High-performance closed-tube PCR based on switchable luminescence probes.
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.
Department of Biotechnology, University of Turku, Turku, Finland. ari.lehmusvuori@utu.fi
Ari
A
Lehmusvuori
Ulla
U
Karhunen
Antti-Heikki
AH
Tapio
Urpo
U
Lamminmäki
Tero
T
Soukka
D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000235:genetics*; D028581:Lanthanoid Series Elements / Q000737:chemistry*; D049408:Luminescent Agents / Q000737:chemistry*; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D019914:Taq Polymerase / Q000378:metabolism
D028581:Lanthanoid Series Elements; D049408:Luminescent Agents; D015345:Oligonucleotide Probes; D004247:DNA; D019914:Taq Polymerase