification for single-stranded dna amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and morphology of the obtained microsphere. A microsphere copolymerized with an acrydite-DNA probe was successfully fabricated. Different methods such as asymmetric PCR, exonuclease digestion, and isolation on streptavidin-coated magnetic beads can be used to synthesize single-stranded DNA (ssDNA). However, these methods cannot efficiently use large amounts of highly purified ssDNA. Here, we describe a microsphere-PCR protocol detailing how ssDNA can be efficiently amplified and separated from dsDNA simply by pipetting from a PCR reaction tube. The amplification of ssDNA can be applied as potential reagents for the DNA microarray and DNA-SELEX (Systematic evolution of ligands by exponential enrichment) processes.

The fabricated polymeric droplet-based microfluidic platform consists of two PDMS layers (Figure 1a). Three kinds of microfluidic channel networks are used for generating microspheres: 1) Flow-focusing geometry as shown in Figure 1b, 2) a serpentine channel for mixing solution I and solution II, and 3) a polymerization channel for microsphere solidification. The height of all channels was 60 &#956;m. The channel length for mixing...

Watch this Scientific Journal Video about A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons Video at JoVE.com

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons Video (Video) | JoVE

This work provides a method for the fabrication of droplet-based microfluidic platforms and the application of polyacrylamide microspheres for microsphere-PCR amplification. The microsphere-PCR method makes it possible to obtain single-stranded DNA amplicons without separating double-stranded DNA.

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

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