Project Details
Projekt
Isothermal Mediator probe assays (Iso M-Tech)
Subject Area
Microsystems
Biochemistry
Measurement Systems
Veterinary Medical Science
Biochemistry
Measurement Systems
Veterinary Medical Science
Term
from 2011 to 2016
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 182656412
Real-time PCRs using different fluorogenic probes or DNA-microarrays are the gold standard to detect multiple nucleic acid sequences in one reaction. These fluorogenic probes are expensive in synthesis and fabrication of sequence-specific DNA-microarrays is time- and cost-intensive. Mediator probe PCR (MP PCR) is a novel method, which firstly reduces costs for fluorogenic probes in real-time PCR and secondly enables fabrication of universal DNA-microarrays: Mediator probes (MP) replace the typically used fluorogenic hydrolysis probes (HP). Since MPs are label-free, synthesis costs are reduced. MPs consist of two sequence regions, a target-sequence specific probe region and a generic sequence, the so-called mediator. During PCR the MP is cleaved and thus the mediator is released. The latter is then detected using fluorogenic mediator-specific probes, termed universal reporter (UR). In summary, the sequence to be detected is independent of the sequence amplified. Thus URs can be used in many different assays, either real-time PCR based or as a UR-microarray. Therefore, the same generic mediators are combined with different target-specific probe regions.Using MP PCR for real-time PCR based detection of multiple nucleic acid sequences of respiratory viruses was demonstrated in the project Mediatorsonden Technologie (M-Tech; FKZ STE 1937/1-1). This included for the first time detection of RNA sequences by RT-MP PCR. Current research objective is the solid-phase based detection using a UR-microarray.Goal of this proposed project is to fasten the detection reaction time, to reduce the demands on thermal stability of the UR-microarray immobilization, and to enable, in future, detection of thermally labile molecules such as proteins. Therefore, M-Tech must be adapted to process amplification isothermally. Previous tests using the isothermal Recombinase Polymerase Amplification (RPA) demonstrated the principle compatibility of RPA-enzymes with the MP cleavage and the signal generation reaction at the UR. However, efficiency of the single reactions is too low, not allowing for complete processing of RPA-based amplification, MP cleavage, and mediator setection at the UR, yet. Major task of the proposed project would be the systematic investigation of single reactions and the interacting oligonucleotides. This can lead to design rules for MPs and URs as well as establishment of reaction protocols for MP RPAs. Both, processing of MP RPA with URs present in a homogeneous reaction volume as well as MP RPAs in combination with a UR-microarray, shall be demonstrated.
DFG Programme
Research Grants
