A new label technology for the detection of specific polymerase chain reaction products in a closed tube.
A novel signal generation principle suitable for real time and end-point detection of specific PCR products in a closed tube is described. Linear DNA probes were labeled at their 5'-ends with a stable, fluorescent terbium chelate. The fluorescence intensity of this chelate is lower when it is coupled to single-stranded DNA than when the chelate is free in solution. The synthesized probes were used in the real time monitoring of PCR using a prototype instrument that consisted of a fluorometer coupled to a thermal cycler. When the probe anneals to a complementary target amplicon, the 5'-->3' exonucleolytic activity of DNA polymerase detaches the label from the probe. This results in an enhanced terbium fluorescence signal. Since terbium has a long excited state lifetime, its fluorescence can be measured in a time-resolved manner, which results in a low background fluorescence and a 1000-fold signal amplification. The detection method is quantitative over an extremely wide linear range (at least 10-10(7)initial template molecules). The label strategy can easily be combined with existing label technologies, such as TaqMan 5'-exonuclease assays, in order to carry out multiplex assays that do not suffer from overlapping emission peaks of the fluorophores.
Nurmi
J
J
Department of Biotechnology, Tykistökatu 6A, 6th Floor, University of Turku, 20520 Turku, Finland. jussi.nurmi@utu.fi
Ylikoski
A
A
Soukka
T
T
Karp
M
M
Lövgren
T
T
D002614:Chelating Agents; D015342:DNA Probes; D018076:DNA, Complementary; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D005470:Fluorometry; D006801:Humans; D009154:Mutation; D009696:Nucleic Acids / Q000032:analysis*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D017430:Prostate-Specific Antigen / Q000235:genetics; D013725:Terbium
D002614:Chelating Agents; D015342:DNA Probes; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D013725:Terbium; D004259:DNA-Directed DNA Polymerase; D017430:Prostate-Specific Antigen