Fluorescence energy transfer by competitive hybridization
AssigneeQuest Diagnostics Incorporated
InventorsChih-Sheng Chiang, Jose F Cuan
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Abstract
A method is provided for detecting the presence of nucleotides or monitoring nucleotide amplification. It utilizes fluorescence energy transfer by competitive hybridization. Competitive hybridization is achieved by using unequal length complementary probes which have a fluorophore on one probe and a quencher on the other. The fluorophore and quencher are juxtaposed in a manner wherein the proximity of the quencher to the fluorophore produces quenching of the fluorescence of the fluorphore.
Claims34
A method for real-time monitoring of nucleic acid amplification comprising:
- amplifying a target nucleic acid and monitoring said target nucleic acid during said amplification using a first oligonucleotide probe and a second oligonucleotide probe,
- said first probe;
- i) is capable of hybridizing to said target nucleic acid;
- ii) comprises a fluorophore;
- and
- iii) is not equal in length to said second probe;
- said second probe;
- i) is capable of hybridizing to said first probe;
- and
- ii) has a quencher molecule which quenches said first probe fluorophore when said first and second probes are hybridized to each other;
- and
- detecting fluorescence of said first probe fluorophore in real-time to monitor amplification, wherein an increase in fluorescence correlates with amplification.
A method for monitoring nucleic acid amplification comprising:
- (i) amplifying a target nucleic acid in a cycling amplification reaction in the presence of a first probe and a second probe;
- said first probe;
- i) is capable of hybridizing to said target nucleic acid;
- ii) comprises a fluorophore;
- and
- iii) is not equal in length to said second probe;
- said second probe;
- i) is capable of hybridizing to said first probe;
- and
- ii) has a quencher molecule which quenches said first probe fluorophore when said first and second probes are hybridized to each other;
- and
- (ii) assessing the amount of amplified target nucleic acid produced by said amplification reaction by detecting the amount of fluorescence of said first probe fluorophore during a plurality of cycles of said amplification reaction, wherein the amount of fluorescence correlates with the amount of amplified target nucleic acid.
Dependencies are inferred from the claim text.