Method for detecting target nucleic acid in a sample

Abstract

The present invention relates to methods for detecting target nucleic acids in a sample. The present invention provides probes, methods, kits and devices that provide accurate, rapid and sensitive multiplex detection, identification and quantification of target nucleic acids in a sample.

Description

[0001] This application is a divisional application of Chinese patent application No. 201780043954.2, filed on May 16, 2017, “Method for detecting target nucleic acid in a sample”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to and the benefit of USSN 62 / 337074, filed May 16, 2016, and USSN 62 / 492889, filed May 1, 2017. The contents of each application are incorporated by reference in their entirety.

[0004] Sequence Listing

[0005] This application contains a sequence listing, which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 16, 2017, is named NATE-032001WO_ST25.txt and is 22780 bytes in size. Background Art

[0006] Although there are currently a variety of methods for detecting nucleic acids in biological samples, there remains a need for improved, accurate, rapid, and sensitive multiplexed detection, identification, and quantification of target nucleic acids. The present invention satisfies this need. Summary of the Invention

[0007] The present invention provides probes, methods, kits and devices that provide accurate, rapid and sensitive multiplex detection, identification and quantification of target nucleic acids in a sample.

[0008] One aspect of the present invention is a method for detecting at least one target nucleic acid in a sample. The method comprises a first step of contacting the sample with at least one probe capable of recognizing and binding to a first specific region of at least one target molecule, wherein the at least one probe comprises a target binding domain and a barcode domain, wherein the target binding domain comprises at least 4 nucleotides, preferably 6 or more nucleotides, and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence; wherein the barcode domain comprises a barcode domain comprising a first attachment region (which comprises a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule, a first complementary nucleic acid molecule of a first reporter complex, or a first hybrid nucleic acid molecule) and at least a second attachment region (which comprises a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule, at least a second complementary nucleic acid molecule of at least a second reporter complex, or at least a second hybrid nucleic acid molecule), wherein the sequence of the first attachment region is different from the sequence of the at least second attachment region. The method further comprises the steps of: (2) binding a first complementary nucleic acid molecule comprising a detectable label or a first complementary nucleic acid molecule comprising a first reporter complex comprising a detectable label to a first attachment region, thereby associating the detectable label with the first attachment region; (3) detecting the detectable label associated with the first attachment region; (4) removing the first detectable label or the first complementary nucleic acid molecule; (5) binding at least a second complementary nucleic acid molecule comprising a detectable label or at least a second complementary nucleic acid molecule of at least a second reporter complex comprising a detectable label to at least a second attachment region, thereby associating the detectable label with the at least second attachment region; and (6) detecting the detectable label associated with the at least second attachment region; wherein the linear or sequential order of the detectable label associated with the first attachment region and the detectable label associated with the at least second attachment region recognizes a specific region of the at least one target molecule, thereby detecting at least one target nucleic acid in the sample. Steps (4) and (5) may occur sequentially or simultaneously.

[0009] In an embodiment, removing the first complementary nucleic acid in step (4) comprises contacting the first attachment region with a first hybrid nucleic acid molecule lacking a detectable label, thereby releasing the binding of the first complementary nucleic acid molecule and allowing the first hybrid nucleic acid molecule lacking a detectable label to bind to the first attachment region, or the change in pH, salt concentration and / or temperature is sufficient to remove the first complementary nucleic acid molecule.

[0010] In an embodiment, the barcode domain may comprise at least a third attachment region comprising a nucleic acid sequence capable of being bound by at least a third complementary nucleic acid molecule, at least a third complementary nucleic acid molecule of at least a third reporter complex, or at least a third hybrid nucleic acid molecule, wherein the sequence of at least the third attachment region is different from the sequence of another attachment region.

[0011] In embodiments, the method may further comprise the steps of: (7) removing the second detectable label or the second complementary nucleic acid molecule; (8) binding at least a third complementary nucleic acid molecule comprising the detectable label or at least a third complementary nucleic acid molecule comprising the detectable label to at least a third attachment region, thereby associating the detectable label with the at least third attachment region; and (9) detecting the detectable label associated with the at least third attachment region; wherein the linear or sequential order of the detectable label associated with the first attachment region, the detectable label associated with the at least second attachment region, and the detectable label associated with the at least third attachment region recognizes a specific region of the at least one target molecule, thereby detecting at least one target nucleic acid in the sample. Steps (7) and (8) may occur sequentially or simultaneously.

[0012] In an embodiment, removing the second complementary nucleic acid in step (7) comprises contacting the second attachment region with a second hybrid nucleic acid molecule lacking a detectable label, thereby releasing the binding of the second complementary nucleic acid molecule and allowing the second hybrid nucleic acid molecule lacking a detectable label to bind to the second attachment region, or the change in pH, salt concentration and / or temperature is sufficient to remove the second complementary nucleic acid molecule.

[0013] In embodiments, the barcode domain may comprise at least a fourth attachment region comprising a nucleic acid sequence capable of being bound by at least a fourth complementary nucleic acid molecule, at least a fourth reporter complex, or at least a fourth hybrid nucleic acid molecule, wherein the sequence of at least the fourth attachment region is different from the sequence of another attachment region. In embodiments, the barcode domain may comprise at least a fifth attachment region comprising a nucleic acid sequence capable of being bound by at least a fifth complementary nucleic acid molecule, at least a fifth reporter complex, or at least a fifth hybrid nucleic acid molecule, wherein the sequence of at least the fifth attachment region is different from the sequence of another attachment region. In embodiments, the barcode domain may comprise at least a sixth attachment region comprising a nucleic acid sequence capable of being bound by at least a sixth complementary nucleic acid molecule, at least a sixth reporter complex, or at least a sixth hybrid nucleic acid molecule, wherein the sequence of at least the sixth attachment region is different from the sequence of another attachment region. In embodiments, the barcode domain may comprise at least a seventh attachment region comprising a nucleic acid sequence capable of being bound by at least a seventh complementary nucleic acid molecule, at least a seventh reporter complex, or at least a seventh hybrid nucleic acid molecule, wherein the sequence of at least the seventh attachment region is different from the sequence of another attachment region.

[0014] In an embodiment, the following steps are repeated: removing the corresponding detectable label or complementary nucleic acid molecule, binding the complementary nucleic acid molecule comprising the detectable label or the complementary nucleic acid molecule of the reporter complex comprising the detectable label to the corresponding attachment area, thereby associating the detectable label with the corresponding attachment area; and detecting the corresponding detectable label associated with the attachment area until each attachment area in the barcode domain has been sequentially bound by a complementary nucleic acid molecule comprising the detectable label and the detectable label of the sequentially bound complementary nucleic acid molecules has been detected, wherein the linear or sequential order of the detectable labels associated with each attachment area identifies a specific region of at least one target molecule, thereby detecting at least one target nucleic acid in the sample.

[0015] In embodiments, the first hybrid nucleic acid molecule lacking a detectable label comprises at least the nucleic acid sequence of the first complementary nucleic acid molecule.

[0016] In an embodiment, the first attachment region may be adjacent to at least one flanking single-stranded polynucleotide or polynucleotide analog.The first hybrid nucleic acid molecule lacking a detectable label may further comprise a nucleic acid sequence that is partially complementary to at least one flanking single-stranded polynucleotide adjacent to the first attachment region.

[0017] In embodiments, the at least second hybrid nucleic acid molecule lacking a detectable label comprises at least the nucleic acid sequence of at least a second complementary nucleic acid molecule.

[0018] In an embodiment, at least the second attachment region may be adjacent to at least one flanking single-stranded polynucleotide or polynucleotide analog.At least the second hybrid nucleic acid molecule lacking a detectable label may comprise a nucleic acid sequence that is partially complementary to at least one flanking single-stranded polynucleotide adjacent to at least the second attachment region.

[0019] In embodiments, the barcode domain may comprise a synthetic backbone comprising a polysaccharide, a peptide, a peptide nucleic acid, a polypeptide, or a polynucleotide selected from single-stranded DNA, single-stranded RNA, or single-stranded PNA.

[0020] In an embodiment, at least one probe may comprise a single-stranded or double-stranded RNA, DNA, PNA or other polynucleotide analog, or PEG spacer between the target binding domain and the barcode domain. The spacer may be double-stranded DNA.

[0021] In an embodiment, the first complementary nucleic acid molecule of the first reporter complex, the at least second complementary nucleic acid molecule, and the at least second complementary nucleic acid molecule of the at least second reporter complex are independently RNA, DNA, PNA, or other polynucleotide analogs.

[0022] In embodiments, the at least third complementary nucleic acid or the at least third complementary nucleic acid of the third reporter complex can be RNA, DNA, PNA or other polynucleotide analogs.

[0023] In embodiments, at least one nucleotide in the target binding domain may be a modified nucleotide or nucleic acid analog. At least two, at least three, at least four, at least five, or at least six nucleotides in the target binding domain may be modified nucleotides or nucleic acid analogs. Each nucleotide in the target binding domain may be a modified nucleotide or nucleic acid analog. At least one modified nucleotide or at least one nucleic acid analog may be a locked nucleic acid (LNA). At least one modified nucleotide or at least one nucleic acid analog may comprise a universal base.

[0024] In an embodiment, the target nucleic acid can first be fixed on a substrate by at least combining the first position of the target nucleic acid with a first capture probe, the first capture probe comprising a first affinity binding reagent that selectively binds to the substrate. In an embodiment, the target nucleic acid is fixed on a substrate by at least combining the first position of the target nucleic acid with the first capture probe after being bound to the probe, the first capture probe comprising a first binding affinity reagent that selectively binds to the substrate. In an embodiment, the first capture probe binds to the target nucleic acid at different positions on the target nucleic acid with at least one probe bound to the target nucleic acid. The target nucleic acid can be extended by applying a force (e.g., gravity, fluid dynamics, electromagnetic force, flow stretching, receding meniscus technology, or a combination thereof) that is sufficient to extend the target nucleic acid that is fixed to the substrate at the first position. The target nucleic acid can be further fixed to the substrate by combining at least the second position of the target nucleic acid with at least the second capture probe, the second capture probe comprising an affinity binding reagent that selectively binds to the substrate. Generally, the second capture probe binds to the target nucleic acid at different positions on the target nucleic acid with at least one probe and the first capture probe bound to the target nucleic acid. The target nucleic acid can be further fixed on the substrate by combining at least a portion of the probe or a complementary nucleic acid molecule or a portion of the reporter complex with at least a third capture probe, and the third capture probe comprises a third affinity binding reagent that selectively binds to the substrate. The target nucleic acid can be further fixed on the substrate by combining a portion of the probe, at least one complementary nucleic acid molecule or a portion of at least one reporter complex with the substrate through a fourth affinity binding reagent. Typical affinity binding reagents include ligands, antigens, carbohydrates, receptors, lectins, antibodies, biotin, avidin, haptens and nucleic acids with known sequences. The target nucleic acid can be fixed on the substrate at about 3-at least 10 positions. Once the second position of the target nucleic acid is fixed on the substrate, the force can be eliminated. In an embodiment, the fixed target nucleic acid is extended.

[0025] In embodiments, the first capture probe may comprise a second affinity reagent.

[0026] In embodiments, the second affinity reagent of the first capture probe is different from the first affinity reagent of at least one probe.

[0027] In embodiments, the first capture probe may further comprise a third affinity reagent that is different from the second affinity reagent.

[0028] In embodiments, the first affinity reagent, the second affinity reagent, and the third affinity reagent are different.

[0029] In embodiments, the number of nucleotides in the target binding domain is equal to the number of different attachment regions in the barcode domain.

[0030] In embodiments, the number of nucleotides in the target binding domain may be at least one greater than the number of different attachment regions in the barcode domain.

[0031] In embodiments, the number of nucleotides in the target binding domain is at least twice the number of attachment regions in the barcode domain.

[0032] In an embodiment, the number of nucleotides in the target binding domain is 8, and the number of attachment regions in the barcode domain is 3.

[0033] In embodiments, the number of nucleotides in the target binding domain may be at least one less than the number of different attachment regions in the barcode domain.

[0034] In embodiments, the target binding domain of the probe comprises at least 6 nucleotides or at least 8 nucleotides.

[0035] In embodiments, the target binding domain of the probe comprises 10-100, 20-60, or 3550 nucleotides.

[0036] In embodiments, at least a first attachment region branches off from a first position on the barcode domain. In embodiments, at least a second attachment region branches off from at least a second position on the barcode domain. In embodiments, each attachment region branches off from a position on the barcode domain. The barcode domain may comprise a first position comprising at least two first attachment regions, wherein at least two first attachment regions comprise the same nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex. The barcode domain may comprise at least a second position comprising two at least second attachment regions, wherein at least two second attachment regions comprise the same nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule or a second complementary nucleic acid molecule of a second reporter complex. The barcode domain may comprise at least a third position comprising two at least third attachment regions, wherein at least two third attachment regions comprise the same nucleic acid sequence capable of being bound by at least a third complementary nucleic acid molecule or a third complementary nucleic acid molecule of a third reporter complex.

[0037] In an embodiment, each position in the barcode field may contain the same number of attachment regions. In an embodiment, at least one position in the barcode field may contain more than one attachment region. Each position in the barcode field may contain more than one attachment region.

[0038] In an embodiment, at least one position in the barcode field may contain a greater number of attachment regions than another position.

[0039] In an embodiment, at least one position on the barcode domain may comprise 1-50 copies of its attachment region, eg, each position on the barcode domain may comprise 1-50 copies of its attachment region.

[0040] In embodiments, at least one probe may comprise multiple copies of a target binding domain operably linked to a barcode domain.

[0041] In embodiments, each reporter complex comprising a detectable label may comprise a complementary nucleic acid molecule directly linked to a primary nucleic acid molecule.

[0042] In embodiments, each reporter complex comprising a detectable label may comprise a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a nucleic acid spacer.

[0043] In embodiments, each reporter complex comprising a detectable label may comprise a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a polymeric spacer having similar mechanical properties as the nucleic acid spacer.

[0044] In an embodiment, each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a cleavable linker.

[0045] In an embodiment, the cleavable linker is photocleavable, chemically cleavable or enzymatically cleavable. Generally, each cleavable linker is independently cleavable from all other linkers.

[0046] In embodiments, the photocleavable linker is cleaved by a light source such as an arc lamp, a laser, a focused UV light source, or a light emitting diode.

[0047] In embodiments, each complementary nucleic acid molecule may comprise between about 8 nucleotides and about 20 nucleotides, such as about 10 nucleotides, about 12 nucleotides, and about 14 nucleotides.

[0048] In an embodiment, each primary nucleic acid molecule can hybridize to at least one secondary nucleic acid molecule, e.g., at least 2 secondary nucleic acid molecules, at least 3 secondary nucleic acid molecules, at least 4 secondary nucleic acid molecules, at least 5 secondary nucleic acid molecules, and at least 6 secondary nucleic acid molecules. One or more secondary nucleic acid molecules can comprise at least one detectable label.

[0049] In embodiments, the secondary nucleic acid molecule may comprise a cleavable linker. For example, the cleavable linker is photocleavable, chemically cleavable, or enzymatically cleavable. In embodiments, the various secondary nucleic acid molecules hybridized to the primary nucleic acid molecule may all comprise the same cleavable linker, no cleavable linker, a combination of various cleavable linkers, or a combination of various cleavable linkers and no cleavable linker.

[0050] In embodiments, each secondary nucleic acid molecule can hybridize to at least one tertiary nucleic acid molecule comprising at least one detectable label, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 tertiary nucleic acid molecules comprising at least one detectable label.

[0051] In an embodiment, at least one secondary nucleic acid molecule may comprise a region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule. In an embodiment, each secondary nucleic acid molecule may comprise a region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may comprise a nucleotide sequence that is directly connected to the complementary nucleic acid molecule of the primary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may be located at the end of the secondary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may comprise between about 8 nucleotides and about 20 nucleotides, for example, about 12 nucleotides.

[0052] 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more and any number of different target nucleic acids.

[0053] In embodiments, the method may further comprise detecting at least one target protein in the sample.

[0054] In embodiments, at least one target protein may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more and any number in between different target proteins.

[0055] The terms "one or more", "at least one", etc. are understood to include, but are not limited to, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42 ,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more and any number in between.

[0056] The terms "plurality", "at least two", "two or more", "at least a second", etc. are understood to include, but are not limited to, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 0, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 0, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 1 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more and any number therebetween. Thus, "at least two marker attachment locations" includes, but is not limited to, 2 marker attachment locations, 4 marker attachment locations, 6 marker attachment locations, 8 marker attachment locations, 10 marker attachment locations or more.

[0057] The present disclosure also provides a method for detecting at least one target nucleic acid in a sample, the method comprising: (1) contacting the sample with at least one probe capable of recognizing and binding to a first specific region of at least one target molecule, wherein the at least one probe comprises: a target binding domain and a barcode domain, wherein the target binding domain comprises at least 4 nucleotides and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence; wherein the barcode domain comprises a barcode domain comprising a first attachment region (which comprises a nucleic acid sequence bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex) and at least a second attachment region (which is bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of at least a second reporter complex); wherein the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex comprises a first detectable label, such that the detectable label is associated with the first attachment region; wherein the at least second complementary nucleic acid molecule or the at least second complementary nucleic acid molecule of the at least second reporter complex comprises a second detectable label, such that the detectable label is associated with the at least second attachment region; wherein the sequence of the first attachment region is different from the sequence of the at least second attachment region; (2) detecting a first detectable label associated with a first attachment region and a second detectable label associated with at least a second attachment region; (3) removing the first detectable label; and (4) detecting the second detectable label associated with at least a second attachment region; wherein the linear or sequential order of the first detectable label associated with the first attachment region and the second detectable label associated with the at least second attachment region identifies a specific region of at least one target molecule, thereby detecting at least one target nucleic acid in a sample.

[0058] Detection in step (4) may include subtracting the signal from the second detectable label associated with at least the second attachment region detected in step (4) to form the signal from the first detectable label associated with the first attachment region and the second detectable label associated with at least the second attachment region detected in step (2).

[0059] The barcode domain may comprise a first attachment region comprising a nucleic acid sequence bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex, at least a second attachment region bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of a second reporter complex, and at least a third attachment region bound by at least a third complementary nucleic acid molecule or at least a third complementary nucleic acid molecule of a third reporter complex; wherein the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex comprises a first detectable label, such that the detectable label is associated with the first attachment region; wherein the second complementary nucleic acid molecule or the second complementary nucleic acid molecule of the at least second reporter complex comprises a second detectable label, such that the detectable label is associated with the at least second attachment region; wherein the third complementary nucleic acid molecule or the third complementary nucleic acid molecule of the at least third reporter complex comprises a third detectable label, such that the detectable label is associated with the at least third attachment region; wherein the sequences of the at least third attachment region, the at least second attachment region, and the at least third attachment region are different; (2) detecting the first detectable label associated with the first attachment region, the second detectable label associated with the at least second attachment region, and the at least third detectable label associated with the third attachment region; (3) removing a first detectable label; (4) detecting a second detectable label associated with at least a second attachment region and at least a third detectable label associated with a third attachment region; (5) removing the second detectable label; and (6) detecting a third detectable label associated with at least a third attachment region; wherein the linear or sequential order of the first detectable label associated with the first attachment region, the second detectable label associated with at least the second attachment region, and the at least third detectable label associated with the third attachment region identifies a specific region of at least one target molecule, thereby detecting at least one target nucleic acid in a sample.

[0060] Detection in step (4) may include subtracting the signal from the second detectable label associated with at least the second attachment zone and the at least third detectable label associated with the third attachment zone in step (4) to form the signal from the first detectable label associated with the first attachment zone, the second detectable label associated with at least the second attachment zone, and the at least third detectable label associated with the third attachment zone detected in step (2).

[0061] Detection in step (6) may include subtracting the signal from at least a third detectable label associated with the third attachment zone in step (6) to form the signal from the second detectable label associated with at least the second attachment zone and the at least third detectable label associated with the third attachment zone detected in step (4).

[0062] The present disclosure also provides a method for detecting at least one target nucleic acid in a sample, the method comprising: (1) contacting the sample with at least one probe capable of recognizing and binding to a first specific region of at least one target molecule, wherein the at least one probe comprises: a target binding domain and a barcode domain, wherein the target binding domain comprises at least 4 nucleotides and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence; wherein the barcode domain comprises a first attachment region (which comprises a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule, a first complementary nucleic acid molecule of a first reporter complex, or a first hybrid nucleic acid molecule) and at least a second attachment region (which comprises a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule, at least a second complementary nucleic acid molecule of at least a second reporter complex, or at least a second hybrid nucleic acid molecule); wherein the sequence of the first attachment region is different from the sequence of the at least second attachment region; (2) binding a first complementary nucleic acid molecule comprising a first detectable label or a first complementary nucleic acid molecule of a first reporter complex comprising a first detectable label to the first attachment region, thereby associating the detectable label with the first attachment region; (3) detecting the first detectable label associated with the first attachment region; (4) removing the first detectable label or the first complementary nucleic acid molecule; (5) allowing at least a second complementary nucleic acid molecule comprising a second detectable label or at least a second complementary nucleic acid molecule of at least a second reporter complex comprising a second detectable label to bind to at least a second attachment zone, thereby associating the detectable label with the at least second attachment zone; and (6) detecting the second detectable label associated with the at least second attachment zone; wherein the linear or sequential sequence of the first detectable label associated with the first attachment zone and the second detectable label associated with the at least second attachment zone recognizes a specific region of the at least one target molecule, thereby detecting at least one target nucleic acid in the sample. Steps (4) and (5) can occur sequentially or simultaneously.

[0063] The barcode domain can comprise at least a third attachment region comprising a nucleic acid sequence capable of being bound by at least a third complementary nucleic acid molecule, at least a third reporter complex, or at least a third hybrid nucleic acid molecule; wherein the sequence of at least the third attachment region is different from the sequence of another attachment region.

[0064] The method may further comprise: (7) removing the second detectable label or the second complementary nucleic acid molecule; (8) binding at least a third complementary nucleic acid molecule comprising a third detectable label or at least a third complementary nucleic acid molecule of at least a third reporter complex comprising a third detectable label to at least a third attachment region, thereby associating the detectable label with the at least third attachment region; and (9) detecting the third detectable label associated with the at least third attachment region; wherein the linear or sequential order of the first detectable label associated with the first attachment region, the second detectable label associated with the at least second attachment region, and the third detectable label associated with the at least third attachment region recognizes a specific region of the at least one target molecule, thereby detecting at least one target nucleic acid in the sample. Steps (7) and (8) occur sequentially or simultaneously.

[0065] Removal of the first complementary nucleic acid in step (4) may comprise: (a) contacting the first attachment region with a first hybrid nucleic acid molecule lacking a detectable label, thereby releasing the binding of the first complementary nucleic acid molecule and allowing the first hybrid nucleic acid molecule lacking a detectable label to bind to the first attachment region, and (b) a change in pH, salt concentration and / or temperature sufficient to remove the first complementary nucleic acid molecule.

[0066] Removal of the second complementary nucleic acid in step (7) may comprise: (a) contacting the second attachment region with a second hybrid nucleic acid molecule lacking a detectable label, thereby releasing the binding of the second complementary nucleic acid molecule and allowing the second hybrid nucleic acid molecule lacking a detectable label to bind to the second attachment region, and (b) a change in pH, salt concentration and / or temperature sufficient to remove the second complementary nucleic acid molecule.

[0067] The barcode domain may comprise at least a fourth attachment region comprising a nucleic acid sequence capable of being bound by at least a fourth complementary nucleic acid molecule, at least a fourth reporter complex, or at least a fourth hybrid nucleic acid molecule; wherein the sequence of at least the fourth attachment region is different from the sequence of another attachment region.

[0068] The barcode domain may comprise at least a fifth attachment region comprising a nucleic acid sequence capable of being bound by at least a fifth complementary nucleic acid molecule, at least a fifth reporter complex, or at least a fifth hybrid nucleic acid molecule; wherein the sequence of at least the fifth attachment region is different from the sequence of another attachment region.

[0069] The barcode domain may comprise at least a sixth attachment region comprising a nucleic acid sequence capable of being bound by at least a sixth complementary nucleic acid molecule, at least a sixth reporter complex, or at least a sixth hybrid nucleic acid molecule; wherein the sequence of at least the sixth attachment region is different from the sequence of another attachment region.

[0070] The barcode domain may comprise at least a seventh attachment region comprising a nucleic acid sequence capable of being bound by at least a seventh complementary nucleic acid molecule, at least a seventh reporter complex, or at least a seventh hybrid nucleic acid molecule; wherein the sequence of at least the seventh attachment region is different from the sequence of another attachment region.

[0071] The following steps are repeated: (a) removing the respective detectable labels or complementary nucleic acid molecules; (b) binding the complementary nucleic acid molecules comprising the detectable labels or the complementary nucleic acid molecules of the reporter complex comprising the detectable labels to the corresponding attachment regions, thereby associating the detectable labels with the corresponding attachment regions; and (c) detecting the corresponding detectable labels associated with the attachment regions, until each attachment region in the barcode domain has been sequentially bound by a complementary nucleic acid molecule comprising a detectable label and the detectable labels of the sequentially bound complementary nucleic acid molecules have been detected, wherein the linear or sequential order of the detectable labels associated with each attachment region identifies a specific region of at least one target molecule, thereby detecting at least one target nucleic acid in the sample.

[0072] The first hybrid nucleic acid molecule lacking a detectable label can comprise at least the nucleic acid sequence of the first complementary nucleic acid molecule.

[0073] The first attachment region can be adjacent to at least one flanking single-stranded polynucleotide or polynucleotide analog.

[0074] The first hybrid nucleic acid molecule lacking a detectable label can further comprise a nucleic acid sequence that is complementary to at least one flanking single-stranded polynucleotide portion adjacent to the first attachment region.

[0075] The at least second hybrid nucleic acid molecule lacking a detectable label can comprise at least the nucleic acid sequence of at least a second complementary nucleic acid molecule.

[0076] At least the second attachment region may be adjacent to at least one flanking single-stranded polynucleotide or polynucleotide analog.

[0077] The at least second hybrid nucleic acid molecule lacking a detectable label can comprise a nucleic acid sequence that is complementary to at least one flanking single-stranded polynucleotide portion adjacent to at least the second attachment region.

[0078] Removing the first detectable label of step (3) can include contacting the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex with a force sufficient to position the first complementary nucleic acid molecule to release the first detectable label.

[0079] Removing the second detectable label of step (5) can include contacting the second complementary nucleic acid molecule, or at least the second complementary nucleic acid molecule of the second reporter complex, with a force sufficient to position the second complementary nucleic acid molecule to release the second detectable label.

[0080] Removing the first detectable label of step (4) can include contacting the first complementary nucleic acid molecule, or at least the first complementary nucleic acid molecule of the first reporter complex, with a force sufficient to position the first complementary nucleic acid molecule to release the first detectable label.

[0081] Removing the second detectable label of step (7) can include contacting the second complementary nucleic acid molecule, or at least the second complementary nucleic acid molecule of the second reporter complex, with a force sufficient to position the second complementary nucleic acid molecule to release the second detectable label.

[0082] At least one of the first complementary nucleic acid molecule, the first complementary nucleic acid molecule of the first reporting complex, the at least second complementary nucleic acid molecule, the at least second complementary nucleic acid molecule of the at least second reporting complex, the at least third complementary nucleic acid molecule, or the at least third complementary nucleic acid molecule of the at least third reporting complex may comprise at least one cleavable linker.

[0083] At least one cleavable linker can be independently selected from photocleavable, chemically cleavable, and enzymatically cleavable. Each cleavable linker can be cleaved independently from all other linkers. The photocleavable linker can be cleaved by a light source selected from an arc lamp, a laser, a focused UV light source, and a light emitting diode. The force can be light.

[0084] The methods of the present disclosure may further comprise washing the probe from the at least one target nucleic acid.Washing may comprise changes in pH, salt concentration, and / or temperature sufficient to remove the probe from the target molecule.

[0085] The method of the present disclosure may further include: (i) contacting the sample with at least a second probe capable of recognizing and binding to a second specific region of at least one target molecule, wherein the second specific region is different from the first specific region of the at least one target molecule; (ii) contacting the sample with at least a second copy of the first probe capable of recognizing and binding to the first specific region of at least one target molecule; or (iii) contacting the sample with at least a third probe capable of recognizing and binding to the first specific region of at least a second target molecule, wherein the at least second target molecule is different from the at least one target molecule; wherein the probe comprises: a target binding domain and a barcode domain, wherein the target binding domain comprises at least 4 nucleotides; and wherein the barcode domain comprises a barcode domain, wherein the barcode domain comprises a first attachment region (which comprises a nucleic acid sequence bound by the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex) and at least a second attachment region (which is bound by the at least second complementary nucleic acid molecule or the at least second complementary nucleic acid molecule of the at least second reporter complex).

[0086] The method of the present disclosure may further include: (i) contacting the sample with at least a second probe capable of recognizing and binding to a second specific region of at least one target molecule, wherein the second specific region is different from the first specific region of the at least one target molecule; (ii) contacting the sample with at least a second copy of the first probe capable of recognizing and binding to the first specific region of at least one target molecule; or (iii) contacting the sample with at least a third probe capable of recognizing and binding to the first specific region of at least a second target molecule, wherein the at least second target molecule is different from the at least one target molecule; wherein the probe comprises: a target binding domain and a barcode domain, wherein the target binding domain comprises at least 4 nucleotides; and wherein the barcode domain comprises a first attachment region (which comprises a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule, a first complementary nucleic acid molecule of a first reporter complex, or a first hybrid nucleic acid molecule) and at least a second attachment region (which comprises a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule, at least a second complementary nucleic acid molecule of at least a second reporter complex, or at least a second hybrid nucleic acid molecule).

[0087] The method may further comprise repeating steps (1)-(6) of claim 3 with at least a second probe, at least a second copy of the first probe, or at least a third probe. The method may further comprise repeating steps (1)-(9) with at least a second probe, at least a second copy of the first probe, or at least a third probe. After washing the probe from the at least one target nucleic acid, steps (1)-(6) or steps (1)-(9) may be repeated up to about 50 times.

[0088] The detectable label can comprise multiple moieties, each of which can be identified by its emission spectrum. The detectable label can comprise quantum dots, fluorescent moieties, colorimetric moieties, or combinations thereof. Preferably, the detectable label comprises a fluorescent moiety. The emission spectrum of each moiety can be the same or different. The emission spectrum of at least one moiety can be different from that of the other moieties. In preferred aspects, the signal is an emission spectrum. In embodiments, the emission spectrum or spectrum of the label is the detectable signal.

[0089] The barcode domain may comprise a synthetic backbone comprising a polysaccharide, a peptide, a peptide nucleic acid, a polypeptide, or a polynucleotide selected from single-stranded DNA, single-stranded RNA, or single-stranded PNA. At least one probe may comprise a single-stranded or double-stranded RNA, DNA, PNA, or other polynucleotide analog, or PEG spacer between the target binding domain and the barcode domain. In a preferred aspect, the spacer is double-stranded DNA.

[0090] The first complementary nucleic acid, the first complementary nucleic acid molecule of the first reporter complex, the at least second complementary nucleic acid molecule, and the at least second complementary nucleic acid molecule of the at least second reporter complex can independently be RNA, DNA, PNA, or other polynucleotide analogs. The at least third complementary nucleic acid or the at least third complementary nucleic acid of the third reporter complex can be RNA, DNA, PNA, or other polynucleotide analogs.

[0091] At least one nucleotide in the target binding domain may be a modified nucleotide or nucleic acid analog. At least two, at least three, at least four, at least five, or at least six nucleotides in the target binding domain may be modified nucleotides or nucleic acid analogs. Every nucleotide in the target binding domain may be a modified nucleotide or nucleic acid analog. Except for the first and last nucleotides, every nucleotide in the target binding domain may be a modified nucleotide or nucleic acid analog.

[0092] At least one modified nucleotide or at least one nucleic acid analogue may be a locked nucleic acid (LNA).At least one modified nucleotide or at least one nucleic acid analogue may comprise a universal base.

[0093] The target nucleic acid can first be immobilized on a substrate before being contacted by a probe by binding at least a first position of the target nucleic acid to a first capture probe, the first capture probe comprising a first affinity binding reagent that selectively binds to the substrate, wherein the first capture probe binds to the target nucleic acid at a different position on the target nucleic acid than at least one probe binds to the target nucleic acid.

[0094] The target nucleic acid is immobilized on a substrate after binding to the probe by binding at least a first position of the target nucleic acid to a first capture probe, the first capture probe comprising a first binding affinity reagent that selectively binds to the substrate, wherein the first capture probe binds to the target nucleic acid at a different position on the target nucleic acid than at least one probe that binds to the target nucleic acid.

[0095] The target nucleic acid can be extended by applying a force sufficient to extend the target nucleic acid fixed to the substrate at the first location. The force can be gravity, fluid dynamics, electromagnetic force, flow stretching, receding meniscus technology, or a combination thereof.

[0096] The target nucleic acid can be further fixed to the substrate by binding at least a second position of the target nucleic acid to at least a second capture probe, wherein the at least second capture probe comprises a second affinity binding reagent that selectively binds to the substrate, wherein the second capture probe binds to the target nucleic acid at a different position on the target nucleic acid than at least one probe and the first capture probe.

[0097] The target nucleic acid can be further immobilized on the substrate by binding at least a portion of the probe or a complementary nucleic acid molecule or a portion of the reporter complex to at least a third capture probe comprising a third affinity binding reagent that selectively binds to the substrate.

[0098] The probe, the at least one complementary nucleic acid, or the at least one reporter complex may comprise a fourth affinity binding reagent.

[0099] The target nucleic acid can be further immobilized on the substrate by binding a portion of the probe, at least one complementary nucleic acid molecule, or a portion of at least one reporter complex to the substrate via a fourth affinity binding reagent.

[0100] Once the second location of the target nucleic acid is fixed to the substrate, the force can be removed.

[0101] Affinity binding reagents can be independently selected from the group consisting of ligands, antigens, carbohydrates, receptors, lectins, antibodies, biotin, avidin, haptens, and nucleic acids of known sequence.

[0102] The first capture probe may comprise a target binding domain comprising 20-60 nucleotides, and wherein the first capture probe binds to the target nucleic acid at a different location on the target nucleic acid than at least one probe that binds to the target nucleic acid. The first capture probe may comprise a target binding domain comprising 35-50 nucleotides.

[0103] The first affinity binding reagent may be different from the second affinity binding reagent.

[0104] At least one of the first affinity binding reagent, the second affinity binding reagent, the third affinity binding reagent, and the fourth affinity binding reagent may be different from the other affinity binding reagents.

[0105] The number of nucleotides in the target binding domain may be at least twice the number of attachment regions in the barcode domain. The number of nucleotides in the target binding domain may be 8, and the number of attachment regions in the barcode domain may be 3. The target binding domain may comprise at least 6 nucleotides. The target binding domain may comprise at least 8 nucleotides. The target binding domain may comprise 10-100 nucleotides. The target binding domain may comprise 20-60 nucleotides. The target binding domain may comprise 35-50 nucleotides.

[0106] Each complementary nucleic acid molecule may comprise between about 8 nucleotides and about 20 nucleotides. Each complementary nucleic acid molecule may comprise about 12 nucleotides. Each complementary nucleic acid molecule may comprise about 14 nucleotides.

[0107] At least a first attachment region may branch out from a first position on the barcode field. At least a second attachment region may branch out from at least a second position on the barcode field. Each attachment region may branch out from a position on the barcode field.

[0108] The barcode domain can comprise a first position comprising at least two first attachment regions, wherein the at least two first attachment regions comprise the same nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex.

[0109] The barcode domain can comprise at least a second position comprising at least two second attachment regions, wherein the at least two second attachment regions comprise the same nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of at least a second reporter complex.

[0110] The barcode domain can comprise at least a third position comprising at least two third attachment regions, wherein the at least two third attachment regions comprise the same nucleic acid sequence capable of being bound by at least a third complementary nucleic acid molecule or at least a third complementary nucleic acid molecule of at least a third reporter complex.

[0111] Each position in the barcode field may contain the same number of attachment zones. At least one position in the barcode field may contain more than one attachment zone. At least one position in the barcode field may contain a greater number of attachment zones than another position.

[0112] At least one probe may comprise multiple copies of a target binding domain operably linked to a barcode domain.

[0113] Each reporter complex that may include a detectable label comprises a complementary nucleic acid molecule directly linked to a primary nucleic acid molecule. Each reporter complex that may include a detectable label comprises a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a nucleic acid spacer. Each reporter complex that may include a detectable label comprises a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a polymer spacer having similar mechanical properties to the nucleic acid spacer. Each reporter complex that may include a detectable label comprises a complementary nucleic acid molecule indirectly linked to a primary nucleic acid molecule via a cleavable linker.

[0114] Cleavable linkers can be independently selected from photocleavable, chemically cleavable, and enzymatically cleavable. Each cleavable linker can be cleaved independently from all other linkers. Photocleavable linkers can be cleaved by a light source selected from an arc lamp, a laser, a focused UV light source, and a light emitting diode.

[0115] Each primary nucleic acid molecule can hybridize to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 secondary nucleic acid molecules.

[0116] One or more secondary nucleic acid molecules may comprise at least one detectable label. Each secondary nucleic acid molecule may hybridize to at least one, at least two, at least three, at least four, at least five, at least six or at least seven tertiary nucleic acid molecules comprising at least one detectable label. At least one secondary nucleic acid molecule may comprise a region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may comprise a nucleotide sequence that is directly connected to the complementary nucleic acid molecule of the primary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may be located at the end of the secondary nucleic acid molecule. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may comprise between about 8 nucleotides and about 20 nucleotides. The region that does not hybridize to the primary nucleic acid molecule and does not hybridize to the tertiary nucleic acid molecule may comprise about 12 nucleotides.

[0117] The present disclosure also provides kits comprising reagents for practicing any of the methods disclosed herein.

[0118] Any of the above aspects and embodiments may be combined with any other aspect or embodiment.

[0119] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0120] Unless the context clearly dictates otherwise, as used herein, the singular form of a word also includes the plural form of the word. For example, the terms "a," "an," and "the" are to be construed as singular or plural, and the term "or" is to be construed as inclusive. For example, "an element" means one or more elements.

[0121] Throughout the specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated element, integer or step or group of elements, integers or steps but not the exclusion of any other element, integer or step or group of elements, integers or steps.

[0122] About can be understood as being within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term "about".

[0123] Although methods and materials similar or equivalent to those described herein can be used for the practice or testing of the present invention, suitable methods and materials are described below. All disclosures, patent applications, patents and other references mentioned herein are combined in their entirety by reference. The references cited herein are not considered to be prior art for the claimed inventions. In the event of a conflict, this specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be restrictive. Other features and advantages of the present invention will be apparent from the following detailed description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0124] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0125] The above and further features will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings.

[0126] Figure 1 Schematic diagram showing exemplary probes of the present invention.

[0127] Figure 2 Schematic diagram showing exemplary probes of the present invention.

[0128] Figure 3 Schematic diagram showing exemplary probes of the present invention.

[0129] Figure 4 Schematic diagram showing exemplary probes of the present invention.

[0130] Figure 5A The method steps of the present invention are illustrated schematically.

[0131] Figure 5B The diagram shows Figure 5A The method steps of the present invention are initially described.

[0132] Figure 5C The diagram shows Figure 5A The method steps of the present invention are initially described.

[0133] Figure 5D The diagram shows Figure 5A The method steps of the present invention are initially described.

[0134] Figure 6 The diagram illustrates an example of a one-step purification process in which the probe and capture probe are added together to the target nucleic acid to form a tripartite complex. The tripartite complex is then purified by immobilizing the affinity reagent to a substrate via the capture probe.

[0135] Figure 7Another example of a one-step purification is illustrated, in which a capture probe comprising a binding moiety is bound to a target nucleic acid, the capture probe-target nucleic acid complex is then immobilized on a substrate via the binding moiety, and the probe then binds to the immobilized complex.

[0136] Figure 8A An example of multi-step purification is illustrated. Here, a probe comprising an affinity reagent is bound to a target nucleic acid, and then the probe-target nucleic acid complex is purified via the probe's affinity reagent (not shown). Subsequently, a capture probe comprising a binding moiety is bound to the complex to form a triple complex. Finally, the triple complex is purified by being immobilized on a substrate via the capture probe binding moiety.

[0137] Figure 8B Another example of multi-step purification is illustrated. Here, a probe comprising an affinity reagent is bound to a target nucleic acid, and the probe-target nucleic acid complex is then purified using the probe's affinity reagent (not shown). A capture probe, whose binding moiety has previously been immobilized on a substrate, captures the purified probe-target nucleic acid complex, thereby forming a purified and immobilized triple complex.

[0138] Figure 8C Another example of multi-step purification is illustrated. Here, a probe comprising an affinity reagent is bound to a target nucleic acid, and then the probe-target nucleic acid complex is purified via the probe's affinity reagent (not shown). Subsequently, a capture probe comprising a binding moiety and an affinity reagent (which is different from the affinity reagent on the probe) is bound to the complex to form a triple complex. The triple complex is purified via the affinity reagent on the capture probe (not shown). Finally, the purified triple complex is immobilized on a substrate via the binding moiety on the capture probe.

[0139] Figure 8D Another example of multi-step purification is illustrated. Here, a capture probe comprising a binding moiety and an affinity reagent is bound to a target nucleic acid, and then the capture probe-target nucleic acid complex is purified via the capture probe's affinity reagent (not shown). Subsequently, the probe is bound to the complex to form a triple complex. Finally, the triple complex is purified by being immobilized on a substrate via the binding moiety of the capture probe.

[0140] Figure 8E Another example of multi-step purification is illustrated. Here, a capture probe and a binding portion comprising a binding moiety and an affinity reagent are bound to a target nucleic acid, and then the capture probe-target nucleic acid complex is purified via the affinity reagent of the capture probe (not shown). Subsequently, a probe comprising an affinity reagent (which is different from the affinity reagent on the capture probe) is bound to the complex to form a triple complex. The triple complex is purified via the affinity reagent on the probe. Finally, the purified triple complex is fixed to a substrate via the binding moiety on the capture probe.

[0141] Figure 9A The initial steps of the method of the present invention are shown.

[0142] Figure 9B Schematic diagram showing a reporter complex comprising a detectable label.

[0143] Figure 9C A plurality of reporter complexes are shown, each reporter complex comprising a detectable label.

[0144] Figure 9D Display Figure 9A Further steps of the method of starting.

[0145] Figure 9E Display Figure 9A Further steps of the method of starting.

[0146] Figure 9F Display Figure 9A Further steps of the method of starting.

[0147] Figure 10 show Figure 9D and Figure 9E Alternative diagram of the steps shown and exemplary data obtained therefrom. The fragment of the probe shown has the sequence of SEQ ID NO: 70.

[0148] Figure 11 Graphic instructions Figure 10 Variation of the method shown. The fragment of the probe shown also has the sequence of SEQ ID NO: 70.

[0149] Figure 12A Various designs of reporter complexes of the invention are shown.

[0150] Figure 12B Display from Figure 12A Fluorescence counts obtained for the indicated reporter complexes.

[0151] Figure 12C Exemplary formulations for constructing reporter complexes of the present invention are shown.

[0152] Figure 13A The reporter complex design including the "extra handle" is shown.

[0153] Figure 13B Fluorescence counts obtained from reporter complexes with an "extra handle" are shown.

[0154] Figure 14A Hybridization kinetics are shown for two exemplary designs of reporter complexes of the invention.

[0155] Figure 14BHybridization kinetics are shown for two exemplary designs of reporter complexes of the invention.

[0156] Figure 15A Description of small barcode probe design.

[0157] Figure 15B Shown are data obtained using the methods of the invention when the probes were provided at lower concentrations.

[0158] Figure 15C Shown are data obtained using the methods of the present invention when the probes were provided at higher concentrations.

[0159] Figure 16A Shown are data obtained using the method of the present invention in which multiple target nucleic acids are detected simultaneously.

[0160] Figure 16B The data obtained using this method were compared to data obtained using probes comprising a detectable label.

[0161] Figure 17A Demonstration of Hyb&Count capture and detection of specific DNA targets.

[0162] Figure 17B Detection of targets in a 100plex capture plate is shown.

[0163] Figure 18 Intensity distribution of multicolor reporters is shown.

[0164] Figure 19 Showing the error rates for the 14-class model (left) and the 10-class model.

[0165] Figure 20 Schematic diagram showing the dual-color reporter probe.

[0166] Figure 21 Shown is the probe hybridization workflow for targeted nucleic acid capture.

[0167] Figure 22 Nucleic acid targeted capture for long-range phasing of haplotypes is shown.

[0168] Figure 23 A diagram illustrating a sequencing cycle using a pre-complexed BC with a cleavable RPTR, also referred to as a complementary nucleic acid molecule comprising a detectable label and a cleavable linker.

[0169] Figure 24 A diagram illustrating the method of using RPTR cutting and image subtraction to identify each RPTR.

[0170] Figure 25 Schematic representation of the construction of a cleavable RPTR probe and showing examples of cleavage modifications.

[0171] Figure 26 Variations in incubation time for hybridization are shown, and the total counts per field were used to determine the relative efficiency of the BC / RPTR complex compared to BC alone followed by RPTR binding in a second step. Figure 27 The BC / RPTR complex has slower binding kinetics than BC alone, but similar binding efficiency can be achieved with longer incubation times.

[0172] Figure 27 The identity of the RPTR can be determined using an image subtraction method. The BRAFex15-BC3 barcode was pre-complexed with a cleavable RPTR and processed for one full cycle. Four features are highlighted from a small portion of the image, and the changes in each fluorescence channel are shown in the bar graph. The RPTR bound to spot 3 (sp3) was first cleaved using the USER enzyme mix (a mixture of uracil DNA glycosylase (UDG) and DNA glycosylase-lyase endonuclease VIII), followed by cleavage of spot 1 (sp1) using exposure to UV light. The RPTR bound to spot 2 (sp2) is not cleavable.

[0173] Figure 28 Demonstrating detection and correct identification of a half-color GY RPTR after cleavage. BC was complexed with one UV-cleavable RPTR and two uncleavable RPTRs and hybridized to an immobilized DNA target on the flow cell surface. The fluorescence intensity of the RPTR was determined before and after UV exposure to cleave a single RPTR to determine the accuracy / extent to which half-color (i.e., GY instead of GG, full-color RPTR) can be detected in the presence of other reporters.

[0174] Figure 29 Shows detection and correct identification of full-color GY RPTR after cutting, used with Figure 6 For comparison, BC was complexed with one UV-cleavable RPTR and two uncleavable RPTRs and hybridized to an immobilized DNA target on the flow cell surface. The fluorescence intensity of the RPTR was determined before and after UV exposure to cleave a single RPTR to determine the accuracy / extent to which full color (i.e., GG) could be detected in the presence of the other reporters. Detailed Description of the Invention

[0176] The present invention provides probes, methods, kits and devices that provide accurate, rapid and sensitive multiplex detection, identification and quantification of target molecules in a sample.

[0177] Probes for detecting one or more nucleic acids in a sample

[0178] The present invention relates to probes comprising a target binding domain and a barcode domain. The target binding domain and the barcode domain are operably connected, for example, covalently linked. The probe is optionally included in a spacer between the target binding domain and the barcode domain. The spacer can be any polymer with appropriate mechanical properties, such as a single-stranded or double-stranded DNA spacer (1-100 nucleotides, for example, 2-50 nucleotides). Non-limiting examples of double-stranded DNA spacers include sequences encompassed by SEQ ID NO: 25-SEQ ID NO: 29. Other exemplary sequences that may be included in the barcode domain are listed with SEQ ID NO: 30-SEQ ID NO: 69.

[0179] Non-limiting examples of probes of the present invention are shown in Figure 1 -5 hits.

[0180] Figure 1 Schematic diagram showing the probes of the present invention. This exemplary probe has a target binding domain of 6 nucleotides. The target binding domain of each probe has a known nucleotide sequence. The barcode domain comprises one or more attachment regions; Figure 1 There are six attachment zones in the barcode domain. The first, third, and fifth attachment zones are annotated. The fifth position contains two attachment zones. Each position on the barcode domain can have multiple attachment zones. For example, a position can have 1-50 attachment zones. Some positions in the barcode domain can have more attachment zones than other positions (as shown here, position 5 relative to positions 1-4 and 6); alternatively, each position in the barcode domain has the same number of attachment zones. Although not shown, each attachment zone contains at least one (i.e., 1-50, e.g., 10-30) copies of a nucleic acid sequence that is capable of reversibly binding to a complementary nucleic acid molecule (RNA or DNA). In Figure 1 In the present invention, the attachment region is necessary to constitute the entire linear polynucleotide molecule that constitutes the barcode domain. The linear sequence of attachment positions and / or the linear sequence of positions identify the specific region of the target nucleic acid to which the target binding domain binds.

[0181] Figure 2A schematic diagram of the probes of the present invention is shown. This exemplary probe has a target binding domain of 5 nucleotides. The target binding domain of each probe has a known nucleotide sequence. The first attachment region is annotated; the first position on the barcode domain contains two first attachment regions that bind to the barcode domain (not necessarily to form the whole). The fourth position on the barcode domain is surrounded and contains a portion of the barcode domain and two fourth attachment regions. The two sixth attachment regions are annotated. Here, each position has two attachment regions; however, each position on the barcode domain can have one attachment region or multiple attachment regions, such as 2-50 attachment regions. Although not shown, each attachment region contains at least one (i.e., 1-50, such as 10-30) copies of a nucleic acid sequence that can reversibly bind to a complementary nucleic acid molecule (RNA or DNA). In Figure 2 In the present invention, the barcode domain is a linear polynucleotide molecule connected / branched by the attachment regions; the attachment regions are not required to constitute the entire polynucleotide molecule. The linear sequence of attachment positions and / or the linear sequence of positions identify the specific region of the target nucleic acid to which the target binding domain binds.

[0182] Figure 3 Another schematic diagram of a probe of the present invention is shown. This exemplary probe has a target binding domain of 4 nucleotides. Each position is shown to have 3 attachment regions connected to / branching from that position.

[0183] Figure 4 Still another schematic diagram of a probe of the present invention is shown. This exemplary probe has a target binding domain of 10 nucleotides. However, only the first 6 nucleotides are specific for the target nucleic acid. The 7th to 10th nucleotides (represented by "n1-n4") are added to increase the length of the target binding domain, thereby affecting the likelihood that the probe will hybridize and remain hybridized to the target nucleic acid. The "n" nucleotides may have universal bases (e.g., inosine, 2'-deoxyinosine (inosine deoxyribonucleotide) derivatives, nitroindole, nitropyrrole (nitroazole) analogs, and hydrophobic aromatic non-hydrogen bonding bases) that can base pair with any of the 4 canonical bases. In an embodiment, the "n" nucleotide may be in a position preceding a specific nucleotide of the target binding domain. In an embodiment, the "n" nucleotide may follow a specific nucleotide of the target binding domain. In Figure 4 4 "n" nucleotides are shown; however, the target binding domain may contain more or less than 4 "n" nucleotides. The target binding domain may lack "n" nucleotides. The second position contains 6 attachment regions that are connected to / branched from the second position of the barcode domain.

[0184] The target binding domain has at least 4 nucleotides, for example at least 4, 5, 6, 7, 8, 9, 10, 11, 12 or more nucleotides. The target binding domain may comprise 10-100, 20-160 or 35-50 nucleotides. The target binding domain is preferably a polynucleotide. The target binding domain is capable of binding to a target nucleic acid.

[0185] The probe may comprise multiple copies of the target binding domain operably linked to a synthetic backbone.

[0186] Probes can be designed to control the probability of hybridization and / or dehybridization and their incidence. Generally, the lower the Tm of the probe, the faster and more likely the probe will hybridize with / from the target nucleic acid. Therefore, using a lower Tm probe will reduce the number of probes that are bound to the target nucleic acid.

[0187] The length of the target binding domain partially affects the probability that the probe will hybridize and remain hybridized to the target nucleic acid. Generally, the longer the target binding domain (the greater the number of nucleotides), the less likely the complementary sequence is to be present in the target nucleic acid. Conversely, the shorter the target binding domain, the greater the probability that the complementary sequence is present in the target nucleic acid. For example, the probability that a tetramer sequence will be located in the target nucleic acid is 1 in 256, while the probability that a hexamer sequence will be located in the target nucleic acid is 1 in 4096. Therefore, compared to a collection of longer probes, a collection of shorter probes is likely to bind to a given stretch of nucleic acid at more locations.

[0188] The term "target nucleic acid" shall mean a nucleic acid molecule (DNA, RNA or PNA) whose presence in a sample is to be determined by the probes, methods and apparatus of the present invention. In general, the terms "target nucleic acid," "nucleic acid molecule," "nucleic acid sequence," "nucleic acid," "nucleic acid fragment," "oligonucleotide," and "polynucleotide" are used interchangeably and are intended to include, but are not limited to, polymeric forms of nucleotides (deoxyribonucleotides or ribonucleotides or analogs thereof) that can have various lengths. Non-limiting examples of nucleic acids include genes, gene fragments, exons, introns, intergenic DNA (including, but not limited to, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), non-coding RNA (ncRNA), cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, sequenced isolated DNA, sequenced isolated RNA, nucleic acid probes, and primers.

[0189] In certain embodiments, the target molecule is not a chromosome. In other embodiments, the target molecule is no larger than 1000 kb (or 1 mb), no larger than 500 kb, no larger than 250 kb, no larger than 175 kb, no larger than 100 kb, no larger than 50 kb, no larger than 20 kb, or no larger than 10 kb. In still other embodiments, the target molecule is isolated from its cellular environment.

[0190] The present method identifies and quantifies the nucleic acid molecules derived from the sample (e.g., sample from an organism), and preferably does not have a conversion (or amplification) step. As an example, for the RNA identification method, the present method does not need to convert the RNA molecule into a DNA molecule (i.e., synthesized through cDNA) before RNA can be identified. Owing to the fact that amplification or conversion is not required, in most cases, when nucleic acid is in a sample or when it derives from a sample, the nucleic acid in the present invention will retain any unique bases and / or epigenetic markers present in the nucleic acid. Such unique bases and / or epigenetic markers are lost in many methods known in the art.

[0191] Target nucleic acid can be obtained from any nucleic acid sample or source, for example any cell, tissue or organism, in vitro, chemical synthesizer etc. Target nucleic acid can be obtained by any art-recognized method. In embodiments, nucleic acid is obtained from a blood sample of a clinical subject. Nucleic acid can be extracted, separated or purified from a source or sample using methods and kits well known in the art.

[0192] As will be appreciated by those skilled in the art, a sample may comprise any number of substances, including but not limited to: cells (including both primary cells and cultured cell lines), cell lysates or extracts (including but not limited to RNA extracts, purified mRNA), tissues and tissue extracts (including but not limited to) of virtually any organism. RNA extracts, purified mRNA), body fluids (including but not limited to blood, urine, serum, lymph, bile, cerebrospinal fluid, interstitial fluid, aqueous or vitreous humor, colostrum, sputum, amniotic fluid, saliva, anal and vaginal secretions, sweat and semen, exudates, transudates (e.g., fluid from an abscess or any other site of infection or inflammation) or fluid from joints (e.g., normal joints or joints affected by diseases such as rheumatoid arthritis, osteoarthritis, gout or septic arthritis), mammalian samples are preferred, and human samples are particularly preferred; environmental samples (including but not limited to air, agricultural, water and soil samples); biological warfare agent samples; research samples, including extracellular fluid, extracellular supernatant from cell culture, inclusion bodies in bacteria, cellular compartments, periplasm, mitochondrial compartments.

[0193] The biomolecule sample can be indirectly derived from a biological sample. For example, when the target molecule of interest is a cellular transcript (e.g., messenger RNA), the biomolecule sample of the present invention can be a sample containing cDNA produced by reverse transcription of the messenger RNA. In another example, the biomolecule sample of the present invention is produced by subjecting a biological sample to fractionation, such as size fractionation or membrane fractionation.

[0194] The biomolecule samples of the present invention can be "natural", that is, not subjected to manipulation or treatment, or "treated", which can include any number of treatments, including exposure to candidate drugs (including drugs), genetic engineering modifications (such as addition or deletion of genes).

[0195] The nucleic acid molecules comprising the target nucleic acid can be fragmented by any means known in the art. Preferably, fragmentation is carried out by enzymatic or mechanical means. Mechanical means can be sonication or physical shearing. Enzymatic means can be carried out by digestion with a nuclease (e.g., deoxyribonuclease I (DNase I)) or one or more restriction endonucleases.

[0196] When the nucleic acid molecule comprising the nucleic acid of interest is an intact chromosome, steps should be taken to avoid fragmenting the chromosome.

[0197] The target nucleic acid can include natural or non-natural nucleotides, including modified nucleotides as are well known in the art.

[0198] The probes of the present invention may have a total length (including the target binding domain, the barcode domain, and any optional domains) of about 20 nm to about 50 nm. The backbone of the probe may be a polynucleotide molecule comprising about 120 nucleotides.

[0199] The barcode domain comprises a synthetic backbone. The synthetic backbone and the target binding domain are operably linked, for example, covalently linked or linked via a linker. The synthetic backbone can comprise any material, for example, a polysaccharide, a polynucleotide, a polymer, a plastic, a fiber, a peptide, a peptide nucleic acid, or a polypeptide. Preferably, the synthetic backbone is rigid. In an embodiment, the backbone comprises "DNA origami" of six DNA double helices (see, for example, Lin et al, "Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA." Nature Chemistry; 2012 Oct; 4(10): 832-9). The barcode can be made of DNA origami bricks (Jungmann et al, "Multiplexed 3D cellular super-resolution imaging with DNA-PAINT and Exchange-PAINT", Nature Methods, Vol. 11, No. 3, 2014).

[0200] The barcode domain comprises a plurality of positions, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more positions. The number of positions may be less than, equal to, or greater than the number of nucleotides in the target binding domain. In an embodiment, it is preferred that the target binding domain comprises additional nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides, in addition to the number of positions in the backbone domain. In an embodiment, the number of nucleotides in the target binding domain is at least twice the number of attachment regions in the barcode domain. In another embodiment, the number of nucleotides in the target binding domain is 8, and the number of attachment regions in the barcode domain is 3. As described above, the length of the barcode domain is not limited, as long as there is enough space for at least 4 positions.

[0201] Each position in the barcode domain comprises at least one attachment region, e.g., 1-50 or more attachment regions. Some positions in the barcode domain may have more attachment regions than others (e.g., a first position may have 3 attachment regions, while a second position may have 2 attachment regions); alternatively, each position in the barcode domain may have the same number of attachment regions. Each attachment region comprises at least one (i.e., 1-50, e.g., 10-30) copies of a nucleic acid sequence capable of reversibly binding to a complementary nucleic acid molecule (RNA or DNA).

[0202] Each attachment region can be linked to a modified monomer (e.g., a modified nucleotide) in the synthetic backbone, such that the attachment region branches from the synthetic backbone. In embodiments, the attachment region is essential for constituting the entirety of the polynucleotide backbone, that is, the backbone is a single polynucleotide, and the attachment region is part of a single polynucleotide sequence. In embodiments, the terms "barcode domain" and "synthetic backbone" are synonymous.

[0203] For each probe, the nucleotide sequence of each attachment region in a position is identical. Thus, in a probe, each first attachment region in the first position has the same nucleotide sequence. Similarly, each ninth attachment region in the ninth position has the same nucleotide sequence.

[0204] In the probe, each attachment region or regions within a position has a unique sequence. Furthermore, the attachment region at a first position comprises a different nucleic acid sequence than the attachment region at a second position. Thus, for the nucleic acid sequence of the attachment region at the first position, there is no binding of a complementary nucleic acid molecule specific for the attachment region at the second position. Furthermore, for the attachment region at the second position, there is no binding of a complementary nucleic acid molecule specific for the attachment region at the third position.

[0205] Each position on the barcode domain can contain one or more (up to 50, preferably 10-30) attachment regions, and thus, each attachment region can bind one or more (up to 50, preferably 10-30) complementary nucleic acid molecules. In an embodiment, at least one position in the barcode domain contains more than one attachment region. In another embodiment, at least one position in the barcode domain contains a greater number of attachment regions than another position. As an example, Figure 1 The probe has a fifth position containing two attachment regions, and Figure 4 The probe in has a second position with 6 attachment regions. In an embodiment, the nucleic acid sequences of the attachment regions at a position are identical, and therefore, the complementary nucleic acid molecules that bind to those attachment regions are identical.

[0206] In alternative embodiments, the nucleic acid sequences of the attachment regions at the locations are different, and thus, the complementary nucleic acid molecules that bind to those attachment regions are different, e.g., each comprising a different nucleic acid sequence and / or detectable label. Thus, in alternative embodiments, a combination of non-identical nucleic acid molecules (e.g., their detectable labels) attached to the attachment regions together provide a code for identifying a nucleotide in a target nucleic acid.

[0207] Table 1 provides exemplary sequences, for illustrative purposes only, for the attachment region of a probe having up to 6 positions in its barcode domain and a detectable label on the complementary nucleic acid to which it binds.

[0208] Table 1:

[0209]

[0210] As shown in Table 1, the nucleic acid sequence of the first attachment region may be one of SEQ ID NO: 1-SEQ ID NO: 4, the nucleic acid sequence of the second attachment region may be one of SEQ ID NO: 5-SEQ ID NO: 8, and the nucleic acid sequence of the third attachment region may be one of SEQ ID NO: 9-SEQ ID NO: 12.

[0211] Table 1 shows that a given attachment zone can bind to one of four possible complementary nucleic acids comprising a detectable label or a reporter complex comprising a detectable label. Thus, if the attachment zone at the first position comprises SEQ ID NO: 1, then the first position can be labeled with GFP; alternatively, if the attachment zone at the first position comprises SEQ ID NO: 2, then the first position can be labeled with RFP. Detectable labels other than GFP, RFP, CFP, and YFP can be used. Additionally, the nucleotide sequence of the attachment zone can differ from those listed in Table 1.

[0212] The probe will have the first, second, and third positions labeled with GFP when the attachment region at the first position comprises SEQ ID NO: 1, the second attachment region at the second position comprises SEQ ID NO: 5, and the third attachment region at the third position comprises SEQ ID NO: 9. The three position GFP codes (i.e., the linear order of the detectable labels) identify the target nucleic acid bound by the target binding site (e.g., GATA3) of the probe.

[0213] However, for example, when the attachment region at the first position comprises SEQ ID NO: 1, the second attachment region at the second position comprises SEQ ID NO: 6, and the third attachment region at the third position comprises SEQ ID NO: 12, the probe will have first, second, and third positions labeled with GFP, RFP, and YFP, respectively. The GFP-RFP-YFP encoding (i.e., the linear sequence of detectable labels) at these three positions identifies the target binding site (e.g., MafB At the same time, the choice of attachment region at each position defines a linear color code that can be generated by the probe backbone, which is associated with a specific target nucleic acid that is complementary to the known nucleotide sequence of the target binding domain.

[0214] Similarly, for example, when the attachment region at the first position comprises SEQ ID NO: 1, the second attachment region at the second position comprises SEQ ID NO: 6, and the third attachment region at the third position comprises SEQ ID NO: 11, the probe will have first, second, and third positions labeled with GFP, RFP, and CFP, respectively. The GFP-RFP-CFP encoding (i.e., the linear sequence of detectable labels) at these three positions identifies the target binding site (e.g., Fat3) bound target nucleic acid.

[0215] At the same time, respectively combined with GATA3, MafB and Fat3 The three probes can be applied to the sample simultaneously and, due to the different linear orders of their detectable labels, can be detected GATA3, MafB and Fat3 The existence and quantity of each.

[0216] In embodiments, the complementary nucleic acid molecule may be bound by a detectable label. In alternative embodiments, the complementary nucleic acid is associated with a reporter complex comprising a detectable label.

[0217] The nucleotide sequence of the complementary nucleic acid is not limited; preferably it lacks substantial homology (eg, 50%-99.9%) to a known nucleotide sequence, which helps avoid undesired hybridization of the complementary nucleic acid and the target nucleic acid.

[0218] Examples of reporter complexes that can be used in the present invention are shown in Figure 9B In this example, complementary nucleic acids are attached to (branching from) a primary nucleic acid molecule, which in turn hybridizes to a plurality of secondary nucleic acid molecules, each of which in turn hybridizes to a plurality of tertiary nucleic acid molecules having one or more detectable labels attached thereto.

[0219] In embodiments, the primary nucleic acid molecule may comprise about 90 nucleotides. The secondary nucleic acid molecule may comprise about 87 nucleotides. The tertiary nucleic acid molecule may comprise about 15 nucleotides.

[0220] Figure 9C Shown is a panel of exemplary reporter complexes. Figure 9C In the upper left panel are four complexes hybridized to probe attachment region 1. There is one type of reporter complex for each possible nucleotide that can be present at nucleotide position 1 of the probe's target binding domain.

[0221] The reporter complex can have various designs. For example, a primary nucleic acid molecule can hybridize to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) secondary nucleic acid molecule. Each secondary nucleic acid molecule can hybridize to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) tertiary nucleic acid molecule. An exemplary reporter complex is shown in FIG. Figure 12AHere, a "4x3" reporter complex has one primary nucleic acid molecule (which is connected to / branched from a complementary nucleic acid molecule) hybridized to four secondary nucleic acid molecules, each of which is hybridized to three tertiary nucleic acid molecules (each containing a detectable label). In this figure, each complementary nucleic acid of the complex is 12 nucleotides long ("12 bases"); however, the length of the complementary nucleic acid is not limited and can be less than 12 or more than 12 nucleotides. The lower right complex contains a spacer between its complementary nucleic acid and its primary nucleic acid molecule. The spacer has been identified as being 20-40 nucleotides long; however, the length of the spacer is non-limiting and can be shorter than 20 nucleotides or longer than 40 nucleotides.

[0222] Figure 12B Displayed from Figure 12A Variable average (fluorescence) counts for the four exemplary reporter complexes shown. Figure 12B In , 10 pM biotinylated target template was attached to the streptavidin-coated flow cell surface, and 10 nM reporter complex was flowed onto the flow cell; after a 1 minute incubation, the flow cell was washed, imaged, and fluorescent features were counted.

[0223] In an embodiment, the reporter complex is "pre-constructed." That is, each polynucleotide in the complex is hybridized before the complex is contacted with the probe. Exemplary formulations for pre-constructing five exemplary reporter complexes are shown in FIG. Figure 12C middle.

[0224] Figure 13A An alternative reporter complex is shown in which the secondary nucleic acid molecule has an "extra handle" that does not hybridize to the tertiary nucleic acid molecule and is at the distal end of the primary nucleic acid molecule. In the figure, each "extra handle" is 12 nucleotides long (a "12-mer"); however, its length is not limited and may be less than 12 or more than 12 nucleotides. In an embodiment, each of the "extra handles" comprises a nucleotide sequence of a complementary nucleic acid; therefore, when the reporter complex comprises an "extra handle", the reporter complex may hybridize to the probe via the complementary nucleic acid of the reporter complex or via the "extra handle". Therefore, the probability of the reporter complex binding to the probe increases. The "extra handle" design can also improve hybridization kinetics. Without being bound by theory, the "extra handle" substantially increases the effective concentration of the complementary nucleic acid of the reporter complex.

[0225] Figure 13B Display usage Figure 12B The method described was derived from variable average (fluorescence) counts of five exemplary reporter complexes with "extra handles".

[0226] Figure 14A and 14BHybridization kinetics and fluorescence intensity are shown for two exemplary reporter complexes. By approximately 5 minutes, total counts begin to plateau, indicating that most of the added reporter complexes have found available targets.

[0227] The detectable moiety, label, or reporter can be bound to the complementary nucleic acid or tertiary nucleic acid molecule in a variety of ways, including direct or indirect attachment of a detectable moiety (e.g., a fluorescent moiety, a colorimetric moiety, etc.). The detectable label can comprise a plurality of detectable moieties, each having a single emission spectrum that may be the same or different. For example, the detectable label can comprise a plurality of fluorophores, each having an emission spectrum that may be the same or different. One skilled in the art can refer to references related to labeling nucleic acids. Examples of fluorescent moieties include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), red fluorescent protein (RFP), umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, cyanine, dansyl chloride, phycocyanin, phycoerythrin, and the like. Fluorescent labels and their attachment to nucleotides and / or oligonucleotides are described in many reviews, including Haugland, Handbook of Fluorescent Probes and Research Chemicals, Ninth Edition (Molecular Probes, Inc., Eugene, 2002); Keller and Manak, DNA Probes, 2nd Edition (Stockton Press, New York, 1993); Eckstein, editor, Oligonucleotides and Analogues: A Practical Approach (IRL Press, Oxford, 1991); and Wetmur, Critical Reviews in Biochemistry and Molecular Biology, 26:227-259 (1991). Specific methodologies suitable for use in the present invention are disclosed in the following sample references: U.S. Patent Nos. 4,757,141, 5,151,507, and 5,091,519. In one aspect, one or more fluorescent dyes are used as labels for the labeled target sequence, for example, fluorescent dyes disclosed in U.S. Patent Nos. 5,188,934 (4,7-dichlorofluorescein dye), 5,366,860 (spectrally resolvable rhodamine dye), 5,847,162 (4,7-dichlororhodamine dye), 4,318,846 (ether-substituted fluorescein dye), 5,800,996 (energy transfer dye), Lee et al. 5,066,580 (xanthine dye), 5,688,648 (energy transfer dye), etc.Labels can also be implemented with quantum dots, such as those disclosed in the following patents and patent publications: U.S. Patents 6,322,901, 6,576,291, 6,423,551, 6,251,303, 6,319,426, 6,426,513, 6,444,143, 5,990,479, 6,207,392, 2002 / 0045045, and 2003 / 0017264. As used herein, the term "fluorescent label" includes a signaling moiety that transmits information through the fluorescent absorption and / or emission properties of one or more molecules. Such fluorescent properties include fluorescence intensity, fluorescence lifetime, emission spectrum characteristics, energy transfer, and the like. Fluorescent labels used herein can include multiple detectable moieties, each having a single fluorescent absorption and / or emission property that can be the same or different. For example, a fluorescent label can include multiple fluorophores, each having an emission spectrum that can be the same or different. In a further non-limiting example, the fluorescent label can comprise any combination of the fluorophores ALEXA FLUOR™ 350, ALEXA FLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, and ALEXA FLUOR™ 647.

[0228] Commercially available fluorescent nucleotide analogs that are readily incorporated into nucleotide and / or oligonucleotide sequences include, but are not limited to, Cy3-dCTP, Cy3-dUTP, Cy5-dCTP, Cy5-dUTP (Amersham Biosciences, Piscataway, NJ), fluorescein-12-dUTP, tetramethylrhodamine-6-dUTP, TEXAS RED™-5-dUTP, CASCADE BLUE™-7-dUTP, BODIPY™ FL-14-dUTP, BODIPY TMR-14-dUTP, BODIPY™ TR-14-dUTP, RHODAMINE GREEN™-5-dUTP, OREGON GREENR™ 488-5-dUTP, TEXAS RED™-12-dUTP, BODIPY™ 630 / 650-14-dUTP, BODIPY™ 650 / 665-14-dUTP, ALEXA FLUOR™ 488-5-dUTP, ALEXA FLUOR™ 532-5-dUTP, ALEXA FLUOR™ 568-5-dUTP, ALEXA FLUOR™ 594-5-dUTP, ALEXA FLUOR™ 546-14-dUTP, Fluorescein-12-UTP, Tetramethylrhodamine-6-UTP, TEXAS RED™-5-UTP, mCherry, CASCADE BLUE™-7-UTP, BODIPY TM FL-14-UTP, BODIPY TMR-14-UTP, BODIPY TM TR-14-UTP, RHODAMINEGREEN™-5-UTP, ALEXA FLUOR™ 488-5-UTP, LEXA FLUOR™ 546-14-UTP (Molecular Probes, Inc. Eugene, OR) etc. Alternatively, the above fluorophores and those mentioned herein can be added during oligonucleotide synthesis using, for example, phosphoramidite or NHS chemistry. Protocols for custom synthesis of nucleotides with other fluorophores are known in the art (see Henegariu et al. (2000) Nature Biotechnol. 18:345). 2-Aminopurine is a fluorescent base that can be directly incorporated into oligonucleotide sequences during their synthesis. Nucleic acids can also be stained a priori with intercalating dyes such as DAPI, YOYO-1, ethidium bromide, cyanine dyes (e.g., SYBR Green) and the like.

[0229] Other fluorophores that can be used for post-synthetic attachment include, but are not limited to, ALEXA FLUOR™ 350, ALEXAFLUOR™ 405, ALEXA FLUOR™ 430, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546, ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 647, BODIPY 493 / 503, BODIPY FL, BODIPY R6G, BODIPY 530 / 550, BODIPY TMR, BODIPY 558 / 568, BODIPY 558 / 568, BODIPY 564 / 570, BODIPY 576 / 589, BODIPY 581 / 591, BODIPY TR, BODIPY 630 / 650, BODIPY 650 / 665, Cascade Blue, Cascade Yellow, Dansyl, Lissamine Rhodamine B, Dock Blue, Oregon Green 488, Oregon Green 514, Pacific Blue, Pacific Orange, Rhodamine 6G, Rhodamine Green, Rhodamine Red, Tetramethylrhodamine, Texas Red (available from Molecular Probes, Inc., Eugene, OR), Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7 (Amersham Biosciences, Piscataway, NJ), etc. FRET tandem fluorophores can also be used, including but not limited to PerCP-Cy5.5, PE-Cy5, PE-Cy5.5, PE-Cy7, PE-Texas Red, APC-Cy7, PE-Alexa dyes (610, 647, 680), APC-Alexa dyes, etc.

[0230] Metallic silver or gold particles can be used to enhance the signal from fluorescently labeled nucleotide and / or oligonucleotide sequences (Lakowicz et al. (2003) BioTechniques 34:62).

[0231] Other suitable labels for oligonucleotide sequences may include fluorescein (FAM, FITC), digoxigenin, dinitrophenol (DNP), dansyl, biotin, bromodeoxyuridine (BrdU), hexahistidine (6xHis), phospho-amino acids (e.g., P-tyr, P-ser, P-thr), etc. In one embodiment, detection is performed using the following hapten / antibody pairs, where each antibody is derivatized with a detectable label: biotin / α-biotin, digoxigenin / α-digoxigenin, dinitrophenol (DNP) / α-DNP, 5-carboxyfluorescein (FAM) / α-FAM.

[0232] Detectable label described herein is spectrally resolvable.About multiple fluorescent labels " spectrally resolvable " means that the fluorescence emission band of label is clear enough, i.e., not overlapping enough, so that the molecular tag connected by each label can be distinguished by a standard photodetection system (such as using a bandpass filter and a photomultiplier tube system, etc.), based on the fluorescence signal produced by the corresponding label, with U.S. Patent No. 4230558, No. 4811218, etc. or at Wheeless et al., pgs.21-76, in FlowCytometry:Instrumentation and Data Analysis (Academic Press, New York, 1985) described system as an example. In one aspect, spectrally resolvable organic dyes (such as fluorescein, rhodamine, etc.) mean that the wavelength emission maximum is at least 20 nm apart, and on the other hand at least 40 nm apart. On the other hand, spectrally resolvable chelated lanthanide compounds, quantum dots, etc. mean that the wavelength emission maximum is at least 10 nm apart, and on the other hand at least 15 nm apart.

[0233] Methods for detecting nucleic acids

[0234] The present invention relates to a method for detecting nucleic acids using the probes of the present invention. An example of the method is shown in Figure 6-11 middle.

[0235] The method comprises reversibly hybridizing at least one probe of the invention to a target nucleic acid immobilized (eg, at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more locations) to a substrate.

[0236] The substrate can be any solid support known in the art, such as coated slides and microfluidic devices, which can fix the target nucleic acid. In certain embodiments, the substrate is a surface, film, bead, porous material, electrode, or array. The target nucleic acid can be fixed on any substrate apparent to those skilled in the art.

[0237] In an embodiment, a target nucleic acid is bound by a capture probe that comprises a domain complementary to a portion of the target nucleic acid. The portion may be a terminal end of the target nucleic acid or non-terminal end.

[0238] Exemplary useful substrates include those comprising a binding moiety selected from a ligand, an antigen, a carbohydrate, a nucleic acid, a receptor, a lectin, and an antibody. The capture probe comprises a binding moiety capable of binding to the binding moiety of the substrate. Exemplary useful substrates comprising a reactive moiety include, but are not limited to, surfaces comprising an epoxy, an aldehyde, gold, a hydrazide, a sulfhydryl, an NHS-ester, an amine, a thiol, a carboxylate, a maleimide, a hydroxymethylphosphine, an imidoester, an isocyanate, a hydroxyl, a pentafluorophenyl ester, a psoralen, a pyridyl disulfide or a vinyl sulfone, polyethylene glycol (PEG), a hydrogel, or a mixture thereof. Such surfaces can be obtained from commercial sources or prepared according to standard techniques. Exemplary useful substrates comprising a reactive moiety include, but are not limited to, OptArray-DNA NHS groups (Accler8), Nexterion Slide AL (Schott), and Nexterion Slide E (Schott).

[0239] In an embodiment, the binding moiety of the capture probe is biotin, and the substrate comprises avidin (e.g., streptavidin). Useful substrates comprising avidin are commercially available and include TB0200 (Accelr8), SAD6, SAD20, SAD100, SAD500, SAD2000 (Xantec), SuperAvidin (Array-It), Streptavidin slides (Cat. No. MPC000, Xenopore), and STREPTAVIDINn slides (Cat. No. 439003, Greiner Bio-one).

[0240] In an embodiment, the binding moiety of the capture probe is avidin (e.g., streptavidin) and the substrate comprises biotin. Useful substrates comprising commercially available biotin include, but are not limited to, Optiarray-Biotin (Accler8), BD6, BD20, BD100, BD500, and BD2000 (Xantec).

[0241] In an embodiment, the binding portion of the capture probe may comprise a reactive portion capable of binding to the substrate by photoactivation. The substrate may comprise a photoreactive portion, or the first portion of the nanoreporter may comprise a photoreactive portion. Some examples of photoreactive portions include aryl azides such as N-((2-pyridyldithio)ethyl)-4-azido salicylamide, fluorinated aryl azides such as 4-azido-2,3,5,6-tetrafluorobenzoic acid, benzophenone-based reagents such as succinimidyl ester of 4-benzoylbenzoic acid, and 5-bromo-deoxyuridine.

[0242] In embodiments, the binding moieties of the capture probes may be immobilized on the substrate via other binding pairs that will be apparent to those skilled in the art.

[0243] Once bound to a substrate, the target nucleic acid can be elongated by applying a force sufficient to extend the target nucleic acid (e.g., gravity, fluid dynamics, electromagnetic forces "electrostretching," flow stretching, receding meniscus techniques, and combinations thereof).

[0244] The target nucleic acid can be bound by a second capture probe comprising a domain complementary to a second portion of the target nucleic acid. This portion can be at the end of the target nucleic acid or not towards the end. Binding of the second capture probe can occur after or during extension of the target nucleic acid, or to a target nucleic acid that has not yet been extended. The second capture probe can have a binding as described above.

[0245] The capture probe may comprise or be associated with a detectable label, ie, a fiducial.

[0246] The capture probe is capable of separating a target nucleic acid from a sample. Here, the capture probe is added to a sample containing the target nucleic acid. The capture probe binds to the target nucleic acid via a region of the capture probe that is complementary to the region of the target nucleic acid. When the target nucleic acid contacts a substrate containing a portion that binds to the binding portion of the capture probe, the nucleic acid becomes fixed to the substrate.

[0247] To ensure that the user "captures" as many target nucleic acid molecules as possible from a highly fragmented sample, it is helpful to include multiple capture probes, each complementary to a different region of the target nucleic acid. For example, there may be three pools of capture probes, the first pool complementary to a region of the target nucleic acid near its 5' end, the second pool complementary to a middle region of the target nucleic acid, and the third pool near its 3' end. This can be summarized as "n regions of interest" per target nucleic acid. In this example, each individual pool of fragmented target nucleic acid is bound to a capture probe containing or bound to a biotin tag. Each pool compartment separates 1 / n of the input sample (where n = the number of different regions in the target nucleic acid). The capture probe binds to the target nucleic acid of interest. The target nucleic acid is then immobilized to an avidin molecule adhered to a substrate via the biotin in the capture probe. Optionally, the target nucleic acid is stretched, for example, by flow or electrostatic forces. All n pools can be stretched and bound simultaneously, or, to maximize the number of fully stretched molecules, pool 1 (which captures the 5' region) can be stretched and bound first; then pool 2 (which captures the middle region of the target) can be stretched and bound; and finally, pool 3 can be stretched and bound.

[0248] The number of different capture probes required is inversely related to the size of the target nucleic acid fragment. In other words, highly fragmented target nucleic acids will require more capture probes. For sample types with highly fragmented and degraded target nucleic acids (e.g., formalin-fixed paraffin-embedded tissues), it may be useful to include multiple capture probe pools. On the other hand, for samples with long target nucleic acid fragments, such as isolated nucleic acids obtained in vitro, a single capture probe at the 5' end may be sufficient.

[0249] The probes or capture probes of the present invention may comprise one or more affinity reagents, each selected from the group consisting of ligands, antigens, carbohydrates, nucleic acids, receptors, lectins, haptens, and antibodies. The affinity reagents enable purification of the complex formed by the probe or capture probe and the target nucleic acid. This purification enriches the concentration of the target nucleic acid to be detected.

[0250] In an embodiment, the affinity reagent is biotin and the purification means (eg, attached to a solid support) comprises avidin (eg, streptavidin).

[0251] In an embodiment, the affinity reagent is avidin (eg, streptavidin) and the purification means (eg, attachment to a solid support) comprises biotin.

[0252] In embodiments, affinity reagent comprises nucleic acid with known sequence.Therefore, the probe comprising affinity reagent or capture probe can be purified from sample using a purification probe comprising nucleic acid complementary to affinity reagent.Similarly, the complex comprising the probe containing affinity reagent or capture probe can be purified from sample using a purification probe comprising nucleic acid complementary to affinity reagent. The affinity portion for the probe and capture probe of the same target nucleic acid can have different nucleic acid sequences. Alternatively, the affinity reagent for probe and the affinity reagent for capture probe (each for identical target nucleic acid) can have the same nucleic acid sequence. Every kind of affinity reagent for every kind of probe in the probe group can have the same nucleic acid sequence. Every kind of affinity reagent for every kind of capture probe in the capture probe group can have the same nucleic acid sequence. Every kind of affinity reagent for every kind of probe in the probe group can have different nucleic acid sequences. Every kind of affinity reagent for every kind of capture probe in the capture probe group can have different nucleic acid sequences.

[0253] In embodiments, the affinity reagent is a hapten and the purification means (eg, attached to a solid support) comprises a protein binding domain (eg, an antibody).

[0254] Figure 5ASchematic diagram of a probe bound to a target nucleic acid is shown. Here, the target nucleic acid comprises a TCAGTG sequence. The barcode domain of the probe is designed to have an attachment region that specifically recognizes the bound TCAGTG with a specific linear color code or "linear sequence of detectable markers". The first complementary nucleic acid pool comprising a detectable marker or reporter complex is shown in the figure above, each member of the pool having a different nucleotide sequence and an associated detectable marker (e.g., a green marker and a cyan marker). As an example, the nucleic acid of the first pool has a sequence complementary to SEQ ID NO: 1-4 of Table 1. In Figure 5A In the example, the first attachment region of the probe comprises one or more nucleotide sequences, which specify that the first position should be labeled with a cyan marker (e.g., the attachment region comprises SEQ ID NO: 3 of Table 1). Thus, only complementary nucleic acids specific for the first attachment position and carrying a cyan marker can bind to the first position of the barcode domain of the probe as shown. The cyan marker is the first color of the linear color code, which identifies the bound target nucleic acid.

[0255] The color associated with the first location is imaged and recorded in the system of the present invention.

[0256] The number of complementary nucleic acid or reporter complex pools is the same as the number of positions in the barcode domain. Thus, for a barcode domain with 6 positions, 6 pools will be cycled over the probe.

[0257] When the capture probe is added to a sample containing a target nucleic acid, the probe may be initially provided with the target nucleic acid (see Figure 6 ). Such probes can be provided at different concentrations, different buffer conditions (such as salt), and different temperatures to improve sensitivity and specificity for the target nucleic acid.

[0258] Capture probes and probes can be provided with affinity reagents for multi-stage purification (see Figure 7 and 8A -8E). You can use either purification method alone or purify from both ends. Purification will improve the specificity and purity of target capture.

[0259] A probe can be provided to a target nucleic acid and initially bound to the target nucleic acid, which completely lacks a complementary nucleic acid comprising a detectable label or a reporter complex comprising a detectable label. Such a probe will be smaller than a probe comprising a detectable complementary nucleic acid. Such a probe can be provided at a higher concentration than a probe comprising a detectable label. Such a small probe will be more rapidly and more effectively bound to the target nucleic acid. Therefore, data can be provided in a fraction of the time required to use a probe comprising a detectable label.

[0260] Alternatively, the probe can be hybridized at its first location to a complementary nucleic acid comprising a detectable label or reporter complex prior to contacting the target nucleic acid with the probe. Thus, when contacted with its target nucleic acid, the probe is capable of emitting a detectable signal from its first location, and it is not necessary to provide a first complementary nucleic acid or reporter complex pool specific to the first location on the barcode domain.

[0261] Figure 5B continue Figure 5A Here, the first complementary nucleic acid (or reporter complex) bound to the first position attachment region of the barcode domain has been replaced with a first hybrid nucleic acid lacking a detectable label. The first hybrid nucleic acid and the lack of a detectable label replace the previously bound complementary nucleic acid containing a detectable label or the previously bound reporter complex. As a result, the first position of the barcode domain no longer emits a detectable signal.

[0262] The hybrid nucleic acid and lack of a detectable label may comprise the same sequence as a previously bound complementary nucleic acid comprising a detectable label or a previously bound reporter complex (e.g., SEQ ID NO: 1-SEQ ID NO: 24). Preferably, the hybrid nucleic acid and lack of a detectable label will be longer than the previously bound complementary nucleic acid comprising a detectable label or the previously bound reporter complex. To this end, the hybrid nucleic acid further comprises a sequence complementary to a single-stranded polynucleotide or polynucleotide analog region adjacent to the attachment region. Without being bound by theory, a hybrid nucleic acid that is longer than its associated complementary nucleic acid comprising a detectable label will have a greater affinity for the barcode domain and will readily displace the complementary nucleic acid comprising a detectable label. Such a hybrid nucleic acid that is longer than its associated complementary nucleic acid is shown in FIG. Figure 10 and 11 middle.

[0263] In an embodiment, a complementary nucleic acid or reporter complex comprising a detectable label can be removed from the attachment zone without being replaced by a hybrid nucleic acid lacking a detectable label. This can occur, for example, by adding a chaotropic agent, increasing the temperature, changing the salt concentration, adjusting the pH, and / or applying a fluid dynamic force. In these embodiments, less reagent is required (i.e., a hybrid nucleic acid lacking a detectable label).

[0264] Figure 5C Continuing with the method of the claimed invention. A second pool of complementary nucleic acids or reporter complexes is shown in the figure above (e.g., having sequences complementary to SEQ ID NOs: 5-8 of Table 1), each member of the pool having a different detectable label and a different nucleotide sequence. In addition, the nucleotide sequence of the complementary nucleic acids of the first pool or the complementary nucleic acids of the reporter complexes is different from that of the second pool. Here, only the complementary nucleic acid from the second pool and comprising a yellow detectable label is bound to the second position of the barcode domain (e.g., a complementary nucleic acid having a sequence complementary to SEQ ID NO: 8 of Table 1).

[0265] The color associated with the second location is imaged and recorded in the system of the present invention.

[0266] In an embodiment, Figure 5C The steps shown continue Figure 5B Here, once the first pool of complementary nucleic acids or reporter complexes ( Figure 5A ) has been replaced by a first hybridized nucleic acid lacking a detectable label ( Figure 5B ), a second pool of complementary nucleic acids or reporter complexes (e.g. Figure 5C as shown). Alternatively, Figure 5C The steps shown are the same as Figure 5B Here, the first hybrid nucleic acid lacking a detectable label ( Figure 5B ) and a second pool of complementary nucleic acids or reporter complexes (e.g. Figure 5C shown).

[0267] Figure 5D continue Figure 5C Here, positions 1 through 5 on the barcode domain are bound by complementary nucleic acids or reporter complexes containing a detectable label, the colors associated with their positions are imaged and recorded, and the complementary nucleic acids have been replaced by hybridized nucleic acids lacking a detectable label. Position 6 of the barcode domain is now bound by a complementary nucleic acid or reporter complex containing a detectable label, identifying the sixth position in the target binding domain as bound to guanine (G).

[0268] The color associated with the sixth position is imaged and recorded in the system of the present invention.

[0269] At this point, the entire linear color code of the probe backbone (i.e., the linear sequence of the detectable labels) has been detected; this linear code is then associated with a specific target nucleic acid that is complementary to the known nucleotide sequence of the target binding domain. As an example, a probe that emits a linear color code of green, cyan, red, yellow, yellow, and red can bind to Fat2 Therefore, if the system of the present invention records the linear color code of green, cyan, red, yellow, yellow, red, the user will know that there is Fat2 .

[0270] Because each color associated with the probe backbone domain is detected sequentially, the probe backbone may not have to be extended to distinguish and resolve each color label. This is an advantage over previous generations of nucleic acid detection probes.

[0271] As described above, the complementary nucleic acid or reporter complex comprising the detectable label can be removed from the attachment zone without being replaced with a hybridized nucleic acid lacking the detectable label.

[0272] If desired, the rate of detectable label exchange can be accelerated by incorporating small single-stranded oligonucleotides that accelerate the rate of detectable label exchange (e.g., "Toe-Hold" Probes; see, e.g., Seeling et al., "Catalyzed Relaxation of a Metastable DNA Fuel"; J. Am. Chem. Soc. 2006, 128(37), pp12211-12220).

[0273] and Figures 5A-5D similar, Figure 9A and 9D -9F shows the method steps of the present invention; however, Figure 9A and 9D -9F clearly shows that the reporter complex (including the detectable label) is bound to the attachment region of the probe. Figure 9D and 9E Fluorescent signals sequentially emitted from probes hybridized to the reporter complex are displayed.

[0274] Figure 10 Overview Figure 9D and 9E The steps are shown. The nucleotide sequence of an exemplary probe is shown in the upper panel of the figure and the important domains of the probe are identified. The probe comprises an optional double-stranded DNA spacer between its target binding domain and its barcode domain. The barcode domain comprises, in sequence, a "Wing 1" portion, an "AR-1" portion, an "AR-1 / Wing 2" portion, an "AR-2" portion, and an "AR-2 / Wing 3" portion. In step 1, "AR-1 detection" hybridizes to the "AR-1" and "AR-1 / Wing 2" portions of the probe. "AR-1 detection" corresponds to a reporter complex comprising a detectable label or a complementary nucleic acid that encodes a first position thymidine. Thus, step 1 corresponds to Figure 9D In step 2, "missing 1" hybridizes to the "flank 1" and "AR-1" portions of the probe. "missing 1" corresponds to the absence of a detectably labeled hybridizing nucleic acid specific for the first attachment region of the probe (e.g., Figure 9E (shown as a black bar covering the first attachment zone) By hybridizing to the "Flank 1" position 5' of the reporter complex or complementary nucleic acid, the hybridizing nucleic acid more efficiently displaces the reporter complex / complementary nucleic acid from the probe. The "Flank" portion is also referred to as the "toehold". In step 3, the "AR-2 detection" hybridizes to the "AR-2" and "AR-2 / Flank 3" portions of the probe. The "AR-2 detection" corresponds to the reporter complex or complementary nucleic acid containing a detectable label that encodes the second position guanine. Therefore, step 3 corresponds to Figure 9E In this embodiment, a hybridizing nucleic acid lacking a detectable label and a complementary nucleic acid / reporter complex comprising a detectable label are provided sequentially.

[0275] Alternatively, a hybridizing nucleic acid lacking a detectable label and a complementary nucleic acid / reporter complex comprising a detectable label are provided simultaneously. This alternative embodiment is shown in Figure 11 In step 2, "lack 1" (hybridized nucleic acid lacking a detectable label) and "AR-2 detection" (reporter complex encoding a guanine at the second position) are provided. This alternative embodiment is comparable to Figure 10 The embodiment shown is more time efficient because it combines two steps into one.

[0276] The detectable labels of the present disclosure can be detected by any means known in the art. For example, the detectable labels can be detected by a system comprising one or more of a microscope, a camera, a microprocessor, and / or a computer system. In one aspect, the camera is a CCD camera. In one aspect, the microscope, camera, and computer system can comprise a complementary metal oxide semiconductor (CMOS chip).

[0277] Multiplex detection of multiple nucleic acids

[0278] In embodiments, multiple nucleic acids are detected simultaneously, i.e., multiplex detection. To this end, a group or a cluster of different probes are provided to a fixed nucleic acid target sample. A group or a cluster of probes preferably comprises at least two, e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more probes.

[0279] A set of probes can be predefined based on the cell type or tissue type to be targeted. For example, if the tissue is breast cancer, the set of probes will include probes for expressed nucleic acids associated with breast cancer cells (e.g., Her2、EGFR and PR ) and / or probes for nucleic acids expressed in normal breast tissue. In addition, the set of probes can be predefined based on the developmental state of the cells or tissues to be targeted.

[0280] NanoString Technologies® nCounter® systems and methods enable the simultaneous multiplexed identification of multiple (800 or more) different target proteins and / or target nucleic acids.

[0281] definition:

[0282] In certain exemplary embodiments, the terms "annealing" and "hybridization" as used herein are used interchangeably to refer to the formation of a stable duplex. In one aspect, a stable duplex means that the duplex structure is not destroyed by stringent washing under conditions such as a temperature about 5°C lower or about 5°C higher than the Tm of the duplex and a low monovalent salt concentration (e.g., less than 0.2 M or less than 0.1 M or a salt concentration known to those skilled in the art). When used in reference to a duplex, the term "perfect match" means that the polynucleotides and / or oligonucleotide chains comprising the duplex form a double-stranded structure with each other such that each nucleotide in each chain undergoes Watson-Crick base pairing with a nucleotide in the other chain. The term "duplex" includes, but is not limited to, pairings of employable nucleoside analogs (such as deoxyinosine, nucleosides with 2-aminopurine bases, PNA, etc.). A duplex "mismatch" between two oligonucleotides means that a pair of nucleotides in the duplex fails to undergo Watson-Crick bonding.

[0283] The term "hybridization conditions" as used herein generally include salt concentrations less than about 1 M, more generally less than about 500 mM and even more generally less than about 200 mM. The hybridization temperature can be as low as 5°C, but is generally greater than 22°C, more generally greater than about 30°C, and typically greater than about 37°C. Hybridization is typically performed under stringent conditions, such as under conditions where the probe specifically hybridizes to its target subsequence. Stringent conditions are sequence-dependent and different in different situations. Longer fragments may require higher hybridization temperatures for specific hybridization. Since other factors may affect the stringency of hybridization, including base composition and complementary chain length, the presence of organic solvents and the degree of base mismatching, the combination of parameters is more important than the absolute measurement of any one alone.

[0284] Typically, stringent conditions are selected to be about 5°C lower than the Tm of a specific sequence at a specific ionic strength and pH. Exemplary stringent conditions include a salt concentration of at least 0.01 M to no more than 1 M Na ion concentration (or other salts) at a pH of 7.0-8.3 and at least 25°C. For example, 5X SSPE (750 mM NaCl, 50 mM sodium phosphate, 5 mM EDTA, pH 7.4) and a temperature of 25-30°C are suitable for allele-specific probe hybridization. For stringent conditions, see, for example, Sambrook, Fritsche and Maniatis, "Molecular Cloning A Laboratory Manual, 2nd Ed." Cold Spring Harbor Press (1989) and Anderson Nucleic Acid Hybridization, 1st Ed., BIOS Scientific Publishers Limited (1999). As used herein, the term "specifically hybridizes to" or "specifically hybridizes to" or similar terms refers to that a molecule substantially binds to, forms a duplex or hybridizes with one or more specific nucleotide sequences under stringent conditions.

[0285] The detectable label associated with a specific position of the probe can be "read" (e.g., its fluorescence is detected) one or more times, and "reading" can be synonymous with the term "base calling." Multiple reads improve accuracy.

[0286] As used herein, a "hybridization and sequencing cycle" refers to all steps required to detect each attachment region on a particular probe or probe population. For example, for a probe capable of detecting six positions on a target nucleic acid, one "hybridization and sequencing cycle" would include at least hybridizing the probe to the target nucleic acid, hybridizing a complementary nucleic acid / reporter complex to each of the six attachment regions on the probe barcode domain, and detecting the detectable label associated with each of the six positions.

[0287] The term "k-mer probe" is synonymous with the probe of the present invention.

[0288] Any device capable of implementing the method and / or recording the results can be used to implement the methods described herein and / or record the results. Examples of usable devices include, but are not limited to, electronic computing devices, including all types of computers. When implementing and / or recording the methods described herein with a computer, a computer program that can be used to configure a computer to implement the method steps can be included in any computer-readable medium that can include a computer program. Examples of usable computer-readable media include, but are not limited to, disks, CD-ROMs, DVDs, ROMs, RAMs, non-transitory computer-readable media, and other memories and computer storage devices. A computer program that can be used to configure a computer to implement the method steps, identify the combined target nucleic acid, and / or record the results can also be provided via an electronic network, for example, via the Internet, an intranet, or other networks.

[0289] "Consumption card" can be incorporated into fluorescence imaging equipment known in the art. Any fluorescence microscope with many different features can implement this readout. For example: wide field lamp, laser, LED, multiphoton, confocal or total internal reflection illumination can be used for excitation and / or detection. A camera (single or multiple) and / or a photomultiplier tube (single or multiple) with filter-based or grating-based spectral resolution (one or more spectrally resolved emission wavelengths) can be used on the emission detection channel of the fluorescence microscope. A standard computer can control the consumption card, the reagents flowing through the card, and the detection through the fluorescence microscope.

[0290] Probes can be detected and quantified using commercially available syringes, software, and systems (e.g., the nCounter® system using nCounter® syringes).

[0291] Additional teachings relevant to the present invention are described in one or more of the following: US 8148512, US 7473767, US 7919237, US 7941279, US 8415102, US 8492094, US 8519115, US 2009 / 0220978, US 2009 / 0299640, US 2010 / 0015607, US 2010 / 0261026, US 2011 / 0086774, US 2011 / 0145176, US 2011 / 0201515, US 2011 / 0229888, US 2013 / 0004482, US 2013 / 0017971, US 2013 / 0178372, US 2013 / 0230851, US 2013 / 0337444, US 2013 / 0345161, US 2014 / 0005067, US 2014 / 0017688, US 2014 / 0037620, US 2014 / 0087959, US 2014 / 0154681, US 2014 / 0162251, and US 14 / 946386, each of which is incorporated herein by reference in its entirety

[0292] Any of the above aspects and implementations may be combined with any other aspects or embodiments disclosed in the Summary and / or Detailed Description sections herein. DETAILED DESCRIPTION

[0293] The present invention also relates to the following embodiments:

[0294] 1. A method for detecting at least one target nucleic acid in a sample, the method comprising:

[0295] (1) contacting the sample with at least one probe capable of recognizing and binding to a first specific region of the at least one target molecule, wherein the at least one probe comprises:

[0296] Target binding domain and barcode domain

[0297] wherein the target binding domain comprises at least 4 nucleotides and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence;

[0298] wherein the barcode domain comprises a barcode domain comprising a first attachment region comprising a nucleic acid sequence bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex; and at least a second attachment region bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of a second reporter complex;

[0299] wherein the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex comprises a first detectable label, such that the detectable label is associated with the first attachment region;

[0300] wherein the at least second complementary nucleic acid molecule or the at least second complementary nucleic acid molecule of the at least second reporter complex comprises a second detectable label, such that the detectable label is associated with the at least second attachment region;

[0301] wherein said sequence of said first attachment region is different from said sequence of said at least second attachment region;

[0302] (2) detecting the first detectable label associated with the first attachment region and the second detectable label associated with the at least second attachment region;

[0303] (3) removing the first detectable label;

[0304] (4) detecting the second detectable label associated with the at least second attachment region;

[0305] wherein the linear or sequential order of the first detectable label associated with the first attachment region and the second detectable label associated with the at least second attachment region recognizes the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

[0306] 2. The method of embodiment 1, wherein the detection of step (4) includes subtracting the signal from the second detectable label associated with the at least second attachment area in step (4) to form the signal from the first detectable label associated with the first attachment area and the second detectable label associated with the at least second attachment area detected in step (2).

[0307] 3. The method of embodiment 1, wherein the barcode domain comprises a barcode domain comprising: a first attachment region comprising a nucleic acid sequence bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporter complex; at least a second attachment region bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of a second reporter complex; and at least a third attachment region bound by at least a third complementary nucleic acid molecule or at least a third complementary nucleic acid molecule of a third reporter complex;

[0308] wherein the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex comprises a first detectable label, such that the detectable label is associated with the first attachment region;

[0309] wherein the second complementary nucleic acid molecule or the second complementary nucleic acid molecule of at least a second reporter complex comprises a second detectable label, such that the detectable label is associated with the at least second attachment region;

[0310] wherein the third complementary nucleic acid molecule or the third complementary nucleic acid molecule of at least a third reporter complex comprises a third detectable label, such that the detectable label is associated with the at least third attachment region;

[0311] wherein the sequences of the at least third attachment region, the at least second attachment region, and the at least third attachment region are different;

[0312] (2) detecting the first detectable label associated with the first attachment zone, the second detectable label associated with the at least second attachment zone, and the at least third detectable label associated with the third attachment zone;

[0313] (3) removing the first detectable label;

[0314] (4) detecting the second detectable label associated with the at least second attachment region and the at least third detectable label associated with the third attachment region;

[0315] (5) removing the second detectable label;

[0316] (6) detecting the third detectable label associated with the at least third attachment region;

[0317] wherein the first detectable label associated with the first attachment region, the second detectable label associated with the at least second attachment region, and the at least third detectable label associated with the third attachment region linearly or sequentially identify the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

[0318] 4. The method of embodiment 3, wherein the detection in step (4) includes subtracting the signal from the second detectable label associated with the at least second attachment area and the at least third detectable label associated with the third attachment area in step (4), forming a signal from the first detectable label associated with the first attachment area, the second detectable label associated with the at least second attachment area, and the at least third detectable label associated with the third attachment area detected in step (2).

[0319] 5. The method of embodiment 3, wherein the detection of step (6) includes subtracting the signal from the at least third detectable label associated with the third attachment zone in step (6) to form the signal from the second detectable label associated with the at least second attachment zone and the at least third detectable label associated with the third attachment zone detected in step (4).

[0320] 6. A method for detecting at least one target nucleic acid in a sample, the method comprising:

[0321] (1) contacting the sample with at least one probe capable of recognizing and binding to a first specific region of the at least one target molecule, wherein the at least one probe comprises:

[0322] Target binding domain and barcode domain

[0323] wherein the target binding domain comprises at least 4 nucleotides and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence;

[0324] wherein the barcode domain comprises a first attachment region comprising a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule, a first complementary nucleic acid molecule of a first reporter complex, or a first hybrid nucleic acid molecule; and at least a second attachment region comprising a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule, at least a second complementary nucleic acid molecule of a second reporter complex, or at least a second hybrid nucleic acid molecule;

[0325] wherein said sequence of said first attachment region is different from said sequence of said at least second attachment region;

[0326] (2) allowing a first complementary nucleic acid molecule comprising a first detectable label or a first complementary nucleic acid molecule of a first reporter complex comprising a first detectable label to bind to the first attachment region, thereby associating the detectable label with the first attachment region;

[0327] (3) detecting the first detectable label associated with the first attachment region;

[0328] (4) removing the first detectable label or the first complementary nucleic acid molecule;

[0329] (5) binding at least a second complementary nucleic acid molecule comprising a second detectable label or at least a second complementary nucleic acid molecule of at least a second reporter complex comprising a second detectable label to the at least second attachment region, thereby associating the detectable label with the at least second attachment region; and

[0330] (6) detecting the second detectable label associated with the at least second attachment region;

[0331] wherein the linear or sequential order of the first detectable label associated with the first attachment region and the second detectable label associated with the at least second attachment region recognizes the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

[0332] 7. The method of embodiment 6, wherein the barcode domain comprises at least a third attachment region comprising a nucleic acid sequence capable of being bound by at least a third complementary nucleic acid molecule, at least a third reporter complex, or at least a third hybrid nucleic acid molecule;

[0333] wherein the sequence of the at least third attachment region is different from the sequence of another attachment region.

[0334] 8. The method of embodiment 6, further comprising:

[0335] (7) removing the second detectable label or the second complementary nucleic acid molecule;

[0336] (8) binding at least a third complementary nucleic acid molecule comprising a third detectable label or at least a third complementary nucleic acid molecule of at least a third reporter complex comprising a third detectable label to the at least third attachment region, thereby associating the detectable label with the at least third attachment region; and

[0337] (9) detecting the third detectable label associated with the at least third attachment region;

[0338] wherein the first detectable label associated with the first attachment region, the second detectable label associated with the at least second attachment region, and the third detectable label associated with the at least third attachment region linearly or sequentially identify the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

[0339] 9. The method according to embodiment 6 or 8, wherein the removing of the first complementary nucleic acid in step (4) or the removing of the second complementary nucleic acid in step (7) comprises:

[0340] (a) contacting the first attachment region with a first hybrid nucleic acid molecule lacking a detectable label, thereby releasing the binding of the first complementary nucleic acid molecule and allowing the first hybrid nucleic acid molecule lacking a detectable label to bind to the first attachment region; or

[0341] (b) a change in pH, salt concentration, and / or temperature sufficient to remove the first complementary nucleic acid molecule.

[0342] 10. The method of embodiment 7, wherein the barcode domain comprises at least a fourth, at least a fifth, at least a sixth, or at least a seventh attachment region comprising a nucleic acid sequence capable of being bound by at least a fourth complementary nucleic acid molecule, at least a fourth reporter complex, or at least a fourth hybrid nucleic acid molecule;

[0343] wherein the sequence of the at least fourth attachment region is different from the sequence of another attachment region.

[0344] 11. The method of embodiment 10, wherein the following steps are repeated:

[0345] (a) removing the corresponding detectable label or complementary nucleic acid molecule;

[0346] (b) binding a complementary nucleic acid molecule comprising a detectable label or a complementary nucleic acid molecule comprising a reporter complex comprising a detectable label to the corresponding attachment region, thereby associating the detectable label with the corresponding attachment region; and

[0347] (c) detecting said corresponding detectable label associated with said attachment region,

[0348] until each attachment region in the barcode domain has been sequentially bound by a complementary nucleic acid molecule comprising a detectable label, and the detectable label of the sequentially bound complementary nucleic acid molecules has been detected,

[0349] wherein the linear or sequential order of the detectable labels associated with each attachment region recognizes the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

[0350] 12. The method of embodiment 6, wherein the first hybrid nucleic acid molecule lacking a detectable label comprises at least the nucleic acid sequence of the first complementary nucleic acid molecule.

[0351] 13. The method of any one of embodiments 1 or 3, wherein the removal of the first detectable label in step (3) or the removal of the second detectable label in step (5) comprises contacting the first complementary nucleic acid molecule or the first complementary nucleic acid molecule of the first reporter complex with a force sufficient to release the first detectable label from the position of the first complementary nucleic acid molecule.

[0352] 14. The method of embodiment 6 or 8, wherein the removal of the first detectable label in step (4) or the removal of the second detectable label in step (7) comprises contacting the first complementary nucleic acid molecule or at least the first complementary nucleic acid molecule of the first reporter complex with a force sufficient to release the first detectable label from the position of the first complementary nucleic acid molecule.

[0353] 15. The method of any one of embodiments 1-8, wherein at least one of the first complementary nucleic acid molecule, the first complementary nucleic acid molecule of the first reporting complex, the at least second complementary nucleic acid molecule, the at least second complementary nucleic acid molecule of the at least second reporting complex, the at least third complementary nucleic acid molecule, or the at least third complementary nucleic acid molecule of the at least third reporting complex comprises at least one cleavable linker.

[0354] 16. The method of embodiment 15, wherein the at least one cleavable linker is independently selected from the group consisting of photocleavable, chemically cleavable, and enzymatically cleavable.

[0355] 17. The method of embodiment 13 or 14, wherein the force is light.

[0356] 18. The method according to any one of embodiments 1-8, further comprising washing the probe from the at least one target nucleic acid.

[0357] 19. The method of embodiment 18, wherein the washing comprises a change in pH, salt concentration and / or temperature sufficient to remove the probe from the target molecule.

[0358] 20. The method of embodiment 19, further comprising:

[0359] (i) contacting the sample with at least a second probe capable of recognizing and binding to a second specific region of the at least one target molecule, wherein the second specific region is different from the first specific region of the at least one target molecule;

[0360] (ii) contacting the sample with at least a second copy of the first probe capable of recognizing and binding to the first specific region of the at least one target molecule; or

[0361] (iii) contacting the sample with at least a third probe capable of recognizing and binding to the first specific region of at least a second target molecule, wherein the at least second target molecule is different from the at least one target molecule;

[0362] wherein the probe comprises:

[0363] Target binding domain and barcode domain

[0364] wherein the target binding domain comprises at least 4 nucleotides; and

[0365] wherein the barcode domain comprises a barcode domain comprising a first attachment region comprising a nucleic acid sequence bound by a first complementary nucleic acid molecule or a first complementary nucleic acid molecule of a first reporting complex; and at least a second attachment region bound by at least a second complementary nucleic acid molecule or at least a second complementary nucleic acid molecule of a second reporting complex.

[0366] 21. The method of embodiment 19, further comprising:

[0367] (i) contacting the sample with at least a second probe capable of recognizing and binding to a second specific region of the at least one target molecule, wherein the second specific region is different from the first specific region of the at least one target molecule;

[0368] (ii) contacting the sample with at least a second copy of the first probe capable of recognizing and binding to the first specific region of the at least one target molecule; or

[0369] (iii) contacting the sample with at least a third probe capable of recognizing and binding to the first specific region of at least a second target molecule, wherein the at least second target molecule is different from the at least one target molecule;

[0370] wherein the probe comprises:

[0371] Target binding domain and barcode domain

[0372] wherein the target binding domain comprises at least 4 nucleotides; and

[0373] The barcode domain comprises a first attachment region comprising a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule, a first complementary nucleic acid molecule of a first reporting complex, or a first hybrid nucleic acid molecule; and at least a second attachment region comprising a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule, at least a second complementary nucleic acid molecule of a second reporting complex, or at least a second hybrid nucleic acid molecule.

[0374] 22. The method of embodiment 20, further comprising repeating steps (1)-(6) of embodiment 3 using the at least second probe, the at least second copy of the first probe, or the at least third probe.

[0375] 23. The method of embodiment 21, further comprising repeating steps (1)-(9) using the at least second probe, the at least second copy of the first probe, or the at least third probe.

[0376] 24. The method according to any one of embodiments 1 to 8, wherein the detectable label comprises a plurality of moieties, each moiety being identifiable by its emission spectrum.

[0377] 25. The method of embodiment 24, wherein the detectable label comprises a quantum dot, a fluorescent moiety, a colorimetric moiety, or a combination thereof.

[0378] 26. The method of embodiment 24, wherein the detectable label comprises a fluorescent moiety.

[0379] 27. The method of embodiment 24, wherein the emission spectrum of each portion is the same or different.

[0380] 28. The method of embodiment 24, wherein the emission spectrum of at least one portion is different from that of the other portions.

[0381] 29. The method of embodiment 4 or 5, wherein the signal is an emission spectrum.

[0382] 30. The method of embodiment 1 or 6, wherein at least one nucleotide in the target binding domain is a modified nucleotide or a nucleic acid analog.

[0383] 31. The method according to embodiment 30, wherein the at least one modified nucleotide or the at least one nucleic acid analog is a locked nucleic acid (LNA).

[0384] 32. The method of embodiment 1 or 6, wherein the target nucleic acid is first fixed to a substrate before being contacted by a probe by binding at least a first position of the target nucleic acid to a first capture probe, wherein the first capture probe comprises a first affinity binding reagent that selectively binds to the substrate, wherein the first capture probe binds to the target nucleic acid at a different position on the target nucleic acid than the at least one probe binds to the target nucleic acid.

[0385] 33. The method of embodiment 1 or 6, wherein the target nucleic acid is fixed to the substrate after binding to the probe by binding at least a first position of the target nucleic acid to a first capture probe, wherein the first capture probe comprises a first binding affinity reagent that selectively binds to the substrate, wherein the first capture probe binds to the target nucleic acid at a different position on the target nucleic acid than the at least one probe binds to the target nucleic acid.

[0386] 34. The method of embodiment 32, wherein the target nucleic acid is elongated by applying a force sufficient to extend the target nucleic acid fixed to the substrate at a first location.

[0387] 35. The method of embodiment 33, wherein the target nucleic acid is elongated by applying a force sufficient to extend the target nucleic acid fixed to the substrate at a first location.

[0388] 36. The method of embodiment 34, wherein the force is gravity, fluid dynamics, electromagnetic force, flow stretching, receding meniscus technology, or a combination thereof.

[0389] 37. The method of embodiment 36, wherein the target nucleic acid is further fixed to the substrate by binding at least a second position of the target nucleic acid to at least a second capture probe, wherein the at least second capture probe comprises a second affinity binding reagent that selectively binds to the substrate, wherein the second capture probe binds to the target nucleic acid at a different position on the target nucleic acid than the at least one probe and the first capture probe are bound to the target nucleic acid.

[0390] 38. The method according to any one of embodiments 1 to 8, wherein each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule directly linked to a primary nucleic acid molecule.

[0391] 39. The method according to any one of embodiments 1 to 8, wherein each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule indirectly linked to the primary nucleic acid molecule via a nucleic acid spacer.

[0392] 40. The method according to any one of embodiments 1 to 8, wherein each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule indirectly linked to the primary nucleic acid molecule via a cleavable linker.

[0393] 41. The method of embodiment 40, wherein the cleavable linker is independently selected from photocleavable, chemically cleavable, and enzymatically cleavable.

[0394] 42. The method of embodiment 38, wherein each primary nucleic acid molecule hybridizes to at least 1, at least 2, at least 3, at least 4, at least 5, or at least 6 secondary nucleic acid molecules.

[0395] 43. The method of embodiment 42, wherein each of the secondary nucleic acid molecules independently comprises a cleavable linker.

[0396] 44. The method of embodiment 43, wherein the cleavable linker is independently selected from photocleavable, chemically cleavable, and enzymatically cleavable.

[0397] 45. The method of embodiment 42, wherein the one or more secondary nucleic acid molecules comprise at least one detectable label.

[0398] 46. ​​The method of embodiment 42, wherein each secondary nucleic acid molecule hybridizes to at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, or at least 7 tertiary nucleic acid molecules comprising at least one detectable label.

[0399] 47. A kit comprising reagents for performing the method of any one of embodiments 1-8. Example

[0400] Example 1: The present invention provides rapid and efficient detection of target nucleic acids

[0401] exist Figure 15A In the PCR amplification assay, the "small barcode" probe contains a barcode sequence in the 30-50 mer range and a target detection sequence. "Probe B" has a specific sequence (30-50 mer) and a universal tag with biotin for deposition onto the surface.

[0402] High concentrations of probes can be provided and applied to samples containing target nucleic acids. Probes of the present invention can be provided at concentrations 10 to 1000 times higher than probes containing detectable labels. Such high concentrations of probes partially provide rapid detection of target nucleic acids.

[0403] exist Figure 15B The probe is provided at 250 pM and in Figure 15C In other experiments using the standard nCounter workflow, probes containing a detectable label were provided at 25 pM. Figure 15A The capture probes were provided at 100 pM and Figure 15B The capture probe was provided at 2.5 nM. In other experiments utilizing the standard nCounter workflow, the capture probe containing a detectable label was provided at 100 pM. Figure 15A and 15B The target nucleic acid was shown to be detectable after 10 minutes. Significant target detection in these experiments was achieved in about 2 hours with the lower probe concentration and in about 30 minutes with the higher probe concentration ( Figure 16A In other experiments using the standard nCounter workflow, probes containing detectable labels required approximately 16.5 hours to detect target nucleic acids.

[0404] Figure 16A Shown are the average counts when simultaneously detecting four target nucleic acids using the probes and methods of the present invention. Here, the four target nucleic acids are Myc (green), Oaz1 (blue), RPL13A (orange), and TubB (red). The probes and capture probes were provided at 2.5 nM, and the target nucleic acid was 100 ng of human reference RNA. Figure 16B The present method was shown to be approximately 9 times more efficient than a method in which the probes were provided with a detectable label (identified in the figure as "Sprint"). Compared to the standard nCounter workflow, these results show a significant increase in efficiency in a much shorter time.

[0405] Example 2: Sample Preparation for Processing FFPE Tissue for Hyb&Count

[0406] First, the nucleic acids to be sequenced are extracted from formalin-fixed paraffin-embedded (FFPE) tissue in a one-step process. One or more 10 μm thick FFPE rolls are heated in a water-based nucleic acid extraction buffer to simultaneously melt the paraffin, decompose the tissue, and release nucleic acids from the cells. Suitable extraction buffers are known in the art and generally contain proteases, detergents such as Triton-100, chelating agents such as EDTA, and ammonium ions. The FFPE rolls and extraction buffer are incubated at 56°C for 30 minutes to separate the paraffin from the tissue and allow proteinase K to digest the tissue structure and expose the embedded cells to detergent to cause cell lysis. The solution is inverted three times at 8-minute intervals to help mix the reagents during the tissue dewaxing and digestion process. After this step, the solution is heated to 98°C to promote the reversal of formaldehyde cross-linking, which further helps extract nucleic acids.

[0407] Once the nucleic acids have been extracted from the FFPE tissue, the solution is filtered using a glass fiber filter (Whatman) with a 2.7 μm pore size to remove tissue debris and solidified paraffin. The resulting solution is a homogeneous, translucent solution containing highly fragmented nucleic acids due to the formalin fixation process and storage conditions. If further fragmentation is required, the DNA can be mechanically sheared using a Covaris focused ultrasound generator. Due to the buffer conditions, extended sonication is required to shear the nucleic acids. Standard settings of 50W peak incident power, 20% duty cycle, and 200 cycles / burst pulses were used for 600 seconds to achieve the maximum increase in captured targets (as shown in the figure). To achieve shorter fragment lengths, emulsified paraffin can be precipitated from the filtrate by centrifugation at 21,000 g and 4°C for 15 minutes. This enables DNA to be sheared to approximately 225 bp.

[0408] Next, target capture is performed by binding the capture probe pair to the target during a rapid hybridization step. The 5' capture probe contains a 3' biotin moiety, allowing the target to bind to the streptavidin-coated flow cell surface during the target deposition process. The 3' capture probe contains a 5' tag sequence (G sequence) that allows binding to the beads during the purification process. The reaction rate is driven by the capture probe concentration, which is added in the low nanomolar range to maximize the reaction rate. The capture probes hybridize to the target in a manner that flanks the region of interest to create a window. For each DNA target, the capture probe set also includes an oligonucleotide (oligo) consisting of the same sequence as the window to hybridize to the antisense strand of the target and prevent reannealing. The solution containing the capture probes is heated to 98°C for 3 minutes to denature the genomic DNA, followed by incubation at 65°C for 15 minutes. NaCl concentrations in the range of 400mM-600mM are used for the hybridization reaction. More than 100 target groups that have been experimentally validated are listed in Table 2, detailing the genes and exons of the targeted DNA regions.

[0409] Table 2

[0410]

[0411]

[0412]

[0413] After the targeted DNA regions bind to the capture probes, they are purified from the rest of the genomic DNA to produce an enriched target solution. Beads coated with antisense oligonucleotides (antisense G sequence) to the 3' capture probe binding sequence are incubated with the capture reaction mixture at room temperature for 15 minutes. After the binding step, the beads are washed 3 times with 0.1 x SSPE to remove non-target DNA and the 5' capture probe containing biotin. After washing, the beads are resuspended in 14 μL of 0.1 x SSPE and then heated at 45°C for 10 minutes to elute the purified DNA targets from the beads. After elution, 1 μL of 5 M NaCl is added to ensure that the capture probe remains bound to the DNA target.

[0414] The final step in the sample preparation process is the deposition of DNA targets onto the flow cell surface, where they can be analyzed using the inventive probes disclosed herein. A syringe pump is used to control the rate at which targets are loaded into the flow cell fluidic channel, allowing all targets time to diffuse across the channel height and bind to the streptavidin surface. This loading method creates a density gradient of targets, where the highest number of molecules per unit area is greatest at the fluidic channel inlet and decreases along the channel length in the direction of fluid flow toward the outlet. For a channel width of 1.6 mm and a height of 40 μm, a flow rate of 0.35 μL / sec achieves quantitative capture within a channel length of approximately 10 mm. Once the targets are bound to the surface via the biotinylated 5' capture probe, a solution of biotinylated oligonucleotides (G-hooks) with the reverse complement of the 3' capture probe binding sequence is injected to immobilize the free ends of the targets, creating a bridge structure with the central ssDNA region as the window of interest. Next, a solution of G-sequence oligonucleotides is added to hybridize to the excess G-hooks on the surface, reducing the amount of ssDNA on the surface.

[0415] To identify the targets that have been enriched, 15-mer probes are designed so that they can specifically bind to a single target in the group. These probes are synthesized with an adapter sequence at the 3' end, allowing them to be connected to a unique barcode oligonucleotide. Each barcode oligonucleotide contains 3 unique reporter binding domains that can bind to the reporter probe for target identification, allowing 64-plex readout using four-color reporter chemistry. These identification probes are injected into the fluid channel and incubated for 1 minute to enable hybridization to the target. Subsequently, a stringent wash of 0.1 x SSPE is used to remove unbound and non-specifically bound oligonucleotides. Based on the unique barcode of the target, 3 rounds of reporter probe hybridization are used to identify the target. The combination of a dual capture probe system that captures selected regions of the genome using target-specific identification probes provides a highly specific system for target enrichment and detection. Figure 17A Specificity is depicted, where a panel of 40 targets were captured and counted from 3 μg of purified, sheared gDNA. Lane 1 shows the counts of typical target detection when all capture probes were used together, compared to lane 2, where no gDNA was present. The specificity of the system was verified by including only capture probes that enriched for targets detected with blue and yellow reporters in lane 3 or with green and red in lane 4.

[0416] This DNA extraction, capture, and detection workflow was applied to three FFPE tissue types: tonsil, lung, and melanoma. For all tissue types, capture and detection of the 100-plex cancer target panel resulted in >95% target identification within 1-log uniformity. Counts of these targets in the three tissue types are shown in Figure 17B middle.

[0417] Example 3: Multicolor Reporter Image Processing for Hyb&Seq

[0418] The image processing pipeline consists of the following steps: background subtraction, registration, feature detection, and classification. In background subtraction, the average background of any given channel is a function of shot noise and exposure. In our system, the blue channel has the highest background level and greater variance. A simple top-hat filter with a circular structuring element of 7 pixels in radius is used to implement local background subtraction.

[0419] For multi-color and multi-cycle feature analysis, the features of interest must be perfectly aligned. The system requires two forms of registration. For the first form, a local affine transformation is applied to all image channels within a single acquisition stack. This transformation is a function of the optical system and is therefore consistent for a given instrument. This function is pre-calculated for each run and applied to every image acquired. For the second form, a global transformation in the form of a rigid shift is calculated using normalized cross-correlation to capture drift of the mechanical gantry during the run.

[0420] The next step is feature detection. Once all images are registered, features are detected using a matched filter, a Laplace of Gaussian (LoG) filter. The filter uses a fixed kernel size (matched to the diffraction limit of the feature) and different standard deviations (matched to the wavelength of the corresponding channel) to match the spot response enhancement. Local maxima are used to identify potential reporter locations. The associated intensity value for each identified feature is retrieved for classification.

[0421] The final step is classification. A Gaussian naïve-Bayes model is used to classify the multicolor reporter intensities. The model assumes that the reporter intensities are independent and follow a normal distribution. The model then calculates the specific features using the maximum a posteriori or MAP rule. (by all channels strength designation) belongs to a certain category Probability of:

[0422]

[0423] The reported dual-color-coded intensity distribution is shown in Figure 18 The figure illustrates an encoding scheme using two dyes, blue and red. In a two-color encoding scenario, there are 6 possible classes (including background). In the implemented system, 4 colors were chosen resulting in 14 potential classes. Note that there is some overlap between the individual half-dye vs. full-dye distributions. Therefore, classification between these classes exhibits a higher error rate, as Figure 19 As shown, xG "and" GG The maximum misclassification rate among the 10-class model was 11.8%. The misclassification rate for the 10-class model was less than 0.2%. Since each reporter requires a maximum of 8 classes, it is straightforward to select the class with the fewest misclassification errors.

[0424] Example 4: Function, design, preparation and testing of dual-color reporter probes

[0425] The dual-color reporter probes bind sequentially to three regions (R1, R2, and R3) of the probe barcode domain. Each region encodes eight "colors" defined by a dual-color fluorescent combination, such as "blue-blue" or "green-yellow." The three sequential "colors" of each probe are reported, which in turn correspond to the three dinucleotide reads that make up the hexamer sequence. The dual-color reporter probes are designed as follows: The dual-color reporter probes are 37 branched DNA oligomer structures designed to accommodate 15 fluorescent dyes for each color, for a total of 30 dyes per reporter probe. The 37 oligomers were divided into three sizes: (1) a 96 nt main branch consisting of two parts, a 12-mer single-stranded DNA sequence that was later used to report the hexamer and six 14-mers that hybridized to the six subbranches, (2) each of the six 89 nt subbranches consisted of two parts, a 14-mer that hybridized to the main branch and five 15-mer repeats that hybridized to five dye oligonucleotides, and (3) each of the five 15 nt dye oligonucleotides had a fluorescent dye modification at the 5' end of the oligonucleotide.

[0426] One of the key design features of the dual-color reporter probe is the different sub-branches and dye oligonucleotide sequences between the four different fluorescent dyes. This prevents "color swapping" or cross-hybridization between different fluorescent dyes. For example, each 15-mer dye oligonucleotide of the Alexa 488 fluorophore or blue corresponds to a sequence that is complementary only to the blue sub-branch. The blue sub-branch further has a different 14-mer sequence that is complementary only to the blue 14-mer sequence on the main branch, but not to yellow, red or green. Therefore, a specific main branch will have a specific dual-color sequence, which indicates which 15 plus 15 dye combinations it will accommodate.

[0427] Another important design feature of the dual-color reporter probe is that the 12 specific sequences on the main branches must meet the following requirements: (1) different 12-mer sequences between R1, R2, and R3, (2) each region encodes 8 different colors with high specificity, (3) high binding efficiency and uniformity among the 8 different colors, and (4) efficient removal of all 12-mers by competing toehold sequences.

[0428] A dual-color reporter probe was prepared as follows. Four fluorescent dyes (B = blue, G = green, Y = yellow, R = red) yielded 10 possible dual-color combinations (BB, BG, BR, BY, GG, GR, GY, RR, YR, YY). Only 8 of the 10 dual-color combinations were used for each of the three barcode regions of the probe, resulting in 24 different reporter probes (8 + 8 + 8 = 24).

[0429] Preparation of dual-color reporter probes occurs in two sequential hybridization steps: (1) dye oligonucleotide to sub-branch and then (2) dye + sub-branch to main branch. Four separate dye-sub-branch reactions were prepared by mixing 100 μM sub-branch and 600 μM dye oligonucleotide in 4.2X SSPE buffer at room temperature for 30 minutes. 24 reporter probes were then prepared using 2 μM main branch, 7.2 μM sub-branch + Dye1, and 7.2 μM sub-branch + Dye2 in 4.8X SSPE. These reactions were heated at 45°C for 5 minutes and then cooled at room temperature for 30 minutes. The 24 Dye + sub-branch-main branch reactions were then combined into 3 different pools (i.e., R1, R2, R3) corresponding to the barcode domain. For example, 8 different dual-color reporter probes (2 μM each) bound to the R1 barcode domain were combined and diluted 10-fold to a final working concentration of 200 nM for each reporter probe.

[0430] The following reporter probes were prepared as standard tests for quality assurance. Each of the three reporter probe pools was tested for binding to its corresponding barcode domain (R1, R2, R3) in three separate flow cells. The modified probe construct (only barcode domain present and fixed on the flow cell) was tested. All eight 12-mers representing each color were multiple, and all eight dual-color reporter probes were expected to be identified as having high color counts.

[0431] A schematic diagram of the dual-color reporter probe is shown in Figure 20 These probes are used in direct probe hybridization workflows for targeted capture of nucleic acids (e.g. Figure 21 shown). Figure 22 The additional power of these probes is demonstrated with respect to their use in identifying haplotypes of interest.

[0432] Example 5: Three dual-color reporter probes and image subtraction

[0433] This example demonstrates the prehybridization of three reporter complexes with sequencing probes in solution prior to binding to surface-immobilized targets. Solution hybridization has been shown to be much more efficient than surface hybridization and can be performed before sequencing experiments, significantly reducing the overall sample response time. The identities of the three reporters are determined by sequentially cleaving the reporters from the sequencing probe (either chemically or optically) and measuring the loss of fluorescence intensity.

[0434] The present disclosure requires hybridization of one of a set of 4096 barcode molecules (BC) to a target molecule immobilized on the surface of a flow cell, also described herein as a probe, wherein a region of the barcode domain can be bound by a complementary nucleic acid molecule comprising a detectable label or a complementary nucleic acid molecule comprising a detectable label reporter complex, one for each possible hexamer sequence. The identity of the barcode and the associated hexamer sequence within the target requires the binding and readout of three dual-color fluorescent reporter probes (RPTRs), also described herein as complementary nucleic acid molecules comprising a detectable label or a complementary nucleic acid molecule comprising a detectable label reporter complex. The RPTRs are flowed into the flow cell to hybridize to the BCs, imaged, and removed by maintaining a foothold in a sequential manner, with three RPTR flow cycles required for each BC readout.

[0435] Figure 23 All three RPTR probes are shown hybridized to the BC before flowing into the flow cell. This BC / RPTR complex can be purified before use to ensure proper formation of close to 100% BC / RPTR complex. The BC / RPTR complex is hybridized to the target on the surface, and an image is taken containing fluorescent signal from all six colors (three dual-color RPTRs). One of the reporters is then cleaved, removing the fluorescent dye from the complex. The cleavage mechanism is discussed in more detail herein. A second image is then taken containing fluorescent signal from only four colors (two dual-color RPTRs).

[0436] like Figure 24 As shown, the identity of the lost RPTR can be determined by comparing the 6-color and 4-color images. Next, the second RPTR is removed using a different cleavage mechanism, and a third image containing fluorescence signals from two colors (one dual-color RPTR) is taken. Again, the identity of the cleaved RPTR is determined by comparing the 2-color and 4-color images. The remaining fluorescence signal identifies the third RPTR to unambiguously identify the BC and, therefore, the hexamer sequence present in the target.

[0437] The cleavable RPTR used for the first two reads of the sequencing cycle was constructed similarly to the non-cleavable version and consisted of 30 dye oligonucleotides hybridized to 6 "daughter branch" oligonucleotides (also described herein as tertiary nucleic acid molecules), which ultimately hybridized to a "main branch" oligonucleotide (also described herein as a secondary nucleic acid molecule), as shown. Figure 25These RPTRs are made cleavable by synthesizing a "main branch" oligonucleotide with one or more of several cleavable modifications (such as photocleavable, chemically cleavable, and enzymatically cleavable) placed between the "main branch" portion that binds the BC and the portion that binds the "sub-branch" and dye. Examples of chemically cleavable modifications include disulfide moieties. Examples of enzymatically cleavable modifications include moieties containing deoxyuracil (dU) (cleavable using the 'USER' enzyme mix from New England Biolabs). The cleavable modifications used for the two RPTRs within one sequencing cycle must be different to enable sequential cleavage.

[0438] The key attributes and advantages of this approach are: (1) BC / RPTR complexes can be prepared prior to the sequencing run, which allows for better control of hybridization (i.e., solution hybridization rather than surface hybridization and much longer hybridization times); (2) this approach has the potential to significantly increase the number of unambiguously identified BCs because (a) the BC / RPTR complexes can be HPLC purified to ensure that each BC has all three RPTRs and (b) the cleavage efficiency is significantly higher than the RPTR hybridization and toehold efficiencies; and (3) the approach is much faster in terms of sequencing run time because (a) it does not require hybridization time for each RPTR to bind to the BC, (b) the cleavage kinetics used to remove the RPTR signal are significantly faster than toeholds, which are also based on hybridization, and (c) it requires far fewer reagent flow steps (8 vs. 14 (current approach), although only 6 flow steps are required if a UV-cleavable linker is used). It also requires taking fewer images (4 vs. 7 images, or 3 vs. 6 images if the final dark image with water wash is omitted).

[0439] Proof of principle experiments were performed using single BCs, UV-cleavable RPTRs, deoxyuracil (dU)-containing RPTRs (cleavable using the 'USER' enzyme mix from New England Biolabs), and standard RPTRs. These components were hybridized into BC / RPTR complexes and hybridized to a synthetic 50-mer BRAF exon 15 target sequence immobilized on a flow cell. Spot identity was determined by first imaging the intact BC / RPTR complex, followed by treatment with the USER enzyme to remove the dU-containing RPTRs and imaging again. Next, the photocleavable RPTRs were cleaved using UV light exposure, and the 50-mer BRAF exon 15 target sequence was hybridized to a synthetic 50-mer BRAF exon 15 target sequence immobilized on a flow cell. Spot identity was determined by first imaging the intact BC / RPTR complex, followed by treatment with the USER enzyme to remove the dU-containing RPTRs and imaging again. Figure 27 The third image was taken as shown. The four cluster features of the image were processed to determine their fluorescence intensity, and simple subtraction correctly identified the identities of the three RPTRs.

[0440] The main potential risk of this approach is the size of the BC / RPTR complex and the slowed hybridization kinetics associated with the surface-immobilized target. The increased size of the BC / RPTR complex relative to BC alone does slow down the binding kinetics; however, Figure 26 As shown, this can be overcome by longer incubation times. The loss of hybridization time can be offset here by reducing the efficiency and speed of other steps (ie, eliminating RPTR hybridization, reducing imaging, reducing flow steps, etc.).

[0441] We also tested whether this image subtraction method could be used to detect half-dye RPTRs. Because these dyes have smaller signals, they may be more difficult to identify reliably. To test this, a set of barcodes with many similarly colored RPTRs (primarily green and yellow) was prepared, where only the spot 1 RPTR was cleavable, as shown in Figure 2. Figure 28 and Figure 29 As shown. Images were taken before and after UV exposure to cut spot 1 RPTR. Both PC-GY and PC-GG were detectable and produced intensity changes similar to those expected based on the amount of dye lost (e.g., GYYYGY RPTR cut to __YYGY would lose 50% of its green and 25% of its yellow). A range of dye colors and classes and associated sequences are shown in the table below.

[0442]

[0443]

[0444]

Claims

1. A method for detecting at least one target nucleic acid in a sample, the method comprising: (1) contacting the sample with at least one probe capable of recognizing and binding to a first specific region of the at least one target molecule, wherein the at least one probe comprises: Target binding domain and barcode domain wherein the target binding domain comprises at least 4 nucleotides and is capable of recognizing and binding to the first specific region of the target nucleic acid, and wherein the target binding domain comprises a known nucleotide sequence; wherein the barcode domain comprises a first attachment region comprising a nucleic acid sequence capable of being bound by a first complementary nucleic acid molecule of a first reporter complex; and at least a second attachment region comprising a nucleic acid sequence capable of being bound by at least a second complementary nucleic acid molecule of at least a second reporter complex; wherein said sequence of said first attachment region is different from said sequence of said at least second attachment region; (2) allowing a first complementary nucleic acid molecule of a first reporter complex comprising a first detectable label to bind to the first attachment region, thereby associating the detectable label with the first attachment region; (3) detecting the first detectable label associated with the first attachment region; (4) removing the first detectable label; (5) binding at least a second complementary nucleic acid molecule of at least a second reporter complex comprising a second detectable label to the at least second attachment region, thereby associating the detectable label with the at least second attachment region; and (6) detecting the second detectable label associated with the at least second attachment region; wherein each reporter complex comprising a detectable label comprises a complementary nucleic acid molecule indirectly linked to the primary nucleic acid molecule via a photocleavable linker, wherein removing the first detectable label in step (4) comprises exposing the first reporter complex to light sufficient to cleave at least one photocleavable linker, thereby releasing the first detectable label, wherein the primary nucleic acid of each reporter complex is hybridized to at least one secondary nucleic acid molecule, and each secondary nucleic acid molecule is hybridized to at least one tertiary nucleic acid molecule and comprises a photocleavable linker, wherein each tertiary nucleic acid molecule comprises at least one detectable label; and The linear or sequential order of the first detectable label associated with the first attachment region and the second detectable label associated with the at least second attachment region recognizes the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

2. The method of claim 1, wherein the barcode domain comprises at least a third attachment region comprising a nucleic acid sequence capable of being bound by at least a third reporter complex; wherein the sequence of the at least third attachment region is different from the sequence of another attachment region.

3. The method of claim 1 or claim 2, further comprising: (7) removing the second detectable label, wherein removing the second detectable label comprises exposing the at least second reporter complex to light sufficient to cleave at least one photocleavable linker, thereby releasing the second detectable label; (8) binding at least a third complementary nucleic acid molecule of at least a third reporter complex comprising a third detectable label to the at least third attachment region, thereby associating the detectable label with the at least third attachment region; and (9) detecting the third detectable label associated with the at least third attachment region; wherein the first detectable label associated with the first attachment region, the second detectable label associated with the at least second attachment region, and the third detectable label associated with the at least third attachment region linearly or sequentially identify the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

4. The method of claim 2, wherein the barcode domain comprises at least a fourth, at least a fifth, at least a sixth, or at least a seventh attachment region comprising a nucleic acid sequence capable of being bound by at least a fourth complementary nucleic acid molecule, at least a fourth reporter complex, or at least a fourth hybrid nucleic acid molecule; wherein the sequence of the at least fourth attachment region is different from the sequence of another attachment region.

5. The method of claim 4, wherein the following steps are repeated: (a) removing the corresponding detectable label or the complementary nucleic acid molecule, wherein removing the corresponding detectable label comprises exposing the corresponding reporter complex to light sufficient to cleave at least one photocleavable linker, thereby releasing the corresponding detectable label; (b) binding a complementary nucleic acid molecule comprising a detectable label reporter complex to the corresponding attachment region, thereby associating the detectable label with the corresponding attachment region; and (c) detecting said corresponding detectable label associated with said attachment region, until each attachment region in the barcode domain has been sequentially bound by a complementary nucleic acid molecule comprising a detectable label, and the detectable label of the sequentially bound complementary nucleic acid molecules has been detected, wherein the linear or sequential order of the detectable labels associated with each attachment region recognizes the specific region of the at least one target molecule, thereby detecting the at least one target nucleic acid in the sample.

6. The method of any one of claims 1-2 and 4-5, wherein the first detectable label comprises a plurality of moieties, each moiety identifiable by its emission spectrum.

7. The method of claim 6, wherein the first detectable label comprises a quantum dot, a fluorescent moiety, a colorimetric moiety, or a combination thereof.

8. The method of claim 6, wherein the first detectable label comprises a fluorescent moiety.

9. The method of claim 6, wherein the emission spectrum of each portion is the same.

10. The method of claim 6, wherein the emission spectrum of each portion is different.

11. The method of claim 6, wherein the emission spectrum of at least one portion is different from the emission spectrum of the other portions.

12. A kit comprising reagents for carrying out the method of any one of claims 1 to 11.

13. Apparatus for carrying out the method according to any one of claims 1 to 11.

Images