Methods for detection of a target nucleic acid by capture
View Patent ↗The invention relates to a method of generating a signal indicative of the presence of a target nucleic acid in a sample, as well as a method of detecting or measuring a target nucleic acid in a sample. The invention comprises forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe having a secondary structure that changes upon binding of the probe to the target nucleic acid and further comprising a binding moiety. The invention also includes the steps of cleaving the cleavage structure with a nuclease to release a nucleic acid fragment to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid in a sample, and detecting and/or measuring the amount of the fragment captured by binding of a binding moiety to a capture element on a solid support.
1. A method of generating a signal indicative of the presence of a target nucleic acid in a sample, comprising
forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe comprising a binding moiety and a secondary structure that changes upon binding of said probe to said target nucleic acid, wherein said binding moiety comprises a region of said probe that is released upon cleavage of said probe with a nuclease and binds to a capture element, and
cleaving said cleavage structure with said nuclease at a cleaving temperature to release a nucleic acid fragment and generate a signal, wherein said secondary structure of said probe is stable at or below said cleaving temperature when not bound to said target nucleic acid, and generation of said signal is indicative of the presence of a target nucleic acid in said sample; and
detecting and/or measuring said signal.
2. A method of detecting or measuring a target nucleic acid comprising the steps of:
forming a cleavage structure by incubating a sample containing a target nucleic acid with a probe comprising a binding moiety and a secondary structure that changes upon binding of said probe to said target nucleic acid, wherein said binding moiety comprises a region of said probe that is released upon cleavage of said probe with a nuclease and binds to a capture element,
cleaving said cleavage structure with said nuclease at a cleavage temperature to release a nucleic acid fragment, wherein said secondary structure of said probe is stable at or below said cleaving temperature when not bound to said target nucleic acid; and
detecting and/or measuring the amount of said fragment captured by binding of said binding moiety to a capture element on a solid support as an indication of the presence of the target sequence in the sample.
3. A method of forming a cleavage structure comprising the steps of:
(a) providing a target nucleic acid,
(b) providing an upstream nucleic acid complementary to said target nucleic acid,
(c) providing a downstream probe having a secondary structure that changes upon binding of said probe to said target nucleic acid and said probe further comprises a binding moiety, wherein said binding moiety comprises a region of said probe that is released upon cleavage of said probe with a nuclease and binds to a capture element, and
(d) annealing said target nucleic acid, said upstream nucleic acid and said downstream probe; and
wherein said cleavage structure can be cleaved with said nuclease at a cleaving temperature, and said secondary structure of said probe is stable at or below said cleaving temperature when not bound to said target nucleic acid.
4. The method of claim 3 wherein the cleavage structure comprises a 5′ flap.
5. A method of generating a signal indicative of the presence of a target nucleic acid in a sample, comprising
forming a cleavage structure by incubating a sample comprising a target nucleic acid with a probe comprising a binding moiety and a secondary structure that changes upon binding of said probe to said target nucleic acid, wherein said binding moiety comprises a tag that binds to a capture element, and
cleaving said cleavage structure with said nuclease at a cleaving temperature to release a nucleic acid fragment and generate a signal, wherein said secondary structure of said probe is stable at or below said cleaving temperature when not bound to said target nucleic acid, and generation of said signal is indicative of the presence of a target nucleic acid in said sample; and
detecting and/or measuring said signal.
6. A method of detecting or measuring a target nucleic acid comprising the steps of:
forming a cleavage structure by incubating a sample containing a target nucleic acid with a probe comprising a binding moiety and a secondary structure that changes upon binding of said probe to said target nucleic acid, wherein said binding moiety comprises a tag that binds to a capture element,
cleaving said cleavage structure with said nuclease at a cleavage temperature to release a nucleic acid fragment, wherein said secondary structure of said probe is stable at or below said cleaving temperature when not bound to said target nucleic acid; and
detecting and/or measuring the amount of said fragment captured by binding of said binding moiety to a capture element on a solid support as an indication of the presence of the target sequence in the sample.
7. The method of claim 1 , 2 , 5 or 6 , further comprising a nucleic acid polymerase, wherein said nucleic acid polymerase and said cleavage structure are incubated under conditions wherein said nucleic acid polymerase forms a primer extension product.
8. The method of claim 1 , 2 , 5 , or 6 , wherein said cleavage structure further comprises a 5′ flap.
9. The method of claim 1 , 2 , 5 or 6 , wherein said cleavage structure further comprises an oligonucleotide primer.
10. The method of claim 1 , 2 , 5 or 6 , wherein said secondary structure is selected from the group consisting of a stem-loop structure, a hairpin structure, an internal loop, a bulge loop, a branched structure, a pseudoknot structure or a cloverleaf structure.
11. The method of claim 1 , 2 , 5 or 6 , wherein said nuclease is a FEN nuclease.
12. The method of claim 1 , 2 , 5 or 6 , wherein said probe further comprises at least one labeled moiety capable of providing a signal.
13. The method of claim 1 , 2 , 5 or 6 , wherein a cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned on said probe to quench the generation of a detectable signal when the probe is not bound to said target nucleic acid.
14. The method of claim 13 wherein said labeled moieties are separated by a site susceptible to nuclease cleavage, thereby allowing the nuclease activity of said nuclease to separate the first interactive signal generating moiety from the second interactive signal generating moiety by cleaving at said site susceptible to nuclease cleavage, thereby generating a detectable signal.
15. The method of claim 13 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
16. The method of claim 1 or 2 , wherein the probe further comprises a reporter.
17. The method of claim 16 , wherein the reporter comprises a tag.
18. The method of claim 5 or 6 , wherein said fragment is captured by binding of said tag to a capture element.
19. The method of claim 17 wherein said fragment is captured by binding of said tag to a capture element.