Team:Freiburg/Results/Immobilization

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Immobilizing DNA

Intro

One of the core components of the DiaCHIP is the copying mechanism that allows production of a protein microarray from a DNA template. The following section summarizes our achievements concerning this procedure. All together we established a cell-free expression system that can be used to translate proteins from DNA immobilized on a PDMS slide. The expressed protein is then immobilized on the opposite glass slide via a specific tag system resulting in a distinct pattern. A microfluidic system is used to flush the slide with an antibody solution. Specific antibody-antigen interactions were successfully detected by imaging reflectometric interference (iRIf), a label-free detection method. For diagnostic applications, the immobilized proteins are antigenic peptides specific for a certain pathogen.

DNA on PDMS

Figure 1: DNA immobilization on activated PDMS. PCR amplification of the expression cassette was performed with an amino-labelled reverse primer and a Cy3-labelled forward primer. Immobilized DNA is visualized by Cy3 fluorescence.

The first step needed for copying the DNA array is to genetically fuse antigen coding sequences to a 10xHis tag that is used for surface immobilization later. The whole expression cassette including promoter and terminator regions is amplified by PCR using an amino-labeled reverse primer. Via this amino group, the DNA is immobilized on an activated PDMS surface. The forward primer used for this PCR is labeled with Cy3. As it is shown in figure 1, spotting the DNA on the activated surface resulted in a distinct pattern visualized by Cy3 fluorescence.
More details about vector design and cloning strategies can be found here.