Methods for detection of a target nucleic acid by forming a cleavage structure with a cleavage resistant probe
First Claim
1. A composition comprising a polymerase, a cleavage agent, an upstream primer and a downstream cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of an overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure, and wherein the cleavage resistant probe comprises a segment complementary to a target, the 5′
- -terminal position of said segment being designated the +1 position, and the position 3′
to the +1 position being designated the +2 position.
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Accused Products
Abstract
The invention relates to compositions and methods for generating a signal indicative of the presence of a target nucleic acid in a sample utilizing a cleavage resistant probe.
30 Citations
37 Claims
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1. A composition comprising a polymerase, a cleavage agent, an upstream primer and a downstream cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of an overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure, and wherein the cleavage resistant probe comprises a segment complementary to a target, the 5′
- -terminal position of said segment being designated the +1 position, and the position 3′
to the +1 position being designated the +2 position. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 24)
- -terminal position of said segment being designated the +1 position, and the position 3′
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23. A composition comprising a polymerase, a cleavage agent, an upstream oligonucleotide and a downstream cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion and said upstream oligonucleotide has at least one 3′
- terminal nucleotide which is non-complementary to a target, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of an overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure.
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25. A composition comprising:
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(i) a cleavage agent (ii) a target nucleic acid, which comprises in 3′
to 5′
order a first region, an extension region, and a second region;(iii) an upstream primer that is at least partially complementary to said first region of said target nucleic acid; (iv) a downstream cleavage resistant probe comprising a 5′
region and a 3′
region, wherein said 3′
region is at least partially complementary to said second region of said target nucleic acid and wherein said 5′
region is not complementary to said target nucleic acid, said cleavage resistant probe also includes a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure; and(v) a polymerase.
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26. A composition comprising:
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(i) a cleavage agent (ii) a target nucleic acid, which comprises in 3′
to 5′
order a first region, an extension region, and a second region;(iii) an upstream oligonucleotide that is at least partially complementary to said first region of said target nucleic acid and comprises at least one 3′
terminal nucleotide which is non-complementary to the target,(iv) a downstream cleavage resistant probe comprising a 5′
region and a 3′
region, wherein said 3′
region is at least partially complementary to said second region of said target nucleic acid and wherein said 5′
region is not complementary to said target nucleic acid, said cleavage resistant probe also includes a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure; and(v) a polymerase.
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27. 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 polymerase, an upstream primer and cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure and cleaving the cleavage structure at a position in said probe that is susceptible to cleavage with a cleavage agent to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid sequence in the sample.
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28. 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 polymerase, an upstream oligonucleotide and cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion and said upstream oligonucleotide has at least one 3′
- terminal nucleotide which is non-complementary to a target, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of an overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure and cleaving the cleavage structure at a position in said probe that is susceptible to cleavage with a cleavage agent to generate a signal, wherein generation of the signal is indicative of the presence of a target nucleic acid sequence in the sample.
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29. 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 polymerase, an upstream primer and cleavage resistant probe and cleaving the cleavage structure at a position in said probe that is susceptible to cleavage with a cleavage agent to release a nucleic acid fragment and detecting and/or measuring the release of the fragment as an indication of the presence of the target sequence in the sample.
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30. A method for detecting a target nucleic acid sequence in a sample comprising:
- mixing
a cleavage agent, an upstream primer, a downstream cleavage resistant probe, wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by said cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by said cleavage agent upon formation of a non-overlapping cleavage structure, a polymerase, and a target nucleic acid under conditions which are permissive for the steps of (i) annealing of the upstream primer and downstream cleavage resistant probe, (ii) extending the upstream primer wherein the polymerase synthesizes a primer extension product and forms a cleavage structure with the downstream cleavage resistant probe, and (iii) cleaving the cleavage structure with the cleavage agent to generate a detectable signal and detecting and/or measuring the signal. - View Dependent Claims (31)
- mixing
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32. A method of detecting a target nucleic acid, comprising:
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(a) providing; an upstream primer that is at least partially complementary to a first region of a target nucleic acid, and a downstream cleavage resistant probe comprising a 5′
region and a 3′
region, wherein the 3′
region is at least partially complementary to a second region of the target nucleic acid and wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by a cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by the cleavage agent upon formation of a non-overlapping cleavage structure;(b) mixing the target nucleic acid and the upstream primer and downstream cleavage resistant probe under conditions which permit formation of a duplex between the target nucleic acid and the upstream primer and the 3′
region of the downstream cleavage resistant probe;(c) subjecting the duplex to a polymerase activity under conditions which permit extension of the upstream primer by polymerization of a nucleic acid strand complementary to a length of the extension region sufficient to form a cleavage structure; (d) providing a cleavage agent under conditions such that cleavage of the cleavage structure occurs at a site located within the downstream cleavage resistant probe in a manner dependent upon the formation of the cleavage structure, thereby permitting cleavage of the downstream cleavage resistant probe; (e) detecting the cleavage of the downstream cleavage resistant probe. - View Dependent Claims (33, 34, 35)
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36. A method of detecting a target nucleic acid, comprising:
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(a) providing; an upstream primer that is at least partially complementary to a first region of a target nucleic acid, an upstream oligonucleotide that is at least partially complementary to an extension region of the target nucleic acid and comprises at least one 3′
terminal nucleotide which is non-complementary to the target, anda downstream cleavage resistant probe comprising a 5′
region and a 3′
region, wherein the 3′
region is at least partially complementary to a second region of the target nucleic acid and wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by a cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by the cleavage agent upon formation of a non-overlapping cleavage structure;(b) mixing the target nucleic acid, the upstream primer, upstream oligonucleotide and downstream cleavage resistant probe under conditions which permit formation of a duplex between the target nucleic acid and the upstream primer, the upstream oligonucleotide and the 3′
region of the downstream cleavage resistant probe;(c) subjecting the duplex to a polymerase activity and cleavage activity under conditions which permit extension of the upstream primer and cleavage of the cleavage resistant probe; and (d) detecting the cleavage of the downstream cleavage resistant probe.
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37. A method of detecting a target nucleic acid, comprising
(a) providing: -
a target nucleic acid, which comprises in 3′
to 5′
order a first region and a second region,a template nucleic acid, which comprises in 3′
to 5′
order a first region and a second region,a first upstream oligonucleotide that is at least partially complementary to said first region of said target nucleic acid, a first cleavage resistant probe comprising a 5′
region and a 3′
region, wherein said 3′
region is complementary to said second region of said target nucleic acid and wherein said 5′
region is not complementary to said target nucleic acid but is at least partially complementary to said first region of said template nucleic acid, and wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by a cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by the cleavage agent upon formation of a non-overlapping cleavage structure;a second cleavage resistant probe comprising a 5′
region and a 3′
region, wherein said 3′
region is at least partially complementary to said second region of said template nucleic acid and said 5′
region is not complementary to said template nucleic acid, and wherein said cleavage resistant probe comprises a cleavage resistant portion and a cleavage susceptible portion, wherein said cleavage resistant portion is not susceptible to cleavage by a cleavage agent upon formation of a overlapping structure and said cleavage susceptible portion is susceptible to cleavage by the cleavage agent upon formation of a non-overlapping cleavage structure;a cleavage agent, and a nucleic acid polymerase; (b) mixing said target nucleic acid, said template nucleic acid, said first said first upstream oligonucleotide, cleavage resistant probe, said second cleavage resistant probe, said cleavage agent and said nucleic acid polymerase under reaction conditions, wherein said reaction conditions permit; forming a duplex between said target nucleic acid and each of said first upstream oligonucleotide and said first cleavage resistant probe, wherein the 5′
region of the first cleavage resistant probe forms a first flap of a first cleavage structure,cleaving the first flap by said cleavage agent, thereby permitting release of the first flap, forming a second duplex with the released first flap, said template nucleic acid, and said second cleavage resistant probe, wherein the 5′
region of said second cleavage resistant probe forms a second flap of a second cleavage structure, andcleaving the second flap by said cleavage agent; and (c) detecting a signal generated from the cleaving of the second flap.
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Specification