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, where the method includes 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. The invention also relates to a method of detecting or measuring a target nucleic acid in a sample, where the method includes forming a cleavage structure by incubating a sample containing a target nucleic acid with a probe having a secondary structure that changes upon binding of the probe to a target nucleic acid and comprising a binding moiety, and cleaving the cleavage structure with a nuclease to generate a cleaved nucleic acid fragment 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 sequence in a sample, comprising
annealing two non-overlapping oligonucleotides to the same strand of said target nucleic acid sequence so as to form a cleavage structure, wherein said cleavage structure comprises duplex and single-stranded nucleic acid, said single stranded nucleic acid comprising a 5′ flap that does not specifically anneal to said target nucleic acid sequence, and
cleaving said cleavage structure with a thermostable FEN- 1 nuclease so as to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
2. A method of detecting or measuring a target nucleic acid sequence in a sample comprising
annealing two non-overlapping oligonucleotides to the same strand of said target nucleic acid sequence so as to form a cleavage structure, wherein said cleavage structure comprises duplex and single-stranded nucleic acid, said single stranded nucleic acid comprising a 5′ flap that does not specifically anneal to said target nucleic acid sequence,
cleaving said cleavage structure with a thermostable FEN- 1 nuclease to release a nucleic acid fragment and
detecting and/or measuring the release of said fragment as an indication of the presence of the target sequence in the sample.
3. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising
forming a cleavage structure comprising duplex and single-stranded nucleic acid, by
incubating a sample comprising a target nucleic acid sequence with a DNA polymerase that substantially lacks 5′ to 3′ exonuclease activity, and
cleaving said cleavage structure with a FEN nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
4. A method of detecting or measuring a target nucleic acid sequence comprising
forming a cleavage structure comprising duplex and single-stranded nucleic acid, by incubating a sample comprising a target nucleic acid sequence with a DNA polymerase that substantially lacks 5′ to 3′ exonuclease activity,
cleaving said cleavage structure with a FEN nuclease to release a nucleic acid fragment and
detecting and/or measuring the release of said fragment as an indication of the presence of the target sequence in the sample.
5. The method of claim 3 or 4 , wherein the FEN nuclease is a flap specific nuclease.
6. The method of claim 3 , or 4 wherein the FEN nuclease is thermostable.
7. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising
forming a cleavage structure comprising duplex and single-stranded nucleic acid, by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase and
cleaving said cleavage structure with a thermostable, flap-specific FEN-1 nuclease to generate a signal, wherein generation of said signal is indicative of the presence of a target nucleic acid sequence in said sample.
8. A method of detecting or measuring a target nucleic acid sequence comprising
forming a cleavage structure comprising duplex and single-stranded nucleic acid, by incubating a sample comprising a target nucleic acid sequence with a nucleic acid polymerase,
cleaving said cleavage structure with a thermostable, flap-specific FEN-1 nuclease to release a nucleic acid fragment and
detecting and/or measuring the release of said fragment as an indication of the presence of the target sequence in the sample.
9. The method of claim 1 , 2 , 3 , 4 , 7 or 8 wherein a cleavage structure is formed comprising at least one labeled moiety capable of providing a signal.
10. The method of claim 1 , 2 , 7 , or 8 wherein a cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal, said labeled moieties being separated by a site susceptible to FEN-1 nuclease cleavage, thereby allowing the nuclease activity of the FEN-1 nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at said site susceptible to FEN-1 nuclease, thereby generating a detectable signal.
11. The method of claim 10 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
12. The method of claim 1 , 2 , 3 , 4 , 7 or 8 wherein a cleavage structure comprises at least one oligonucleotide primer.
13. The method of claim 7 or 8 wherein said nucleic acid polymerase substantially lacks 5′ to 3′ exonuclease activity.
14. The method of claim 7 or 8 wherein the nucleic acid polymerase is a DNA polymerase.
15. The method of claim 7 or 8 wherein the nucleic acid polymerase is thermostable.
16. A polymerase chain reaction process for simultaneously forming a cleavage structure comprising duplex and single-stranded nucleic acid, wherein said single stranded nucleic acid comprises a flap, amplifying a target nucleic acid sequence in a sample and cleaving said cleavage structure comprising:
(a) mixing in any order an upstream oligonucleotide primer complementary to a region in one strand of the target nucleic acid sequence and a downstream labeled probe complementary to a region in the same strand of the target nucleic acid sequence, wherein the upstream primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and the downstream probe contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and
(b) detecting a nucleic acid which is produced in a reaction comprising amplification of said target nucleic acid sequence and cleavage thereof wherein a nucleic acid polymerase is a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers to a target nucleic acid sequence, (ii) extending the primers of step (a) wherein said nucleic acid polymerase synthesizes primer extension products, and wherein the primer extension product of the primer of step (a) partially displaces the downstream probe of step (a) to form a cleavage structure comprising duplex and single stranded nucleic acid; and (iii) cleaving said cleavage structure employing a FEN nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments.
17. The polymerase chain reaction process of claim 16 wherein said nucleic acid polymerase substantially lacks 5′ to 3′ exonuclease activity.
18. A method of forming a cleavage structure comprising duplex and single-stranded nucleic acid, comprising providing a target nucleic acid sequence, providing an upstream primer complementary to said target nucleic acid sequence, providing a downstream probe complementary to said target nucleic acid sequence, extending the 3′ end of the upstream primer with a DNA polymerase that substantially lacks 5′ to 3′ exonuclease activity; and displacing the 5′ end of the downstream probe so as to form said cleavage structure.
19. A method for detecting a target sequence in a sample, comprising the steps of annealing two non-overlapping oligonucleotides to the same strand of said target nucleic acid sequence so as to form a cleavage structure on said target sequence that is cleavable by a FEN- 1 endonuclease, cleaving said cleavage structure with a cleavage agent under conditions such that at least a 5′ nucleotide is removed from a nucleic acid molecule contained in said cleavage structure and detecting cleavage of said cleavage structure, wherein detection of said cleavage is indicative of the presence of said target sequence.
20. The method of claim 19 , wherein said target sequence comprises a secondary structure that is spanned by at least one nucleic acid molecule contained in said cleavage structure.
21. The method of claim 19 , wherein said cleavage structure comprises at least one non-nucleotide constituent.
22. The method of claim 21 , wherein said non-nucleotide constituent is positioned in said cleavage structure to permit cleavage of said cleavage structure, such that, if said non-nucleotide constituent was not present in said position, said cleavage structure would not be cleaved by said cleavage agent.
23. The method of claim 3 or 4 wherein a cleavage structure is formed comprising a pair of interactive signal generating labeled moieties effectively positioned to quench the generation of a detectable signal, said labeled moieties being separated by a site susceptible to FEN nuclease cleavage, thereby allowing the nuclease activity of the FEN nuclease to separate the first interactive signal generating labeled moiety from the second interactive signal generating labeled moiety by cleaving at said site susceptible to FEN nuclease, thereby generating a detectable signal.
24. The method of claim 1 , 2 , or 19 , wherein said two non-overlapping oligonucleotides anneal to said target so they are separated by less than 20 nucleotides.
25. The method of claim 24 , wherein said two non-overlapping oligonucleotides anneal to said target so they are separated by 15 nucleotides.
26. The method of claim 24 , wherein said two non-overlapping oligonucleotides anneal to said target so they are separated by 10 nucleotides.
27. The method of claim 24 , wherein said two non-overlapping overlapping oligonucleotides anneal to said target so they are separated by 5 nucleotides.
28. The method of claim 1 , 2 or 19 wherein said method further comprises incubating said sample with a polymerase.
29. The method of claim 28 , wherein said polymerase substantially lacks 5′ to 3′ exonuclease activity.
30. The method of claim 28 , wherein said polymerase is a DNA polymerase.
31. The method of claim 28 , wherein said polymerase is thermostable.
32. The method of claim 1 , 2 or 19 wherein said method further comprises annealing a third oligonucleotide to the same strand of said target nucleic acid sequence, said third oligonucleotide being a primer.
33. The method of claim 1 , 2 or 19 further comprising incubating said sample with a deoxyribonucleoside triphosphate, wherein said annealing step is performed in the presence of said deoxyribonucleoside triphosphate.
34. The method of claim 33 , wherein said deoxyribonucleoside triphosphate is one or more of dATP, dTTP, dGTP, and dCTP.
35. The method of claim 31 further comprising incubating said sample with a deoxyribonucleoside triphosphate, wherein said annealing step is performed in the presence of said deoxyribonucleoside triphosphate.
36. The method of claim 35 , wherein said deoxyribonucleoside triphosphate is one or more of dATP, dTTP, dGTP, and dCTP.
37. The method of claim 32 further comprising incubating said sample with a dNTP deoxyribonucleoside triphosphate, wherein said annealing step is performed in the presence of said deoxyribonucleoside triphosphate.
38. The method of claim 37 wherein said deoxyribonucleoside triphosphate is one or more of dATP, dTTP, dGTP, and dCTP.
39. The method of claim 1 , 2 , 7 , 8 or 19 , wherein said thermostable FEN-1 is Archaeglobus fulgidus FEN-1 , Methanococcus jannaschii FEN- 1, Pyrococcus furiosus FEN- 1, or Pyrococcus horikoshii FEN-1.
40. The method of claim 3 , 4 or 16 , wherein said FEN is Archaeglobus fulgidus FEN-1, Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1, or Pyrococcus horikoshii FEN-1.