High-performance closed-tube PCR based on switchable luminescence probes

Abstract

We introduce a switchable lanthanide luminescence reporter technology based closed-tube PCR for the detection of specific target DNA sequence. In the switchable lanthanide chelate complementation based reporter technology hybridization of two nonfluorescent oligonucleotide probes to the adjacent positions of the complementary strand leads to the formation of a highly fluorescent lanthanide chelate complex. The complex is self-assembled from a nonfluorescent lanthanide chelate and a light-harvesting antenna ligand when the reporter molecules are brought into close proximity by the oligonucleotide probes. Outstanding signal-to-background discrimination in real-time PCR assay was achieved due to the very low background fluorescence level and high specific signal generation. High sensitivity of the reporter technology allows the detection of a lower concentration of amplified DNA in the real-time PCR, resulting in detection of the target at the earlier amplification cycle compared to commonly used methods.