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DNA-based Detection of Proteins.


Starick,  Stephan
Mechanisms of Transcriptional Regulation (Sebastiaan H. Meijsing), Dept. of Computational Molecular Biology (Head: Martin Vingron), Max Planck Institute for Molecular Genetics, Max Planck Society;

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Starick, S. (2010). DNA-based Detection of Proteins. Diploma Thesis, Universität Kassel, Kassel.

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Analyzing signaling pathways (SP) in small amount of cells or even in single cells is very challenging. Following information transmitting proteins requires a sensitive assay with outraging detection rates to distinguish modified vs. unmodified proteins. Proteins have to be detected over a wide concentration range. A temporal resolution of protein based answers in SP is needed additionally. The small quantities of proteins used in SP makes it necessary to improve signal amplification in immunoassays by DNA based detection of proteins. The outraging epitope recognition of monoclonal antibodies (AB) is combined with the rolling circle amplification (RCA) for signal amplification. AB’s are labeled with short DNA sequences serving as starting points for the RCA. Free primary amines of AB’s are used for conjugation to SH modified DNA. Conjugation could successfully be established, using Sulfo-SMCC as a conjugation reagent. Proof of conjugation succeeded with Cy3-labeled primer for visualizing the conjugation product. Readout of immunoassays using the DNA-AB conjugates is switched from a protein based one to DNA based readout. In contrast to established PCR-based signal amplification the RCA works at constant temperatures, not disrupting the non covalent antibody antigene interaction in immunoassays. The concatemeric RCA-Product is a key feature of this assay. The increased mass of DNA on the AB can be detected in multiple ways. A direct way for signal detection is the fluorescent dye incorporation. For indirect detection of the RCP, interaction of labeled probes is the method of choice. Fluorescent labeled Streptavidin interacts with the biotinylated RCP. The RCA for amplification of a circular template DNA could be expanded to DNA-AB conjugation products. The functionality of the AB after conjugation process was validated and subsequent RCA on membranes need to be optimized. The established method can be expanded to biosensing tests for examples in life sciences and more important in healthcare, using the protein microarrays for substance detection only present in small amounts of sample or in high dilutions.