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.
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.
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.
1. Fabrication of a PDMS Microfluidic Platform
2. Production of Polyacrylamide Oligo-Microspheres
3. Performing Polyacrylamide Oligo-Microspheres Counts
4. Performing DNA Hybridization on the Surface of Polyacrylamide Oligomicrosphere
Note: An identical DNA probe with a 5’-NH2-group instead of 5’-acrytide modification is added into solution I and tested for Ap-containing microspheres in parallel. DNA hybridization results are shown in Figure 2. The Cy3-labeled complementary oligonucleotide probes (cAp) solution should be placed in a dark room.
5. Asymmetric PCR for Amplifying ssDNA
6. Microsphere-PCR for Amplifying ssDNA
Note: This section describes the protocol for amplifying ssDNA in a PCR reaction tube. Microsphere-PCR reactions were performed in 50 μL of reaction volume. The detailed sequences used to amplify ssDNA are listed in Table 5. In this case, Ap on the surface of microspheres can anneal to random DNA templates in a complementary manner. This is a very important step for producing complementary DNA strands (antisense DNA strand, Figure 3). The DNA extended is used as a template for microsphere-PCR amplification.
7. Confocal Microscopy Acquisition
Note: The results of microsphere-DNA probe hybridization are imaged under a confocal microscope. Image analysis is performed using ImageJ.
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 polymerization were 74.35 mm and 94.45 mm, respectively. The widths of the microchannel for two immiscible fluid flows and the one for mineral oil flow were 100 μm and 200 μm, respectively. The orifice structure used for microsphere formation was of 50 μm length and 25 μm width. The angle of the diffuser structure was 37°. A lab-based pneumatic control system for continuous flow of the mineral oil and two syringe-pumps for the solution flow (Figure 1c) were used for generating microspheres in the microfluidic platform (Figure 1d). The production speed of microspheres was about 30 microspheres per second when the flow rate of solutions and applied pressure were at 0.6 mL/h and 108 kPa, respectively (Table 2). Its diameter in the micro channel is 78.7 ± 2.5 μm. The on-flow synthesis of microspheres can be successfully manipulated using the microfluidic device. Bead sizes were measured after swelling. The average diameter of the resulting microspheres was 150.4 ± 12.8 μm. The size variations were about 8.5%. The microspheres undergo swelling in water, resulting in an enormous size increase.
The copolymerizable property of microsphere can be varied. We incorporated a 5'-acrydite-DNA probe into the microsphere solution (solution I, see Table 1). Following co-polymerization, a complementary DNA probe is labeled with fluorescent dye, Cy3, at room temperature for 1 h. Confocal microscopy can be used to prove the synthesis of copolymerizable oligomicrospheres as well as functional hybridization on the surface of microspheres. Fluorescent images of the microsphere are shown in Figure 2. If microspheres are correctly functionalized with the 5'-acrydite-DNA probe, they should result in coverage of fluorescent activity on the surface during the hybridization experiment. If copolymerization did not occur correctly, the optical microsphere image would exhibit internal-contamination inside the microspheres. As shown in Figure 2, there is no interference of random-interior orientation. This result allowed us to carry out the DNA probe presentation in a 3-dimensional (3-D) arrangement and microsphere-PCR. It should be noted that an identical DNA probe with a 5'-NH2-group instead of a 5'-acrytide modification did not copolymerize during on-flow synthesis of microsphere8.
When working with microspheres, manipulating the 3-D surface with a DNA oligo-probe is a much faster process14. Therefore, an on-flow microsphere synthesis platform can provide a tool for ssDNA amplification and purification, as outlined in Figure 3. The anti-sense DNA template (-, complementary template) can be extended by adding a random DNA template (+, template). In this case, the initially copolymerized 5'-Ap provides a 3'-OH end for DNA polymerization after annealing to its complementary template (76 nt). Microsphere-PCR can be performed in a single PCR microtube using functionalized microspheres, a random DNA template, and a Tag-forward primer. In order to distinguish the resultant ssDNA amplicons from random DNA template (+ template, 76 nt), the forward primer has an additional 24 nucleotide tag. If the forward primer does not have an additional tag sequence, it is very hard to recognize between ssDNA amplicons and the initial random DNA template.
An asymmetric PCR experiment was performed. We were able to observe dsDNA contaminants as shown in Figure 4. In most cases, it is necessary to isolate ssDNA using streptavidin-coated magnetic beads and exonuclease digestion. However, microsphere-PCR makes efficient use of a single primer (Tag-forward primer) to accumulate ssDNA in aqueous phase. It is expected that dsDNA contaminants will attach to the surface of microspheres. Therefore, ssDNA amplicons can be obtained through pipetting without the need for centrifugation. The resultant ssDNA was demonstrated by comparing it to the synthetic size markers (76 nt ssDNA and 100 nt ssDNA) through gel electrophoresis analysis.
Figure 1: The microfluidic platform. (a) Fabricated microfluidic platform, (b) enlarged view of the flow-focusing geometry, (c) experimental set-up, and (d) Captured images showing continuous generation of microspheres. 1: Micro channel structure for flow focusing geometry, 2: for mixing solutions, 3: for microsphere solidification. Please click here to view a larger version of this figure.
Figure 2: Fluorescent readout of DNA hybridization on the surface of microspheres. 5'-Acrydite-modified DNA probes (Ap) were capable of hybridizing with complementary Cy3 labeled DNA probes (cAp). Please click here to view a larger version of this figure.
Figure 3: Illustration of the microsphere-PCR protocol. Please click here to view a larger version of this figure.
Figure 4: Comparison between conventional asymmetric PCR and microsphere-PCR. M; ssDNA marker (76mer and 100mer), Lane 1; Asymmetric PCR, Lane 2; Microbeads-PCR. Reprinted with permission from previous work8. Please click here to view a larger version of this figure.
Reagent | Volume in mix (μL) | Final Concentration | |
Solution I | 40% Acrylamide:bis solution (19:1) | 25 | 10% |
100 μM Acrydite probe (Ap, 5’-Acrydite-(TTTTTTT, linker sequence) AGA TTG CAC TTA CTA TCT-3’) | 10 | 10 μM | |
5X TBE buffer (Tris-base-EDTA) | 10 | 0.5X | |
Water | 5 | – | |
Solution II | 20% Ammonium persulfate | 50 | 10% |
Solution III | TEMED (N,N,N′,N′-Tetramethylethylenediamine) | 8 | 0.40% |
Mineral oil | 1000 | – |
Table 1. Reagent mix components for on-flow polyacrylamide microsphere synthesis.
Operational condition | Microsphere production speed |
|
Solution flowrate | Oil pressure | |
(mL/h) | (kPa) | ( / sec) |
0.4 | 82 | 7.1 |
0.5 | 94 | 13.3 |
0.6 | 108 | 28.9 |
0.7 | 116 | 36.5 |
Table 2. Summary of operational conditions and produced microsphere.
DNA probe | Sequences |
Cy3 labeled complementary oligonucleotide probe (cAp) | 5’-Cy3-AGATAGTAAGTGCAATCT-3’ |
Tag (first 24 nt)-forward primer | 5’- GGT AAT ACG ACT CAC TAT AGG GAG ATA CCA GCT TAT TCA ATT-3’ |
Table 3. Sequence information of DNA probes.
Reagent | Volume for 1x reaction (μL) | Final concentration |
Random DNA template | 1 | 1 ng/μL |
Tag-forward primer | 1 | 0.4 μM |
Reverse primer | 1 | 0.02 μM |
10X Taq buffer | 5 | 1X |
10 mM of dNTP | 4 | 2.5 mM |
Ex taq (1000U) | 0.2 | 1 U |
Water | 37.8 | – |
Total | 50 | – |
Table 4. Asymmetric PCR reagent mix components in 20 μL reaction.
Template | Sequence | Length (nt) |
Random DNA template | 5’- ATA CCA GCT TAT TCA ATT (Random sequence, 40 mer) AGA TAG TAA GTG CAA TCT-3’ | 76 |
Tag-Forward primer (Tag-F) | 5’- GGT AAT ACG ACT CAC TAT AGG GAG ATA CCA GCT TAT TCA ATT-3’ | 42 |
Reverse primer | 5’- AGA TTG CAC TTA CTA TCT-3’ | 18 |
Table 5. Sequence information for Microsphere-PCR.
Reagent | Volume for 1x reaction (μL) | Final concentration |
Ap-Microspheres | ~25 microspheres | – |
Random DNA template | 1 | 1 ng/μL |
Tag-Forward primer | 1 | 0.4 μM |
10X Taq buffer | 5 | 1X |
10 mM dNTP | 4 | 2.5 mM |
Ex taq (1000U) | 0.2 | 1 U |
Water | 37.8 | – |
Total | 50 | – |
Table 6. Microsphere-PCR reagent mix components in 50 μL reaction.
Scan mode | Plane |
Scaling | X: 2.49 μm, Y: 2.49 μm |
Stack size | X: 1272.79 μm, Y: 1272.79 μm |
Scan zoom | 0.7 |
objective | EC Plan-Neofluar 10 x / 0.3 M27 |
Average | 1 |
Pinhole | Ch2: 104 um |
Filters | Ch3: LP 420 |
Beam splitters | MBS: HFT 488 / 543, DBS1: mirror, DBS2: NFT 515, FW1: None |
Wavelength | 543 nm 100.0 % |
Table 7. Settings for the confocal microscope.
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. This method applies unequal amounts of primer (e.g., 20: 1 ratio) in order to generate large amounts of ssDNA. However, it is very difficult to optimize every amplification reaction to yield ssDNA. Thus, the byproducts (dsDNA) must be eliminated from the resultants4.
In order to generate ssDNA without additional separation steps, we produced polyacrylamide microspheres to expose DNA probes based on the acrydite copolymerization method. Our DNA attachment method was easily adapted by using a droplet-based microfluidic platform. Copolymerized oligomicrospheres having microscale diameters were successfully produced. Consequently, microsphere-PCR was used to amplify the ssDNA in a single-tube reaction. Of course, to adapt this procedure for other PCR experiments, commonly necessary adjustments (e.g., changing the amplification cycle, annealing temperature, and template sequences) are required. In conclusion, microsphere-PCR has been detailed here, making it available for bioassay development, DNA sequencing, and DNA microarray analysis.
The authors have nothing to disclose.
This study is supported by a project entitled "Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0011642)" 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 & 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.
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 |