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 forming a cleavage structure by incubating a sample with a nucleic acid polymerase and a thermostable FEN-1 nuclease, wherein said cleavage structure comprises a 5′ flap that does not specifically anneal to said target nucleic acid sequence, and cleaving said cleavage structure with said 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 forming a cleavage structure by incubating a sample with a nucleic acid polymerase and a thermostable FEN-1 nuclease, wherein said cleavage structure comprises a 5′ flap that does not specifically anneal to said target nucleic acid sequence, cleaving said cleavage structure with said thermo stable 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, 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 thermostable 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.
4. A method of detecting or measuring a target nucleic acid sequence comprising forming a cleavage structure, 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 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.
5. A method of generating a signal indicative of the presence of a target nucleic acid sequence in a sample, comprising forming a cleavage structure, 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.
6. A method of detecting or measuring a target nucleic acid sequence comprising forming a cleavage structure, 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.
7. The method of claim 1 , 2 , 3 , 4 , 5 or 6 wherein a cleavage structure is formed comprising at least one labeled moiety capable of providing a signal.
8. The method of claim 1 , 2 , 3 , 4 , 5 , or 6 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.
9. The method of claim 8 wherein said pair of interactive signal generating moieties comprises a quencher moiety and a fluorescent moiety.
10. The method of claim 1 , 2 , 3 , 4 , 5 or 6 wherein a cleavage structure comprises at least one oligonucleotide primer.
11. The method of claim 1 , 2 , 3 4 , 5 or 6 wherein said nucleic acid polymerase substantially lacks 5′ to 3′ exonuclease activity.
12. The method of claim 1 , 2 , 5 or 6 wherein the nucleic acid polymerase is a DNA polymerase.
13. The method of any one of claims 1 , 2 , 3 , 4 , 5 or 6 wherein the polymerase is thermostable.
14. A polymerase chain reaction process for simultaneously forming a cleavage structure, 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, wherein said single stranded nucleic acid comprises a flap; and (iii) cleaving said cleavage
structure employing a thermo stable FEN-1 nuclease as a cleavage agent for release of labeled fragments from said cleavage structure thereby creating detectable labeled fragments.
15. The polymerase chain reaction process of claim 14 wherein said nucleic acid polymerase substantially lacks 5′ to 3′ exonuclease activity.
16. A method of forming a cleavage structure, 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 thermostable 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.
17. The method of claim 1 , 2 , 3 , 4 , 5 or 6 further comprising incubating said sample with a deoxyribonucleoside triphosphate.
18. The method of claim 17 , wherein said deoxyribonucleosidetriphosphate is one or more of dATP, dTTP, dGTP and dCTP.
19. The method of claim 1 , 2 , 3 , 4 , 5 or 6 wherein said thermostable FEN-1 is Archaeglobus fulgidus FEN-1 , Methanococcus jannaschii FEN-1 , Pyrococcus furiosus FEN-1, or Pyrococcus horikoshii FEN-1.