Team:TU Dresden/DipGene

Testing for the presence of a specific sequence of DNA is needed in many contexts, from biological laboratories, to tests for genetic diseases or viruses that integrate into the human genome to testing for the absence or presence of antibiotic resistance in pathogens. State of the art methods include PCR and whole genome sequencing, both of which are expensive, slow and require advanced technologies. These limitations make genetic testing inaccessible to most of humanity.

We aim to provide a fast and easy tool for detecting any nucleic acid sequence of interest from microbial samples and human cells. By combining a novel DNA extraction method with a newly designed fusion protein, it will be possible to obtain a visual color readout within minutes, which will indicate the presence or absence of the sequence of interest. The method will be designed to be in the field applicable, this means, it will be cheap, fast and not require advanced technologies. Ideally it will not even require electricity. By doing so, we will be able to open a new scope of diagnostic application to a broad audience.

The basic steps of the procedure:

In the first step DNA will be extracted directly from the raw cell lysate by immobilizing it on a cellulose paper (based on a method developed by Zou et al. 2017).

After a short washing step DNA of a concentration around 40 ng/µl and of good purity will be left on the paper (260/280 ~ 1.8).
In the second step, the cellulose paper, is dipped into a dCas9-HRP (horseradish peroxidase) solution. The dCas9 will recognize and bind to the specific DNA sequence that is given by its guideRNA.

In the third and last step the strip will be transferred to a solution of the HRP substrate. If the fusion protein bound to the DNA it will be transferred to the third solution and there lead to a colour change of the substrate. Like this the colour change will only occur when the sequence of interest is present.

In order to design the fusion protein, the RCF25 translational fusion standard from the Freiburg team 2008 will be used. The restriction enzymes NgoMIV and AgeI leave a compatible overhang that ensures that the downstream protein sequence will still be read in frame.

For future development of our diagnostic technique the basic idea of the biobrick assembly comes in handy. As can be seen in the picture on the left dCas9 surrounded with the prefix and suffix of the RCF25 standard can be fused to any reporter protein of interest flanked by the same prefix and suffix sequences. With each reporter catalyzing a colour reaction for a different substrate, one could test for several different disease genes at once.
For laboratory applications it could addionally be of interest to fuse a variety of Cas-proteins to reporters, which can be used to target either RNA or DNA as needed.



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