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

Summary

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.

Abstract

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.

Introduction

Single-stranded DNA (ssDNA) has been extensively considered as a molecular recognition element (MRE) due to its intrinsic properties for DNA-DNA hybridization^1,2. The development of ssDNA synthetic systems can lead to biological applications such as DNA microarrays³, oligotherapeutics, diagnostics, and integrated molecular sensing based on complementary interactions^4,5.

To date, micrometer-scale polymer particles have been successfully demonstrated using microfluidic devices. Several microfluidic tech....

Protocol

1. Fabrication of a PDMS Microfluidic Platform

1. 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 master using the typical photolithography process (See Supplemental Figures). This master is comprised of the negative photoresist SU-8 mo....

Results

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 μm. The channel length for mixing and .......

Discussion

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........

Materials

List of materials used in this article

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