Nucleic acid target detection using a detector, a probe and an inhibitor
The present invention generally pertains to methods for detecting the presence or absence of a particular nucleic acid sequence. The present invention generally relates to incorporating a detector into a target nucleic acid, adding an oligonucleotide probe, polymerase enzyme and an inhibitor to the reaction, and detecting interference of the oligonucleotide probe with the inhibitor as an indication of the presence of a particular target nucleic acid sequence as well as kits encompassing the same.
1. A method for detecting a target nucleic acid comprising:
a) incorporating a detector oligonucleotide conjugated to a fluorophore or a quencher into a target nucleic acid to form a reaction, said incorporating comprising:
i. ligation; or
ii. by amplification using a PCR primer comprising (i) a target-specific sequence on the 3′ end of the primer (ii) a universal nucleic acid sequence complementary to the inhibitor and (iii) the fluorophore or quencher;
b) adding an oligonucleotide probe to the reaction;
c) adding an inhibitor oligonucleotide to the reaction such that the inhibitor oligonucleotide hybridizes to the incorporated detector, wherein the inhibitor oligonucleotide is conjugated to a quencher if the detector oligonucleotide is conjugated to a fluorophore and wherein the inhibitor oligonucleotide is conjugated to a fluorophore if the detector oligonucleotide is conjugated to a quencher and wherein hybridization of the inhibitor oligonucleotide to the detector oligonucleotide quenches signal from the fluorophore; and
d) detecting interference of the oligonucleotide probe with the inhibitor oligonucleotide as an indication of the presence of the target nucleic acid, thereby detecting a target nucleic acid, wherein the detecting comprises performing a strand displacement reaction with a polymerase that extends the oligonucleotide probe, displaces the inhibitor oligonucleotide, and no longer quenches the fluorophore to generate a fluorescent signal if the oligonucleotide probe hybridizes to the target nucleic acid.
2. A method according to claim 1 , wherein the target nucleic acid is single stranded.
3. A method according to claim 1 , wherein a single nucleotide within the target nucleic acid is detected.
4. A method according to claim 1 , wherein a sequence of 6-8 nucleotides within the target nucleic acid is detected.
5. A method according to claim 1 , wherein the entire target nucleic acid is detected.
6. A method according to claim 1 , wherein a variance within the target nucleic acid is detected.
7. A method according to claim 1 , wherein a mutation within the target nucleic acid is detected.
8. A method according to claim 1 , wherein a polymorphism within the target nucleic acid is detected.
9. A method according to claim 1 , wherein the detector oligonucleotide is conjugated to a fluorophore.
10. A method according to claim 1 , wherein the inhibitor oligonucleotide is conjugated to a quencher.
11. A method according to claim 1 , wherein the detector oligonucleotide is conjugated to a quencher.
12. A method according to claim 1 , wherein the inhibitor oligonucleotide is conjugated to a fluorophore.
13. A method according to claim 1 , wherein the detector oligonucleotide is conjugated to an epitope.
14. A method according to claim 1 , wherein the inhibitor is conjugated to an epitope.
15. A method according to claim 1 , wherein more than one target nucleic acid is detected.
16. A method according to claim 1 , wherein the method takes place within an emulsion.
17. A method according to claim 16 , wherein the emulsion is in a microfluidic device.
18. The method of claim 1 , wherein the detector oligonucleotide has a 5′ end and the fluorophore or quencher is conjugated to the 5′ end of the detector oligonucleotide.
19. The method of claim 1 , wherein the incorporating comprises ligation.
20. The method of claim 1 , wherein the incorporating comprises amplification using a PCR primer comprising a target-specific sequence on the 3′ end of the primer and a universal nucleic acid sequence complementary to the inhibitor on the 5′ end of the primer.