Team:Freiburg/Results/Immobilization

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Sabine kannst du das bitte korrigieren usw. Ich hab einfach mal versucht was zu schreiben, aber ich hab echt nicht so viel Plan ^^ (LS 13/9)
Überarbeitet (jb 20150915)

Cell-free expression of immobilized DNA

An important part of the DiaCHIP is the possibility to ship and store information encoded in DNA to produce protein-arrays on demand. Therefore, DNA has first to be fixed on silicone slides that later form the upper side of our microfluidic chamber. Using cell-free expression mix, the DNA can then be transcribed and translated into tagged proteins. These can bind the specific surface on the glass slide forming the lower part of the chamber.

Immobilizing DNA on a PDMS surface

Figure 1: Schematic of the APTES/PDITC surface.

Immobilizing DNA on a surface can be accomplished similar to the immobilization of proteins, if the DNA is tagged to an amino group. Therefore, we amplified our DNA templates by PCR using an amino-labeled revers primer and a Cy3-labeled forward primer. The Cy3-label enables us to detect the DNA after binding to the PDMS surface using an appropriate microarray scanner. To show the correct amplification of our constructs, an agarose-gel analysis was performed, confirming the right length of the DNA sequences (figure 1). As DNA has to be fixed on a flow cell consisting of the silicone PDMS (Polydimethylsiloxane), this silicone is first activated using oxygen plasma. Coupling of DNA is achieved using the cross-linker PDITC after binding of the silane APTES to the silicone. (Bild: layers)

Figure 2: Immobilization of DNA onto a PDMS slide. A: Top view on the slide indicating the spotting pattern. B: Microarray scanner measurement of Cy3 fluorescence.

Coupling of DNA to the PDMS slide was achieved usign a DNA concentration of 25 ng/µl spotted directly onto the slide (figure 2A). The slide was subsequently incubated over night and the DNA-solution was dried afterwards at 60°C. After washing the slide, binding was confirmed by measuring the Cy3 fluorescence in a microarray scanner (figure 2B). The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide, thereby confirming that the spotted DNA is responsible for the fluorescence signal.

Cell-free expression of GFP from spotted DNA

Figure 3: Cell-free expressed GFP confirmed by fluorescence microscopy

To confirm, that DNA was not only bound to the PDMS slide but is also suited for cell-free expression, we flushed the microfluidic chamber described above with cell-free expression mix. After incubation for two hours at room temperature the expressed GFP was be detected using a standard fluorescence microscope (figure 3). More details on vector design and cloning strategies to generate the needed DNA can be found here.