Cell-Free Expression of Immobilized DNA

An important part of the DiaCHIP is the possibility to ship and store information encoded by DNA to produce protein arrays on demand. Therefore, DNA has to be fixed on silicone slides first that will form one side of our microfluidic chamber. Using a cell-free expression mix, the DNA can then be transcribed and translated into proteins. If the coding sequence of the protein is genetically fused to a one of a tag, they can bind to a specific surface on the glass slide forming the other part of the chamber.

Immobilizing DNA on a PDMS Surface

Figure 1: Scheme of the APTES/PDITC surface.

Immobilizing DNA on a surface can be accomplished in a similar way as the immobilization of proteins if the DNA exhibits 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 flowcell consisting of the silicone PDMS (Polydimethylsiloxane), this silicone is first activated using oxygen plasma. Coupling of DNA is achieved using the crosslinker PDITC after binding of the silane APTES to the activated silicone. (Bild: layers)

Figure 2: Immobilization of DNA on 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 using a DNA concentration of 25 ng/µl spotted directly onto the slide (figure 2 (A)). The slide was subsequently incubated overnight 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 2 (B)). The resulting fluorescence pattern clearly corresponds to the spotting pattern on the slide, thereby confirming that the spotting of DNA was successful.

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 our cell-free expression mix. After incubation for two hours at room temperature the expressed GFP was detected using a standard fluorescence microscope (figure 3). More details on the vector design and cloning strategies to generate the needed DNA can be found here.