Capture moieties for nucleic acids and uses thereof
First Claim
1. A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe comprising:
- (i) a first nucleic acid sequence complementary to the target sequence; and
(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions.
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Accused Products
Abstract
A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe includes: a first nucleic acid sequence complementary to the target sequence (target-binding sequence); and a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex. In use, the target and target-bind sequence hybridize to form a duplex. A probe can be used to detect a molecule containing the target sequence, act as a primer for synthesis or amplification, etc.
42 Citations
95 Claims
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1. A unimolecular probe for hybridization to a molecule comprising a target nucleic acid sequence, the probe comprising:
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(i) a first nucleic acid sequence complementary to the target sequence; and
(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 29, 30, 31, 32, 33, 34, 35, 36, 37, 43, 44, 45, 46, 47, 49, 50, 52, 53, 62, 64, 68, 69, 70, 71, 72, 73, 91, 93, 94, 95)
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20. A unimolecular probe for detecting the presence or absence in a sample of a target molecule comprising a target nucleic acid, the probe comprising a molecule selected from the group consisting of:
A-B-C-D-E-F-G, D-E-F-G, and E-F-G, wherein;
(i) each of A, C, D, E and G is a nucleic sequence;
(ii) E and G are complementary to each other and covalently linked to each other by F so as to form a first intramolecular duplex under a defined set of conditions;
(iii) (a) for the molecule E-F-G, the entirety of sequence E and at least a portion of F together form a nucleic acid sequence substantially complementary to the target nucleic acid sequence of the target molecule, and the molecule E-F-G and the target molecule hybridize with each other such that the probe and target molecule form an intermolecular duplex under said conditions;
(b) for each of the molecules A-B-C-D-E-F-G and D-E-F-G, (1) the entirety of sequence E and at least a portion of D, or (2) the entirety of sequence E and at least a portion of D and at least a portion of F, together form a nucleic acid sequence substantially complementary to the target nucleic acid sequence of the target molecule, and each of the molecules A-B-C-D-E-F-G and D-E-F-G, and the target molecule, hybridize with each other such that the probe and target molecule form an intermolecular duplex under said conditions;
; and
(c) A and C of the molecule A-B-C-D-E-F-G are complementary to each other and covalently linked to each other by B so as to form a second intramolecular duplex under said set of conditions. - View Dependent Claims (21, 22, 23, 24, 25, 26, 27, 28, 38, 39, 40, 41, 42, 92)
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48. A kit for determining the presence in a sample of a first molecule -comprising a target nucleic acid sequence, the kit comprising:
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(1) a probe for determining the presence of the first molecule, the probe comprising;
(i) a first nucleic acid sequence complementary to the target sequence; and
(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions;
(2) a second molecule comprising a control nucleic acid sequence, the control nucleic acid sequence being different from the target nucleic acid sequence; and
(3) a probe for determining the presence of the second molecule, the probe comprising;
(i) a third nucleic acid sequence complementary to the control sequence; and
(ii) a fourth nucleic acid sequence complementary to a portion of the third nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under said set of conditions; and
wherein;
(iii) hybridization of the control and third sequences to form an intermolecular duplex occurs under said set of conditions.
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51. A composition comprising a mixture of a plurality of microparticles, each microparticle having a probe attached thereto for detection of a target nucleic acid sequence, each probe comprising a single molecule comprising:
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(i) a first tag sequence complementary to the target sequence; and
(ii) a nucleic acid second sequence complementary to a portion of the first tag sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and tag sequences to form an intermolecular duplex occurs under said set of conditions. - View Dependent Claims (54, 55, 56, 57, 58, 59, 60)
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61. A repertoire of oligonucleotide tag complements, each complement belonging to a population of like complements and being a unimolecular moiety comprising:
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(i) a first complement sequence complementary to a target tag sequence; and
(ii) a nucleic acid second sequence complementary to a portion of the first complement sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the complement and tag sequences to form an intermolecular duplex occurs under said set of conditions;
wherein there are at least ten said populations of tag complements, each tag of a population having a said complement sequence different from the complement sequence of the tags in every other population in the repertoire.
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63. A kit for sorting and identifying polynucleotides, the kit comprising:
a solid phase support having one or more spatially discrete regions, each such region having a uniform population of substantially identical probes covalently attached, wherein each probe is a unimolecular moiety comprising;
(i) a first tag sequence complementary to a target sequence of said polynucleotide to be identified; and
(ii) a nucleic acid second sequence complementary to a portion of the first tag sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and tag sequences to form an intermolecular duplex occurs under said set of conditions. - View Dependent Claims (65, 66, 67)
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74. A method for detecting a single-stranded nucleic acid target sequence, comprising:
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(a) providing a nucleic acid capture moiety comprising a unimolecular moiety comprising;
(i) a first nucleic acid sequence complementary to the target sequence; and
(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a defined set of conditions; and
wherein;
(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions;
(b) forming a reaction mixture comprising the single-stranded target nucleic acid and nucleic acid capture moiety under conditions such that the duplex of (a)(iii) forms;
(c) detecting the presence or absence of the duplex of (b);
such that the target single-stranded nucleic acid is detected.
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75. A method of amplifying a nucleic acid molecule, the method comprising:
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(a) providing a sample containing the nucleic acid molecule to be amplified;
(b) providing first and second probes, wherein;
(i) the first probe comprises a unimolecular capture moiety comprising;
(1) a forward primer; and
(2) a nucleic acid sequence complementary to a portion of the forward primer and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for amplifying the nucleic acid molecule;
(ii) the second probe comprises a unimolecular capture moiety comprising;
(3) a reverse primer, and (4) a nucleic acid sequence complementary to a portion of the reverse primer and capable of hybridization therewith to form a first intramolecular duplex under said set of conditions;
wherein;
(c) exposing the sample and first and second probes to said conditions so as to amplify the nucleic acid molecule.
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76. A method of synthesizing a nucleic acid molecule, the method comprising:
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(a) providing a sample containing or suspected of containing the nucleic acid to be synthesized;
(b) providing a probe comprising a unimolecular capture moiety comprising;
(1) a first nucleic acid sequence complementary to a sequence of the nucleic acid molecule to be synthesized so as to act as primer for said synthesis; and
(2) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for said synthesis; and
(c) exposing the sample and probe to said conditions so as to synthesize the nucleic acid molecule.
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77. A method of detecting first and second target sequences comprising first and second target nucleic acid sequences, the method comprising:
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(a) providing a sample that may contain the first and second sequences;
(b) providing first and second probes, wherein;
(i) the first probe comprises a unimolecular capture moiety comprising;
(1) a first nucleic acid sequence complementary to the first target sequence; and
(2) a nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form an intramolecular duplex therewith under a defined set of conditions;
(ii) the second probe comprises a unimolecular capture moiety comprising;
(3) a second nucleic acid sequence complementary to the second target sequence; and
(4) a nucleic acid sequence complementary to a portion of the second nucleic acid sequence and capable of hybridization therewith to form an intramolecular duplex therewith under said set of conditions;
wherein;
(iii) hybridization of the first target and first nucleic acid sequences to form a first intermolecular duplex occurs under said set of conditions, and hybridization of the second target and second nucleic acid sequences to form a second intermolecular duplex occurs under said set of conditions; and
(iv) the first and second target sequences differ from each other by at least one nucleotide base; and
(c) detecting the absence or presence of each of the first and second intermolecular duplexes. - View Dependent Claims (78, 79, 80, 81, 82)
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83. A process for amplifying nucleic acids comprising:
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(1) providing a nucleic acid template;
(2) hybridizing at least one primer to the 3′
-end of the template, wherein the primer comprises;
(i) a nucleic acid sequence complementary to said 3′
-end of the template and having a free 3′
-hydroxyl;
(ii) a nucleic acid sequence complementary to a portion of the nucleic acid sequence of (i) and capable of hybridization therewith to form a first intramolecular duplex under a set of conditions suitable for amplifying the nucleic acid molecule; and
(3) amplifying the primer-template hybrid using at least one temperature-stable polymerase enzyme to produce linear amplification products. - View Dependent Claims (84, 85)
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86. A process of claim 970, wherein the primer further comprises a modified nucleotide located immediately upstream of the nucleic acid sequence complementary to the 3′
- -end of the template so as to preclude extension of the 3′
-end of the template.
- -end of the template so as to preclude extension of the 3′
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87. A method of diagnosing a disease comprising:
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(1) obtaining a nucleic acid template from a biological sample;
(2) mixing, under a defined set of condition, at least one primer, specific for the disease associated with the expression of one or more genes, with the sample, the primer comprising;
(i) a first nucleic acid sequence complementary to the template; and
(ii) a second nucleic acid sequence complementary to a portion of the first nucleic acid sequence and capable of hybridization therewith to form a first intramolecular duplex under the set of conditions; and
wherein;
(iii) hybridization of the target and first sequences to form an intermolecular duplex occurs under said set of conditions; and
(3) detecting the presence or absence of the intermolecular duplex of (2)(iii) in order to determine the presence or absence of the template in the biological sample of (1).
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88. A method of diagnosing a disease comprising:
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(1) obtaining a nucleic acid template from a biological sample;
(2) mixing at least one primer, specific for the disease associated with the expression of one or more genes, with the sample to form a primer-template hybrid, the primer comprising;
(i) a nucleic acid sequence complementary to a 3′
-end of the template and having a free 3′
-hydroxyl;
(ii) a nucleic acid sequence complementary to a portion of the nucleic acid sequence of (i) and capable of hybridization therewith to form an intramolecular duplex under conditions in which the primer-template hybrid forms; and
(3) amplifying the primer-template hybrid using at least one temperature-stable polymerase enzyme to produce amplification products. - View Dependent Claims (89, 90)
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Specification