The Lightup Probes
We have constructed light-up probes for specific nucleic acid detection in homogeneous solution. The LightUp probe is a peptide-nucleic acid (PNA) linked to an asymmetric cyanine reporter dye. When added to a sample, the LightUp probe binds to complementary nucleic acid becoming intensively luminescent. Designing the LightUp probe with a melting temperature between the annealing and elongation temperatures of the PCR reaction, the LightUp probe can be present during PCR reporting the amount of product formed in real-time. Owing to the very high affinity of PNA for nucleic acid, the LightUp probe out-competes efficiently reannealing of the dsDNA product. Using LightUp probed real-time PCR we can determine the number of DNA molecules in biological samples with a precision of +/- 20% with a sensitivity that only is limited by sample preparation and the contamination problem.
1. N. Svanvik, J. Nygren, G. Westman & M. Kubista. Light-up Probes Thiazole Orange Conjugated PNA for Detection of Nucleic Acid in Homogeneous Solution. Anal. Biochem. 281, 26-35 (2000).
M. Kubista, N. Svanvik, G. Westman, A. Stålberg and Ulrica Sehlstedt1
Department of Molecular Biotechnology. Chalmers University of Technology,