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Team:EPFL/Microneedles


DNA
Extraction

Abstract

Extracting and isolating pathogenic DNA from plant tissues is currently limited by cumbersome multistep protocols that mostly require a laboratory environment. To address this challenge, a rapid plant DNA extraction method was employed using a disposable polymeric microneedle (MN) patch. By applying a MN patch on a plant leaf, amplification-ready DNA can be extracted within a minute. MN-extracted DNA was used for amplification of plant plastid DNA without purification. MN extraction achieved 100% detection rate of the vines DNA compared to DNeasy chemical and mechanical extraction method. This simple, cell-lysis-free, and purification-free DNA extraction method is a transformative approach that facilitates rapid sample preparation for molecular diagnosis of the vine plant diseases directly in the field.


Schematic of the conventional DNeasy extraction which involve 17 steps and takes about 1 to 1,5 hours


Schematic of microneedles extraction which only involve 3 steps and takes about 1 min

Principle

PVA gel and its derivatives are highly swell-able materials that can rapidly absorb 10−30% its own weight in water within minutes. Swelling-driven capillary flow is considered to be one of the main forces to concentrate intracellular DNA molecules around the microneedle tips. The fracture force of a polymeric MN patch can be up to ∼1 N/needle, strong enough to insert into skin and plant tissues without breaking. The MN patch performs two roles during the process:

(1) it penetrates deep into plant tissue in a minimally invasive fashion to break hard-to-lyse plant cell walls and release encapsulated nucleic acid materials;

(2) it absorbs and concentrates DNA and other molecules on the surface of the MN tips during the needle retraction. After MN retraction, plant DNA can be released from the needle tips by rinsing with elution buffer.

Methods

Fabrication of a Microneedle Patch

All MN patches used for DNA extraction were fabricated using polydimethylsiloxane molds. These molds were fabricated by laser ablation, and the dimension of each mold is approximately 10 mm × 10 mm, which has 15 × 15 arrays of microneedle conical cavities. The height of each cavity is 800 μm, and the diameters of the tip and base are 10 and 300 μm, respectively. To fabricate the microneedle patches, 0.5 mL of poly(vinyl alcohol) (30− 70 kDa, 10 wt %) solution was added to each silicone mold. After that, the molds are placed in a centrifuge for 20 min at 40°C at 4000 rpm to draw the PVA solution into the cavities and achieve the desired viscosity. These molds were then kept overnight at 25°C in a chemical hood vacuum. After being dried, the microneedle patches were carefully separated from the molds and stored at 25°C in a sealed Petri dish. The patches ought to be used within a month after their fabrication date.

FIGURE - A picture of one of our Microneedle patches.

MN Patch-Based DNA Extraction

There were two simple steps for MN patch-based DNA extraction from a fresh plant leaf. First, a fresh MN patch is gently placed on the surface of the leaf of interest. Then, a puncture force is delivered to the patch by finger pressing for a few seconds. Finally, the patch is peeled off and rinsed with 100 μL of TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0). For each extraction, a new MN patch was used. MN- extracted solutions were used each time without further purification. Please refer to the previous video for an illustrated procedure.

FIGURE - A picture of a leaf after application of a Microneedle patch. The extraction was performed three weeks before (left); one week before (right).

DNA Extraction with the DNeasy Plant Pro Kit

The protocol consists of multiple steps. Samples are added to the tissue disruption tube, which contains a specially shaped bead and a buffer for rapid homogenization. Cell lysis and DNA release occur by mechanical and chemical methods. Released genomic DNA is cleared of PCR inhibitors using QIAGEN’s second-generation​ IRT and then captured on a silica membrane in a spin column format. DNA is then washed and eluted from the membrane and is ready for PCR and other downstream applications. ​

PCR Amplification

After the extraction, all amplifications were performed by Polymerase Chain Reaction (PCR) using 1 µL of DNA , 25 µL of Q5 master mix and 2,5 µL of each primer in a 50 µL PCR reaction. We used EC_PCR_fwd/rev primer to amplify an amplicons of 214 bp of the vine gene.​

Gel Electrophoresis

After amplification, gel electrophoresis was performed to visualize the amplified PCR products. For that, agarose, 10× (TE) buffer, midori green gel stain ,gel pilot 6X loading dye,5 µL GelPilot 50 bp l DNA adder (Qiagen)were used. All PCR-amplified products were visualized in 2% agarose gel.

Results

We compared our DNA extraction method using microneedles with the conventional extraction method, by characterizing the quantity and purity of DNA extracted with both methods.

Nanodrop analysis

We used the Nanodrop to analyze the DNA extracted with a microneedle patch (red). The positive control (green) is EC synthetic DNA sequence and the negative control (blue) is the elution buffer applied on an unused microneedle patch.
As expected, the negative control absorption at 260nm is close to zero, no DNA is present in the microneedle patch. In contrast, the positive control has the typical DNA Nanodrop readout.
The graph of DNA extracted by microneedle patch shows a peak of absorption at 260nm, we can also see that that values absorption values at 230nm and 280nm are lower that the value at 260nm. Overall, the graph is similar to the positive control but with lower values. This indicates that DNA was extracted by the microneedle patch.

PCR amplification

We amplified different EC DNA by PCR and ran a gel electrophoresis to analyze the results. Bands are observed in all the lanes containing DNA, no band is present in the control. This shows that the microneedle patch successfully extracted DNA from the plant, which was then amplified by PCR.

Conclusion

We demonstrated that microneedle patches can be used to extract DNA from leaves in less than 1 minute. The extracted DNA could be then amplified by PCR and analyzed by gel electrophoresis. We can also note that the conventional method takes 1 to 2 hours and requires a lab and is therefore more than 100 times more time consuming than microneedle extraction, which is non neglectable.

References

Extraction of Plant DNA by Microneedle Patch for Rapid Detection of Plant Diseases, June 9, 2019, Rajesh Paul, Amanda C. Saville et al., [link to paper]

Preparation of Polyvinyl Alcohol (PVA) Solutions, 2019, Silverson., [link to website]

Dissolution behaviorof polyvinyl alcohol in waterand its effect on the physical morphologies of PLGAscaffolds, 2014, Adeyinka Aina, Andrew Morris et al., [link to paper]

A scalable fabrication process of polymer microneedles, 2012, Sixing Yang, Yan Feng, Lijun Zhang, Nixiang Chen, Weien Yuan, and Tuo Jin., [link to paper]