We designed, built and tested our prototype for DNA extraction on a grapevine leaf.
We demonstrated that the DNA extracted using our prototype can be amplified by RPA. Lane 5 of the gel corresponds to the amplification by RPA of grapevine DNA (EC) using the extraction done in the video as the template.
We have demonstrated that phytoplasma DNA sequences can be detected at low concentration in Microneedle extract. Our limit of detection test shows an amplification of Flavescence Dorée sequences using an initial template concentration as low as 10 copies/μl (lane 7).
The microneedle extract solution has therefore been shown not to inhibit the RPA reaction, as the limit of detection for Flavescence Dorée is the same in nuclease free water as in microneedle extract (see Amplification page).
We did an in vitro transcription reaction of our RPA products (lanes 1, 4 and 7) using T7 polymerase that generated RNA which was detected by electrophoresis in a denaturing gel. This gel demonstrate that RPA product can successfully be transcribed, therefore creating a toehold trigger RNA sequence.
The fluorescence curve shows that the BN toehold expresses GFP in presence of the trigger dsDNA. When there is no trigger, small GFP expression can be observed due to leakage. This leakage is very small compared to the expression of the activated toehold.
We expressed the enzyme CDO into OnePot PURE, it reacts with the substrate catechol which creates a yellow color. We used two controls, one with CDO but no catechol, the other with catechol but no CDO. The yellow color can be seen after 30 minutes of incubation, and it becomes brighter one hour after the start of the reaction. There are no colors in both controls, this proves that the color is indeed created by the reaction of CDO with catechol and not by self-oxidation of catechol.
Although we have not proven yet that our test works as a whole, we have shown that every individual part works and that most of them can be linked. We showed that amplification by RPA works on plant DNA extracted by the microneedle patches. Our experiments also proved that the DNA amplified by RPA can successfully be transcribed into the trigger RNA in order to activate the toehold switches. Finally it was shown that the toeholds can be activated in the cell-free system OnePot PURE and generate a fluorescent readout. We could also independently assess that the enzyme CDO creates a yellow color when it reacts with catechol.
To have a fully functional test, we would only need to show that phytoplasma DNA can consistently be extracted in sufficient quantities for amplification using our prototype, as well linking the activation of toeholds with the generation of a signal based on a CDO system.