Characterizing biomarker-specific identifiers from highly multiplexed lobed cut assays

Abstract

Techniques for characterizing samples, such as nucleic acid samples from subjects, include, but are not limited to, methods, compositions, kits, systems, reagents, and reaction mixtures, particularly by characterizing biomarker-specific identifiers generated in multilobe-cut assays of nucleic acid samples.

Description

[0001] Priority Statement

[0002] This application claims priority to U.S. Provisional Application No. 63 / 581,624, filed September 8, 2023, and U.S. Provisional Application No. 63 / 593,807, filed October 27, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This document provides compositions and methods for detecting multiple target sequences using petal-shaped oligonucleotides and ligandable molecules comprising a circularizable vector molecule (e.g., a padlock probe or dumbbell probe) and an oligonucleotide pair (e.g., a “vector pair”) that forms a linear construct upon ligation with the petal-shaped oligonucleotide. In some embodiments, bridging oligonucleotides (e.g., DNA, RNA, or chimeric oligonucleotides) are used to align the ends of the vector and cleavage petal sequences to form one or more ligandable nicks. The methods described herein can be used to detect mixtures of cleaved probe lobes, such as cleavage lobes generated in multiplex and highly multiplexed petal-shaped cleavage assays (including multiplex PCR-petal assays), using circular and / or circularizable molecules, or using individual oligonucleotides or vector pairs, to form a linear molecule upon ligation. Embodiments of this technology include amplification and / or sequencing techniques for detecting and / or counting cleavage lobes generated in petal-shaped cleavage assays. Background Technology

[0004] For example, for diagnostic purposes, the detection of target nucleic acids typically requires high specificity, such as the ability to distinguish nucleic acids that may differ by a single nucleotide, and high sensitivity, such as the ability to detect target nucleic acids that may be present in very low copy numbers. There is a need in the art for assays that combine specific identification of target nucleic acids with signal amplification to produce sensitive reports of the presence of target nucleic acids. Summary of the Invention

[0005] The techniques described herein provide methods, compositions, kits, systems, reagents, and reaction mixtures for characterizing samples (e.g., nucleic acid samples from subjects), particularly by characterizing cleavage lobes containing marker-specific identifiers, such as cleavage lobes generated in multilobe cleavage assays.

[0006] Exemplary implementations of this technology include, but are not limited to, the following:

[0007] 1. A method comprising:

[0008] a) A reaction mixture comprising or suspected to comprise a population of different target nucleic acids, multiple valve-shaped oligonucleotides, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises:

[0009] i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and

[0010] ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion;

[0011] In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed.

[0012] In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to generate a population of cleavage flaps;

[0013] b) Providing a population of linker molecules, wherein the linker molecules comprise sequences of the population of the cleavage lobes and / or sequences complementary to the population of the cleavage lobes, and

[0014] c) Characterize the population of the linker molecules to characterize the population of the different target nucleic acids present in the reaction mixture.

[0015] 2. The method as described in embodiment 1, wherein the connecting molecule is a cyclic molecule.

[0016] 3. The method as described in embodiment 1, wherein the connecting molecule is a linear molecule.

[0017] 4. The method of embodiment 1, wherein providing the population of linking molecules comprises treating the population of cleavage flaps with a linkable carrier molecule formulation, wherein the cleavage flaps are linked to the linkable carrier molecules to form the population of linking molecules, each linking molecule comprising a carrier molecule sequence and a cleavage flap sequence.

[0018] 5. The method of any one of embodiments 1-4, wherein the reaction mixture is an amplification reaction mixture, and wherein the population of different target nucleic acids is contained in the amplified nucleic acids generated in the amplification reaction mixture.

[0019] 6. The method of embodiment 5, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

[0020] 7. The method of any one of embodiments 1-6, wherein characterizing the population of the linker molecules includes counting the different cleavage sequences or their complementary sequences present in the population of the linker molecules.

[0021] 8. The method of embodiment 7, wherein characterizing the population of linker molecules includes sequencing the cleavage lobe sequence or its complement sequence in the population of linker molecules.

[0022] 9. The method of any one of embodiments 1-8, wherein the valve endonuclease is a FEN-1 endonuclease.

[0023] 10. The method of embodiment 9, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture contains Mg. ++ Lobe-shaped assay buffer.

[0024] 11. The method as described in embodiment 10, wherein the stringent FEN-1 endonuclease is selected from the group consisting of: Archaeocystis scintillans ( Archaeoglobus fulgidus (Afu) FEN-1, Vibrio flavogenes ( Pyrococcus furiosus (Pfu) FEN-1 and mixed-virus Archaeococcus ( Archaeoglobus veneficus (Ave) FEN-1 and its variants, as well as CLEAVASE 2.0 nuclease.

[0025] 12. The method of any one of embodiments 1-11, wherein each linker in the group of linkers comprises one or more of the following:

[0026] i) A first bridging oligonucleotide complementary arm located at the 3′ end of the first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of the second vector sequence, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm.

[0027] ii) A pair of amplification primer binding sites configured to amplify a portion of the cleavage lobe sequence of the ligation molecule;

[0028] iii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification;

[0029] iv) At least one unique molecular tag;

[0030] v) Polynucleotide linkers; and / or

[0031] vi) Polymerization terminator, preferably an internal spacer region.

[0032] 13. The method of any one of embodiments 1-12, further comprising the step of amplifying the region of the linker molecule containing the cleavage lobe sequence to form a population of lobe amplicones.

[0033] 14. The method of embodiment 13, further comprising sequencing the lobe amplicon to generate sequencing reads.

[0034] 15. The method of any one of embodiments 4-14, wherein the connectable carrier molecule comprises one or more of a dumbbell probe, a padlock probe, a carrier pair and / or a linear carrier molecule.

[0035] 16. The method of any one of embodiments 4-15, wherein the connectable vector molecule comprises a pair of vectors, wherein each pair of vectors comprises a first bridging oligonucleotide complementary arm located at the 3′ end of a first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of a second vector sequence, and wherein step b) comprises contacting a population of the cleavage flaps with the connectable vector molecule formulation and a population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

[0036] 17. The method of embodiment 16, wherein each of the linkable carrier molecules at the 5′ end of the second carrier sequence comprises a 5′ blocking group.

[0037] 18. The method of any one of embodiments 4-15, wherein the connectable carrier molecule is a dumbbell probe, wherein each dumbbell probe contains a bridging sequence complementary to the cleavage lobe sequence.

[0038] 19. The method of any one of embodiments 1-15 and 17-18, wherein the linker molecule comprises a circular molecule, the method further comprising the step of using rolling circle replication to replicate the circular molecule comprising a cleavage lobe sequence to form a population of lobe replicons.

[0039] 20. The method of any one of embodiments 1-19, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 10 unique valve oligonucleotides.

[0040] 21. The method of embodiment 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 20 unique valve oligonucleotides.

[0041] 22. The method of embodiment 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 30 unique valve oligonucleotides.

[0042] 23. The method of embodiment 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises 40 to 1000 unique valve oligonucleotides.

[0043] 24. The method of any one of embodiments 1-23, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

[0044] 25. The method of embodiment 24, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is 13 to 100 nucleotides in length.

[0045] 26. The method of any one of embodiments 1-25, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

[0046] 27. The method of any one of embodiments 1-26, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

[0047] 28. The method of embodiment 27, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid.

[0048] 29. The method of embodiment 28, wherein the oligonucleotide comprises a flap oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

[0049] 30. The method of embodiment 28 or embodiment 29, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

[0050] 31. The method of embodiment 30, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

[0051] 32. The method of any one of embodiments 28 to 31, wherein the reference nucleic acid comprises methylation. B3GALT6 DNA.

[0052] 33. A composition comprising:

[0053] i) One or both of the following:

[0054] a) Groups of cleavage flaps; and

[0055] b) A group of linker molecules, each linker molecule containing a cleavage valve sequence or its complementary sequence, wherein each cleavage valve sequence or its complementary sequence contains a nucleotide sequence indicating a target nucleic acid;

[0056] as well as

[0057] ii) Ligase.

[0058] 34. The composition of embodiment 33, further comprising a population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

[0059] 35. The composition of embodiment 33 further comprises a linker molecule, wherein each linker molecule comprises at least one linker molecule sequence.

[0060] 36. The composition of embodiment 33 or embodiment 35 further comprises a group of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

[0061] 37. The composition as described in embodiment 36, further comprising one or more of the following:

[0062] i) A first linkable carrier molecule and a second linkable carrier molecule, the second linkable carrier molecule comprising a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the first linkable carrier molecule and the second linkable carrier molecule in a complex, the complex comprising:

[0063] I) A first connectable slit, comprising the 3' end of the first connectable molecule and the 5' end of the cutting flap, and

[0064] II) An invasive cleavage substrate comprising the 3' end of the cleavage flap and the 5' end of the second connectable carrier molecule comprising the 5' blocking group.

[0065] The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups;

[0066] ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions;

[0067] iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications;

[0068] iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease;

[0069] v) Mg ++ Lobe-shaped assay buffer solution;

[0070] vi) Amplification reagents;

[0071] vii) Lobe-like assay reagent; and / or

[0072] viii) Ligases, including those from Chlorella. (Chlorella) Viral PBCV-1 DNA ligase.

[0073] 38. The composition of any one of embodiments 33-37, wherein each linker in the group of linker molecules comprises one or more of the following:

[0074] i) A pair of amplification primer binding sites configured as part of a cleavage lobe sequence in an amplification ligand molecule;

[0075] ii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification;

[0076] iii) At least one unique molecular tag;

[0077] iv) Polynucleotide linkers; and / or

[0078] v) Polymerization terminator, preferably an internal spacer region.

[0079] 39. The composition of any one of embodiments 33 to 38, further comprising a population of amplification products, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in a linker molecule of the population of linker molecules.

[0080] 40. A method comprising:

[0081] a) Providing in the mixture a bridging oligonucleotide, a valve sequence oligonucleotide, preferably a cleaved valve oligonucleotide, a first linker molecule, and a second linker molecule containing a 5′ blocking group, wherein the bridging oligonucleotide comprises:

[0082] i) Internal segments complementary to the said lobe sequence oligonucleotides,

[0083] ii) The 3' end complementary to the 3' end of the first connectable carrier molecule, and

[0084] iii) The 5' end complementary to the 5' end of the second connectable carrier molecule.

[0085] In the mixture, the bridging oligonucleotide hybridizes with the lobe sequence oligonucleotide, the first linker molecule, and the second linker molecule to form a complex, the complex comprising:

[0086] I) A first connectable nick, comprising the 3′ end of the first connectable molecule and the 5′ end of the valve sequence oligonucleotide, and

[0087] II) Invasive cleavage of a substrate comprising the 3′ end of the said lobe sequence oligonucleotide and the 5′ end of the second linkable vector molecule comprising the said 5′ blocking group;

[0088] b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate cleaves the 5′ blocking group of the second linkable carrier molecule and creates a second linkable nick; and

[0089] c) Treat the complex with a ligase, wherein the first ligase and the second ligase are joined to form a linker molecule.

[0090] 41. The method of embodiment 40, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

[0091] 42. The method of embodiment 40 or 41, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

[0092] 43. The method of embodiment 42, wherein the RNA portion comprises one or more nucleotide modifications.

[0093] 44. The method of embodiment 43, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modifications.

[0094] 45. The method of embodiment 43 or embodiment 44, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

[0095] 46. ​​The method of embodiment 43, wherein the 3′ end and adjacent portion of the inner segment of the bridging oligonucleotide comprises DNA.

[0096] 47. The method of any one of embodiments 40-46, further comprising detecting the linker molecule in a nucleic acid detection assay.

[0097] 48. The method of any one of embodiments 40-47, wherein the ligase comprises Chlorella virus PBCV-1 DNA ligase.

[0098] 49. A method comprising:

[0099] a) A reaction mixture comprising or suspected to comprise a population of different target nucleic acids, multiple valve-shaped oligonucleotides, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises:

[0100] i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and

[0101] ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion;

[0102] In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed.

[0103] In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to generate a population of cleavage flaps;

[0104] b) Providing a population of cyclic molecules, wherein the cyclic molecules comprise sequences of the population of the cleavage lobes and / or sequences complementary to the population of the cleavage lobes, and

[0105] c) Characterize the population of the cyclic molecules to characterize the population of the different target nucleic acids present in the reaction mixture.

[0106] 50. The method of embodiment 49, wherein characterizing the population of the cyclic molecules includes detecting hybridization of the cleavage flap with the cyclic molecules.

[0107] 51. The method of embodiment 49, wherein providing the population of the cyclic molecules includes processing the population of the cleavage lobes, wherein the 5′ and 3′ ends of individual cleavage lobes are joined together to form cyclic molecules.

[0108] 52. The method of embodiment 49, wherein providing the population of cyclic molecules comprises treating the population of cleavage flaps with a cyclizable molecular formulation, wherein the cleavage flaps are linked to the cyclizable molecules to form the population of cyclic molecules, each cyclic molecule comprising a cyclizable molecular sequence and a cleavage flap sequence.

[0109] 53. The method of any one of embodiments 49-52, wherein the reaction mixture is an amplification reaction mixture, and wherein the population of different target nucleic acids is contained in the amplified nucleic acids generated in the amplification reaction mixture.

[0110] 54. The method of embodiment 53, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

[0111] 55. The method of any one of embodiments 49-54, wherein characterizing the population of the cyclic molecules comprises counting the different cleavage sequences or their complementary sequences present in the population of the cyclic molecules.

[0112] 56. The method of embodiment 55, wherein characterizing the population of the cyclic molecules includes sequencing the cleavage sequence or its complementary sequence in the population of the cyclic molecules.

[0113] 57. The method of any one of embodiments 49-56, wherein the valve endonuclease is a FEN-1 endonuclease.

[0114] 58. The method of embodiment 57, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture contains Mg ++ Lobe-shaped assay buffer.

[0115] 59. The method of embodiment 58, wherein the strict FEN-1 endonuclease is selected from the group consisting of: Archaeococcus scintillans (Afu) FEN-1, Pfu FEN-1 and Ave FEN-1 and their variants, and CLEAVASE 2.0 nuclease.

[0116] 60. The method of any one of embodiments 49-59, wherein each cyclic molecule in the group of said cyclic molecules comprises one or more of the following:

[0117] i) A pair of bridging oligonucleotide complementary arms located at the 5′ and 3′ ends of the cyclizable molecule, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm;

[0118] ii) A pair of amplification primer binding sites configured to amplify a portion of the cleavage lobe sequence of a circular molecule;

[0119] iii) Primer binding sites configured to amplify the circular molecule via rolling circle amplification;

[0120] iv) At least one unique molecular tag;

[0121] v) Polynucleotide linkers; and / or

[0122] vi) Polymerization terminator, preferably an internal spacer region.

[0123] 61. The method of any one of embodiments 49-60, further comprising the step of amplifying the region of the cyclic molecule containing the cleavage lobe sequence to form a population of lobe amplicones.

[0124] 62. The method of embodiment 61, further comprising sequencing the lobe amplicon to generate sequencing reads.

[0125] 63. The method of any one of embodiments 52-62, wherein the cyclizable molecule comprises a dumbbell probe and / or a padlock probe.

[0126] 64. The method of any one of embodiments 52-63, wherein each of the cyclizable molecules comprises a pair of bridging oligonucleotide complementary arms located at the 5′ end and the 3′ end of the cyclizable molecule, and wherein step b) comprises contacting a population of the cleavage flaps with the cyclizable molecule formulation and the population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 5′ end complementary to the 5′ end of the cyclizable molecule, and a 3′ end complementary to the 3′ end of the cyclizable molecule.

[0127] 65. The method of embodiment 64, wherein each of the cyclizable molecules comprises a 5′ blocking group.

[0128] 66. The method of any one of embodiments 52-65, wherein the cyclizable molecule is a dumbbell probe, wherein each dumbbell probe comprises a bridging sequence complementary to the cleavage lobe sequence.

[0129] 67. The method of any one of embodiments 49-66, further comprising the step of using rolling ring replication to replicate the circular molecule containing the cleavage lobe sequence to form a population of lobe replicons.

[0130] 68. The method of any one of embodiments 49-67, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 10 unique valve oligonucleotides.

[0131] 69. The method of embodiment 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 20 unique valve oligonucleotides.

[0132] 70. The method of embodiment 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 30 unique valve oligonucleotides.

[0133] 71. The method of embodiment 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises 40 to 1000 unique valve oligonucleotides.

[0134] 72. The method of any one of embodiments 49-71, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

[0135] 73. The method of embodiment 72, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is 13 to 100 nucleotides in length.

[0136] 74. The method of any one of embodiments 49-73, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

[0137] 75. The method of any one of embodiments 49-74, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

[0138] 76. The method of embodiment 75, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid.

[0139] 77. The method of embodiment 76, wherein the oligonucleotide comprises a flap oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

[0140] 78. The method of embodiment 76 or embodiment 77, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

[0141] 79. The method of embodiment 78, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

[0142] 80. The method of any one of embodiments 76 to 79, wherein the reference nucleic acid comprises methylation. B3GALT6 DNA.

[0143] 81. A composition comprising:

[0144] i) One or both of the following:

[0145] a) Groups of cleavage flaps; and

[0146] b) A group of circular molecules, each circular molecule containing a cleavage sequence or its complement, wherein each cleavage sequence or its complement contains a nucleotide sequence indicating a target nucleic acid;

[0147] as well as

[0148] ii) Ligase.

[0149] 82. The composition of embodiment 81, further comprising a population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

[0150] 83. The composition of embodiment 81 further comprises cyclizable molecules, wherein each cyclic molecule comprises a cyclizable molecular sequence.

[0151] 84. The composition of embodiment 81 or embodiment 83 further comprises a group of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the cyclizable molecule, and a 5′ end complementary to the 5′ end of the cyclizable molecule.

[0152] 85. The composition as described in embodiment 84, further comprising one or more of the following:

[0153] i) A cyclizable carrier molecule containing a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the cyclizable molecule to form a complex, the complex comprising:

[0154] I) A first connectable slit, comprising the 3' end of the cyclizable molecule and the 5' end of the cleavage flap, and

[0155] II) An invasive cleavage substrate comprising the 3′ end of the cleavage flap and the 5′ end of the cyclizable molecule comprising the 5′ blocking group;

[0156] The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups;

[0157] ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions;

[0158] iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications;

[0159] iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease;

[0160] v) Mg ++ Lobe-shaped assay buffer solution;

[0161] vi) Amplification reagents;

[0162] vii) Lobe-like assay reagent; and / or

[0163] viii) Ligases, including the Chlorella virus PBCV-1 DNA ligase.

[0164] 86. The composition according to any one of embodiments 81-85, wherein each cyclic molecule in the group of said cyclic molecules comprises one or more of the following:

[0165] i) A pair of amplification primer binding sites configured to amplify a circular molecule containing a cleavage lobe sequence;

[0166] ii) Primer binding sites configured to amplify circular molecules via rolling circle amplification;

[0167] iii) At least one unique molecular tag;

[0168] iv) Polynucleotide linkers; and / or

[0169] v) Polymerization terminator, preferably an internal spacer region.

[0170] 87. The composition of any one of embodiments 81-86, further comprising a population of amplification products, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in a circular molecule within the population of said circular molecules.

[0171] 88. A method comprising:

[0172] a) Providing a bridged oligonucleotide, a valve sequence oligonucleotide, preferably a valve-cleaving oligonucleotide, and a cyclizable molecule containing a 5′ blocking group in the mixture, wherein the bridged oligonucleotide comprises:

[0173] i) Internal segments complementary to the said lobe sequence oligonucleotides,

[0174] ii) The 3′ end complementary to the 3′ end of the cyclizable molecule, and

[0175] iii) The 5' end complementary to the 5' end of the cyclizable molecule.

[0176] In the mixture, the bridging oligonucleotide hybridizes with the valve sequence oligonucleotide and the cyclizable molecule to form a complex, the complex comprising:

[0177] I) A first connectable nick, comprising the 3′ end of the cyclizable molecule and the 5′ end of the valve sequence oligonucleotide, and

[0178] II) Invasive cleavage of a substrate comprising the 3′ end of the said valve sequence oligonucleotide and the 5′ end of the said cyclizable molecule comprising the said 5′ blocking group;

[0179] b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate is cleaved to remove the 5′ blocking group and create a second connectable nick; and

[0180] c) Treat the complex with a ligase such that the first ligase and the second ligase are joined to form a cyclic molecule.

[0181] 89. The method of embodiment 88, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

[0182] 90. The method of embodiment 88 or 89, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

[0183] 91. The method of embodiment 90, wherein the RNA portion comprises one or more nucleotide modifications.

[0184] 92. The method of embodiment 91, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modification.

[0185] 93. The method of embodiment 91 or embodiment 92, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

[0186] 94. The method of embodiment 93, wherein the 3′ end and the adjacent portion of the inner segment of the bridging oligonucleotide comprise DNA.

[0187] 95. The method as described in any one of embodiments 93-94, further comprising detecting the cyclic molecule in a nucleic acid detection assay.

[0188] 96. The method of any one of embodiments 88-95, wherein the ligase comprises Chlorella virus PBCV-1 DNA ligase.

[0189] 97. A method comprising:

[0190] a) A reaction mixture comprising or suspected to comprise at least one target nucleic acid, at least one valve-shaped oligonucleotide, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises:

[0191] i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and

[0192] ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion;

[0193] In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed.

[0194] In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to produce at least one cleavage flap.

[0195] b) Provide at least one linker molecule, wherein the linker molecule comprises a sequence of the cleavage lobe and / or a sequence complementary to the cleavage lobe, and

[0196] c) Characterize the at least one linker molecule to characterize the at least one target nucleic acid present in the reaction mixture.

[0197] 98. The method of embodiment 97, wherein the connecting molecule is a cyclic molecule.

[0198] 99. The method of embodiment 97, wherein the connecting molecule is a linear molecule.

[0199] 100. The method of embodiment 97, wherein providing the linker molecule comprises treating the cleavage flap with a formulation comprising a linkable carrier molecule, wherein the cleavage flap is linked to the linkable carrier molecule to form a linker molecule, each linker molecule comprising a carrier molecule sequence and a cleavage flap sequence.

[0200] 101. The method of any one of embodiments 97-100, wherein the reaction mixture is an amplification reaction mixture, and wherein the at least one target nucleic acid is contained in at least one amplified nucleic acid generated in the amplification reaction mixture.

[0201] 102. The method of embodiment 101, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

[0202] 103. The method of any one of embodiments 97-102, wherein characterizing the at least one linker molecule comprises counting different cleavage sequences or their complementary sequences if present in the reaction mixture.

[0203] 104. The method of embodiment 103, wherein characterizing the at least one linker molecule comprises sequencing a cleavage lobe sequence or its complementary sequence if present in the reaction mixture.

[0204] 105. The method of any one of embodiments 97-104, wherein the valve endonuclease is a FEN-1 endonuclease.

[0205] 106. The method of embodiment 105, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture contains Mg ++ Lobe-shaped assay buffer.

[0206] 107. The method of embodiment 106, wherein the strict FEN-1 endonuclease is selected from the group consisting of: Archaeococcus scintillans (Afu) FEN-1, Pfu FEN-1 and Ave FEN-1 and their variants, and CLEAVASE 2.0 nuclease.

[0207] 108. The method as described in any one of embodiments 97-107, wherein each linker molecule comprises one or more of the following:

[0208] i) A first bridging oligonucleotide complementary arm located at the 3′ end of the first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of the second vector sequence, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm.

[0209] ii) A pair of amplification primer binding sites configured to amplify a portion of the cleavage lobe sequence of the ligation molecule;

[0210] iii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification;

[0211] iv) At least one unique molecular tag;

[0212] v) Polynucleotide linkers; and / or

[0213] vi) Polymerization terminator, preferably an internal spacer region.

[0214] 109. The method of any one of embodiments 97-108, further comprising the step of amplifying the region of the at least one linker molecule containing the cleavage lobe sequence to form at least one lobe amplicon.

[0215] 110. The method of embodiment 109, further comprising sequencing the at least one lobe amplicon to generate sequencing reads.

[0216] 111. The method of any one of embodiments 100-110, wherein the connectable carrier molecule is selected from dumbbell probes, padlock probes, carrier pairs and / or linear carrier molecules.

[0217] 112. The method of any one of embodiments 100-111, wherein the connectable vector molecule comprises a vector pair, the vector pair comprising a first bridging oligonucleotide complementary arm located at the 3′ end of a first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of a second vector sequence, and wherein step b) comprises contacting the at least one cleavage flap with the connectable vector molecule and the bridging oligonucleotide, wherein the bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

[0218] 113. The method of embodiment 112, wherein the connectable carrier molecule comprising the 5′ end of the second carrier sequence contains a 5′ blocking group.

[0219] 114. The method of any one of embodiments 100-111, wherein the connectable carrier molecule is a dumbbell probe comprising a bridging sequence complementary to the cleavage lobe sequence.

[0220] 115. The method of any one of embodiments 97-111 and 114, wherein the linker molecule is a cyclic molecule, the method further comprising the step of using rolling circle replication to replicate the cyclic molecule containing the cleavage lobe sequence to form a lobe replicon.

[0221] 116. The method of any one of embodiments 97-115, wherein the target-specific portion of the valve oligonucleotide in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

[0222] 117. The method of embodiment 116, wherein the target-specific portion of the valve oligonucleotide in the reaction mixture is 13 to 100 nucleotides in length.

[0223] 118. The method of any one of embodiments 97-117, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

[0224] 119. The method of any one of embodiments 97-118, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

[0225] 120. The method of embodiment 119, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid, the reference nucleic acid preferably comprising methylation. B3GALT6 DNA.

[0226] 121. The method of embodiment 120, wherein the oligonucleotide comprises a flap oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

[0227] 122. The method as described in embodiment 120 or embodiment 121, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

[0228] 123. The method of embodiment 122, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

[0229] 124. A composition comprising:

[0230] i) One or both of the following:

[0231] a) at least one cutting flap; and

[0232] b) At least one linker molecule comprising a cleavage lobe sequence or its complementary sequence, wherein the cleavage lobe sequence or its complementary sequence comprises a nucleotide sequence indicating a target nucleic acid;

[0233] as well as

[0234] ii) Ligase.

[0235] 125. The composition of embodiment 124 further comprises at least one bridging oligonucleotide, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

[0236] 126. The composition of embodiment 124 further comprises at least one linker molecule, wherein each linker molecule comprises at least one linker molecule sequence.

[0237] 127. The composition of embodiment 124 or embodiment 126 further comprises at least one bridging oligonucleotide, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

[0238] 128. The composition as described in embodiment 127, further comprising one or more of the following:

[0239] i) A first linkable carrier molecule and a second linkable carrier molecule, the second linkable carrier molecule comprising a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the first linkable carrier molecule and the second linkable carrier molecule in a complex, the complex comprising:

[0240] I) A first connectable slit, comprising the 3' end of the first connectable molecule and the 5' end of the cutting flap, and

[0241] II) An invasive cleavage substrate comprising the 3' end of the cleavage flap and the 5' end of the second connectable carrier molecule comprising the 5' blocking group.

[0242] The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups;

[0243] ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions;

[0244] iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications;

[0245] iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease;

[0246] v) Mg ++ Lobe-shaped assay buffer solution;

[0247] vi) Amplification reagents;

[0248] vii) Lobe-like assay reagent; and / or

[0249] viii) Ligases, including the Chlorella virus PBCV-1 DNA ligase.

[0250] 129. The composition according to any one of embodiments 124-128, wherein the linker molecule comprises one or more of the following:

[0251] i) A pair of amplification primer binding sites configured as part of a cleavage lobe sequence in an amplification ligand molecule;

[0252] ii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification;

[0253] iii) At least one unique molecular tag;

[0254] iv) Polynucleotide linkers; and / or

[0255] v) Polymerization terminator, preferably an internal spacer region.

[0256] 130. The composition of any one of embodiments 124 to 129 further comprises at least one amplification product, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in the linker molecule.

[0257] 131. A method comprising:

[0258] a) Providing in the mixture a bridging oligonucleotide, a valve sequence oligonucleotide, preferably a cleaved valve oligonucleotide, a first linker molecule, and a second linker molecule containing a 5′ blocking group, wherein the bridging oligonucleotide comprises:

[0259] i) Internal segments complementary to the said lobe sequence oligonucleotides,

[0260] ii) The 3' end complementary to the 3' end of the first connectable carrier molecule, and

[0261] iii) The 5' end complementary to the 5' end of the second connectable carrier molecule.

[0262] In the mixture, the bridging oligonucleotide hybridizes with the lobe sequence oligonucleotide, the first linker molecule, and the second linker molecule to form a complex, the complex comprising:

[0263] I) A first connectable nick, comprising the 3′ end of the first connectable molecule and the 5′ end of the valve sequence oligonucleotide, and

[0264] II) Invasive cleavage of a substrate comprising the 3′ end of the said lobe sequence oligonucleotide and the 5′ end of the second linkable vector molecule comprising the said 5′ blocking group;

[0265] b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate cleaves the 5′ blocking group of the second linkable carrier molecule and creates a second linkable nick; and

[0266] c) Treat the complex with a ligase, wherein the first ligase and the second ligase are joined to form a linker molecule.

[0267] 132. The method of embodiment 131, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

[0268] 133. The method as described in embodiment 131 or 132, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

[0269] 134. The method of embodiment 133, wherein the RNA portion comprises one or more nucleotide modifications.

[0270] 135. The method of embodiment 134, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modifications.

[0271] 136. The method as described in embodiment 134 or embodiment 133, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

[0272] 137. The method of embodiment 134, wherein the 3′ end and adjacent portion of the inner segment of the bridging oligonucleotide comprises DNA.

[0273] 138. The method of any one of embodiments 131-137, further comprising detecting the linker molecule in a nucleic acid detection assay.

[0274] definition

[0275] To facilitate understanding of this technology, several terms and phrases are defined below. Further definitions will be provided throughout the specific implementation.

[0276] Throughout the specification and claims, unless the context clearly specifies otherwise, the following terms have the meanings explicitly applicable herein. As used herein, the phrase "in one embodiment" does not necessarily refer to the same embodiment, although it may refer to the same embodiment. Furthermore, as used herein, the phrase "in another embodiment" does not necessarily refer to different embodiments, although it may refer to different embodiments. Therefore, as described below, various embodiments of the present technology can be readily combined without departing from the scope or spirit of the present technology.

[0277] Additionally, as used herein, unless the context clearly specifies otherwise, the term "or" is an inclusive "or" operator and is equivalent to the terms "and / or". Unless the context clearly specifies otherwise, the term "based on" is not exclusive and allows for basing on other factors not described. Furthermore, throughout the specification, the meanings of "an / a" and "the / described" include plural references. The meaning of "in" includes both "in" and "on".

[0278] As used herein, the transitional term “comprising” is synonymous with “including,” “containing,” and “characterized in,” and is inclusive or open-ended, and does not exclude additional unlisted elements or methodological steps.

[0279] As used in the claims of this application, the transitional phrase “consistently of…” limits the scope of the claims to the specified materials or steps “and those materials or steps that do not substantially affect the basic and novel features of the claimed invention,” as discussed in In re Herz, 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976). For example, a composition “consistently of the listed elements” may contain unlisted contaminants, which, although present, do not alter the function of the listed composition compared to the pure composition (i.e., a composition “consisting of the listed ingredients”).

[0280] As used in the claims of this application with respect to a group or plurality of elements each having a particular listed feature, for example, "each oligonucleotide," "each lobe," and "each linker molecule" in the group of oligonucleotides, lobes, and linkers are respectively understood to relate to the group of those elements having the listed features, but do not exclude the presence of similar elements without the listed features. For example, in a mixture having "a group of oligonucleotides each having a 5′ blocking group," the mixture may also contain oligonucleotides without a 5′ blocking group.

[0281] The term "primer" refers to an oligonucleotide, whether naturally occurring (e.g., a fragment of nucleic acid from a restriction digest) or synthetically produced, that, when placed under conditions that induce the synthesis of a primer extension product complementary to the nucleic acid template strand (e.g., in the presence of a nucleotide and an inducer such as DNA polymerase, and at suitable temperature and pH), can act as the starting point for synthesis. For maximum amplification efficiency, primers are preferably single-stranded, but double-stranded primers are also possible. If double-stranded, the primers are first treated to separate their strands before being used to prepare the extension product. Preferably, the primer is an oligodeoxyribonucleotide. Typically, the primer is long enough to initiate the synthesis of the extension product in the presence of an inducer. The exact length of the primer will depend on a variety of factors, including temperature, primer source, and the method used.

[0282] As used herein, the term "nucleic acid assay" refers to any method for determining the nucleotide composition of a nucleic acid of interest. Nucleic acid assays include, but are not limited to, sequencing, probe hybridization, structure-specific cleavage assays (e.g., "INVADER" flap assays or invasive cleavage assays (Hologic, Inc.), described in, for example, U.S. Patent Nos. 5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and 6,872,816, Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et al., PNAS, USA, 97:8272 (2000); and combined PCR / invasive cleavage assays (Hologic, Inc.). Inc., for example in U.S. Patent Publications 2006 / 0147955 and 2009 / 0253142 (each of which is incorporated herein by reference in its entirety for all purposes); enzyme mismatch cleavage (e.g., Variagenics, U.S. Patents 6,110,684, 5,958,692, and 5,851,770, which are incorporated herein by reference in their entirety); polymerase chain reaction (PCR) as described above; branching hybridization. (e.g., Chiron, U.S. Patents 5,849,481, 5,710,264, 5,124,246, and 5,624,802, which are incorporated herein by reference in their entirety); rolling circle replication (e.g., U.S. Patents 6,210,884, 6,183,960, and 6,235,502, which are incorporated herein by reference in their entirety); variants of rolling circle amplification referred to as “RAM amplification” (see, for example, US 5,942,391, which is incorporated herein by reference in its entirety; Fan Li et al., J ClinMicrobiol. Dec. 2005; 43(12): 6086–6090).); NASBA (e.g., U.S. Patent No. 5,409,818, which is incorporated herein by reference in its entirety); molecular beacon technology (e.g., U.S. Patent No. 6,150,097, which is incorporated herein by reference in its entirety); electronic sensor technology (U.S. Patent Nos. 6,248,229, 6,221,583, 6,013,170, and 6,063,573, which are incorporated herein by reference in their entirety); cyclic probe technology (e.g., U.S. Patent Nos. 5,403,711, 5,011,769, and 5,660,988, which are incorporated herein by reference in their entirety); Dade Behring signal amplification methods (e.g., U.S. Patents 6,121,001, 6,110,677, 5,914,230, 5,882,867, and 5,792,614, all of which are incorporated herein by reference in their entirety); ligase chain reaction (e.g., Barany Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich hybridization methods (e.g., U.S. Patent 5,288,609, which is incorporated herein by reference in its entirety).

[0283] As used herein, the terms “sequencing,” “DNA sequencing,” and “RNA sequencing” in relation to nucleic acid analysis broadly refer to any method for determining the nucleotide or nucleobase sequence of a nucleic acid strand or region of a nucleic acid strand. Sequencing methods include, but are not limited to, methods that use chemicals or enzymes to break nucleic acid strands in a nucleobase-specific manner (e.g., “Maxam-Gilbert” sequencing, Maxam AM, Gilbert W (February 1977). Proc. Natl. Acad. Sci. USA. 74 (2): 560–64 Methods involving extension using templated primers in the presence of chain-terminating reagents (e.g., dideoxynucleotides), such as “Sanger” sequencing (Sanger F et al.). Proc. Natl. Acad. Sci. USA. 74 (12): 5463–77 (1977) Smith LM et al. Nature. 321 (6071): 674–679 (1986); and circular sequencing (see, for example, Murray V). Nucleic Acids Res The method used in .17(21):8889 (1989)); and the method in which each nucleotide added to the extended chain by polymerase is monitored at the time of its addition (“sequencing-by-synthesis” or “SBS”; see, for example, Nyren P et al., Analytical Biochemistry. 208 (1): 171 (1993 These references are incorporated herein by reference in their entirety for all purposes.

[0284] The terms "next-generation sequencing" and "NGS" refer to any parallelized method, preferably highly parallelized, for nucleic acid sequencing, including but not limited to sequencing-by-synthesis and sequencing-by-ligation platforms. NGS sequencing platforms include, but are not limited to, the following: massively parallel signature sequencing (Lynx Therapeutics); 454 pyrosequencing (454 Life Sciences / Roche Diagnostics); solid-phase reversible dye terminator sequencing (Solexa / Illumina™); SOLiD™ technology (Applied Biosystems); ion semiconductor sequencing (Ion Torrent™); and DNA nanosphere sequencing (CompleteGenomics, BGI Americas). Descriptions of some NGS platforms can be found in the following literature: Shendure et al., “Next-generation DNA sequencing,” Nature, 2008, Vol. 26, No. 10, pp. 135-1145; Mardis, “The impact of next-generation sequencing technology on genetics,” Trends in Genetics, 2007, Vol. 24, No. 3, pp. 133-141; Su et al., “Next-generation sequencing and its applications in molecular diagnostics,” Expert Rev Mol Diagn, 2011, 11(3):333-43; and Zhang et al., “The impact of next-generation sequencing on genomics,” J Genet Genomics, 201, 38(3): 95-109. Nanopore sequencing, such as that provided by Oxford Nanopore Technologies, is described in, for example, Clarke J et al. Nature Nanotechnology. 4(4): 265–70 (2009); and U.S. Patent Nos. 5,795,782, 2, 6,015,714, 6,627,067, 7,238,485, and 7,258,838; the Ion Torrent technology commercialized by Life Technologies is further described, for example, in Rusk N, Nat Methods. 8(1): 44 (2011); and Dewey FE et al. , Circulation.125 (7): 931–944 (2012). Further description of a high-throughput sequencing method for combinatorial indexing of amplified nucleic acids produced in embodiments of the present technology is provided in US 2023 / 0193356. As used herein, the term “nanopore” for polymer analysis refers, for example, a nanoscale pore in a membrane configured to allow single strands of a polymer (e.g., DNA or RNA) to pass through in a manner that allows analysis of each individual monomeric unit in the strand as the polymer passes through the pore. Thus, “nanopore sequencing” for nucleic acid polymer analysis refers to the detection of the base sequence in a nucleic acid strand by passing the nucleic acid strand through a nanopore. See, for example, Soni et al., 2007. Clin Chem 53: 1996-2001, which is incorporated herein by reference in its entirety. In a typical implementation, an electric field or molecular motor is used to drive a nucleic acid chain through a specialized nanopore configured to detect characteristic changes in current at the nanopore, changes specific to different nucleotides in the polynucleotide chain. See, for example, U.S. Patent No. 6,015,714, January 18, 2000, to Baldarelli et al., and U.S. Patent No. 8,673,556, March 18, 2014, to Akeson et al., and Y. Feng et al., Nanopore-based Fourth-generation DNA Sequencing Technology. Genomics, Proteomics & Bioinformatics , 13 (1): 4-16 (2015), these references are individually incorporated into this paper.

[0285] As used herein, the term "nanopore device" refers to a device suitable for characterizing monomeric subunits of polymer molecules (e.g., nucleotides in polynucleotides) by means of methods including passing polynucleotides through nanopores. Nanopore devices include, but are not limited to, the MinION® real-time sequencer from Oxford Nanopore Technologies, Inc.

[0286] As used herein, “rolling circle amplification” refers to the in vitro rolling circle replication of circular nucleic acids using strand displacement DNA polymerase to form DNA molecules containing tandem repeats of sequences complementary to the circular nucleic acid, as described in, for example, U.S. Patents 6,210,884, 6,183,960, 6,235,502, 5,942,391, 6,316,229, and 7,862,999; and M. Ali et al., “Rolling circle amplification: a versatile tool for chemicalbiology, materials science, and medicine.” Chemical Society Reviews 43 (10):3324–3341 (2014). Rolling circle amplification can be performed using suitable cyclizable molecules as templates, which interact with the target (if present) to form a structure that can be amplified by rolling circle amplification.

[0287] As used herein, “rolling circle transcription” or “RCT” refers to the in vitro rolling circle transcription of circular nucleic acids using RNA polymerase to form RNA molecules containing tandem repeats of sequences complementary to the circular nucleic acid, as described in, for example, by SL Daubendiek et al. Rolling Circle RNA Synthesis: Circular Oligonucleotides as Efficient Substrates for T7 RNA Polymerase J Am Chem Soc. 1995;117:7818–7819. Cyclic oligonucleotides do not require a canonical promoter sequence to serve as a template for rolling circle transcription.

[0288] The term "circularizable probe," also referred to herein as "circularizable molecule," is used in the broadest sense and refers to any oligonucleotide or probe that can form or cause to form a circular structure (e.g., a covalently closed circular chain of nucleic acid) in the presence of a target nucleic acid or a product indicating the presence of a target nucleic acid (e.g., a cleavage flap). In some embodiments, the circularizable molecule is a padlock probe that forms a circular structure under suitable conditions in the presence of a target (e.g., a cleavage flap sequence) and a ligase. In some embodiments, the circularizable molecule is a dumbbell probe that forms a closed loop-stem-loop structure (e.g., a loop structure comprising two stem-loops linked by a stem duplex) under suitable conditions in the presence of a target (e.g., a cleavage flap sequence) and a ligase. In some embodiments, the circularizable molecule is configured to include features that restrict ligation, preferably features that are removable or repairable in the presence of the target nucleic acid (e.g., a cleavage flap). For example, in some implementations, when a cyclizable molecule is combined with a cleavage flap, one or both of the cyclizable molecule or the cleavage flap must be modified, for example, with a nuclease (such as a polymerase or nuclease) to form a connectable cleavage.

[0289] As used herein, the term "circular molecule" in relation to the use of nucleic acids refers to a single-stranded or double-stranded nucleic acid, wherein at least one strand is in the form of a covalently closed loop lacking a free 3′ and 5′ end. Embodiments of circular molecules include the synthesis of oligonucleotides in a circular form prior to use ("pre-circularization"), for example, as a template for rolling circle replication, such as... Figure 3 As shown. In some embodiments, the cyclizable probe is used in its linear form and is linked as a step in the detection assay to form a cyclized molecule, for example, as... Figure 6A As shown. Cyclic molecules can be pre-cyclized, or they can be cyclized as part of a reaction or assay.

[0290] As used in this article, the term "linear" in relation to nucleic acid molecules refers to a nucleic acid strand with 5′ and 3′ ends that are not covalently attached to each other, such that the nucleic acid strand does not form a loop.

[0291] The term "stem-loop" in nucleic acid terminology refers to a polynucleotide internal structure formed by the hybridization of two regions of the same nucleic acid strand to form a double-stranded stem. One end of this double-stranded stem has an unpaired region, i.e., a loop connecting the two double-stranded regions. The loop typically contains at least three nucleotides.

[0292] As used herein, the term "dumbbell probe" refers to a probe comprising a stem-loop, preferably forming a loop-stem-loop structure upon hybridization with a complementary nucleic acid (e.g., a cleavage valve). In some embodiments, the dumbbell probe is provided as a pre-formed circular molecule, suitable for use as a template, for example, in an RCA reaction initiated by a cleavage valve molecule. In some embodiments, the circulizable dumbbell probe forms a circular structure only under suitable conditions (appropriate ligation conditions) and in the presence of a target (e.g., a cleavage valve sequence). In some embodiments, the dumbbell probe includes a bridging sequence located between two stem-loops, which is complementary to the valve sequence of the valve oligonucleotide. In some such embodiments, hybridization of the dumbbell probe with the valve portion of an uncleaved valve oligonucleotide forms a complex that cannot ligate to form a circular molecule (see, for example...). Figure 5B (bottom right). When the valve oligonucleotide is cleaved, for example, when an invasive cleavage structure containing a probe is treated with a valve endonuclease, the hybridization of the dumbbell probe with the resulting cleavage valve sequence forms a complex that can link to form a cyclic molecule (see, for example...). Figure 5B(Lower left). In some embodiments, the stem of the dumbbell probe contains a bridging sequence comprising 10-20 unpaired nucleotides complementary to the lobe sequence or the cleavage lobe sequence. In some embodiments, the stem contains additional bases at the 5′ and / or 3′ of the bridging sequence that are not complementary to the lobe or cleavage lobe sequence. In some embodiments, the dumbbell probe additionally comprises a loop containing any suitable number of nucleotides, wherein the base sequence in the loop is substantially not complementary to the lobe or cleavage lobe sequence. In a preferred embodiment, the dumbbell probe comprises two stem-loops separated by the bridging sequence.

[0293] As used herein, the term "carrier pair" refers to a carrier or flap carrier comprising a pair of molecules, wherein the first carrier oligonucleotide contains a first bridging complementary region complementary to the 5' end sequence or end of the bridging oligonucleotide, and the second carrier oligonucleotide contains a second bridging complementary region complementary to the 3' end or end of the bridging oligonucleotide. In a preferred embodiment, the carrier pair hybridizes with the bridging oligonucleotide such that a third oligonucleotide (e.g., a cleavage flap) can hybridize with an internal segment of the bridging oligonucleotide, such that the third oligonucleotide is located between the two oligonucleotides of the carrier pair, preferably forming a connectable cleavage, or may be made connectable by an additional step (e.g., cleaving the 5' flap from one of the oligonucleotides of the carrier pair).

[0294] In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and an invasive cleavage assay is used to detect the amplified nucleic acid to produce a cleavage flap sequence. In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and an invasive cleavage assay is performed simultaneously. In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and an invasive cleavage assay is performed to produce a cleavage flap sequence, and the invasive cleavage assay is followed by further amplification and / or sequencing steps. Assays configured for use in combination with amplification assays for detection assays (e.g., invasive cleavage assays) are described in U.S. Patent No. 9,096,893 B2 (Sequence No. 13 / 941,122), which is incorporated herein by reference in its entirety for all purposes. See also Allavi HT et al. J of Clin Microbio 2006; Vol. 44, No. 9: 3443-3447. Further extended invasive cutting detection configurations known as the Quarts method are described in U.S. Patents 8,361,720, 8,715,937, and 8,916,344, which are incorporated herein by reference in their entirety for all purposes.

[0295] In some embodiments, the target nucleic acid is amplified (e.g., by PCR), and the amplified nucleic acid is detected using an invasive cleavage assay. Cleavage of the amplified target in the invasive cleavage structure results in the formation of a cleavage flap sequence, which is then detected, for example, by using a ligation-compatible vector (e.g., a padlock probe, a dumbbell probe, one or a pair of oligonucleotides in a vector pair) hybridized with and / or ligated to the cleavage flap sequence. In some embodiments, ligation of the cleavage flap sequence to a ligation-compatible vector (e.g., a padlock probe, a dumbbell probe) results in the formation of a closed loop or linear structure containing vector ends, the presence of which is further amplified and / or detected (e.g., by using the closed loop or linear ligation product as a template for PCR, RCA, RCT, sequencing, etc.).

[0296] As used herein, the terms “invasive cleavage” and “overlapping cleavage” are used interchangeably and refer to cleavage structures that typically include: a template nucleic acid (e.g., a target nucleic acid), an upstream nucleic acid (e.g., an invasive oligonucleotide, or a 3′ portion of a template strand that folds back and hybridizes to form a hairpin, or a 3′ portion of a padlock probe), and iii) a downstream nucleic acid (e.g., a probe, a target-specific region of a petal-shaped oligonucleotide, or a 5′ portion of a template strand that folds back and hybridizes to form a hairpin, or a 5′ portion of a padlock probe), wherein the upstream and downstream nucleic acids anneal to adjacent regions of the template strand (e.g., the target nucleic acid) (i.e., regions of the template strand adjacent to each other on the strand, not separated by intervening nucleotides or base pairs) to form an upstream double helix and a downstream double helix, as shown below. Figure 1 As shown, an overlap is formed between the 3' end of the upstream nucleic acid and the double strand formed between the downstream nucleic acid and the template nucleic acid. In some embodiments, an "invasive cleavage structure" refers to... Figure 1 The cleavage structure shown comprises a petal-shaped oligonucleotide, a target nucleic acid, and an upstream invasive oligonucleotide. The upstream invasive oligonucleotide forms an upstream double strand with the target nucleic acid, and the target-specific region of the petal-shaped oligonucleotide forms a downstream double strand with the target nucleic acid. The petal-shaped oligonucleotide also contains a petal sequence, which is cleaved in the presence of a suitable structure-specific endonuclease.

[0297] In some implementations, "invasive cutting structure" refers to, for example... Figure 2 The cut structure shown contains a petal oligonucleotide and a target nucleic acid, wherein the 3′ portion of the petal oligonucleotide contains a portion of the target nucleic acid that hybridizes to form an upstream double strand and an intrusive portion overlapping the 3′ end.

[0298] In some embodiments, the 3′ portion of the upstream nucleic acid (e.g., an invasive oligonucleotide) overlapping the downstream duplex is a single nucleotide. In some embodiments, the 3′ portion of the upstream nucleic acid overlapping the downstream duplex is a non-base chemical portion, such as an aromatic ring structure, for example, as disclosed in, for example, U.S. Patent No. 6,090,543, which is incorporated herein by reference in its entirety.

[0299] As used herein, the term "upstream double-stranded structure" for invasive cleavage structures refers to a double-stranded structure formed between the template strand and a hybridized upstream nucleic acid strand having a 3' end, and the term "downstream double-stranded structure" refers to a double-stranded structure formed between the template strand and a hybridized downstream nucleic acid strand having a 5' end, such that the overlapping flap endonuclease substrate is formed from adjacent hybridization regions of the upstream and downstream nucleic acid strands. In some embodiments, the upstream and downstream nucleic acids are part of the same strand of a polynucleotide, such that the invasive cleavage structure comprises two nucleic acid strands. See, for example... Figure 2 This illustrates the upstream and downstream double-stranded regions of an invasive cleavage structure formed by two nucleic acid chains. In some embodiments, one or more of the nucleic acid chains in the invasive cleavage structure may be attached to each other via non-nucleic acid chemical bonds (e.g., multi-carbon chains). See, for example, U.S. Patent No. 8,445,238, which is incorporated herein by reference in its entirety. In some embodiments, the two nucleic acid chains are attached to each other via nucleic acid backbone regions to form a padlock or dumbbell probe. Preferably, the nucleic acid backbone in the padlock dumbbell probe is substantially non-complementary to the target nucleic acid.

[0300] "Legular oligonucleotides" refer to oligonucleotides that can be cleaved by a lever endonuclease in detection assays (such as invasive cleavage assays). In some embodiments, the lever oligonucleotide comprises at least one target-specific region (also referred to herein as a target-specific sequence) and a lever sequence (also referred to herein as a lever moiety), preferably a 5′ lever moiety or sequence. At least one target-specific region is complementary to the target of interest (e.g., a target nucleic acid), while the lever moiety is preferably an arm (e.g., a 5′ arm) that is not substantially complementary to the target of interest (e.g., does not bind to the target of interest under the assay conditions using the lever oligonucleotide). The lever sequence is not limited to any particular length or sequence. In a preferred embodiment, the 5′ lever moiety of the lever oligonucleotide is selected such that secondary structures in the lever moiety of the lever oligonucleotide do not degrade the cleavage efficiency of the lever oligonucleotide. See, for example, C. Spiro et al., Molecular Cell, Vol. 4, No. 6, December 1999, pp. 1079-1085. Thus, in some embodiments, the lever sequence is selected to have a sequence that does not form significant secondary structures under cleavage reaction conditions. In some embodiments, the length and / or sequence of the lobe sequence are selected to reduce this secondary structure. In some embodiments, the length of the lobe sequence is preferably less than about 200 nucleotides, more preferably less than about 175 nucleotides. In some embodiments, the lobe sequence is 1 to about 150 nucleotides, preferably 1 to about 100 nucleotides, and more preferably about 5 to 75 nucleotides. In a preferred embodiment, the length of the 5′ lobe portion is at least 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, or 75 nucleotides. In some embodiments, the length of the 5′ lobe portion is preferably 6 to 45 nucleotides, more preferably 7 to 40 nucleotides, more preferably 8 to 35 nucleotides, more preferably 8 to 30 nucleotides, more preferably 8 to 25 nucleotides, more preferably 9 to 25 nucleotides, more preferably 10 to 20 nucleotides, and more preferably 10 to 15 nucleotides.

[0301] Cleavage of a valve-like oligonucleotide (e.g., by a valve-like endonuclease) typically produces a cleavage valve sequence, also referred to herein as a "cleavage valve," "cleavage valve segment," "cleavage valve portion," "cleavage arm," "cleavage marker-specific identifier arm," "cleavage target-specific marker," or "cleavage target-specific identifier." In a preferred embodiment, the valve-like oligonucleotide forms an invasive cleavage structure with one or more other nucleic acids (e.g., a target or template nucleic acid and an invasive oligonucleotide). For example, in some embodiments, the valve-like oligonucleotide forms an invasive cleavage structure with a target nucleic acid strand, wherein the upstream duplex contains the invasive oligonucleotide, and the downstream duplex contains the target-specific region of the valve-like oligonucleotide. Figure 1 The embodiment is illustrated schematically. In some embodiments, the valve-like oligonucleotide contains two target-specific regions and therefore does not require a separate invasive oligonucleotide to form an invasive cleavage structure. For example, in some embodiments (including...) Figure 2 In the illustrated embodiment, the valve-like oligonucleotide comprises a first target-specific region (or "valve portion") and a second target-specific region (or "invasive portion"), each binding to different but adjacent regions on the target nucleic acid, thereby forming upstream and downstream duplexes in the invasive cleavage structure. In a preferred embodiment, the cleavage valve comprises a 5′ valve sequence and a 5′ nucleotide that is a cleavage site for a valve-like endonuclease, or is composed of such a sequence. Figure 2 As shown.

[0302] As used herein, the term "valve endonuclease" or "FEN" refers to a structure-specific lysozyme that cleaves valve-like structures of nucleic acids (e.g., invasive cleavage structures). Valve endonucleases include, for example, the 5′ exonuclease domain of DNA polymerase I protein from eukaryotes and the FEN-1 protein from eukaryotes and archaea (Kaiser MW et al. (1999) J. Biol. Chem., 274:21387), as well as engineered endonucleases described in, for example, US 7,935,800, WO 2003 / 073067, WO 02 / 070755 and US Patent No. 7,122,364, which are incorporated herein by reference in their entirety for all purposes. Valve endonucleases can also cleave other structures, such as pseudo-Y structures, 5′ protrusions, and notched structures. See, for example, Shen, B., BioEssays 27:717-729 (2005) and Finger, LD., SubcellBiochem. 62:301–326 (2012), each of which is incorporated herein by reference in its entirety. Reviews of valve endonucleases are found in Ceska and Savers (Trends Biochem. Sci. 1998 23:331-336), Liu et al. (Annu. Rev. Biochem.2004 73: 589-615), and Tsutakawa S et al. (Cell v145:198-211 (2011)), each of which is incorporated herein by reference in its entirety.

[0303] As used herein, the term “FEN-1” refers to a non-polymerase valve-shaped endonuclease derived from eukaryotic or archaea organisms, such as that encoded by the FEN-1 (valve-shaped structure-specific endonuclease 1) gene. See, for example, Kaiser et al., ibid., WO 02 / 070755 and U.S. Patent No. 7,122,364, which are incorporated herein by reference in their entirety for all purposes. The term “FEN-1 activity” refers to any enzymatic activity of a FEN-1 enzyme. FEN-1 endonucleases also include modified FEN-1 proteins, such as chimeric proteins containing portions of FEN-1 enzymes from different organisms, and enzymes containing one or more mutations (e.g., substitution, deletion, insertion, etc.), as described in WO 02 / 070755 and U.S. Patent No. 7,122,364. The term “FEN1” is used interchangeably with “FEN-1” herein. The preferred FEN-1 endonuclease is a thermostable endonuclease, preferably derived from or derived from archaea.

[0304] When used in relation to enzymes (such as valve endonucleases), the term "thermally stable" means that the enzyme is functional or active (i.e., capable of catalysis) at elevated temperatures (i.e., about 55°C or higher) and retains its functionality or activity after exposure to highly elevated temperatures (e.g., about 90°C or higher).

[0305] As used herein, the term "strict FEN-1" refers to FEN-1 enzymes typically derived from archaea that, under suitable conditions, cleave invasive structures but are substantially inactive when cleaving non-invasive structures, such as notched structures (single-stranded regions with a target or template strand between the upstream and downstream duplexes), Y-shaped structures (with 5′ lobes but no overlap between the upstream and downstream duplexes), and pseudo-Y-shaped structures (with 5′ lobes and a non-duplex template strand upstream of the downstream duplex). Furthermore, strict FEN-1 endonucleases do not cleave invasive structures formed on or around the cleavage site of a target strand composed of RNA nucleotides or their analogues (e.g., 2′-O-methyl nucleotides). See, for example, U.S. Patent No. 7,045,289 to Allavi et al. For example, when Mg ++ When it is the dominant or sole divalent cation in the cleavage buffer (e.g., in a flap assay buffer), Afu FEN-1 and Pfu FEN-1 is strict FEN-1. Examples of solution conditions under which a strict FEN-1 enzyme will exhibit strict selective cleavage behavior include, but are not limited to, solutions containing 10 mM MOPS, pH 7.5, 5 mM MgSO4, and 100 mM KCl (see US 7,045,289) and solutions containing 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 7.5 mM MgCl2, and 0.8 mM KCl.

[0306] As used herein, the terms "PCR-valve assay" and "PCR-invasive cleavage assay" are used interchangeably and refer to an assay configuration that combines PCR target amplification and amplified DNA detection by forming a first overlapping cleavage structure containing the amplified target DNA and a second overlapping cleavage structure containing a 5′ valve cleaved from the first overlapping cleavage structure, as well as an optional reporter oligonucleotide. In some embodiments, the reporter oligonucleotide is labeled.

[0307] In some embodiments, the reporter oligonucleotide comprises a linear molecule. In some embodiments, successful cleavage of the valve sequence from the valve oligonucleotide and subsequent ligation of the cleavage sequence to the reporter molecule results in the formation of a suitable template, which can be further amplified and / or detected, for example by PCR, transcription, sequencing, etc.

[0308] In some embodiments, the reporting oligonucleotide includes a cyclizable molecule. In some embodiments, successful cleavage of the valve sequence from the valve oligonucleotide and subsequent ligation of the cleaved valve sequence to the cyclizable molecule results in the formation of a suitable template, which can be further amplified and / or detected, for example by PCR, RCA, RCT, sequencing, etc.

[0309] In some embodiments, the cyclizable molecule includes a padlock probe. In some embodiments, the padlock probe includes a 5' end connected to the 3' end of the cutting flap and a 3' end connected to the 5' end of the cutting flap.

[0310] The terms “bridge” and “sandwich” are used interchangeably, both referring to oligonucleotides that align the ends of connectable molecules (e.g., cleavage flaps, or cleavage flaps and cyclizable vectors, or the ends of cleavage flaps and vector pairs) into a connectable conformation.

[0311] In some embodiments, the bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 5′ segment complementary to the first target region of the padlock probe, and a 3′ segment complementary to the second target region of the padlock probe. In some embodiments, the bridging oligonucleotide hybridizes with the first and second target regions of the padlock probe, leaving the inner segment of the bridging oligonucleotide available for binding (e.g., hybridization), such as binding to the flap sequence or the cleavage flap sequence. In some embodiments, the cleavage flap sequence hybridizes with the bridging oligonucleotide to generate a ligation-capable nick. After ligation with a suitable ligase, a closed circular structure is formed, which can then be amplified and / or detected by, for example, PCR, RCA, RCT, sequencing, etc.

[0312] In some implementations, the valve sequence hybridizes with the bridging oligonucleotide regardless of whether it is cleaved from the valve oligonucleotide (e.g., regardless of whether it is cleaved from the target-specific region of the valve oligonucleotide). If the valve oligonucleotide is not cleaved, the valve sequence may be able to hybridize with the bridging oligonucleotide, but the connection at a cleavage site is blocked, and a closed loop structure is not formed.

[0313] In some embodiments, the cyclizable molecule comprises a dumbbell probe. In some embodiments, the dumbbell probe includes a bridge sequence complementary to the cleavage valve sequence, thereby hybridizing with the cleavage valve sequence. In some embodiments, the cleavage valve sequence hybridizes with the bridge sequence, thereby generating a ligable cleavage. After ligation with a suitable ligase, a closed circular structure is formed (e.g., forming a dumbbell probe containing two stem-loops at either end), which can then be amplified and / or detected by, for example, PCR, RCA, RCT, sequencing, etc. In contrast, if no cleavage occurs, the valve sequence may be able to hybridize with the bridge sequence, but ligation at a cleavage is prevented, and therefore a closed circular structure is not formed.

[0314] In some embodiments, the cleavage flap acts as a cyclizable molecule. For example, in some embodiments, the cleavage flap is treated with a ligase to form a cyclizable molecule by connecting the 5′ end of the cleavage flap to the 3′ end.

[0315] In some preferred embodiments, a single-stranded ligase, such as CIRCLIGASE™ ssDNA ligase, is used. CIRCLIGASE™ ligase is a thermostable ligase that forms a phosphodiester bond between the 5′ phosphate and 3′ hydroxyl group of the same or different single DNA or RNA strands (LGC Biosearch, Technologies, Teddington, Middlesex, TW11 0LY, UK or Epicentre Biotechnologies, Madison, WI). CIRCLIGASE II™ is ATP-independent. Preferably, the cleavage flap to be circularized is single-stranded and has a length of at least about 10 nucleotides, preferably about 15 nucleotides. Nucleic acid loops with 13 or more nucleotides have been shown to serve as templates for rolling circle replication. (See, for example, MGMMohsen and ET Kool, Acc Chem Res. 2016 Nov 15; 49(11): 2540–2550, which are incorporated herein by reference in their entirety for all purposes.)

[0316] In some embodiments, the cleavage valve sequence hybridizes with the bridging oligonucleotide and is subsequently ligated to the padlock probe to form a closed circular structure. Thus, the bridging oligonucleotide facilitates the hybridization and stabilization of the cleavage valve sequence for subsequent ligation to the padlock probe and the generation of a closed circular structure. In some embodiments, the closed circular structure is amplified and / or detected, for example, by PCR, RCA, RCT, sequencing, etc. In contrast, if the target nucleic acid is absent and therefore no cleavage of the valve oligonucleotide occurs (e.g., the cleavage valve sequence is not released), the valve oligonucleotide sequence hybridizes with the bridging oligonucleotide (e.g., the valve sequence hybridizes with the inner segment of the bridging oligonucleotide) but cannot ligate to the padlock probe, and therefore no closed circular structure is formed. For example, in some embodiments, the valve sequence hybridizes with the inner segment of the bridging oligonucleotide, but the target-specific region within the oligonucleotide (still intact and ligated to the valve sequence) interferes with successful ligation.

[0317] In some embodiments, the cleavage valve sequence hybridizes with the bridging oligonucleotide and is subsequently ligated to the vector pair to form a linear linker structure. Thus, the bridging oligonucleotide promotes the hybridization and stabilization of the cleavage valve sequence for subsequent ligation to the vector pair, generating a linear linker molecule. In some embodiments, the linear linker molecule is amplified and / or detected, for example, by PCR, transcription, sequencing, etc. However, if the target nucleic acid is absent and therefore no cleavage of the valve oligonucleotide occurs (e.g., the cleavage valve sequence is not released), the valve oligonucleotide sequence hybridizes with the bridging oligonucleotide (e.g., the valve sequence hybridizes with the inner segment of the bridging oligonucleotide), but cannot ligate to the two members of the vector pair, and therefore does not form a complete linear linker molecule. For example, in some embodiments, the valve sequence hybridizes with the inner segment of the bridging oligonucleotide, but target-specific regions within the oligonucleotide (still intact with the valve sequence) interfere with successful ligation, for example, such as… Figure 5C As shown.

[0318] The techniques used to form linear linker molecules are not limited to the use of carrier pairs. For example, in some embodiments, a cleavage lobe sequence hybridizes with a bridging oligonucleotide and is subsequently ligated to a single carrier oligonucleotide to form a linear structure.

[0319] For example, ligation via DNA ligase typically requires a 3′ end of a nucleic acid strand with a 3′ OH group and a 5′ end of a nucleic acid strand with a 5′ phosphate group. In some embodiments, the valve-like oligonucleotide, such as synthetic oligonucleotides, lacks the 5′ phosphate. For example, the phosphoramidite process for synthesizing oligonucleotides typically produces oligonucleotides with a 5′ OH group. Therefore, in some embodiments where the 5′ end of the valve-like oligonucleotide will participate in the ligation reaction, the valve-like oligonucleotide is enzymatically phosphorylated, for example using a nucleic acid kinase (e.g., phage T4 polynucleotide kinase). In some embodiments, the valve-like oligonucleotide is phosphorylated prior to the valve assay reaction, while in other embodiments, the cleavage valves generated in the valve assay reaction are phosphorylated after the valve assay reaction and before the ligation reaction.

[0320] In the exemplary PCR-valve assay used herein, the assay reagent comprises a mixture of: DNA polymerase, FEN-1 endonuclease, a master probe (e.g., a valve-shaped oligonucleotide) containing a portion complementary to the target nucleic acid and a 5′ arm, and a ligandable molecule (e.g., a cyclizable molecule, a padlock probe, a dumbbell probe, a linear vector, or one or a pair of vector oligonucleotides). In some embodiments, the target nucleic acid is amplified by PCR, and the amplified nucleic acid is detected simultaneously (i.e., detection is performed during target amplification). In some embodiments, the target nucleic acid is amplified by PCR, and the amplified nucleic acid is subsequently detected (e.g., detection is performed after target amplification).

[0321] PCR-valve assays include: the Quarts assay, described in U.S. Patent Nos. 8,361,720, 8,715,937, 8,916,344, and 9,212,392; and the amplification assay of U.S. Patent No. 9,096,893 (e.g., as described in that patent). Figure 1 (As shown). LQAS assays are flap assays using probe oligonucleotides with longer target-specific regions (long probe quantitative amplification signals, "LQAS"), while TELQAS assays combine LQAS probe oligonucleotides with a pre-amplification step (target enrichment of long probe quantitative amplification signals), as described in U.S. Patent Nos. 10,648,025 and WO 2020 / 112869, each of which is incorporated herein by reference in its entirety. In some embodiments, PCR-flap assays are performed in a manner similar to the Quarts and LQAS assays mentioned above, except that the FRET cassette or FRET reporter molecule is not used to detect cleavage (e.g., not to detect cleavage flap sequences), but instead a ligandable molecule (such as a circularizable molecule, padlock probe, dumbbell probe, linear vector, or one or a pair of oligonucleotides of a vector pair) is used to detect cleavage. Exemplary conditions for analyzing DNA and RNA target nucleic acids using PCR-flap assays are provided, for example, in U.S. Patent Nos. 10,648,025 and WO 2021 / 041726. Each of the aforementioned patents and patent disclosures is incorporated herein by reference in its entirety for all purposes.

[0322] As used herein, the term "PCR-flap assay reagent" refers to one or more reagents used in a PCR-flap assay to detect a target sequence, the reagent comprising nucleic acid molecules capable of participating in the amplification of the target nucleic acid and the formation of a flap-shaped cleavage structure in the presence of the target sequence in a mixture containing a DNA polymerase, a flap-shaped endonuclease (e.g., FEN-1 endonuclease), and a ligase-compatible molecule (e.g., a circularizable molecule, a padlock probe, a dumbbell probe, a linear vector, or one or a pair of oligonucleotides of a vector pair). In some embodiments, the reagent further comprises a bridging oligonucleotide. In some embodiments, the reagent further comprises a ligase.

[0323] As used herein, the term "padlock probe" refers to any oligonucleotide probe that can be used to form a nicked loop by hybridizing with a nucleic acid strand and to form a covalently closed circular molecule by treatment including a ligase. See, for example, M. Nilsson et al., "Padlock probes: circularizing oligonucleotides for localized DNA detection." Science265 (5181): 2085–2088 (1994). In some embodiments, the padlock probe requires modification before ligation, such as digestion with endonucleases or exonucleases (e.g., to remove the flap sequence), polymerase extension (e.g., to fill the gap), or other treatments to produce a circular probe containing a ligation-compatible cleavage. Preferably, the modification to produce the ligation-compatible cleavage depends on the padlock probe hybridizing with the intended target nucleic acid. Padlock probes are described in U.S. Patent Nos. 5,854,033 (Lizardi), WO99 / 49079 (Landegren), and 5,871,921 (Landegren and Kwiatkowski). A form of padlock probe called an inversion probe is described in U.S. Patent No. 6,858,412 (Willis et al.). An inversion probe is a padlock probe containing a cleavage site in the probe backbone, allowing the circularized probe to be cleaved to form a linear product, which can then be amplified and detected. In some embodiments, the padlock probe is used to detect the amplified target via PCR-valve assay, wherein the padlock probe comprises a 5′ end attached to a first end (e.g., a 5′ or 3′ end) of the cleavage valve sequence and a 3′ end attached to a second end of the cleavage valve sequence, thereby forming a closed circular structure. In some embodiments, the formation of the closed circular structure is aided by using a nick-filling oligonucleotide (e.g., the cleavage valve sequence), which hybridizes with the padlock probe along with a nucleic acid strand positioned at the ends of both the nick-filling oligonucleotide and the padlock probe to form a ligation nick at each end of the nick-filling oligonucleotide. After ligation, the circular nucleic acid comprises the padlock probe and the nick-filling sequence.

[0324] In some implementations, the padlock probe and the nick-filling oligonucleotide hybridize together with the target nucleic acid, and in other implementations, the padlock probe and the nick-filling oligonucleotide hybridize together with a non-target oligonucleotide referred to as a "bridge" or "sandwich" oligonucleotide. See, for example, Kuhn et al., Nucleic Acids Research, 2002, Vol. 30, No. 2: 574 (2002); Yixiao Cui et al., "Cyclization of secondarily structured doligonucleotides to single-stranded rings by using TaqDNA ligase at hightemperatures,” RSC. Adv. 8:18972-79 (2018); I. Tabuchi et al., “An EfficientLigation Method in the Making of an in vitro Virus for in vitro ProteinEvolution” Biol. Proced. Online ; 4(1): 49-54. (2002); M. Szemes et al., Diagnostic application of padlock probes - multiplex detection of plantpathogens using universal microarrays" Nucleic Acids Research 33(8): e70(2005); and Lohman et al., U.S. Patent No. 10,597,650, which are incorporated herein by reference in their entirety for all purposes. In some embodiments, closed circular structures are amplified and / or detected, for example, by PCR, RCA, RCT, sequencing, etc.

[0325] Bridging oligonucleotides may include DNA or RNA nucleotides or analogues thereof, such as 2′-O-methylnucleotides, 2′-O-methoxyethylnucleotides, phosphate thioester nucleotides, or nucleotides containing base analogues, or combinations thereof, such as chains containing one or more sites or regions of native DNA or RNA bases containing 2′-O-methylnucleotides. In some embodiments, for example when hybridizing with complementary nucleic acid chains, the nucleotides used in the bridging oligonucleotides may be selected to provide an A-type helical form or a B-type helical form or a combination thereof. For example, hybridization of an RNA or RNA analog chain with DNA typically provides an A-type structure, while hybridization of a DNA or DNA analog chain with DNA typically provides a B-type structure, and when hybridizing with a DNA chain, a chimeric chain having, for example, RNA and DNA regions can provide a double helix where the A-type region is transformed into a B-type region. See, for example, Shaw et al., "Recognition of the unique structure of DNA:RNA hybrids," Biochimie 90:1026-1039 (2008), which is incorporated herein by reference in its entirety for all purposes.

[0326] As used herein, regarding cleavage of invasive cleavage structures, the term "target cleavage site" refers to one or more preferred sites on a nucleic acid structure (e.g., an invasive cleavage structure) where a structure-specific nuclease (e.g., FEN-1 endonuclease) that recognizes the structure as a cleavage substrate makes cleavage. For example, as discussed by Kaiser et al., 5′ valve-like endonucleases (including FEN-1 endonuclease) typically cleave invasive cleavage structures in downstream nucleic acids, generally located after the first base-paired nucleotide, i.e., at a position one nucleotide deep into the downstream double strand. Target cleavage sites are selected such that cleavage at that site releases a cleavage valve sequence, which can then be used for subsequent detection. For example, in some embodiments, the valve sequence is located within a valve-like oligonucleotide such that when the invasive cleavage structure is cleaved at the target cleavage site, the valve-like oligonucleotide is cleaved to generate (release) a cleavage valve from the target-specific portion of the oligonucleotide. Thus, the cleavage valve sequence can be used for hybridization.

[0327] In some embodiments, the cleavage site in the invasive cleavage structure causes cleavage of the lobe sequence to result in a cleavage lobe sequence containing an extra base compared to the uncleaved lobe sequence. For example, in some embodiments, the cleavage site causes cleavage of the lobe sequence to add a 5′ terminal base of the target-specific region to the cleavage lobe sequence. Thus, in some embodiments, the lobe sequence (when not cleaved from the lobe oligonucleotide) contains 14 bases, and cleavage by an endonuclease results in a cleavage lobe sequence containing 15 bases. In a preferred embodiment, the invasive cleavage structure may be in Mg ++ The flap assay buffer is passed through the FEN-1 endonuclease (“FEN-1, “ ... Afu FEN-1” cuts at the target cleavage site, as discussed below. See also, for example, U.S. Patent No. 6,562,611 to Kaiser et al., and Kaiser MW et al. (1999) J. Biol. Chem., 274:21387, which are incorporated herein by reference in their entirety for all purposes.

[0328] As used herein, the terms "first paired nucleotide" and "first paired base" for the FEN-1 cleavage site of an invasive cleavage structure, such as a padlock probe or a flap oligonucleotide, refer to the 5′ outermost base paired nucleotide of the probe or oligonucleotide when hybridizing in the downstream duplex of the cleavage structure, such as, for example... Figure 1 and Figure 2 As shown.

[0329] The detection of cleavage lobes is not limited to any particular method. In some embodiments, for example, the cleavage lobes can be used to form additional cleavage structures, such as in conjunction with a reporter molecule like a FRET box, or to form a promoter that enables transcription to function as a reporter signal. See, for example, U.S. Patent No. 6,872,816, which is incorporated herein by reference for all purposes. In some embodiments, the cleavage lobes are used as primers for subsequent amplification. In some embodiments, the cleavage lobes specifically hybridize with circular molecules to induce, for example, rolling circle replication, as a means of creating a signal representing the presence of the target nucleic acid in a sample. In some embodiments, the cleavage lobes hybridize with bridging oligonucleotides or bridging sequences to form connectable cleavages at both ends of a padlock or dumbbell probe, which are then ligated. For example, the cleavage lobe sequence can be ligated to a matching padlock or dumbbell probe to form a closed circular structure for each lobe sequence, and each of these can then be amplified for subsequent detection of target-specific cleavage events. In some embodiments, the cleavage flap hybridizes with bridging oligonucleotides, for example, with DNA oligonucleotides, RNA oligonucleotides, or with chimeric molecules containing both RNA and DNA motifs, such that they can be linked to a vector pair of oligonucleotides to form a linearly linked product. See, for example... Figure 14 .

[0330] As used herein, "methylation" refers to cytosine methylation at position C5 or N4 of cytosine, position N6 of adenine, or other types of nucleic acid methylation. DNA amplified in vitro is typically unmethylated because typical in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA" or "methylated DNA" can also refer to amplified DNA with an unmethylated or methylated original template, respectively.

[0331] Therefore, as used herein, "methylated nucleotide" or "methylated nucleotide base" refers to a nucleotide base containing a methyl moiety that is not present in a typically accepted nucleotide base. For example, cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide, while 5-methylcytosine is. In another example, thymine contains a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this document, thymine is not considered a methylated nucleotide when it is present in DNA, as thymine is a typical nucleotide base of DNA.

[0332] As used in this article, "methylated nucleic acid molecule" refers to a nucleic acid molecule containing one or more methylated nucleotides.

[0333] As used herein, the “methylation state,” “methylation profile,” and “methylation status” of a nucleic acid molecule refer to the presence or absence of one or more methylated nucleotide bases in the nucleic acid molecule. For example, a nucleic acid molecule containing methylated cytosine is considered methylated (e.g., the methylation state of the nucleic acid molecule is methylated). A nucleic acid molecule that does not contain any methylated nucleotides is considered unmethylated. In some embodiments, a nucleic acid may be characterized as “unmethylated” if it is not methylated at a specific locus (e.g., a locus of a specific single CpG dinucleotide) or a specific combination of loci, even if it is methylated at other loci in the same gene or molecule.

[0334] The methylation status of a specific nucleic acid sequence (e.g., a gene marker or DNA region as described herein) can indicate the methylation status of each base in the sequence, or the methylation status of a subgroup of bases within the sequence (e.g., one or more cytosines), or information about the methylation density of a region within the sequence, while providing or not providing precise information about the location where methylation occurs within the sequence. As used herein, the terms “marker gene” and “marker” are used interchangeably to refer to DNA, RNA, or protein (or other sample component) associated with a condition (e.g., cancer or other disease), regardless of whether the marker region is in a coding region of DNA. Markers may include, for example, regulatory regions, flanking regions, intergenic regions, etc. Similarly, the term “marker” used with respect to any component of a sample (e.g., protein, RNA, carbohydrate, small molecules, etc.) refers to a component that can be determined (e.g., measured or otherwise characterized) in the sample and is associated with a condition of a subject or a sample from a subject. The term “methylation marker” refers to a gene or DNA in which the methylation status of a gene or DNA is associated with a condition (e.g., cancer or other disease).

[0335] The methylation state of a nucleotide locus in a nucleic acid molecule refers to the presence or absence of methylated nucleotides at a specific locus. For example, when the nucleotide at the 7th nucleotide position in a nucleic acid molecule is 5-methylcytosine, the methylation state of cytosine at that 7th nucleotide position is methylated. Similarly, when the nucleotide at the 7th nucleotide position in a nucleic acid molecule is cytosine (not 5-methylcytosine), the methylation state of cytosine at that 7th nucleotide position is unmethylated.

[0336] Methylation status can optionally be represented or indicated by a "methylation value" (e.g., representing methylation frequency, fraction, proportion, percentage, etc.). Methylation values ​​can be generated, for example, by quantifying the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing the amplification profile after a bisulfite reaction, or by comparing bisulfite-treated and untreated nucleic acids. Therefore, the value of the methylation value represents the methylation status and can thus be used as a quantitative indicator of the methylation status of multiple copies across a locus. This is particularly useful when it is necessary to compare the methylation status of sequences in a sample with a threshold or reference value.

[0337] As used in this article, “methylation frequency” or “methylation percentage (%)” refers to the number of molecules or loci that are methylated relative to the number of molecules or loci that are not methylated.

[0338] Therefore, methylation status describes the state of methylation of nucleic acids (e.g., genomic sequences). Additionally, methylation status refers to the characteristics of methylation-associated nucleic acid segments at a specific genomic locus. Such characteristics include, but are not limited to, whether any of the cytosine (C) residues in this DNA sequence are methylated, the location of methylated C residues, the frequency or percentage of methylated C in any particular region of the nucleic acid, and allelic methylation differences due to, for example, different allelic origins. The terms “methylation status,” “methylation profile,” and “methylation condition” also refer to the relative, absolute, or pattern of methylated or unmethylated C in any particular region of nucleic acids in a biological sample. For example, if cytosine (C) residues within a nucleic acid sequence are methylated, it may be described as “hypermethylated” or “highly methylated,” while if cytosine (C) residues within a DNA sequence are not methylated, it may be described as “unmethylated,” “hypomethylated,” or “lowly methylated.” Similarly, if a cytosine (C) residue in a nucleic acid sequence is methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be highly methylated or have increased methylation compared to the other nucleic acid sequence. Alternatively, if a cytosine (C) residue in a DNA sequence is not methylated compared to another nucleic acid sequence (e.g., from a different region or from a different individual), the sequence is considered to be hypomethylated or have decreased methylation compared to the other nucleic acid sequence. Additionally, as used herein, the term "methylation pattern" refers to the aggregate site of methylated and unmethylated nucleotides on a nucleic acid region. When the number of methylated and unmethylated nucleotides is the same or similar throughout a region, but the positions of the methylated and unmethylated nucleotides differ, two nucleic acids may have the same or similar methylation frequency or methylation percentage, but different methylation patterns. When sequences differ in the degree of methylation (e.g., one sequence is more or less methylated than another), frequency, or pattern, the sequences are referred to as "differentially methylated," "differentially methylated," or "having different methylation states." The term "differential methylation" refers to a difference in the level or pattern of nucleic acid methylation in a positive sample of cancer or other disease compared to the level or pattern of nucleic acid methylation in a negative sample of cancer or other disease. It can also refer to a difference in levels or patterns between patients whose cancer or other disease has recurred after surgery or other treatment and those who have not. Differential methylation and specific levels or patterns of DNA methylation are prognostic and predictive biomarkers, for example, once the correct cutoff values ​​or predictive features are defined.

[0339] Methylation state frequencies can be used to describe nucleotide loci or nucleic acid regions in a population of individuals or a sample from a single individual. For example, a nucleotide locus with a methylation state frequency of 50% is methylated in 50% of cases and unmethylated in 50% of cases. Such frequencies can be used, for example, to describe the degree of methylation of nucleotide loci or nucleic acid regions in a population of individuals or a collection of nucleic acids. Therefore, when the methylation in a first population or pool of nucleic acid molecules differs from the methylation in a second population or pool of nucleic acid molecules, the methylation state frequency of the first population or pool will differ from the methylation state frequency of the second population or pool. Such frequencies can also be used, for example, to describe the degree of methylation of nucleotide loci or nucleic acid regions in a single individual. For example, such frequencies can be used to describe the degree of methylation or unmethylation at nucleotide loci or nucleic acid regions in a group of cells from a tissue sample.

[0340] As used in this article, a "nucleotide locus" refers to the location of a nucleotide within a nucleic acid molecule. A methylated nucleotide locus refers to the location of a methylated nucleotide within a nucleic acid molecule.

[0341] Typically, methylation of human DNA occurs at dinucleotide sequences containing adjacent guanine and cytosine, with cytosine located at the 5′ of guanine (also known as the CpG dinucleotide sequence). In the human genome, most of the cytosine within a CpG dinucleotide is methylated, but in specific CpG-rich genomic regions (called CpG islands), some cytosine remains unmethylated (see, for example, Antequera et al. (1990)). Cell 62: 503–514).

[0342] As used herein, a “CpG island” refers to a G:C-rich region of genomic DNA containing an increased number of CpG dinucleotides relative to the total genomic DNA. CpG islands can be at least 100, 200, or more base pairs long, with a G:C content of at least 50% and an observed CpG frequency to expected frequency ratio of 0.6; in some cases, CpG islands can be at least 500 base pairs long, with a G:C content of at least 55% and an observed CpG frequency to expected frequency ratio of 0.65. (See Gardiner-Garden et al. (1987)). J. Mol. Biol.The method provided in 196: 261–281 calculates the ratio of observed CpG frequencies to expected frequencies. For example, the ratio of observed CpG frequencies to expected frequencies can be calculated using the formula R = (A × B) / (C × D), where R is the ratio of observed CpG frequencies to expected frequencies, A is the number of CpG dinucleotides in the analyzed sequence, B is the total number of nucleotides in the analyzed sequence, C is the total number of C nucleotides in the analyzed sequence, and D is the total number of G nucleotides in the analyzed sequence. Methylation status is typically determined in CpG islands, for example, in promoter regions. However, it should be understood that other sequences in the human genome are also prone to DNA methylation, such as CpA and CpT (see Ramsahoye (2000)). Proc. Natl. Acad. Sci. USA 97: 5237–5242; Salmon and Kaye (1970) Biochim. Biophys. Acta. 204: 340–351; Grafstrom (1985) Nucleic Acids Res. 13: 2827–2842; Nyce (1986) Nucleic Acids Res. 14: 4353–4367; Woodcock (1987) Biochem. Biophys. Res. Commun. 145: 888-894).

[0343] As used herein, the terms “methylcytosine”, “methyl C”, “methylated cytosine”, “methylated C” and “meC” are used interchangeably and cover both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC).

[0344] As used herein, the terms "modified cytosine" or "modified C" refer to cytosine nucleobases with side groups or other modifications at the base moiety compared to standard cytosine nucleotides. Modified cytosines include, but are not limited to, 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC), 5-carboxycytosine (5caC), and 5-formylcytosine (5fC).

[0345] As used herein, a "methylation-specific reagent" refers to a reagent that modifies the nucleotides of a nucleic acid molecule according to the methylation state of the nucleic acid molecule. In a particular embodiment, the term refers to a compound or composition or other preparation, or a collection or sequence thereof, that can alter the nucleotide sequence of a nucleic acid molecule in a manner that reflects the methylation state of the nucleic acid molecule. Methods of treating nucleic acid molecules with such reagents may include contacting the nucleic acid molecule with the reagent, with additional steps if necessary, to achieve the desired nucleotide sequence alteration. Such methods can be applied in a manner that modifies unmethylated nucleotides (e.g., each unmethylated cytosine) into different nucleotides. For example, in some embodiments, the reagent can deaminate unmethylated cytosine nucleotides to produce deoxyuridine residues. An exemplary reagent is a bisulfite reagent.

[0346] Additionally, treatment with "methylation-specific reagents" can be used to modify methylated nucleotides into different nucleotides. For example, methylated cytosine (including 5mC and 5hmC) in DNA can be converted by combining oxidation with a deca-11 translocation (TET) family dioxygenase with reduction with a borane derivative (e.g., pyridineborane and 2-methylpyridineborane (pic-BH3)), a process referred to herein as TAPS (TET-assisted pyridineborane sequencing). See, for example, the TAPS method combining TET enzyme oxidation with borane derivative reduction, described in, for example, US 2020 / 0370114A1, application sequence 16 / 960,510, filed July 7, 2020, which is incorporated herein by reference for all purposes. In embodiments of the TAPS method, methylated cytosine is converted to dihydrouracil. Other methods for converting methylated C include, for example:

[0347]

[0348] (Loise Williams et al., Enzymatic Methyl-seq: The next generation of methylome analysis, New England Biolabs Expressions 2019. Feature article)

[0349] In a preferred embodiment, a methylation-specific reagent modifies one of the four commonly found nucleotides in a nucleic acid molecule (C, G, T, and A in DNA and C, G, U, and A in RNA) such that the reagent modifies one nucleotide without modifying the other three. The resulting nucleotide is not limited to the four commonly found nucleotides listed above and may include modified or variant forms, such as purine or pyrimidine structures, including, for example, nucleobase analogs discussed herein. In a preferred embodiment, the resulting nucleotide is recognized by a DNA-modifying enzyme (e.g., DNA polymerase) as one of the commonly found nucleotides listed above and can be used as a template for strand replication. Nucleotide transformations performed by any of the methods described herein can be detected by determining the sequence of the resulting strand, for example using standard sequencing methods, or by identifying the nucleobases at selected locations by probing single or several specific nucleotide positions.

[0350] As used herein, the term "transformation" in relation to nucleotides or DNA strands refers to the treatment of a nucleotide or DNA strand with one or more reagents under conditions where some nucleotides have been converted to other nucleotides. For example, in bisulfite transformation, cytosine bases in DNA are typically deaminated, resulting in uracil bases at the transforming locus. Although less efficient, bisulfite can also deaminate 5-methylcytosine bases, resulting in thymine bases at the transforming locus. "Bisulfite-treated" and "bisulfite-transformed" are used interchangeably herein to refer to DNA or nucleotide sites exposed to a bisulfite reagent under conditions where cytosine is typically converted to uracil. In bisulfite-free TAPS, methylated cytosine is selectively converted to dihydrouracil (DHU), while unmethylated C is not transformed. DHU nucleotides pair with A nucleotides, not G nucleotides, making them easily distinguishable from unmethylated C bases in the transformed DNA strand.

[0351] As used herein, the term "poor conversion" in relation to the conversion of nucleotides after treatment with reagents and / or conditions that convert some nucleotides to others refers to nucleotides whose conversion rate is lower (preferably less than 10%, more preferably less than 1%) under a given treatment compared to the conversion rate of nucleotides expected to be converted under the same treatment. For example, bisulfite-mediated deamination of cytosine is significantly slowed by the presence of 5-methyl, and thus the rate of 5-methylcytosine deamination to thymine is about two orders of magnitude lower than the rate of cytosine deamination to uracil (see, for example, Hayatsu et al. Biochemistry 18:4:632-37 (1979); Hayatsu, Proc. Jpn. Acad 84(8):321-330 (2008), each of which is incorporated herein by reference). Therefore, it is said that under bisulfite treatment conditions commonly used to convert cytosine to uracil, 5-methylcytosine is poorly converted compared to cytosine.

[0352] The term "bisulfite reagent" refers to a reagent, as disclosed herein, that can be used to distinguish between methylated and unmethylated CpG dinucleotide sequences, including bisulfites, metabisulfites, monohydrogen sulfites, or combinations thereof. The methods of treatment are known in the art (e.g., PCT / EP2004 / 011715 and WO 2013 / 116375, each incorporated herein by reference in its entirety). In some embodiments, the bisulfite treatment is carried out in the presence of a denaturing solvent (such as, but not limited to, n-alkylene glycol or diethylene glycol dimethyl ether (DME)), or in the presence of dioxane or dioxane derivatives. In some embodiments, the denaturing solvent is used at a concentration between 1% and 35% (v / v). In some embodiments, the bisulfite reaction is carried out in the presence of a scavenger, such as, but not limited to, chromium derivatives, such as 6-hydroxy-2,5,7,8,-tetramethylchromium-2-carboxylic acid or trihydroxybenzoic acid and its derivatives, such as gallic acid (see: PCT / EP2004 / 011715, which is incorporated herein by reference in its entirety). In some preferred embodiments, the bisulfite reaction comprises treatment with ammonium monohydrogen sulfite (also known as ammonium bisulfite), for example, as described in WO 2013 / 116375.

[0353] The term "methylation assay" refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.

[0354] The terms “target” and “template” are used interchangeably for nucleic acid chains with flap-like structures (e.g., invasive cleavage structures) and refer to nucleic acid chains to which upstream and downstream nucleic acids or nucleic acid regions hybridize to form invasive cleavage structures. The target nucleic acid can be isolated from a sample (e.g., a patient sample) or can be a replicating or amplified nucleic acid chain. For example, in a PCR-flap assay, the replicating DNA chain generated in the amplification reaction mixture is also a target nucleic acid, on which an invasive cleavage structure is formed by hybridization of a flap-like oligonucleotide with the target chain of a neighboring upstream nucleic acid (e.g., an upstream invasive oligonucleotide or a primer that becomes an invasive oligonucleotide by polymerase extension). While the template chain can be used as a template for primers extended by polymerase, as in the case of a PCR-flap endonuclease assay, the use of the term “template” is not limited to the polymerization assay or reaction. When used in relation to ligation, the template refers to a nucleic acid chain to which the 3′ and 5′ ends of a complementary strand hybridize to form a ligation site.

[0355] As used herein, the term "3′ blocker" refers to, for example, a 3′-end modification on an oligonucleotide that, when present at the 3′ end of the upstream nucleic acid in a cleavage structure, inhibits cleavage by a flap-shaped endonuclease that invasively cleaves the structure. For example, 3′ amines, 3′ phosphates (3′-PO4), 3′ biotin, 3′ C6 (a 6-carbon-diol spacer, also known as "3′ hexanediol"), and 3′ dideoxynucleotides inhibit flap-shaped cleavage if present in the upstream nucleic acid. See, for example, Kaiser et al., ibid.

[0356] As used herein, the term "5′ blocking group" refers to a 5′-end modification or portion that prevents the phosphate at the 5′ end of a nucleic acid strand or a region of the nucleic acid strand (e.g., the 5′ end of the double-stranded portion of the padlock probe in a downstream duplex when the 5′ end of the padlock probe contains an unpaired 5′ lobe sequence) from being used for enzymatic ligation, for example, ligation to a hydroxyl group at the 3′ end of the nucleic acid strand (e.g., the 3′ terminal nucleotide of an adjacent double-stranded portion of the padlock probe). Exemplary 5′ blocking groups include, but are not limited to, carbon chains (e.g., hexane), non-standard nucleotides, baseless nucleotides (e.g., ribose or deoxyribose lacking nucleobases), dye tags, lobe-shaped nucleic acid sequences, hairpin lobes, etc. In a preferred embodiment, the 5′ blocking group is attached to the 5′ position of the nucleotide via a phosphate ester (e.g., a phosphodiester bond) (e.g., the 5′ end of the double-stranded portion of the padlock probe in a downstream duplex). In a particularly preferred embodiment, the 5′ blocking group is attached to the 5′ position of the nucleotide, which is the 5′ of the FEN-1 cleavage site in an invasive cleavage structure.

[0357] As used in this article, the term "Mg" ++ "Loop-shaped assay buffer" refers to a buffer solution for lobular endonucleases, which contains Mg... ++As the dominant or essentially sole divalent cation in the buffer solution, Mg2+ causes the assay reactions performed in the buffer solution (e.g., clasp assays) to exhibit Mg2+. ++ The presence of certain characteristics (e.g., some valve-shaped endonucleases exhibit high specificity for invasive cleavage structures compared to, for example, Y-shaped or pseudo-Y-shaped structures), and does not show characteristics associated with the presence of other divalent cations (e.g., with or without Mg). ++ Using Mn ++ (Alternative cleavage activities of associated flap endonucleases), such as those described above by Kaiser et al., for example. In some embodiments, the flap assay buffer has very low or no KCl compared to the standard PCR buffer and contains elevated Mg2+. ++ (For example, the flap assay buffer of this technology contains >6mM, preferably >7mM, more preferably 7.5mM Mg) ++ PCR buffer typically contains approximately 1.5 to 2.5 mM Mg. ++ ).

[0358] As used herein, the term "low-biased amplification buffer" refers to an amplification buffer configured to exhibit low inter-target variability in amplification of different targets co-amplified in multiplex amplification reactions (e.g., multiplex pre-amplification reactions performed before subsequent amplification reactions such as PCR-flap assays). Amplification bias between different targets can be assessed by measuring the amplification efficiency of different targets at known concentrations. In some embodiments of this technique, the low-biased amplification buffer is a buffer suitable for PCR-flap assays and containing a high magnesium concentration (e.g., at least 6 mM, preferably 6 to 10 mM, preferably 7 to 9 mM, preferably about 7.5 mM as the final concentration in the reaction mixture), in contrast to PCR buffers that typically contain a final magnesium concentration of about 1 to 4 mM in the reaction mixture. In a preferred embodiment, the low-biased amplification buffer comprises 3-(n-morpholino)propanesulfonic acid (MOPS) buffer, and in some preferred embodiments, the low-biased amplification buffer comprises 7.5 mM MgCl2, 10 mM MOPS, 0.3 mM Tris-HCl (pH 8.0), 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. In this document, the terms "PCR-flap assay buffer" and "low-biased amplification buffer" are used interchangeably.

[0359] Mg used in low-bias amplification buffer ++The range of amounts includes any concentration covered by the above range. For example, low-biased amplification buffer may contain 6, 6.1, 6.2, 6.5, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, or 11.0 mM Mg ++ Or more, or any fractional concentration in between. These concentrations are for illustrative purposes only and are not limitations.

[0360] As used in this article, "low-biased amplification buffer" is different from buffer containing more than approximately 4 mM Mg. ++ The PCR buffer, but it also contains ingredients to reduce high Mg levels. ++ Reagents that provide concentration-dependent helical stabilization, such as DMSO, (NH4)2SO4, and betaine, are sometimes referred to as enhancers. For a discussion of such enhancers, see, for example, Raser et al., An efficient and economic enhancer mix for PCR, Biochemical and Biophysical Research Communications 347(2006) 747–751; O. Henegariu et al., “Multiplex PCR: Critical Parameters and Step-by-Step Protocol,” BioTechniques 23:504-511 (September 1997); And Qiagen PCRBrochure “Maximizing PCR and RT-PCR Success,” 3rd Edition (Document 1104683, 10 / 2016), these documents are incorporated herein by reference in their entirety. In a preferred embodiment, the low-biased amplification buffer is substantially free of (NH4)2SO4. In some embodiments, the low-biased amplification buffer is substantially free of ammonium ions. In some embodiments, the low-biased amplification buffer is substantially free of DMSO and / or betaine.

[0361] As used herein, the term "substantially free" regarding components excluded from a composition (e.g., reaction mixture, buffer, etc.) means that the composition is not formulated to contain the excluded component, but may contain trace amounts of that component, such as remnants from a previous reaction step, or concentrated stock solutions of small amounts of added reaction components (e.g., enzymes in storage solutions). For example, small amounts of glycerol or other substances may be introduced into a reaction mixture (e.g., a PCR flap assay reaction mixture) by adding an aliquot of an enzyme (e.g., a polymerase) containing substances for stable storage, but such substances are typically diluted sufficiently in the reaction mixture so that trace amounts of glycerol and other substances do not alter the intended function of the reaction mixture; for example, trace amounts of (NH4)2SO4, betaine, or DMSO are not expected to alter the low-biased multiplex amplification results observed when using the low-biased amplification buffer described herein. Such trace substances are generally not reported in the literature as part of the final reaction mixture formulation. Therefore, a composition that is “substantially free” of the listed components may contain a certain level of the excluded components such that, even if present, the function of the listed composition is not altered compared to a pure composition (i.e., a composition that is completely free of the excluded components).

[0362] As used herein, the terms "lobe assay reagent" or "invasive cleavage assay reagent" refer to the collection of all reagents required for performing a lobe assay or invasive cleavage assay on a substrate. As is known in the art, lobe assays typically involve oligonucleotides and lobe endonucleases for forming invasive cleavage structures. Lobe assay reagents may optionally contain a target nucleic acid or template nucleic acid bound to a lobe oligonucleotide and an invasive oligonucleotide (or a lobe oligonucleotide having an invasive oligonucleotide region). In a particularly preferred embodiment, the lobe assay reagent comprises Mg ++ The flap assay buffer, as discussed herein. In some embodiments, the flap assay reagent further comprises a ligase (e.g., a cyclizable molecule, padlock probe, dumbbell probe, linear vector, or one or a pair of oligonucleotides of a vector pair) that hybridizes to the flap sequence. In some embodiments, the flap assay reagent further comprises a bridging oligonucleotide. In some embodiments, the bridging oligonucleotide comprises a sequence complementary to the flap sequence. For example, in some embodiments, the bridging oligonucleotide comprises an inner segment complementary to the flap sequence. In some embodiments, the bridging oligonucleotide comprises additional nucleotides at the 3′ and 5′ (e.g., the 3′ and 5′ of the inner segment) of a sequence complementary to the flap sequence, which are complementary to the first and second target regions of the ligase (e.g., a cyclizable molecule, padlock probe, dumbbell probe, linear vector, or one or a pair of oligonucleotides of a vector pair). In some embodiments, the flap assay reagent further comprises a ligase.

[0363] As used herein, the term "FRET" refers to fluorescence resonance energy transfer, a process in which energy is transferred partially (e.g., from a fluorophore) to itself or from a fluorophore to a non-fluorophore (e.g., a quencher molecule). In some cases, FRET involves the excited donor fluorophore transferring energy to a lower-energy acceptor fluorophore via short-range (e.g., about 10 nm or less) dipole-dipole interactions. In other cases, FRET involves a loss of donor fluorescence energy and an increase in acceptor fluorophore fluorescence. In other forms of FRET, energy may be exchanged from the excited donor fluorophore to a non-fluorescent molecule (e.g., a quencher molecule). FRET is known to those skilled in the art and has been described (see, for example, Streyer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995, Methods Enzymol., 246:300; Orpana, 2004 Biomol Eng 21, 45-50; Olivier, 2005 Mutant Res 573, 103-110, each of which is incorporated herein by reference in its entirety).

[0364] As used herein, the term "FRET system" refers to a pair or set of parts that together act as donor-acceptor or donor-quencher conjugates for FRET-based molecular (e.g., valvular oligonucleotides or assay reporter molecules) analysis. The parts of the FRET system available in this technique are not limited to these arrangements and may, for example, be interchanged in position, located on different parts of the illustrated molecule, or may contain the same or different additional donor or acceptor parts.

[0365] As used herein, the phrase “substantially non-complementary” for the use of probe lobes or arms means that the lobes are sufficiently non-complementary to not selectively hybridize with nucleic acid sequences (e.g., target nucleic acids or amplified DNA) under specified annealing conditions or stringent conditions, encompassing the terms “substantially non-complementary” and “completely non-complementary”.

[0366] As used herein, the term “signal” refers to any detectable effect, such as an effect that will be caused or provided by the action or accumulation of a component or product in a assay reaction.

[0367] As used herein, the term "real-time" in relation to nucleic acid amplification or signal amplification detection refers to the detection or measurement of the accumulation of products or signals during the reaction, such as during incubation or thermal cycling. This detection or measurement may occur continuously, at multiple discrete points during the amplification reaction, or in combination. For example, in polymerase chain reaction (PCR), detection (e.g., fluorescence detection) may occur continuously throughout or part of the thermal cycling, or instantaneously at one or more points during one or more cycles. In some embodiments, real-time detection of PCR is accomplished by determining the fluorescence level at the same point (e.g., a time point in the cycle, or a temperature step in the cycle) in each of multiple cycles or within each cycle. Real-time detection of amplification may also be referred to as detection "during" the amplification reaction.

[0368] As used herein, the term "nucleic acid abundance" refers to the amount of a specific target nucleic acid sequence present in a sample or aliquot. This amount is typically expressed as mass (e.g., µg), mass per unit volume (e.g., µg / µL), copy number (e.g., 1000 copies, 1 attomole), or copy number per unit volume (e.g., 1000 copies / mL, 1 attomole / µL). Nucleic acid abundance can also be expressed as an amount relative to a standard of known concentration or copy number. Measurements of nucleic acid abundance can be based on any basis understood by those skilled in the art as a suitable quantitative expression of nucleic acid abundance, including the physical density, optical density, refractive properties, staining properties of the sample, or based on the intensity of a detectable label (e.g., a fluorescent label).

[0369] In the context of nucleic acids, the term "amplifying" refers to the process of generating multiple copies of a polynucleotide or a portion of a polynucleotide, typically starting from a small number of polynucleotides (e.g., a single polynucleotide molecule), where the amplification product or amplicon is generally detectable. Polynucleotide amplification encompasses a variety of chemical and enzymatic processes. During polymerase chain reaction (PCR) or ligase chain reaction (LCR; see, for example, U.S. Patent No. 5,494,810; which is incorporated herein by reference in its entirety), the generation of multiple copies of DNA from one or more copies of a target or template DNA molecule is a form of amplification. Other types of amplification include, but are not limited to: allele-specific PCR (see, for example, U.S. Patent No. 5,639,611, which is incorporated herein by reference in its entirety), assembly PCR (see, for example, U.S. Patent No. 5,965,408, which is incorporated herein by reference in its entirety), helicase-dependent amplification (see, for example, U.S. Patent No. 7,662,594, which is incorporated herein by reference in its entirety), hot-start PCR (see, for example, U.S. Patent Nos. 5,773,258 and 5,338,671, which are each incorporated herein by reference in their entirety), sequence-specific PCR, inverse PCR (see, for example, Triglia et al. (1988) Nucleic Acids Res., 16:8186, which is incorporated herein by reference in its entirety), and ligation-mediated PCR (see, for example, Guilfoyle, R.).Nucleic Acids Research, 25:1854-1858 (1997); U.S. Patent No. 5,508,169 (these references are incorporated herein by reference in their entirety), methylation-specific PCR (see, for example, Herman et al., (1996) PNAS 93 (13) 9821-9826; which are incorporated herein by reference in their entirety), small primer PCR, multiplex ligation-dependent probe amplification (see, for example, Schouten et al., (2002) Nucleic Acids Research 30(12): e57; which are incorporated herein by reference in their entirety), multiplex PCR (see, for example, Chamberlain et al., (1988) Nucleic Acids Research 16(23) 11141-11156; Ballabio et al., (1990) Human Genetics 84(6) 571-573; Hayden et al., (2008) BMC Genetics 9:80; these references are incorporated herein by reference in their entirety, each cited individually), nested PCR, overlap extension PCR (see, for example, Higuchi et al., (1988) Nucleic Acids Research 16(15) 7351-7367; these are incorporated herein by reference in their entirety), real-time PCR (see, for example, Higuchi et al., (1992) Biotechnology 10:413-417; Higuchi et al., (1993) Biotechnology 11:1026-1030; these references are incorporated herein by reference in their entirety, each cited individually), reverse transcription PCR (see, for example, Bustin, SA (2000) J. Molecular Endocrinology 25:169-193; these are incorporated herein by reference in their entirety), solid-phase PCR, thermally asymmetric staggered PCR, and falling PCR (see, for example, Don et al., Nucleic Acids Research (1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814; Hecker et al., (1996) Biotechniques 20(3) 478-485; these references are incorporated herein by reference in their entirety. Polynucleotide amplification can also be achieved using digital PCR (see, for example, Kalinina et al., Nucleic Acids Research).25; 1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA. 96; 9236-41, (1999); International Patent Publication No. WO05023091A2; U.S. Patent Application Publication No. 20070202525; these documents are each incorporated herein by reference in their entirety. In some embodiments, a portion of the target nucleic acid is copied during amplification, and in some embodiments, non-target polynucleotides are amplified in response to the presence of the target nucleic acid (e.g., cleavage flaps, ligation products, rolling circle replication products, etc.).

[0370] The term "polymerase chain reaction" ("PCR") refers to the methods described in KB Mullis U.S. Patents 4,683,195, 4,683,202, and 4,965,188, which describe a method for increasing the concentration of a target sequence fragment in a mixture of genomic or other DNA or RNA without cloning or purification. This process for amplifying the target sequence comprises: introducing a large excess of two oligonucleotide primers into a DNA mixture containing the desired target sequence, followed by a precise thermal cycling process in the presence of DNA polymerase. The two primers are complementary to the corresponding strands of the double-stranded target sequence. To achieve amplification, the mixture is denatured, and then the primers are annealed to their complementary sequences within the target molecule. After annealing, the primers are extended with polymerase to form a new pair of complementary strands. The denaturation, primer annealing, and polymerase extension steps can be repeated multiple times (e.g., denaturation, annealing, and extension constitute a "cycle"; multiple "cycles" may exist) to obtain a high concentration of the amplified segment of the desired target sequence. The length of the amplified fragment of the desired target sequence is determined by the relative positions of the primers, and therefore this length is a controllable parameter. Due to the reproducibility of this process, the method is called a “polymerase chain reaction” (“PCR”). Because the desired amplified fragment of the target sequence becomes the dominant sequence in the mixture (in terms of concentration), they are called “PCR-amplified” and are “PCR products” or “amplifiers.” Those skilled in the art will understand that the term “PCR” encompasses many variations of the originally described methods, such as real-time PCR, nested PCR, reverse transcription PCR (RT-PCR), single-primer PCR, and arbitrary-primer PCR.

[0371] As used herein, the term "primer annealing" refers to conditions that allow oligonucleotide primers to hybridize with the template nucleic acid strand, preferably sufficient for extension by DNA polymerase. Primer annealing conditions vary with primer length and sequence and are typically based on T0 values ​​determined or calculated for the primer. m For example, the annealing step in amplification methods involving thermal cycling involves lowering the temperature after the thermal denaturation step to a T value based on the primer sequence. mThe temperature is maintained sufficiently for the time required for this annealing process.

[0372] As used herein, the term "amplifiable nucleic acid" refers to nucleic acid that can be amplified by any amplification method. It is expected that "amplifiable nucleic acid" will typically include a "sample template".

[0373] The terms "amplifier" or "amplification product" refer to nucleic acid segments, usually DNA, generated through an amplification process (such as PCR). The term is also used to refer to RNA segments generated using RNA polymerase amplification methods (such as NASBA, TMA, etc.).

[0374] The term "amplification plot" used in thermal cycling amplification reactions refers to a plot showing the amplified signal (e.g., fluorescence signal) relative to the number of cycles. When used in relation to non-thermal cycling amplification methods, the amplification plot typically refers to a plot showing the change in signal accumulation over time.

[0375] The term “baseline” used in amplification mapping refers to the signal detected from the assembled amplification reaction before incubation, or in the case of PCR, during the initial cycle when the signal has hardly changed.

[0376] As used in this article, the term "C" refers to real-time detection during the amplification reaction of thermal cycling. t "Threshold cycling" or "threshold loop" refers to the number of cycles of a detected signal (e.g., fluorescence) that exceeds a fixed threshold.

[0377] As used herein, the terms “template-free control” and “target-free control” (or “NTC”) refer to reactions or samples that do not contain the template or target nucleic acid. They are used to verify amplification quality.

[0378] As used herein, the term "sample template" refers to the nucleic acids derived from a sample used for analysis of the presence of a "target". In contrast, "background template" is used to refer to nucleic acids other than the sample template that may or may not be present in the sample. The presence of a background template is usually unintentional. It may be the result of residues or due to the presence of nucleic acid contaminants that are being attempted to be purified from the sample. For example, nucleic acids from an organism other than the nucleic acid to be detected may be present as background in the test sample.

[0379] As used herein, the terms "patient" or "subject" refer to an organism to be subjected to the various tests provided by this technology. The term "subject" includes animals, preferably mammals, including humans. In a preferred embodiment, the subject is a primate. In an even more preferred embodiment, the subject is a human. Further regarding diagnostic methods, the preferred subject is a vertebrate subject. The preferred vertebrate is warm-blooded; the preferred warm-blooded vertebrate is a mammal. The preferred mammal is most preferably a human. As used herein, the term "subject" includes both human and animal subjects. Therefore, this document provides for veterinary therapeutic use. Thus, this technology provides for the diagnosis of mammals, mammals such as humans, and those mammals of importance due to their endangerment, such as the Siberian tiger; mammals of economic importance, such as animals raised on farms for human consumption; and / or animals of social importance to humans, such as animals kept as pets or in zoos. Examples of such animals include, but are not limited to: carnivores, such as cats and dogs; swine, including domestic pigs, meat pigs, and wild boars; ruminants and / or ungulates, such as cattle, steers, sheep, giraffes, deer, goats, bison, and camels; pinnipeds; and horses. Therefore, diagnosis and treatment of livestock are also provided, including but not limited to domestic pigs, ruminants, ungulates, horses (including racehorses), etc. The subject matter disclosed in this invention also includes systems for diagnosing conditions (such as cancer or other diseases) in subjects. Such systems may be provided, for example, as commercial kits that can be used to screen for the risk of conditions (such as cancer or other diseases) or to diagnose conditions (such as cancer or other diseases) in subjects from whom biological samples have been collected. Exemplary systems provided according to the technology of this invention include assessing the methylation status of the biomarkers described herein.

[0380] The term "sample" is used in its broadest sense. In one sense, it can refer to animal cells, tissues, or fluids. In another sense, it refers to specimens or cultures obtained from any source, as well as biological and environmental samples. Biological samples can be obtained from plants or animals (including humans) and encompass, for example, fluids, solids, tissues, and gases. Environmental samples include environmental materials such as surface substances, soil, water, and industrial samples. These examples should not be construed as limiting the types of samples applicable to this invention. As used herein with respect to samples, the term "sample" taken from a source or subject (e.g., from a patient) is not limited to a single physical specimen but also encompasses samples taken in multiple parts; for example, a "sample" of blood may be taken from two, three, four, or more different blood collection tubes or other blood collection devices (e.g., bags), or combinations of different blood collection devices. A sample "suspected of containing" a marker or other material may or may not contain that marker.

[0381] This technology is not limited by the type of biological sample used or analyzed. This technology can be used with a wide variety of biological samples, including but not limited to: tissues (e.g., organs (e.g., heart, liver, brain, lungs, stomach, intestines, spleen, kidneys, pancreas, and reproductive organs), glands, skin, and muscle), cells (e.g., blood cells (e.g., lymphocytes or erythrocytes), muscle cells, tumor cells, and skin cells), or gaseous, bodily fluid (e.g., blood or portions thereof, serum, plasma, urine, semen, saliva, etc.) or solid (e.g., feces) samples obtained from humans (e.g., adults, infants, or embryos) or animals (e.g., cattle, poultry, mice, rats, dogs, pigs, cats, horses, etc.). In some embodiments, the biological sample may be a solid food and / or feed product and / or ingredient, such as dairy products, vegetables, meat and meat by-products, and waste. Biological samples can be obtained from a wide range of domesticated animal categories as well as wild or rewilded animals, including but not limited to animals such as ungulates, bears, fish, lagomorphs, rodents, pinnipeds, etc.

[0382] Biological samples also include biopsy tissues and tissue sections (e.g., biopsy tissues or sections of tumors, growths, rashes, infected tissues, or paraffin-embedded sections), medical or hospital samples (e.g., including but not limited to blood samples, saliva, oral swabs, cerebrospinal fluid, pleural fluid, breast milk, colostrum, lymph, sputum, vomit, bile, semen, oocytes, cervical cells, amniotic fluid, urine, feces, hair, and sweat), laboratory samples (e.g., subcellular fractions), and forensic samples (e.g., blood or tissue containing nucleic acids (e.g., splashes or residues), hair, and skin cells), as well as archaeological samples (e.g., fossilized organisms, tissues, or cells).

[0383] Environmental samples include, but are not limited to, environmental materials such as surface materials, soil, water (e.g., fresh or seawater), algae, lichens, geological samples, airborne materials containing nucleic acids, crystals, and industrial samples, as well as samples obtained from food and dairy processing instruments, apparatus, equipment, appliances, disposable and non-disposable items.

[0384] Samples can be prepared by any desired or suitable method. In some embodiments, nucleic acids are analyzed directly from bodily fluids, feces, or other samples using the method described in U.S. Patent No. 9,000,146, which is incorporated herein by reference in its entirety for all purposes.

[0385] However, the above examples should not be construed as limiting the types of samples applicable to this technology (e.g., samples suspected of containing target sequences, genes, or templates (e.g., whose presence or absence can be determined using compositions and methods of this technology)).

[0386] As used in this article, the term "nucleic acid" refers to any polynucleotide in which nucleosides are linked together by a sugar-phosphate backbone.

[0387] As used in this article, the terms “nucleic acid sequence” and “nucleic acid molecule” refer to oligonucleotides, nucleotides or polynucleotides and their fragments or portions. This term encompasses sequences of analogues including DNA and RNA nucleotides, including those listed above, and also including but not limited to: 4-acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine, 5-(carboxyhydroxy-methyl)uracil, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine, 7-methylguanine, 5-methylaminomethyluracil. The following are listed: pyrimidines, 5-methoxy-amino-methyl-2-thiouracil, β-D-mannosyl-Q nucleoside, 5′-methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, methyl uracil-5-oxyacetic acid, uracil-5-oxyacetic acid, oxybutyroxyglycoside, pseudouracil, Q nucleoside, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid, pseudouracil, Q nucleoside, 2-thiocytosine, 2,6-diaminopurine, and pyrazolo[3,4-d]pyrimidines, such as the guanine analog 6-amino1H-pyrazolo[3,4d]pyrimidine 4(5H)one (ppG or PPG, also known as Super G) and the adenine analog 4-amino1H-pyrazolo[3,4d]pyrimidine (ppA or PPA). The xanthine analog 1H-pyrazolo[5,4d]pyrimidine 4(5H)-6(7H)-dione (ppX) can also be used. When present in oligonucleotides, these base analogs enhance hybridization and improve mismatch discrimination. All tautomerisms of naturally occurring bases, modified bases, and base analogs can be included in the oligonucleotide conjugates of this technique. Other modifying bases that can be used in this technology include: 6-amino-3-prop-1-ynyl-5-hydropyrazolo[3,4-d]pyrimidin-4-one, PPPG; 6-amino-3-(3-hydroxyprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, HOPPPG; 6-amino-3-(3-aminoprop-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, NH2PPPG; 4-amino-3-(prop-1-ynyl)pyrazolo[3,4-d]pyrimidin, PPPA; 4-amino-3-(3-hydroxyprop-1-ynyl)pyrazolo[3,4-d]pyrimidin, HOPPPA; 4-amino-3-(3-aminoprop-1-ynyl)pyrazolo[3,4-d]pyrimidin, NH2PPPA;3-Propyl-1-ynylpyrazolo[3,4-d]pyrimidin-4,6-diamino, (NH2)2PPPA; 2-(4,6-diaminopyrazolo[3,4-d]pyrimidin-3-yl)ethyn-1-ol, (NH2)2PPPAOH; 3-(2-aminoethynyl)pyrazolo[3,4-d]pyrimidin-4,6-diamine, (NH2)2 PPPANH2; 5-prop-1-ynyl-1,3-dihydropyrimidin-2,4-dione, PU; 5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidin-2,4-dione, HOPU; 6-amino-5-prop-1-ynyl-3-dihydropyrimidin-2-one, PC; 6-amino-5-(3-hydroxyprop-1-ynyl)-1,3-dihydropyrimidin-2-one, HOPC; and 6-amino-5-(3-aminoprop-1-ynyl)-1,3-dihydropyrimidin-2-one, NH2PC; 5-[4-amino-3-(3-methoxyprop-1-ynyl)pyrazol[3,4-d]pyrimidinyl]-2-(hydroxymethyl)oxacyclopentan-3-ol, CH3 OPPPA; 6-amino-1-[4-hydroxy-5-(hydroxymethyl)oxacyclopentan-2-yl]-3-(3-methoxypropyl-1-ynyl)-5-hydropyrazolo[3,4-d]pyrimidin-4-one, CH3 OPPPG; 4,(4,6-diamino-1H-pyrazolo[3,4-d]pyrimidin-3-yl)-but-3-yn-1-ol, Super A; 6-amino-3-(4-hydroxy-but-1-ynyl)-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-one; 5-(4-hydroxy-but-1-ynyl)-1H-pyrimidin-2,4-dione, Super T; 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPAI); 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPABr); 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine ((NH2)2PPACl); 3-iodo-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPAI); 3-bromo-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPABr); and 3-chloro-1H-pyrazolo[3,4-d]pyrimidine-4-ylamine (PPACl). The term covers base analogs that provide alternative hydrogen-bonded configurations (e.g., Iso-C and Iso-G, as well as other non-standard base pairs, as described in S. Benner’s U.S. Patent No. 6,001,983);Non-hydrogen-bonded analogs (e.g., nonpolar aromatic nucleoside analogs, such as 2,4-difluorotoluene, described in BA Schweitzer and ET Kool, J. Org. Chem., 1994, 59, 7238-7242; BA Schweitzer and ET Kool, J. Am. Chem. Soc., 1995, 117, 1863-1872); “universal” bases, such as 5-nitroindole and 3-nitropyrrole; and universal purine and pyrimidine (such as “K” and “P” nucleotides, respectively, P. Kong et al., Nucleic Acids Res., 1989, 17, 10373-10383; P. Kong et al., Nucleic Acids Res., 1992, 20, 5149-5152). Nucleotide analogs also include nucleotides and polynucleotides having one or more modified or substituted sugar and / or phosphate moieties, including but not limited to phosphate thioester nucleotides, phosphonate nucleotides, 2'-substituted sugars (e.g., 2'-O-methylribose), and / or substituted sugars (e.g., arabinose). Nucleotide analogs include modified forms of deoxyribonucleotides and ribonucleotides.

[0388] Nucleic acid sequences or molecules can be genomic or synthetically derived DNA or RNA, and can be single-stranded or double-stranded, representing sense or antisense strands. Therefore, nucleic acid sequences can be dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, ssDNA made from dsDNA (e.g., through melting, denaturation, helicase, etc.), type A, type B, or type Z DNA, triple-stranded DNA, RNA, ssRNA, dsRNA, mixed ss and dsRNA, ssRNA made from dsRNA (e.g., through melting, denaturation, helicase, etc.), messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), catalytic RNA, snRNA, microRNA, or protein nucleic acid (PNA).

[0389] This technology is not limited by the type or source of the nucleic acid used (e.g., sequence or molecule (e.g., target sequence and / or oligonucleotide)). For example, nucleic acid sequences can be amplified or created by synthesizing (e.g., polymerization (e.g., primer extension (e.g., RNA-DNA hybridization primer technology)) and reverse transcription (e.g., RNA to DNA)) and / or amplifying (e.g., polymerase chain reaction (PCR), rolling circle amplification (RCA), nucleic acid sequence-based amplification (NASBA), transcription-mediated amplification (TMA), ligase chain reaction (LCR), cycling probe technology, Q-β replicase, strand displacement amplification (SDA), branched DNA signal amplification (bDNA), hybridization capture, and helicase-dependent amplification).

[0390] Unless otherwise stated herein, the terms “nucleotide” and “base” are used interchangeably in relation to nucleic acid sequences and refer to nucleosides comprising a sugar molecule and a nucleobase, with an optional 5′ phosphate. A “nucleobase” is a heterocyclic nitrogenous base, such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or their heterocyclic derivatives, analogs, or tautomers. Nucleobases may be naturally occurring or synthetic. Non-restrictive examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted with methyl or bromine at the 8-position, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazoxanthine, 7-deazoguanine, 7-deazoadenine, N4-vinylcytosine, 2,6-diaminopurine, N6-vinyl-2,6-diaminopurine, 5-methylcytosine, 5-(C3-C 6)-Alkyne cytosine, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylpyrazine, 6-dihydrothymidine, 5,6-dihydrouracil, 4-methyl-indole, ethylene adenine; and non-naturally occurring nucleobases, described in U.S. Patent Nos. 5,432,272 and 6,150,510 and PCT Application WO The entire contents of the following references are incorporated herein by reference: WO 92 / 002258, WO 93 / 10820, WO 94 / 22892 and WO 94 / 24144, and Fasman (“Practical Handbook of Biochemistry and Molecular Biology”, pp. 385-394, 1989, CRC Press, Boca Raton, LO).

[0391] As used herein, the term “non-standard” with respect to nucleotides and nucleobases (e.g., in oligonucleotides) refers to nucleotides other than adenine, guanine, cytosine, thymine, and uracil deoxyribonucleotides and ribonucleotides. Non-standard nucleotides include analogues and derivative nucleobases, including but not limited to those described above in the definitions of “nucleic acid sequence” and “nucleotide”.

[0392] As used herein, the term "standard" for nucleotides and nucleobases (e.g., in oligonucleotides) refers to adenine, guanine, cytosine, thymine, and uracil deoxyribonucleotides and ribonucleotides.

[0393] As used herein, the term "oligonucleotide" is defined as a molecule containing two or more nucleotides (e.g., deoxyribonucleotides or ribonucleotides), preferably at least 5 nucleotides, more preferably at least about 10–15 nucleotides, and even more preferably at least about 15 to 30 nucleotides or longer (e.g., oligonucleotides are typically less than 200 residues in length (e.g., 15 to 100 nucleotides); however, as used herein, the term is also intended to cover longer polynucleotide chains). The exact size will depend on a variety of factors, which in turn depend on the final function or use of the oligonucleotide. Oligonucleotides are generally referred to by their length. For example, a 24-residue oligonucleotide is called a "24-mer". Oligonucleotides can form secondary and tertiary structures through self-hybridization or hybridization with other polynucleotides. Such structures can include, but are not limited to, duplexes, hairpins, crosses, bends, and triplexes. Oligonucleotides can be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or combinations thereof. In some embodiments, oligonucleotides that form invasive cleavage structures are generated in a reaction (e.g., by extending primers in an enzymatic extension reaction).

[0394] Since mononucleotides are formed into oligonucleotides through a reaction in which the 5′ phosphate of a mononucleotide's pentose ring attaches to its adjacent 3′ oxygen in one direction via a phosphodiester bond, the end of an oligonucleotide is called a "5′ end" or "5′ terminus" if the 5′ phosphate at one end is not attached to the 3′ oxygen of the pentose ring, and a "3′ end" or "3′ terminus" if the 3′ oxygen at one end is not attached to the 5′ phosphate of the subsequent pentose ring. As used herein, nucleic acid sequences, even within larger oligonucleotides, may be described as having both 5′ and 3′ ends. Unlike the 3′ and 5′ ends of nucleic acid molecules, the 3′ and 5′ ends of nucleic acid sequences do not need to be located at the 3′ and 5′ ends of the molecular chain. If, when moving along the nucleic acid chain in a 5′ to 3′ direction, the 3′ end of a first region precedes the 5′ end of a second region, then the first region along the nucleic acid chain is said to be upstream of the other region.

[0395] When two different non-overlapping oligonucleotides anneal to different regions of the same linear complementary nucleic acid sequence, and the 3′ end of one oligonucleotide points to the 5′ end of the other oligonucleotide, the former can be called the “upstream” oligonucleotide and the latter the “downstream” oligonucleotide.

[0396] As used herein, the “base pairing rule” refers to the rules for nucleotide base pairing established by Watson and Crick. In standard Watson-Crick geometry, large purine (A and G bases) pair with small pyrimidine (T and C) bases, where A pairs with T (or U) and G pairs with C, such that AT base pairs are substantially the same size as GC base pairs. This means that the rungs of the DNA ladder formed by AT (or AU) or GC base pairs have the same length throughout the entire helix. Under standard Watson-Crick rules for base pairing, AT and AU base pairs form two hydrogen bonds between their base moieties, and GC base pairs form three hydrogen bonds.

[0397] Nucleotides can pair in non-standard ways, such as in "wobbly" base pairings, where one or more hydrogen bonds are formed, but the geometry of the base pair does not conform to the standard Watson-Crick geometry. The most common wobble base pair is the GT (or U) base pair, where a single hydrogen bond is formed between G and T (or G and U). Non-Watson-Crick base pairings also include base pairs formed by inosine (I) with C, T, or A; purine base pairs AG (or I), AA, and GC; and the trans-Watson-Crick base pair AT, where the T ring is rotated 180° relative to a normal Watson-Crick pair. See, for example, Hyone-Myong Eun. Enzymology Primer for Recombinant DNA Technology, Academic Press, (1996)。

[0398] As used herein, “standard base pair” refers to a base pair having a standard Watson-Crick geometry and encompasses AT, AU, and GC base pairs. The term covers base pairings of modified analogues of these nucleotides where the modification (e.g., an attached dye or other motif) substantially does not alter the hydrogen bonding between the bases (e.g., the number, position, or acceptor / donor pair of hydrogen bonds). As used herein, “non-standard base pair” refers to a pairing between nucleotides (e.g., native nucleotides, nucleotide analogues) that does not fall under the category of standard base pairings having a standard Watson-Crick geometry.

[0399] As used herein, the terms “complementarity” or “complementarity” are used to refer to polynucleotides (e.g., sequences of two or more nucleotides, such as oligonucleotides or target nucleic acids) that are related by base pairing rules. For example, the sequence “5′-AGT-3′” is complementary to the sequence “3′-TCA-5′”. Complementarity can be “partial,” where only some nucleic acid bases match according to base pairing rules. Alternatively, there may be “complete” or “full” complementarity between nucleic acid bases. The degree of complementarity between nucleic acid chains has a significant impact on the efficiency and strength of hybridization between nucleic acid chains. This is particularly important in amplification reactions and in detection methods that rely on the association of two or more nucleic acid chains. Either term can also be used to refer to individual nucleotides, especially in the context of polynucleotides. For example, one may note the complementarity or lack thereof of a particular nucleotide within an oligonucleotide with a nucleotide within another nucleic acid sequence (e.g., a target sequence), in contrast to or compared to the complementarity between the rest of the oligonucleotide and the nucleic acid sequence.

[0400] As used herein, a complementary sequence to a nucleic acid sequence refers to an oligonucleotide that is in "antiparallel association" when aligned with the nucleic acid sequence such that the 5' end of one sequence pairs with the 3' end of another. As discussed above, nucleotide analogs may be included in the nucleic acids of this technique, and include, for example, inosine, 7-denitroguanine, iso-C, and iso-G. Complementarity need not be perfect; stable duplexes may contain mismatched base pairs or unmatched bases. Those skilled in the art of nucleic acid technology can empirically determine duplex stability by taking into account several variables, including, for example, the length of the oligonucleotide, its base composition and sequence, ionic strength, and the incidence of mismatched base pairs.

[0401] As used herein, the term “label” refers to any part that is detectable or can cause a detectable reaction (e.g., a chemical substance). In some preferred embodiments, the detection of a label provides quantifiable information. A label can be any known detectable part, such as a radiolabel (e.g., a radionuclide), a ligand (e.g., biotin or avidin), a chromophore (e.g., a dye or particle that imparts a detectable color), a hapten (e.g., digoxigenin), a mass label, latex beads, metal particles, paramagnetic labels, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent labels), or a fluorescent compound. A label may also include phosphor particles for detection. In a particularly preferred embodiment, the phosphor particles are upconversion phosphor particles (see, for example, Ostermayer, FW Preparation and properties of infrared-to-visible conversion phosphors. Metall. Trans. 752, 747–755

[1971] ). In some embodiments, rare-earth-doped ceramic particles are used as phosphor particles. Phosphor particles can be detected by any suitable method, including but not limited to upconversion phosphor technology (UPT), in which upconversion phosphors convert low-energy infrared (IR) radiation into high-energy visible light. While the invention is not limited to any particular mechanism, in some embodiments, UPT upconverts infrared light into visible light via multiphoton absorption and subsequent dopant-dependent phosphorescence emission. See, for example, U.S. Patent No. 6,399,397, June 4, 2002, to Zarling et al.; van De Rijke et al., Nature Biotechnol. 19(3):273-6

[2001] ; Corstjens et al., IEE Proc. Nanobiotechnol. 152(2):64

[2005] , each of which is incorporated herein by reference in its entirety.

[0402] As used herein, the term “different” in relation to a signal (e.g., a signal from a different label) means a signal that can be distinguished from another, for example by means of spectral properties (such as fluorescence emission wavelength, color, absorbance, mass, size, fluorescence polarization properties, charge, etc.) or by means of its ability to interact with another part (such as a chemical reagent, enzyme, antibody, etc.).

[0403] Labeling can be directly or indirectly linked to oligonucleotides or other biomolecules. Direct labeling can be achieved through bonds or interactions that link the label to the oligonucleotide, including covalent bonds or non-covalent interactions (such as hydrogen bonding, hydrophobic and ionic interactions), or by forming chelates or complexes. Indirect labeling can be achieved by using bridging portions or "connectors," such as antibodies or other oligonucleotides that are directly or indirectly labeled.

[0404] The marker can be used alone or in combination with portions of the emission spectrum of the marker (e.g., a luminescent marker) that can suppress (e.g., quench), excite, or transfer (e.g., shift) the marker (e.g., a luminescent marker).

[0405] "Polymerase" is an enzyme commonly used to ligate 3′-OH 5′-triphosphate nucleotides, oligomers, and their analogues. Polymerases include, but are not limited to, template-dependent DNA-dependent DNA polymerases, DNA-dependent RNA polymerases, RNA-dependent DNA polymerases, and RNA-dependent RNA polymerases. Polymerases include, but are not limited to, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase 1, Klenow fragment, and thermophilic aquatic bacteria (…). Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, Vent DNA polymerase (New England Biolabs), Deep Vent DNA polymerase (New England Biolabs) Bst Large fragment of DNA polymerase, Stoeffel fragment, 9°N DNA polymerase Pfu DNA polymerase, Tfl DNA polymerase, RepliPHI Phi29 polymerase, Tli DNA polymerase, eukaryotic DNA polymerase β, telomerase, Therminator polymerase (New England Biolabs), KOD HiFi DNA polymerase (Novagen), KOD1 DNA polymerase, Q-β replicase, terminal transferase, AMV reverse transcriptase, M-MLV reverse transcriptase, Phi6 Reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered through bioprospecting, and polymerases referenced in US 2007 / 0048748, US Patent Nos. 6,329,178, 6,602,695, and 6,395,524 (incorporated by reference). These polymerases include wild-type, mutant isotypes, and genetically engineered variants.

[0406] "DNA polymerase" is an enzyme that produces DNA from deoxyribonucleotide monomers (dNTPs). As used herein, "bacterial DNA polymerase" refers to Pol A-type DNA polymerases (repair polymerases) from eubacteria, including but not limited to those from *Escherichia coli*. E. coli DNA polymerase I from *Thermophyton floccosum* (a type of fungus) Thermus aquaticus Taq DNA polymerase and from Thermomyces genus ( Thermus Other members of the family and DNA Pol I enzymes of other eubacterial species, etc.

[0407] As used herein, a nucleic acid "ligase" is an enzyme capable of catalyzing the formation of a covalent bond between the 5' end and the 3' end of a nucleic acid strand. In some embodiments, the ligase catalyzes the formation of a phosphodiester bond between a 3' hydroxyl group and a 5'-phosphoryl group, for example, at a cleavage site in a duplex nucleic acid strand. Ligases can catalyze bond formation on other substrates. For example, the *E. coli* RtcB ligase ligates single-stranded RNA or DNA with a 3'-phosphate or a 2',3'-cyclic phosphate to the 5'-OH group of the single-stranded RNA. Exemplary ligases include, for example, T4 DNA ligase, T7 DNA ligase, T4 RNA ligase, Taq DNA ligase, Electroligase® T4 DNA ligase (NEB); HiFi Taq DNA ligase (NEB), Hi-T4™ DNA ligase (NEB), Salt-T4® DNA ligase (NEB), SplintR® ligase (also known as PBCV-1 DNA ligase or Chlorella virus DNA ligase; NEB), Escherichia coli RtcB ligase; CIRCLIGASE ss DNA ligase, CIRCLIGASE II ss DNA ligase, and AMPLIGASE thermostable ligase.

[0408] As used herein, the term "target" in relation to a nucleic acid strand or a site on a nucleic acid strand refers to a type of nucleic acid or a nucleic acid sequence or structure that is attempted to be separated from other nucleic acids, for example, through probe binding, amplification, separation, capture, etc. For example, when used in relation to polymerase chain reaction, "target" refers to a region of nucleic acid bound by primers used in the polymerase chain reaction, while in assays where the target DNA is not amplified, such as in some embodiments of invasive cleavage assays, the target includes a site where a probe and an invasive oligonucleotide (e.g., INVADER oligonucleotide) bind to form an invasive cleavage structure, thereby allowing the detection of the target nucleic acid. "Segment" is defined as a region of nucleic acid within a target sequence. As used in relation to double-stranded nucleic acids, the term "target" is not limited to a specific strand (e.g., the coding strand) of a double-stranded target, but can be used to refer to one or both strands of, for example, a double-stranded gene or reference DNA.

[0409] Therefore, as used herein, for example when used to describe nucleic acids (such as DNA or RNA), "non-target" refers to nucleic acids that may be present in a reaction but are not detected or characterized by the reaction. In some embodiments, non-target nucleic acids may refer to nucleic acids present in a sample that do not contain, for example, a target sequence, while in other embodiments, non-target may refer to exogenous nucleic acids, i.e., nucleic acids not derived from samples containing or suspected of containing target nucleic acids, and which are added to the reaction, for example, to normalize the activity of an enzyme (e.g., polymerase), thereby reducing the variability of enzyme performance in the reaction.

[0410] As used herein, the terms “cell-free” and “circulating cell-free” for nucleic acids derived from blood are used interchangeably and refer to nucleic acids found in the blood but not in cells within the blood, such as DNA and RNA species. As used herein, the term “nucleic acid extracted from blood” refers to the nature and location of the nucleic acids prior to sample collection from the subject and prior to extraction from the blood sample.

[0411] As used herein, the term "marker" refers to a substance (e.g., nucleic acid or nucleic acid region or protein) that can be used, for example, to distinguish abnormal cells (e.g., diseased or cancerous cells) from normal cells (non-diseased or non-cancer cells) based on the presence, absence, or condition (e.g., methylation status) of the marker substance. As used herein, "normal" methylation of a marker refers to the degree of methylation typically seen in normal cells, such as in non-diseased or non-cancer cells.

[0412] As used herein, the term "amplification reagents" refers to those reagents (deoxyribonucleoside triphosphates, buffers, etc.) required for amplification, in addition to primers, nucleic acid templates, and amplification enzymes. Typically, amplification reagents are placed and contained in a reaction vessel along with other reaction components.

[0413] "Reaction mixture" is a mixture of reagents (e.g., oligonucleotides, target nucleic acids, enzymes, etc.) in a combination and / or site where a reaction can occur, such as in a single reaction vessel, at a location in a fluid device, at a location on a surface, etc.

[0414] As used herein, a "multiplex" reaction is a reaction that operates on multiple targets (2, 3, 4, 5, 6, 7, 8...20, 30, 100, 1000, 100,000, etc.) in a single reaction mixture (e.g., RCA, RCT, ligation, PCR, PCR-valve assay). A multiplex reaction differs from a reaction in which each reaction mixture operates on one target analyte. As used herein, the term "highly multiplexed" refers to a reaction that operates on at least 6, preferably at least 10, more preferably at least 20 or more different targets (e.g., different genes or gene regions) in a single reaction mixture.

[0415] As used herein, the term "nested" in relation to target nucleic acid analysis using consecutive rounds of specific PCR refers to a second (or subsequent) round of amplification of the target nucleic acid using a primer pair, wherein one or both primers in the second primer pair anneal to a site in the target sequence within the region defined by the initial primer pair, such that the second primer pair is considered "nested" within the first primer pair. When only one primer in the second primer pair anneales to a site within the region defined by the initial primer pair, the second amplification reaction is referred to as "semi-nested." The terms "nested" and "semi-nested" can be used to refer to the second (or subsequent) primer pair (e.g., "nested primers") and to an amplification reaction performed using nested primers (e.g., "nested PCR-petal assay").

[0416] As used herein, when referring to the use of nucleic acid detection or analysis, the term "control" refers to a nucleic acid with known characteristics (e.g., known sequence, known copy number per cell) used for comparison with an experimental target (e.g., nucleic acid at an unknown concentration). The control can be endogenous, preferably a constant gene, against which the test or target nucleic acids in the assay can be normalized. This normalization controls for inter-sample variations that may occur in areas such as sample handling, assay efficiency, etc., and allows for accurate inter-sample data comparisons. Controls can also be external. For example, in quantitative assays (such as qPCR, Quarts, etc.), a "calibrator" or "calibration control" is a nucleic acid with a known sequence, such as a portion of the experimental target nucleic acid, and whose concentration or concentration series is known (e.g., a series of dilution control targets used to generate calibration curves in quantitative PCR). Typically, calibration controls are analyzed using the same reagents and reaction conditions as the experimental DNA. In some embodiments, the measurement of the calibrator is performed simultaneously with the experimental assay, for example, in the same thermal cycler. In a preferred embodiment, a single plasmid may contain multiple calibrators, making it easy to provide different calibrator sequences in equimolar amounts. In a particularly preferred embodiment, the plasmid calibrators are digested, for example with one or more restriction enzymes, to release the calibrator portions from the plasmid vector. See, for example, WO 2015 / 066695, which is incorporated herein by reference.

[0417] As used herein, the term "fish DNA" refers to a large quantity (e.g., genomic) of DNA isolated from fish, such as that described in U.S. Patent No. 9,212,392. Large quantities of purified fish DNA are commercially available, for example, in the form of cod and / or herring sperm DNA (Roche Applied Science, Mannheim, Germany) or salmon DNA (USB / Affymetrix). "Fish DNA" is distinct from any specific gene from fish that is in an isolated form, such as that which has been synthesized alone or isolated from other DNA in the fish genome.

[0418] As used herein, the “sensitivity” of a given biomarker (or a group of biomarkers used together) refers to the percentage of samples that report DNA methylation values ​​above a threshold distinguishing tumor samples from non-tumor samples. In some embodiments, a positive result is defined as reporting histologically confirmed tumor formation with DNA methylation values ​​above a threshold (e.g., a disease-related range), and a false negative result is defined as reporting histologically confirmed tumor formation with DNA methylation values ​​below a threshold (e.g., a non-disease-related range). Thus, a sensitivity value reflects the probability that a DNA methylation measurement of a given biomarker obtained from a known lesion sample falls within the disease-related measurement range. As defined herein, the clinical relevance of a calculated sensitivity value represents an estimate of the probability that a given biomarker can be detected in a subject with a clinical condition.

[0419] As used herein, the “specificity” of a given biomarker (or a group of biomarkers used together) refers to the percentage of non-tumor samples that report DNA methylation values ​​below a threshold distinguishing tumor samples from non-tumor samples. In some embodiments, a negative result is defined as a histologically confirmed non-tumor sample reporting DNA methylation values ​​below a threshold (e.g., within a disease-free range), and a false positive result is defined as a histologically confirmed non-tumor sample reporting DNA methylation values ​​above a threshold (e.g., within a disease-related range). Thus, a specificity value reflects the probability that a DNA methylation measurement of a given biomarker obtained from a known non-tumor sample falls within a non-disease-related measurement range. As defined herein, the clinical relevance of a calculated specificity value represents an estimate of the probability that a clinical condition is not present when the given biomarker is applied to a patient without the clinical condition.

[0420] As used herein, “selected nucleotide” refers to one of the four typically present nucleotides in a nucleic acid molecule (C, G, T, and A for DNA, and C, G, U, and A for RNA), and may include methylated derivatives of the typically present nucleotide (e.g., when C is a selected nucleotide, both methylated and unmethylated C are included in the meaning of selected nucleotide), while methylated selected nucleotide specifically refers to a normally methylated nucleotide, and unmethylated selected nucleotide specifically refers to a nucleotide normally present in an unmethylated form.

[0421] The term "methylation-specific restriction enzyme" refers to a restriction enzyme that selectively digests nucleic acids based on the methylation state of the nucleic acid recognition site. In the case of restriction enzymes that specifically cleave unmethylated or hemimethylated recognition sites (methylation-sensitive enzymes), cleavage will not occur (or the cleavage efficiency will be significantly reduced) if the recognition site is methylated on one or both strands. In the case of restriction enzymes that specifically cleave only when the recognition site is methylated (methylation-dependent enzymes), cleavage will not occur (or the cleavage efficiency will be significantly reduced) if the recognition site is unmethylated. Preferably, a methylation-specific restriction enzyme has a recognition sequence containing a CG dinucleotide (e.g., a recognition sequence such as CGCG or CCCGGG). For some embodiments, it is further preferred to be a restriction enzyme that will not cleave if the cytosine in the dinucleotide is methylated at carbon C5.

[0422] The terms “selective binding” and “specific binding” (or selective or specific binding, hybridization, annealing, etc.) used in relation to interactions between oligonucleotides or other nucleic acids are used interchangeably herein and refer to hybridization or base pairing with sufficient sequence selectivity such that, under conditions of selective or specific binding, the oligonucleotide or nucleic acid will preferentially hybridize with a specific nucleic acid (e.g., a target nucleic acid with a specific nucleotide sequence) and will substantially not bind with non-target nucleic acids (e.g., nucleic acids with a nucleotide sequence that is slightly or completely different from that of the target nucleic acid).

[0423] The term "probe" or "oligonucleotide probe" refers to an oligonucleotide (e.g., a nucleotide sequence) capable of hybridizing with another oligonucleotide of interest, whether naturally occurring (e.g., in purified restriction digests) or produced through synthesis, recombination, or PCR amplification. Probes can be single-stranded or double-stranded. Probes can be used to detect, identify, and isolate specific gene sequences (e.g., "capture probes"). It is envisioned that in some embodiments, any probe used in this invention can be labeled with any "reporter molecule" to make it detectable in any detection system, including but not limited to enzyme-based (e.g., ELISA and enzyme-based histochemical assays), fluorescent, radioactive, and luminescent systems. This invention is not intended to be limited to any particular detection system or label. Probe oligonucleotides include, for example, "valve oligonucleotides," which, when used with respect to valve assays, refers to oligonucleotides that interact with target nucleic acids in the presence of invasive oligonucleotides to form cleavage structures, as well as padlock probes that interact with nucleic acid chains to form invasive cleavage structures. In some embodiments, valve oligonucleotides comprise a single-stranded 5′ valve portion, typically a 5′ portion, forming several nucleotides within the invasive cleavage structure. As used herein, the terms “probe oligonucleotide” and “lobular oligonucleotide” are used interchangeably in relation to invasive cleavage structures, and refer to oligonucleotides that form downstream duplexes and are cleaved in invasive cleavage structures.

[0424] The term "invasive oligonucleotide" refers to an oligonucleotide that hybridizes with a target nucleic acid at a location near the hybridization region between the probe and the target nucleic acid, wherein the 3' end of the invasive oligonucleotide contains a portion (e.g., a chemical portion, or one or more nucleotides) overlapping the hybridization region between the probe and the target. The 3' terminal nucleotide of the invasive oligonucleotide may be complementary to a corresponding nucleotide in the target, but is not required to be. In some embodiments, the invasive oligonucleotide contains a sequence at its 3' end that is substantially identical to the sequence located at the 5' end of the portion of the probe oligonucleotide annealed to the target strand. In some embodiments, such as in PCR-flap assays, primers used for amplification may also be used as invasive oligonucleotides carrying the probe.

[0425] As used herein, the term "kit" refers to any delivery system for delivering materials. In the context of nucleic acid purification systems and reaction assays, such delivery systems include systems that allow the storage, transport, or delivery of reagents and devices (e.g., dissociative salts, particles, buffers, denaturants, oligonucleotides, filters, etc. in appropriate containers) and / or supporting materials (e.g., sample processing or storage containers, written instructions for performing procedures, etc.) from one location to another. For example, a kit may contain one or more shells (e.g., boxes) containing the relevant reaction reagents and / or supporting materials. As used herein, the term "aliphatic kit" refers to a delivery system comprising two or more separate containers, each containing a sub-component of all kit components. The containers may be delivered together or separately to the intended recipient. For example, a first container may contain materials and buffers for sample collection, while a second container contains capture oligonucleotides and denaturants. The term "alternate kit" is intended to cover, but is not limited to, kits containing analyte-specific reagents (ASRs) regulated under Section 520(e) of the Federal Food, Drug, and Cosmetic Act. In fact, any delivery system containing two or more separate containers, provided that each container contains a sub-part of all kit components, is included in the term "alternate kit." In contrast, "combination kit" refers to a delivery system that contains all components of the reaction assay in a single container (e.g., a single box containing each of the desired components). The term "kit" includes both aliquot kits and combination kits.

[0426] As used herein, the term "system" refers to a collection of articles of writing used together for a particular purpose. In some embodiments, the articles of writing include instructions for use, which are provided as information on, for example, on the articles themselves, on paper, on a network (e.g., on a website or URL), or on recordable media (e.g., floppy disks, CDs, DVDs, flash drives, etc.). In some embodiments, the instructions for use direct the user to an online location, such as a website for viewing, listening to, and / or downloading the instructions for use. In some embodiments, the instructions for use or other information are provided as an application ("app"), such as an application for a computer or for a mobile device (such as a smartphone). Attached Figure Description

[0427] Figure 1A schematic diagram of an exemplary invasive cleavage structure formed using a petal-shaped oligonucleotide as described herein is provided. In this embodiment, the petal-shaped oligonucleotide includes a target-specific region and a 5′ petal sequence, and a separate oligonucleotide (e.g., an invasive oligonucleotide) is added to form the invasive cleavage structure. In this embodiment, the invasive oligonucleotide hybridizes with a nucleic acid strand (e.g., target DNA) to form an upstream duplex, wherein a single nucleotide is unpaired at the 3′ end of the invasive oligonucleotide. A downstream oligonucleotide (e.g., the target-specific region of the petal-shaped oligonucleotide) hybridizes with a nucleic acid strand (e.g., target DNA) to form a downstream duplex. In the illustrated embodiment, the downstream oligonucleotide includes an unpaired 5′ petal (e.g., a petal sequence). As shown, the upstream and downstream duplexes are adjacent to each other such that there is no gap in the base pairing of the target strand between the upstream and downstream duplexes. Figure 1 The structure shown is a substrate for cleavage by the FEN-1 endonuclease. As illustrated, cleavage typically occurs after the first paired base of the oligonucleotide in the downstream double strand. Therefore, cleavage at the cleavage site results in the cutting of the flap sequence from the flap oligonucleotide, thereby generating the cleaved flap sequence. In this embodiment, the cleaved flap sequence contains one additional base compared to the uncleaved flap sequence. For example, in some embodiments, the flap sequence contains 14 bases, and the cleaved flap sequence contains 15 bases.

[0428] Figure 2 A schematic diagram of another exemplary invasive structure formed using a padlock probe comprising a petal portion and an invasive portion as described herein is shown. In this embodiment, the petal portion acts as a petal-shaped oligonucleotide and includes a target-specific region that hybridizes to a first portion of the target nucleic acid and a second target-specific region that hybridizes to a second portion of the target nucleic acid. For example, Figure 2 The exemplary petal-shaped oligonucleotide shown hybridizes with a nucleic acid strand (e.g., a target nucleic acid). The petal-shaped oligonucleotide is a non-linkable padlock probe comprising a target-specific 5′ region (e.g., a first target-specific region) that forms a downstream duplex with the target strand and a target-specific 3′ end (e.g., a second target-specific region) that forms an adjacent upstream duplex with the target strand. In this embodiment, the first target-specific region is adjacent to a petal sequence (e.g., a 5′ petal sequence or a 5′ arm), as shown. The petal-shaped oligonucleotide also comprises a linker backbone sequence connecting the two target-specific domains. The backbone sequence is located in… Figure 2 The structure is shown as a gray line. Cleavage with the FEN endonuclease removes one nucleotide from the 5′ lobe and the downstream double strand (e.g., one nucleotide from the first target-specific region). Following cleavage, the cleavage lobe sequence can be detected using any of the methods discussed herein, and the remaining portion of the cleavage probe forms a ligation site that can be joined to form a loop structure.

[0429] Figure 3This is a schematic diagram illustrating how the methods described herein are used to determine the presence of a target nucleic acid in a sample based on whether a cleavage of the petal sequence (e.g., the 5′ arm) of the petal oligonucleotide disclosed herein occurs. In this embodiment, the invading oligonucleotide is shown to form an invasive cleavage structure with the target sequence and the petal oligonucleotide disclosed herein, but it should be understood that the petal oligonucleotide itself (e.g., Figure 2 (As described in the text) may include a first target-specific region and a second target-specific region, so that no invasive oligonucleotide is required to form an invasive cleavage structure. For example... Figure 3 As shown, successful cleavage results in the formation of a cleavage lobe sequence, which typically contains an extra base (e.g., a terminal base containing the target-specific region) compared to the uncleaved lobe sequence. Successful cleavage produces a cleavage lobe sequence that can be used as a primer, for example, to replicate a circular template (bottom left). In contrast, uncleavage produces a lobe structure that remains attached to the target-specific region, thus preventing the replication of the circular template.

[0430] Figure 4 This is a schematic diagram illustrating how the petal-shaped oligonucleotides described herein and their methods of use (e.g., in petal assays) enable multiplex detection of multiple targets in a sample. Different colors (e.g., gray, black, white) represent different sequences available for use with the petal-shaped oligonucleotides disclosed herein (e.g., different petal sequences, different sequences of target-specific regions), which are equally successful in generating cleavage petal sequences and, for example, are used as primers for extension onto circular templates (e.g.,...). Figure 4 (as shown) or used to form closed loop structures (e.g., as shown) Figures 5A-6C The illustrated implementation scheme), or used in conjunction with oligonucleotide vector pairs to form a linear vector structure (e.g. Figure 14 (As shown in one embodiment), it can then be amplified and / or detected and / or sequenced.

[0431] Figure 5A This is a schematic diagram illustrating an exemplary implementation involving a suitable probe that generates a template containing a cleavage lobe sequence (if present in the sample), wherein the template is suitable for rolling circle amplification. Figure 5A As shown, in some embodiments, suitable probes include padlock probes. In the embodiment shown in the figure, the invasive cleavage structure is formed using an invasive oligonucleotide and a single valve-shaped oligonucleotide containing a valve sequence (“arm 1”). However, it should be understood that the invasive cleavage structure can also use, as well as... Figure 2The illustrated form of a petal oligonucleotide comprising two target-specific regions, wherein the petal portion and the invasive portion of the petal oligonucleotide each bind to a distinct but adjacent region on the target nucleic acid to form an invasive cleavage structure. Successful cleavage at the target site releases the cleavage petal sequence, along with one nucleotide from the target-specific region of the petal oligonucleotide. In this exemplary embodiment using a padlock probe, the cleavage petal sequence hybridizes with a bridging oligonucleotide, which also hybridizes with both ends of the padlock probe, forming two connectable cleavages. In some embodiments, the bridging oligonucleotide includes an inner segment complementary to the cleavage petal sequence, and 3′ and 5′ ends complementary to the first and second target regions of the padlock probe. However, when the petal oligonucleotide is intact (e.g., when the petal is not cleaved from the petal oligonucleotide), the petal sequence of the oligonucleotide hybridizes with the bridging oligonucleotide to form only one connectable cleavage, and this petal cannot connect to the padlock probe to form a cyclized molecule.

[0432] Figure 5B Another exemplary embodiment using a dumbbell probe is shown. The dumbbell probe comprises two stem-loop structures separated by a single-stranded region (referred to as a “bridge sequence”) complementary to the cleavage lobe sequence. In some embodiments, a closed loop structure is not properly formed until both ends of the cleavage lobe sequence are successfully ligated to the probe. The cleavage lobe sequence (“arm 1” plus one nucleotide of the target-specific region of the lobe oligonucleotide) hybridizes with the bridge sequence and forms a ligation-capable cleavage. However, when the lobe oligonucleotide is intact (e.g., when the lobe sequence is not cleaved from the lobe oligonucleotide), the lobe sequence of the oligonucleotide hybridizes with the bridge sequence, but the lobe cannot ligate to form a circular dumbbell probe.

[0433] Figure 5CAnother exemplary embodiment using a carrier pair comprising two carrier oligonucleotides is shown. In the embodiment illustrated, the invasive cleavage structure is formed using an invasive oligonucleotide and a single valve-shaped oligonucleotide containing a valve sequence (“arm 1”). Successful cleavage at the target site releases the cleavage valve sequence, along with one nucleotide of the target-specific region of the valve-shaped oligonucleotide. In this exemplary embodiment using the carrier pair, the cleavage valve sequence hybridizes with a bridging oligonucleotide, which also hybridizes with each of the two oligonucleotides in the carrier pair (carrier 1 and carrier 2), forming two connectable cleavages. In some embodiments, the bridging oligonucleotide comprises an inner segment complementary to the cleavage valve sequence, and 3′ and 5′ ends complementary to a first target region in the first carrier oligonucleotide and a second target region in the second carrier oligonucleotide. Upon ligation, a linearly linked product is formed comprising the first carrier oligonucleotide, the cleavage valve, and the second carrier oligonucleotide. However, when the petal oligonucleotide is intact (e.g., when the petal is not cleaved from the petal oligonucleotide), the petal sequence of the oligonucleotide hybridizes with the bridging oligonucleotide to form only one connectable cleavage, and the petal can be connected to only one oligonucleotide of the vector pair (e.g., vector 1 in the illustrated embodiment).

[0434] Figure 6A This is a schematic diagram showing the connection of incisions formed by a sequence of cutting flaps, such as... Figure 5A and Figure 5B As shown, this results in the formation of a closed loop structure, which serves as a template for rolling circle amplification. In implementations using padlock probes, the ligation of the ligation leads to the appropriate formation of a closed loop structure, which can subsequently be detected. In implementations using dumbbell probes, the ligation of the ligation results in the formation of a closed loop structure in the form of a loop-stem-loop, which serves as a suitable template for, for example, PCR, RCA, RCT, sequencing, etc.

[0435] Figure 6B This is a schematic diagram illustrating an embodiment of intrastranded ligation at the end of the cleavage flap sequence (cleavage arm 1). In one embodiment (left), a splint oligonucleotide is used to align the 5′ and 3′ ends of the cleavage flap to form a nick that can be ligated by a DNA ligase (e.g., HiFi Ampligase, T4 DNA ligase, etc.). In another embodiment (right), a single-strand ligase (e.g., T4 RNA ligase, CircLigase™ ssDNA ligase) is used to ligate the 5′ and 3′ ends of the cleavage flap to each other without the use of splint oligonucleotides. In both embodiments, intrastranded ligation at the end of the cleavage flap results in the formation of a closed circular molecule, which serves as a suitable template for subsequent detection (e.g., PCR, RCA, RCT, sequencing, etc.).

[0436] Figure 6CThis is a schematic diagram illustrating an implementation scheme where hybridization of the cleavage flap with the splint oligonucleotide does not produce a connectable cleavage but rather an overlapping cleavage structure. For example, cleavage with a flap endonuclease removes the flap, creating a connectable cleavage. The ligation of the ends of the cleavage flap results in the formation of a closed circular molecule, which serves as a suitable template for subsequent detection (e.g., PCR, RCA, RCT, sequencing, etc.).

[0437] Figure 7A This is a schematic diagram illustrating the pre-amplification steps performed before cutting assays. As shown, multiple targets can be amplified to generate libraries of different amplicones. The amplicones can then be cut and assayed, and subsequently detected, for example, by PCR, RCA, RCT, or sequencing.

[0438] Figure 7B This is a schematic diagram illustrating the pre-amplification implementation scheme, where one of the target-specific primers in the pre-amplification reaction contains a universal tag sequence that is the same for multiple different targets. Following the pre-amplification step, PCR-valve assays are performed using an original target-specific primer and probe, along with universal primers. Figure 7A Similar to the implementation scheme shown, multiple different targets can be pre-amplified together to generate libraries of different amplicones. The amplicones can be subjected to flap-cut assays and subsequent detection, such as by PCR, RCA, RCT, or sequencing.

[0439] Figure 7C This is a schematic diagram illustrating the pre-amplification implementation scheme, where one of the target-specific primers in the pre-amplification reaction contains a universal tag sequence that is the same for multiple different targets. Following the pre-amplification step, PCR-lobe assays are performed using nested target-specific primers and probes, as well as universal primers. Figure 7A Similar to the implementation scheme shown, multiple different targets can be pre-amplified together to generate libraries of different amplicones. The amplicones can be subjected to flap-cut assays and subsequent detection, such as by PCR, RCA, RCT, or sequencing.

[0440] Figure 8 This is a schematic diagram of an exemplary cyclizable molecule complexed with a valve-specific bridging oligonucleotide that hybridizes with a cleavage valve to form two connectable nicks. The cyclizable molecule contains an internal spacer region (iSp18) to terminate any polymerization around the cyclization probe. The hybridization arm (called the “Hyb arm”) is a complementary region between the bridging oligonucleotide and the cyclizable molecule. Ad1 and Ad2 are adaptor sequences used for cyclization-specific amplification. A unique molecular identifier (UMI) is used to quantify unique ligation events. A sample index is used for sample-specific barcoding to provide additional indexing capabilities, allowing for higher levels of multiplex analysis in a single sequencing run.

[0441] Figure 9A This is a schematic diagram illustrating an exemplary method described herein for generating and subsequently detecting target-specific cleavage flap sequences. Target-specific flap cleavage has single-base resolution and can be used, but is not limited to, detecting single nucleotide variants, single nucleotide polymorphisms, or methylation-specific transformation sequences.

[0442] Figure 9B This is a schematic diagram illustrating a second exemplary method described herein for generating and subsequently detecting target-specific cleavage lobe sequences.

[0443] Figure 10 This graph illustrates how CIP / PNK addition improves assay sensitivity after generating cleft sequences based on PCR-flap assays. This sensitivity is determined by reads of methylated DNA markers during sequencing. Similar methods for reducing residual polymerase activity after PCR-flap assays include, but are not limited to, co-incubation with proteases, ligation at low temperatures, or engineering bridging oligonucleotides to have nucleotide modifications that inhibit DNA polymerase activity.

[0444] Figure 11 This is a bar graph showing the reduction of non-specific assay signals after adding exonucleases following the ligation of the cleavage flap sequence into the circularized probe in a PCR-flap assay. Shaded bars indicate conditions without exonuclease addition. Hollow bars indicate conditions treated with exonucleases.

[0445] Figures 12A-12C Images are provided illustrating the performance of different DNA ligases for the cleavage flap assay produced in an LQAS PCR-flap assay performed without the use of a FRET box, as described in Example 2. Figure 12A It provides bioanalytical gel images of the final product after cleavage of petal oligonucleotides, ligation, and amplification and enrichment with cyclized probes.

[0446] Figure 12B A graph comparing the background reads generated in reactions using different DNA ligases in the ligase-free control ((-) ligase) and the template-free control (TE) is provided.

[0447] Figure 12C The results show differences in sequencing quality based on the DNA ligase used (generated using an Illumina MiSeq instrument).

[0448] Figure 13 It is a bar chart showing the correlation between the concentration of bridging oligonucleotides and background reads.

[0449] Figure 14A schematic diagram is provided showing a chimeric bridging oligonucleotide comprising a 3′ portion of 2′-O-methyl RNA and a 5′ portion of DNA. The bridging oligonucleotide is shown as hybridizing with the complementary 3′ end of the first vector (“vector 1”) of the vector pair to form a first nick with a cleavage flap (“cFLAP”). The use of RNA in this portion of the bridging oligonucleotide prevents the extension of the 3′ end of the first vector by Taq DNA polymerase remnants from the PCR-flap reaction. The 5′ portion of the second vector (“vector 2”) of the vector pair is complementary to the DNA portion of the chimeric bridging oligonucleotide and contains an activation tail with a non-ligable 5′ OH group. When the 5′ portion of vector 2 with the 5′ activation tail hybridizes with the bridging oligonucleotide at the 3′ end of the adjacent cleavage flap, an invasive cleavage structure is formed, which is cleaved by a flap endonuclease (e.g., FEN-1 endonuclease remnants from PCR-flap amplification), thereby releasing the activation tail and forming a second nick. Both the first and second nicks can be ligated using, for example, SPLINTR DNA ligase. In the illustrated embodiment, vector 1 and vector 2 are provided as independent oligonucleotides in a vector pair, and their ligation produces a linear ligation product. In some embodiments, a first vector sequence and a second vector sequence may be provided at both ends of a single cyclizable molecule, such that the ligation of the two nicks forms a cyclized molecule.

[0450] Figure 15 A schematic diagram is provided of the 3′ and 5′ portions of the vector pair and the chimeric bridging oligonucleotide that hybridizes with the cleavage flap (“cFlap”). As described above, the 5′ end of the flap vector (vector 2) contains a non-attached 5′ OH group and includes an activation tail that forms a cleavable structure with cFlap. The right figure shows different possible designs for the 5′ portion of the flap vector of vector 2, for use with bridging oligonucleotides having a universal hybridization arm or a base-specific hybridization arm. This is referred to as “…” The different vector 2 designs of the “activated universal arm” represent a mixture of four different vectors, each having the same universal hybridization arm and each having a different base (e.g., A, G, C, or T) in the activation tail. In the universal hybridization arm subplot, the bridging oligonucleotide contains a T nucleotide that pairs with the 3′ terminal A nucleotide base in the cleavage lobe. This terminal A more effectively promotes cleavage of the activation lobe, which has a base-paired A nucleotide at the 5′ cleavage site in the activation lobe (the “A” in the activation lobe in the top structure of this subplot). Other vector 2 activation lobes (e.g., having G, C, or T in the activation lobe) are also cleaved, but with reduced efficiency. While not limited to any specific mechanism of action, the reduced cleavage may be because the G, C, and T nucleotides at the 5′ cleavage site in the activation lobe do not form a standard base pair with the T nucleotide at the corresponding position in the bridging oligonucleotide.

[0451] Figure 15The base-specific hybridization arm diagram is illustrated in the following embodiment, wherein each of the vectors 2 is designed with a different base-specific hybridization arm, such that the activation tail (including the nucleotide at the 5′ cleavage site) of each vector 2 region specifically hybridizes with the corresponding sequence in the bridging oligonucleotide. This concept is... Figure 16 Further details will be provided later.

[0452] Figure 16 This is a schematic diagram depicting the cleavage when using a mixture of four different vector 2 designs (e.g., having A, G, C, or T in the activation tail) that use the same universal hybridization arm sequence (left figure), or a mixture of four different vector 2 designs (e.g., having A, G, C, or T in the activation tail), each with different base-specific hybridization arms. For the universal sequence design, as shown in the left figure, each universal vector 2 region will hybridize with the universal portion of the bridging oligonucleotide. As described above, the universal vector 2 region having an A in the activation tail that pairs with the T shown in the universal bridging oligonucleotide will be cleaved more efficiently than other G, C, or T forms of the universal vector 2 design. For the base-specific vector 2 sequence design, as shown in the right figure, each vector 2 portion specifically hybridizes with a bridging oligonucleotide having a matching complementary hybridization arm. As described above, the complementarity between the base-specific hybridization arms of the bridging oligonucleotide and the corresponding vector 2 oligonucleotide ensures that, in the illustrated embodiment, only the vector 2 oligonucleotide having an A in the activation tail will hybridize with the bridging oligonucleotide having a T at the corresponding position in the bridging oligonucleotide.

[0453] Figure 17 This is a bar chart showing the number of reads generated using either the vector 2 design with a universal hybridization arm or the vector 2 design with a base-specific hybridization arm. The base-specific vector 2 is designed to produce preferred invasive cutting structures for each of four different base-specific activation lobes, thereby improving overall cutting efficiency and signal generation capability, for example, in multiplex analysis of biomarkers.

[0454] Figure 18 This is a graph showing the components used in flap amplification reactions (e.g., LQAS, TELQAS). It shows the concentrations of invasive and non-invasive primers that lead to asymmetric amplification. (Decrease) B3GALT6 The amplification efficiency can be improved by reducing the concentration of primers (invasive oligonucleotides, non-invasive oligonucleotides) and / or probes (e.g., lobular oligonucleotides) in pre-amplification, TELQAS, or LQAS.

[0455] Figure 19 The comparison shows FLAP-Seq reads (y-axis) versus TELQAS chains (x-axis) from 40 clinical samples. Cancer-specific biomarkers are shown as solid circles, while... B3GALT6Presented as a hollow triangle. Cancer-specific methylated DNA markers (MDM) fall within the linear range of FLAP-Seq assays. Comparatively, B3GALT6 It has a much higher chain count in cfDNA and falls outside the linear range of FLAP-Seq.

[0456] Figure 20 The multiplex amplification curves (grey curves) of cancer-specific MDM without primer titration and the universal MDM reference DNA after primer titration are shown. B3GALT6 The multiplex amplification curves (black curves) are shown. Titration of universal MDM primers reduces amplification efficiency, resulting in a rightward shift of the amplification curve. Cancer-specific MDM amplification is unaffected by primer titration of universal MDM.

[0457] Figure 21 The comparison shows FLAP-Seq reads (y-axis) versus TELQAS chains (x-axis) from 40 clinical samples. Cancer-specific MDM biomarkers are shown as solid circles, while... B3GALT6 The reference DNA appears as a hollow triangle. Reduced primer concentrations are used for amplification. B3GALT6 Cancer-specific MDM and B3GALT6 The results fell within the linear range of the FLAP-Seq assay. Detailed Implementation

[0458] This document provides techniques relating to valve-like oligonucleotides, particularly methods for analyzing cleavage lobes generated by target-dependent cleavage of valve-like oligonucleotides via valve-like endonucleases. Embodiments of this technique provide methods and compositions for multiplexing cleavage lobes and for counting cleavage lobes, which indicate, for example, the presence of a specific target nucleic acid in a mixture of different target nucleic acids and non-target nucleic acids. In some embodiments, this technique provides methods for distinguishing and counting cleavage lobes associated with multiple different target nucleic acids, wherein the cleavage lobes are generated in a single cleavage reaction mixture. In a particular embodiment, counting the cleavage lobes includes, for example, sequencing the cleavage lobes using next-generation sequencing.

[0459] In this detailed description of the various embodiments, numerous specific details are set forth for illustrative purposes to provide a thorough understanding of the disclosed embodiments. However, those skilled in the art will understand that these different embodiments may be practiced with or without these specific details. In other instances, structures and apparatus are shown in block diagram form. Furthermore, those skilled in the art will readily appreciate that the particular order in which the methods are presented and performed is illustrative, and that such order is expected to vary while remaining within the spirit and scope of the various embodiments disclosed herein.

[0460] target nucleic acid

[0461] This technology can be used to determine any type of target nucleic acid, such as DNA, RNA, etc. In a preferred embodiment, the target nucleic acid comprises DNA, including but not limited to cDNA, amplified DNA, genomic DNA, circulating cell-free DNA, etc. In some embodiments, the DNA is treated with a methylation-specific reagent as defined above. For example, in some embodiments, the target DNA (e.g., DNA extracted from a sample) is treated with a bisulfite reagent, causing unmethylated cytosine to be converted to deoxyuridine. For example, in some embodiments, the target nucleic acid is a cancer-specific methylated DNA marker (MDM). In some embodiments, the treated target nucleic acid strand is replicated or amplified, for example by PCR, prior to hybridization with one or more oligonucleotides that form a cleavage structure with the target nucleic acid. For example, for target DNA treated with a bisulfite reagent, the U in the transformed target DNA sequence is replaced by a T in the replicated strand. Embodiments of this technology are equally applicable to such treated, replicated target DNA.

[0462] Similarly, sequence variations induced by other reagents (e.g., the TAPS technique described above) as well as sequence variations unrelated to methylation (e.g., single nucleotide polymorphisms (SNPs), mutations, insertions, and deletions) can be determined using similar design strategies, such as distinguishing them from reference nucleic acids.

[0463] Target nucleic acids can be prepared using any standard method used to purify or isolate nucleic acids from a nucleic acid source (e.g., from a sample). For example, kits for isolating nucleic acids from different types of samples (e.g., environmental samples, biological samples, medical samples, etc.) are available from commercial suppliers such as Qiagen, Promega Corp., and ThermoFisher Scientific.

[0464] Lobe-shaped oligonucleotides

[0465] Implementations of this technology include the use of a petal oligonucleotide having at least one target-specific region (also referred to herein as a target-specific sequence) and a petal sequence (preferably a 5′ petal sequence).

[0466] At least one target-specific region is sufficiently complementary to the target of interest (e.g., the target nucleic acid) to hybridize with the target strand under cleavage reaction conditions, while the lobe sequence is typically an arm (e.g., a 5′ arm), which is preferably substantially non-complementary to the target strand of interest, such that it generally does not hybridize with the target nucleic acid, for example, under cleavage reaction conditions. Preferably, the lobe sequence is long enough that, once cleaved from the lobe oligonucleotide, the cleavage lobe can specifically hybridize with the complementary nucleic acid strand of, for example, a ligation template (e.g., a bridging oligonucleotide or a FRET cassette), under conditions for detecting the cleavage lobe. For example, the cleavage lobe segment preferably hybridizes with suitable specificity under ligation conditions, selectively annealing to a matching lobe-specific ligation template, such that it can be ligated to an adjacent oligonucleotide hybridizing with the same ligation template. In some embodiments, the cleavage lobe segment hybridizes with suitable specificity under ligation conditions, selectively annealing to a matching lobe-specific bridging sequence to form a ligable nick with the 5′ and 3′ ends of the padlock probe, for example as... Figure 5A and Figure 5B As shown, or with Figure 5C and Figure 14 The 3′ end of vector 1 and the 5′ end of the oligonucleotide of vector 2, as shown in the vector pair, form a connectable nick. The length of the 5′ lobe sequence is generally preferably less than about 200 nucleotides, more preferably less than about 175 nucleotides. In some embodiments, the lobe sequence is 1 to about 150 nucleotides, preferably 1 to about 100 nucleotides, and more preferably about 5 to 75 nucleotides. In a preferred embodiment, the length of the 5′ lobe portion is at least 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, or 75 nucleotides. In some embodiments, the length of the 5′ lobe portion is preferably 6 to 45 nucleotides, more preferably 7 to 40 nucleotides, more preferably 8 to 35 nucleotides, more preferably 8 to 30 nucleotides, more preferably 8 to 25 nucleotides, more preferably 9 to 25 nucleotides, more preferably 10 to 20 nucleotides, and more preferably 10 to 15 nucleotides.

[0467] The target-specific region of the valve oligonucleotide is not limited to any particular length or sequence. Preferably, the target-specific region of the valve oligonucleotide is selected to selectively hybridize with a specific target strand under the conditions to be performed. For example, in some embodiments, the sequence is selected to form an invasive cleavage structure with the target nucleic acid and the upstream invasive oligonucleotide sequence under conditions useful or optimal for cleavage with a thermostable FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, for example, at a temperature between about 25°C and 75°C, between about 40°C and about 75°C, or at any temperature preferably between about 45°C and 70°C, preferably between about 50°C and 70°C, preferably between about 55°C and 70°C. In a preferred embodiment, the valve oligonucleotide is in Mg ++ Buffer solution, preferably high Mg ++ Buffer solution (e.g., preferably higher than 4 mM Mg) ++ More preferably, at least about 5, 6, 7, 8, 9, 10 or 11 mM Mg ++ Hybridization with the target nucleic acid occurs in the presence of [specific region]. In some preferred embodiments, the target-specific region forms a downstream double strand with the target strand, the length of which is at least 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 or 35 base pairs, preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 bp, preferably 7 to 15 bp, preferably 8 to 14, preferably 9 to 13, preferably 10 to 12 bp.

[0468] Invasive oligonucleotides and sequences

[0469] Implementations of this technology include using an invasive oligonucleotide or invasive sequence to form a FEN-1 cleavable structure with a petal oligonucleotide. In some embodiments of this technology, the invasive sequence is provided on a molecule different from the petal oligonucleotide, and in some oligonucleotides, a single molecule (e.g., a padlock probe) comprises a 3′ portion acting as the invasive oligonucleotide and a 5′ portion acting as the petal oligonucleotide, wherein the 3′ and 5′ portions are linked, preferably covalently, via the backbone portion of the molecule. The length and sequence of the 5′ and 3′ portions can be selected for selective hybridization with a specific target strand, thereby forming an invasive cleavable structure under the conditions of the assay to be performed. For example, in some embodiments, the sequence is selected to form an invasive cleavable structure with the target nucleic acid under conditions useful or optimal for cleavage with a thermostable FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, such as the temperatures and conditions discussed above with respect to the petal oligonucleotide. While the upstream duplex is not limited to a specific length, in some preferred embodiments, the invasive oligonucleotide or invasive sequence on the 3′ portion of the padlock probe forms an upstream duplex of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 bp in length with the target (see [link to documentation]). Figure 1 See, for example, Kaiser MW et al. (1999) J. Biol. Chem., 274:21387.

[0470] Comparison

[0471] In some embodiments, the assay utilizes a control, also referred to herein as a "reference" (e.g., "reference nucleic acid"). In some embodiments, the assay utilizes a control / reference, such as a DNA marker of generalized methylation. B3GALT6 In some implementations, the target nucleic acid is a cancer-specific methylated DNA biomarker, and the assay uses a control of universally methylated DNA biomarkers. B3GALT6 In some embodiments, the reaction mixture comprises oligonucleotides specific to the target nucleic acid (e.g., lobular oligonucleotides, invasive oligonucleotides, and optionally non-invasive oligonucleotides) and oligonucleotides specific to the target nucleic acid. B3GALT6 Oligonucleotides with specificity (e.g., lobular oligonucleotides, invasive oligonucleotides, and optionally non-invasive oligonucleotides). In some embodiments, the concentration of an equivalent oligonucleotide relative to the target nucleic acid (e.g., cancer-specific MDM) is... BGALT6 The concentration of one or more specific oligonucleotides is diluted. For example, in some embodiments, the concentration of the valve oligonucleotide targeting the target nucleic acid is diluted relative to the concentration of the valve oligonucleotide targeting the target nucleic acid in the reaction mixture. B3GALT6The concentration of the lobular oligonucleotides is diluted. As another example, in some embodiments, the concentration of the invasive oligonucleotide targeting the target nucleic acid is reduced relative to the concentration of the invasive oligonucleotide targeting the target nucleic acid in the reaction mixture. B3GALT6 The concentration of the invasive oligonucleotide is diluted. As yet another example, in some embodiments, the concentration of the non-invasive oligonucleotide targeting the target nucleic acid is diluted relative to the concentration of the non-invasive oligonucleotide targeting the target nucleic acid in the reaction mixture. B3GALT6 The concentration of the non-invasive oligonucleotides is diluted. In some implementations, the concentration of the oligonucleotides is... B3GALT6 Multiple oligonucleotides with specificity are diluted. For example, in some embodiments, the concentration of oligonucleotides targeting the target nucleic acid is reduced compared to the concentration of an equivalent oligonucleotide targeting the target nucleic acid. B3GALT6 One or more oligonucleotides (e.g., lobular oligonucleotides, invasive oligonucleotides, non-invasive oligonucleotides) are diluted by at least 50%. In some embodiments, the concentration of the oligonucleotide relative to the equivalent concentration of the target nucleic acid is... B3GALT6 The specific oligonucleotides were diluted to concentrations of 0.2, 0.1, or 0.5.

[0472] In some implementations, oligonucleotides (e.g., lobular oligonucleotides, invasive oligonucleotides, non-invasive oligonucleotides) are compared with a reference nucleic acid (e.g., B3GALT6 It is specific, binding to regions in the target nucleic acid that have one or more mismatched base pairs (e.g., mismatched base pairs between an oligonucleotide and its target region), thereby weakening (e.g., reducing) the amplification efficiency of the reference nucleic acid.

[0473] Lobe-like cutting reaction

[0474] In some aspects, this document provides compositions comprising a plurality of the valve-like oligonucleotides described herein. In some embodiments, the compositions described herein can be used for valve-like assays in which the target strand is not replicated, and in some embodiments, the compositions described herein can be used for amplified valve-like assays in which the target strand is replicated, for example, with a polymerase. For example, in some embodiments, when a target nucleic acid and a suitable valve-like endonuclease are present in a sample, a valve sequence is cleaved from the valve-like oligonucleotide, and conditions are suitable for valve-like endonuclease to perform valve-like cleavage, thereby generating a cleaved valve sequence. The cleaved valve sequence can then be amplified and / or detected, thereby serving as a marker of the target nucleic acid in the sample. Thus, in some aspects, this document provides a method for multiplex detection of multiple target nucleic acids in a sample, the method comprising contacting the sample with a composition described herein. In some embodiments, the target nucleic acid is RNA, and cleavage to release the valve comprises using a structure-specific 5′ nuclease suitable for the RNA target strand, as described, for example, in WO 2003 / 073067 and US 7,851,150. In other embodiments, the target nucleic acid strand is DNA, and cleavage to release the valve includes the use of a valve-shaped endonuclease, preferably FEN-1 endonuclease.

[0475] In some embodiments, this document provides compositions comprising multiple lobe-like oligonucleotides. In some embodiments, each lobe-like oligonucleotide comprises a lobe sequence and at least one target-specific region. In some embodiments, at least one target-specific region comprises a nucleotide sequence that binds to a target nucleic acid in a sample. In some embodiments, each lobe-like oligonucleotide binds to a different target nucleic acid if present in the sample. For example, in some embodiments, each lobe comprises a unique target-specific region such that each target-specific region binds to a different target nucleic acid in the sample. Therefore, the compositions described herein can be used for multiplex detection of multiple targets in a given sample. In some embodiments, each lobe-like oligonucleotide comprises a single target-specific region. In some embodiments, each lobe-like oligonucleotide comprises more than one target-specific region. For example, in some embodiments, each lobe-like oligonucleotide comprises a first target-specific region and a second target-specific region, wherein the lobe portion and the invasive portion of the lobe-like oligonucleotide each bind to different but adjacent regions on the target nucleic acid to form an invasive cleavage structure, such as... Figure 2 As shown. This type of lobular oligonucleotide is particularly advantageous for forming invasive cleavage structures without the addition of separate invasive oligonucleotides.

[0476] One aspect of this technology is that, compared to structures such as notched structures (with a single-stranded notch between the upstream and downstream duplexes), Y-shaped structures (with 5′ lobes but no overlap between the upstream and downstream duplexes), and pseudo-Y-shaped structures (with 5′ lobes and a non-duplex template strand located upstream of the downstream duplex), it offers advantages in enzyme pairing, such as... Figure 1 The invasive cleavage structure shown exhibits maximum specificity under conditions using the FEN-1 endonuclease. Therefore, in a preferred embodiment, the cleavage of the hybridized petal oligonucleotides is performed in Mg... ++ The assay is performed in a lobular assay buffer. As used herein, the term "Mg" is... ++ "Loop-shaped assay buffer" refers to a buffer solution for lobular endonucleases, which contains Mg... ++ As the dominant or essentially sole divalent cation in the buffer, cleavage reactions carried out in the buffer (e.g., cleavage of invasive oligonucleotide / lobe oligonucleotide pairs or hybridized padlock probes that form invasive cleavage structures) exhibit Mg... ++ It possesses the characteristics of presence, and does not exhibit the characteristics associated with the presence of other divalent cations (e.g., with or in place of Mg). ++ Using Mn ++ (Alternative cleavage activities of associated valve endonucleases, such as those described above by Kaiser et al., for example.)

[0477] In some implementations, the flap assay buffer has very low or no KCl compared to standard PCR buffer, and contains elevated Mg2+. ++ (For example, the flap assay buffer of this technology contains >6mM, preferably >7mM, more preferably 7.5mM Mg) ++ PCR buffer typically contains approximately 1.5 to 2.5 mM Mg. ++ In a preferred embodiment, the cleavage is performed in a flap assay buffer comprising 7.5 mM MgCl2, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. While this technique is not limited to any particular FEN endonuclease, in a preferred embodiment, the cleavage reaction comprises a thermostable FEN-1 endonuclease selected from the following: CLEAVASE 2.0 nuclease (Hologic, Inc.), *Afu* FEN-1, *Pfu* FEN-1, and *Ave* FEN-1 (see, for example, WO 02 / 070755).

[0478] In some embodiments, the composition comprises at least 5 unique lobular oligonucleotides. In some embodiments, the composition comprises at least 10 unique lobular oligonucleotides. In some embodiments, the composition comprises at least 20 unique lobular oligonucleotides. In some embodiments, the composition comprises at least 30 unique lobular oligonucleotides. In some embodiments, the composition comprises at least 40 unique lobular oligonucleotides. In some embodiments, the composition comprises at least 45 unique lobular oligonucleotides, and in some embodiments, the number of unique lobular oligonucleotides corresponds to the number of target nucleic acids to be detected in the sample. In some embodiments, the composition comprises multiple copies of a given lobular oligonucleotide to ensure binding to the target nucleic acid (if present in the sample). For example, in some embodiments, the composition comprises at least 5 unique lobular oligonucleotides (e.g., at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, or at least 45 unique lobular oligonucleotides) and contains multiple copies of each unique lobular oligonucleotide.

[0479] In some embodiments, the composition further comprises a valve-shaped endonuclease. In some embodiments, the valve-shaped endonuclease is a FEN-1 endonuclease, preferably a strictly FEN-1 endonuclease.

[0480] In some embodiments, the composition further comprises a connectable molecule. In some embodiments, the connectable molecule is also referred to herein as a "carrier" or "lobe carrier." In some embodiments, the connectable molecule is a cyclizable molecule, such as a dumbbell probe or padlock probe, while in some embodiments, the connectable molecule forms a linear chain upon connection, such as when connected to a cutting lobe.

[0481] In some embodiments, the vector or flap vector comprises a pair of molecules (“vector pair”), wherein the first vector oligonucleotide contains a first bridging complement region complementary to the 5′ end (e.g., 5′ end sequence) of the bridging oligonucleotide, and the second vector oligonucleotide contains a second bridging complement region complementary to the 3′ end (e.g., 3′ end sequence) of the bridging oligonucleotide, such that a third oligonucleotide (e.g., a cleavage flap) hybridizing with an inner segment of the bridging oligonucleotide is located between the two oligonucleotides of the vector pair, for example, as shown below. Figure 14 This is shown schematically. Figure 14 The following implementation scheme is shown, wherein carrier 1 and carrier 2 can be provided as independent carrier molecules of a carrier pair, or can be provided as cyclizable carrier molecules by being linked by a main chain (not shown).

[0482] In some embodiments, the connectable cyclizable molecule is a dumbbell probe. In some embodiments, each dumbbell probe contains a bridging sequence complementary to the cleavage lobe sequence. Thus, under suitable conditions, a given dumbbell probe hybridizes with the cleavage lobe sequence to generate a connectable nick (e.g., Figure 5B As shown, the cut is connected under suitable conditions to generate a dumbbell probe containing a closed loop-stem-loop structure, which can then be amplified and / or detected, for example, by PCR, RCA, RCT, sequencing, etc.

[0483] In some embodiments, the linkable cyclizable molecule is a padlock probe. In some embodiments, each padlock probe comprises a first target region complementary to the 5′ end (e.g., the 5′ end sequence) of the bridging oligonucleotide and a second target region complementary to the 3′ end (e.g., the 3′ end sequence) of the bridging oligonucleotide. Thus, under suitable conditions, a given padlock probe hybridizes with the bridging oligonucleotide, wherein a single-stranded segment (i.e., the middle segment) remains between the 3′ and 5′ ends of the probe, which, under suitable conditions, hybridizes with a lobe sequence or a cleavage lobe sequence.

[0484] In some embodiments, the composition further comprises a plurality of bridging oligonucleotides, such as bridging oligonucleotides for aligning the cleavage lobe with one or more linkable molecules (e.g., linear carrier oligonucleotides or carrier pairs, or cyclizable molecules, such as padlock probes or dumbbell probes). In some embodiments, each bridging oligonucleotide comprises an internal segment complementary to the lobe sequence. For example, in some embodiments, each bridging oligonucleotide comprises an internal segment of 10-20 bases in length complementary to the lobe sequence. In some embodiments, each bridging oligonucleotide comprises an internal segment complementary to the cleavage lobe sequence. For example, in some embodiments, each bridging oligonucleotide comprises, for example, an internal segment of 10-20 bases in length complementary to the cleavage lobe sequence, preferably complementary to the full length of the cleavage lobe sequence. In some embodiments, each bridging oligonucleotide additionally comprises a 5′ end sequence and a 3′ end sequence that are not complementary to the lobe sequence or the cleavage lobe sequence and therefore do not bind to the lobe sequence or the cleavage lobe sequence. In some embodiments, the 5′ end sequence and the 3′ end sequence each comprise at least three bases. In some embodiments, the 5′ end sequence and the 3′ end sequence each contain 3-30 bases. In some embodiments, the 5′ end sequence and the 3′ end sequence each contain 10-25 bases. In some embodiments, the 5′ end sequence and the 3′ end sequence each contain about 18 bases. In some embodiments, the 3′ end sequence and the 5′ end sequence contain the same number of nucleotides, and in some embodiments, they contain different numbers of nucleotides.

[0485] In some embodiments, the cleavage flaps act as cyclizable molecules. In some embodiments, bridging oligonucleotides are used to align the 3′ and 5′ ends of the cleavage flaps to achieve ligation. For example, in some embodiments, each cleavage flap contains a first region complementary to the 5′ end (e.g., the 5′ end sequence) of the bridging oligonucleotide and a second region complementary to the 3′ end (e.g., the 3′ end sequence) of the bridging oligonucleotide. Thus, under suitable conditions, a given cleavage flap hybridizes with the bridging oligonucleotide at its 5′ and 3′ end sequences, preferably forming a ligation-compatible cleavage. In some embodiments, the 5′ and 3′ end sequences each contain at least three bases. In some embodiments, the 5′ and 3′ end sequences each contain 3-30 bases. In some embodiments, the 5′ and 3′ end sequences each contain 10-25 bases. In some embodiments, the 5′ and 3′ end sequences each contain about 18 bases. In some embodiments, the 3′ and 5′ end sequences contain the same number of nucleotides, and in some embodiments, they contain different numbers of nucleotides. In some implementations, the cleavage lobes may be connected in a non-circular manner (e.g., end to end) to form a linear chain containing two or more cleavage lobes in a continuous chain.

[0486] In some embodiments, each ligandable molecule (e.g., a linear vector or vector pair, or a circularizable molecule such as a padlock probe, dumbbell probe, or cleavage flap) also includes a primer binding site, for example, for amplification. For example, in some embodiments, each ligandable molecule includes a primer binding site, such as in PCR, RCA, RCT, sequencing, etc. The sequence primer binding site can be any suitable nucleotide sequence that serves as a hybridization site for initiating a sequencing reaction. In some embodiments, each ligandable molecule (e.g., a linear vector or vector pair, or a circularizable molecule such as a padlock probe, dumbbell probe, or cleavage flap) includes a unique sequencing barcode (e.g., a unique molecular identifier, UMI) that can be determined by sequencing to help determine which particular ligandable molecule among a plurality of ligandable molecules binds to and ligates to a given cleavage flap sequence, or which particular flap oligonucleotides are cleaved in the cleavage reaction. In some embodiments, the UMI contains 6-50 nucleotides. In some embodiments, the UMI contains 10-40 nucleotides, 10-30 nucleotides, or 10-20 nucleotides. For example, in some embodiments, the UMI contains 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. The nucleic acid molecule may contain one UMI or multiple different UMI sequences. In some embodiments, each linker molecule or lobe portion of a lobe probe, for example, contains one or more sequencing adaptors (e.g., binding sites for sequencing primers).

[0487] In some implementations, each ligable vector molecule or each ligable vector pair also includes an index sequence. As used herein, the terms "index sequence" and "indexing sequence" are used interchangeably and refer to a unique nucleotide sequence that can be used for sample barcode labeling. For example, an index sequence can be used to identify which molecular population (e.g., linear or circular linkers formed in a ligation assay or replicated, for example, in an amplification assay) is associated with which particular sample or patient. Each molecule in a given molecular population contains the same index sequence, and each molecular population contains an index sequence that is different from every other molecular population. For example, molecules in population "A" contain index sequence "A", molecules in population "B" contain index sequence "B", and so on.

[0488] In some embodiments, the composition comprises a plurality of ligandable vector molecules or pairs of vectors, wherein each ligandable vector pair contains the same number of nucleotides (e.g., of the same size). For example, in some embodiments, the composition comprises a plurality of linker molecules, wherein each linker molecule contains the same number of nucleotides (e.g., of the same size) and contains the same components except for the UMI, which is a randomized sequence unique to each linker molecule, thereby enabling specific identification of a given linker molecule during sequencing (e.g., after ligation of the cleavage lobe sequence). In some embodiments, the size (e.g., number of bases) of each UMI for each probe is the same (e.g., 6-20 bases), but the sequence of the UMI for each linker molecule is different. In some embodiments, the composition comprises a plurality of linker molecules, some of which are of a different size than the other linker molecules in the composition. For example, in some embodiments, the composition comprises a first subgroup of linker molecules containing a first number of total bases, a second subgroup of linker molecules containing a second number of total bases, a third subgroup of linker molecules containing a third number of total bases, and so on. Any suitable number of linker molecule subgroups can be used, and any suitable number of bases can be used for different linker molecule subgroups. In some embodiments, the first linkable molecular subunit has N bases, the second linkable molecular subunit has N-1 bases, the third linkable molecular subunit has N-2 bases, the fourth linkable molecular subunit has N-3 bases, and so on. In some embodiments, multiple carrier oligonucleotides or carrier pairs (e.g., used to form multiple linear linker products) can be used instead of multiple linkable molecules to provide the above-described elements.

[0489] In some embodiments, the composition comprises a plurality of ring probes, such as... Figure 4The circular template shown contains each circular probe with the same number of nucleotides (e.g., of the same size). For example, in some embodiments, the composition comprises multiple circular probes, each containing the same number of nucleotides (e.g., of the same size) and the same components except for the UMI, which is a randomized sequence unique to each circular probe, enabling specific identification of a given circular probe during sequencing (e.g., after extending the cleavage sequence as a primer). In some embodiments, the size (e.g., number of bases) of each UMI for each probe is the same (e.g., 6-20 bases), but the sequence of the UMI for each circular probe is different. In some embodiments, the composition comprises multiple circular probes, some of which are different in size from the other circular probes in the composition. For example, in some embodiments, the composition comprises a first subgroup of circular probes containing a first number of total bases, a second subgroup of circular probes containing a second number of total bases, a third subgroup of circular probes containing a third number of total bases, and so on. Any suitable number of circular probe subgroups can be used, and any suitable number of bases can be used for different circular probe subgroups. In some implementations, the number of bases in the first circular probe subgroup is N, the number of bases in the second circular probe subgroup is N-1, the number of bases in the third circular probe subgroup is N-2, the number of bases in the fourth circular probe subgroup is N-3, and so on.

[0490] In some embodiments, the linkable molecule (e.g., a cyclizable molecule or a linear carrier molecule or carrier pair) contains a 3′-terminal ribonucleotide. In some embodiments, the 3′-terminal ribonucleotide present on the linkable molecule prevents polymerase, such as residual polymerase remaining after PCR-flap assays (e.g., the LQAS reaction described above), from extending the molecule.

[0491] In some embodiments, the composition further comprises a ligase, preferably a high-fidelity ligase. In some embodiments, the composition further comprises a phosphate source to be added to the cleavage lobe sequence to facilitate ligation. In some embodiments, the lobe sequence is phosphorylated prior to cleavage. In some embodiments, the composition further comprises Mg. 2+ In some embodiments, the composition further comprises one or more primer pairs for amplifying the target nucleic acid. In some embodiments, the composition further comprises nucleic acid extracted from a biological sample obtained from a subject.

[0492] In some aspects, this document provides multiplexing methods for detecting multiple target nucleic acids in a sample. In some embodiments, the method for multiplexing multiple target nucleic acids in a sample includes contacting the sample with a composition comprising a plurality of valve-shaped oligonucleotides as described herein.

[0493] In some embodiments, methods for multiplex detection of multiple targets include contacting a sample containing or suspected of containing a target nucleic acid with a composition comprising multiple valve-shaped oligonucleotides under suitable conditions (e.g., in a buffer suitable for DNA sequence-specific hybridization, e.g., in the presence of monovalent or divalent cations), such that the target-specific region hybridizes with the target sequence. In some embodiments, the hybridization of the target-specific region with the target nucleic acid occurs in the presence of a nuclease, preferably in the presence of a strictly thermostable FEN-1 enzyme, such that cleavage of the valve sequence occurs when the hybridization of the target-specific region with the target nucleic acid forms an invasive cleaving structure.

[0494] One aspect of this technology is that, compared to structures such as notched structures (with a single-stranded notch between the upstream and downstream duplexes), Y-shaped structures (with 5′ lobes but no overlap between the upstream and downstream duplexes), and pseudo-Y-shaped structures (with 5′ lobes and a non-duplex template strand located upstream of the downstream duplex), it offers advantages in enzyme pairing, such as... Figure 1 The invasive cleavage structure shown exhibits maximum specificity when using a flap-shaped endonuclease. Therefore, in a preferred embodiment, the cleavage of the hybridized padlock probe is performed in Mg... ++ The assay is performed in a lobular assay buffer. As used herein, the term "Mg" is... ++ "Loop-shaped assay buffer" refers to a buffer solution for lobular endonucleases, which contains Mg... ++ As the dominant or essentially sole divalent cation in the buffer solution, cleavage reactions carried out in the buffer solution (e.g., cleavage of hybrid padlock probes that form invasive cleavage structures) exhibit Mg... ++ It possesses the characteristics of presence, and does not exhibit the characteristics associated with the presence of other divalent cations (e.g., with or in place of Mg). ++ Using Mn ++ (Alternative cleavage activities of associated valve endonucleases, such as those described above by Kaiser et al., for example.)

[0495] In some implementations, the flap assay buffer has very low or no KCl compared to standard PCR buffer, and contains elevated Mg2+. ++ (For example, the flap assay buffer of this technology contains >6mM, preferably >7mM, more preferably 7.5mM Mg) ++ PCR buffer typically contains approximately 1.5 to 2.5 mM Mg. ++In a preferred embodiment, the cleavage is performed in a flap assay buffer comprising 7.5 mM MgCl2, 10 mM MOPS, 0.3 mM Tris-HCl, pH 8.0, 0.8 mM KCl, 0.1 μg / μL BSA, 0.0001% Tween-20, and 0.0001% IGEPAL CA-630. While this technique is not limited to any particular FEN endonuclease, in a preferred embodiment, the cleavage reaction comprises a thermostable FEN-1 endonuclease selected from the following: Cleavase 2.0 nuclease (Hologic, Inc.), *Afu* FEN-1, *Pfu* FEN-1, and *Ave* FEN-1 (see, for example, WO 02 / 070755).

[0496] In some embodiments, the cleavage lobe sequence is ligated to a circularizable molecule to form a closed loop structure that can be subsequently amplified and / or detected. In some embodiments, the circularizable molecule is a padlock probe. In some embodiments, the circularizable molecule is a dumbbell probe. In some embodiments, the 3′ and 5′ ends of the cleavage lobe are ligated together to form a closed loop structure that can be subsequently amplified and / or detected.

[0497] In some embodiments, the cleavage lobe sequence is ligated to a linear carrier molecule, preferably a pair of linear carrier molecules, to form a linear linker molecule comprising the carrier and the lobe sequence, which can then be amplified and / or detected. In some embodiments, the 3′ end of the lobe is ligated to a carrier molecule, and in some embodiments, the 3′ and 5′ ends of the cleavage lobe are ligated to different carrier molecules in a carrier pair.

[0498] In some embodiments, the cleavage flap sequence hybridizes with the bridging oligonucleotide. The bridging oligonucleotide may be present in a composition comprising multiple flap oligonucleotides as described above, or may be added to the sample after contact with the composition. In some embodiments, the cleavage flap sequence hybridizes with the inner segments of the bridging oligonucleotide, as described above, while the 3′ and 5′ ends of the bridging oligonucleotide do not hybridize with the cleavage flap sequence. In some embodiments, the uncleaved flap sequence (e.g., the flap sequence of an uncleaved flap oligonucleotide) can hybridize with the bridging oligonucleotide even in the absence of endonuclease cleavage.

[0499] In some embodiments, the linker molecule is present in the composition or added to the sample after contact with the composition. In some embodiments, the linker molecule (e.g., a padlock probe, a dumbbell probe) generates a closed loop structure containing a cleavage sequence, while in other embodiments, the linker molecule generates a linear linker product containing a cleavage sequence.

[0500] In some embodiments, the cyclizable molecule is a padlock probe, and each padlock probe comprises a first target region complementary to the 5′ end (e.g., a 5′ end sequence) of the bridging oligonucleotide and a second target region complementary to the 3′ end (e.g., a 3′ end sequence) of the bridging oligonucleotide. Thus, under suitable conditions, a given padlock probe hybridizes with the bridging oligonucleotide, while, as described above, the internal segment of the padlock probe hybridizes with the cleavage flap sequence. In some embodiments, the cyclizable molecule is a dumbbell probe, and each dumbbell probe comprises a bridging sequence complementary to the cleavage flap sequence, such that the cleavage flap sequence hybridizes with the bridging sequence of the dumbbell probe.

[0501] In some embodiments, the linker molecule comprises a pair of carrier oligonucleotides, similar to the ends of the padlock and dumbbell probes described above. For example, in some embodiments, the linker molecule comprises a first carrier oligonucleotide and a second carrier oligonucleotide, the first carrier oligonucleotide containing a first target region complementary to the 5′ end (e.g., the 5′ end sequence) of the bridging oligonucleotide, and the second carrier oligonucleotide containing a second target region complementary to the 3′ end (e.g., the 3′ end sequence) of the bridging oligonucleotide. Thus, under suitable conditions, a given carrier pair hybridizes to each end of the bridging oligonucleotide, and the internal segments of the bridging oligonucleotide hybridize to the cleavage flap sequence.

[0502] In some implementations, the cleavage lobe sequence can hybridize with a circularizable molecule and be used as a primer, for example, for replicating a circular template (e.g. Figure 3 (As shown). In some embodiments, the circularizable molecule may have already been circularized prior to hybridization with the cleavage lobe sequence. In some embodiments, the cleavage lobe sequence may hybridize with a linear nucleic acid strand, for example, to act as a primer for replication using the linear nucleic acid strand as a template.

[0503] Linkage of molecules

[0504] In some embodiments, the cleavage lobe sequence is ligated to a lobe-like vector, such as a cyclizable molecule or a vector oligonucleotide pair, thereby forming a closed circular linker or a linear linker, respectively. Ligation of the 5′ end to the 3′ end of the nucleic acid can occur when both ends pair with adjacent nucleotide bases of complementary sequences, wherein the 5′ terminal nucleotide has a 5′ phosphate and the 3′ terminal nucleotide has a 3′ hydroxyl group. The corresponding terminal nucleotide in the template strand pairs with the bases of the adjacent nucleotide to form a nucleic acid duplex containing the cleavage. The ligation of the two ends at the cleavage can be catalyzed by a DNA ligase. Therefore, providing the conditions for ligation will typically include providing a DNA ligase and reaction conditions under which the DNA ligase ligates the two ends to form a continuous nucleic acid strand, thereby closing the cleavage. Preferably, a high-fidelity ligase is used under conditions that maximize mismatch discrimination. For example, a suitable amount of HiFi Taq DNA ligase (New England Biolabs) can be used, preferably in a buffer of 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NAD, and 0.1% Triton® X-100 (at 25°C and pH 8.5), and incubated at 37–75°C for 10–60 min, or by thermal cycling at 95°C for 30 s to 1 min, followed by annealing / ligation at a ligation temperature selected for the specific probe used for 1–5 min. Several other ligases are commercially available, including Ampligase (Epicentre), which is suitable when 1 U of enzyme is added and incubated at 55°C in ligase buffer for 1 hour. In some implementations, ligation is performed at 37°C for 30 min in the presence of the target DNA (e.g., the cleavage flap sequence) using 10 mM Tris-acetate at pH 7.5, 10 mM magnesium acetate, 50 mM NaCl, 1 mM ATP, 1 µg / mL BSA, and 0.2 U / µL T4 DNA ligase (Amersham Pharmacia Biotech, Uppsala, Sweden). See, for example, Nilsson et al., Nucl. Acids Res. 30(14):e66 (2002) and Lohman, G. et al. (2016) Nucleic Acids Res. 44(2):e14-e14 DOI: 0.1093 / nar / gkv898, which are incorporated herein by reference in their entirety.

[0505] In some embodiments, the cleft sequence is ligated to a ligase-compatible molecule (e.g., a padlock probe, a dumbbell probe, a linear vector oligonucleotide, or a vector pair) in the same buffer where cleft endonuclease cleavage occurs. In some embodiments, the sample is contacted with a composition comprising a cleft oligonucleotide, a FEN endonuclease, a bridging oligonucleotide, a padlock probe or a linear vector oligonucleotide or vector pair, and a ligase. In some embodiments, the sample is contacted with a composition comprising a cleft oligonucleotide, a FEN endonuclease, a dumbbell probe, and a ligase.

[0506] In some embodiments, the cleaved padlock probe or vector oligonucleotide or vector pair is ligated in the same buffer in which the FEN cleavage occurs, while in other embodiments, the cleavage reaction mixture is desalted or diluted prior to ligation, for example, to provide buffer conditions more suitable for the selected ligase. In some embodiments, the reaction conditions for cleavage are selected to be compatible with the selected ligase, thereby allowing the cleaved probe to be used in the ligation reaction without dilution or desalting.

[0507] For example, in some implementations, cleavage and ligation are performed in a buffer containing MgCl2 and ATP (e.g., 10 mM MOPS, pH 7.5, 2 mM MgCl2, 20 μg / ml tRNA, 1 mM ATP; see Kaiser et al., ibid.).

[0508] In some embodiments, the reaction mixture comprises a target nucleic acid, a vector probe (e.g., a padlock probe, a dumbbell probe, a linear vector oligonucleotide, or a vector pair), a FEN endonuclease, and a ligase. HiFi has been shown. Taq DNA ligase can be used as an alternative to buffers, for example, in cases where activity and fidelity are reduced. Taq DNA ligase buffer. HiFi... (The sentence is incomplete and requires more context to translate accurately.) Taq DNA ligase can also be used in various polymerase buffers, but its activity and fidelity are reduced to a certain extent:

[0509]

[0510] (See www.) <dot>neb <dot>com / faqs / 2016 / 03 / 18 / can-hifi-taq-dna-ligase-be-used-in-other-buffers)

[0511] 1 Supplemented with 1mM NAD

[0512] 1X HiFi Taq DNA ligase buffer:

[0513] 20mM Tris-HCl; 150mM KCl; 10mM MgCl2; 10mM DTT; 1mM NAD; 0.1% Triton® X-100; (at 25℃, pH 8.5)

[0514] Phi29 Reaction buffer solution:

[0515] 50mM Tris-HCl pH 7.5, 10mM MgCl2, 10mM (NH4)SO4, 4mM dithiothreitol (at 25℃, pH 7.5)

[0516] Standard Taq reaction buffer conditions:

[0517] 10 mM Tris-HCl; 50 mM KCl; and 1.5 mM MgCl2 (at 25 °C, pH 8.3).

[0518] Exemplary flap assay reaction buffer:

[0519] 7.5mM MgCl2, 10mM MOPS, 0.3mM Tris-HCl, pH 8.0, 0.8mM KCl, 0.1μg / μL BSA, 0.0001% Tween-20 and 0.0001% IGEPAL CA-630

[0520] The expected similarity of these buffers suggests that this ligase, along with other ligases, is compatible with conditions suitable for DNA polymerase and FEN-1 endonuclease activity. For example, as noted above, Kaiser performed cleavage and ligation in a buffer containing 10 mM MOPS, pH 7.5, 2 mM MgCl2, 20 μg / ml tRNA, and 1 mM ATP (see Kaiser et al., ibid.).

[0521] In some embodiments, the cleavage lobe sequence is linked to a cyclizable molecule, a linear carrier oligonucleotide, or a carrier pair in a separate reaction, which occurs after cleavage by a lobe endonuclease. For example, in some embodiments, the invasive cleavage assay is performed by contacting a sample with a composition comprising a lobe oligonucleotide and a lobe endonuclease, the contact conditions being such that if the target nucleic acid is present in the sample and the lobe sequence is cleaved from the lobe oligonucleotide, an invasive cleavage structure comprising the lobe oligonucleotide and the target nucleic acid is formed. In some embodiments (e.g., as...) Figure 1 (As shown), the composition comprises an invasive oligonucleotide that serves as an upstream oligonucleotide in the invasive cleavage structure. In some embodiments, the invasive oligonucleotide is designed to be complementary to a portion of the target nucleic acid strand that is not bound to a target-specific region of the valve-like oligonucleotide. In some embodiments (e.g., as shown) Figure 2 As shown, the lobular oligonucleotide comprises both a lobular portion and an intrusive portion, wherein the lobular portion and the intrusive portion of the lobular oligonucleotide bind to different but adjacent regions on the target nucleic acid, thereby forming upstream and downstream duplexes in the invasive cleavage structure.

[0522] Connection of circumsizable cutting lobes

[0523] In some implementations, the cleavage flap is treated under suitable conditions such that the 5′ and 3′ ends of the cleavage flap chain hybridize with splint oligonucleotides, thereby aligning the ends of the cleavage flap to form a connectable cleavage, for example, as... Figure 6B The left figure schematically illustrates the ligation of the nick, where a circular valve is formed. Therefore, the conditions provided for ligation will be the same as or similar to those for ligating the aforementioned circularizable molecules. For example, ligation typically involves providing a DNA ligase and reaction conditions under which the DNA ligase ligates the two ends to form a continuous nucleic acid chain, thereby closing the nick. In a preferred embodiment, a high-fidelity ligase is used under conditions that maximize mismatch discrimination. For example, a specific amount of HiFi Taq DNA ligase (New England Biolabs) is used, preferably in a buffer of 20 mM Tris-HCl, 150 mM KCl, 10 mM MgCl2, 10 mM DTT, 1 mM NAD, and 0.1% Triton® X-100 (at 25°C, pH 8.5), and incubated at 37–75°C for 10–60 min, or by thermal cycling at 95°C for 30 s to 1 min, followed by annealing / ligation at a ligation temperature selected for the specific probe used for 1–5 min. Several other ligases are commercially available, including Ampligase (Epicentre), which is suitable for incubation at 55°C for 1 hour with 1 U of enzyme added. In some embodiments, ligation is performed at 37°C for 30 min in the presence of the target DNA (e.g., the cleavage sequence) using 10 mM Tris-acetate at pH 7.5, 10 mM magnesium acetate, 50 mM NaCl, 1 mM ATP, 1 µg / mL BSA, and 0.2 U / µL T4 DNA ligase (Amersham Pharmacia Biotech, Uppsala, Sweden). See, for example, Nilsson et al., Nucl. Acids Res. 30(14):e66 (2002) and Lohman, G. et al. (2016) Nucleic Acids Res. 44(2):e14-e14 DOI: 0.1093 / nar / gkv898, which are incorporated herein by reference in their entirety. In another example, the cleavage flap can be assembled in a reaction containing splint oligonucleotides and ligated in a process including assembling the cleavage flap and the isolated oligonucleotides in 50 mM Tris, pH 7.5, and 10 mM MgCl2. After denaturation at 75 °C for 5 min, the solution was allowed to slowly reach room temperature. ATP, DTT, and BSA were added to final concentrations of 100 μM, 10 mM, and 25 μg / ml, respectively. The reaction was initiated by adding T4 DNA ligase to a final concentration of 800 U / ml. The reaction mixture was incubated at room temperature for, for example, 12 h, followed by heat inactivation of the ligase. (See, for example, JS Hartig et al., Nucleic Acids Res. 2004; 32(19):e152).In an embodiment where the cleavage flap hybridizes with the splint oligonucleotide to produce another flap-like structure, the complex can be treated with a flap-like endonuclease (e.g., FEN-1 endonuclease) to remove the 5′ flap and leave a cleavage opening for connection.

[0524] In some implementations, a ligase capable of joining single-stranded ends (e.g., T4 RNA ligase, CIRCLIGASE™ ssDNA ligase, or CIRCLIGASE II™ ssDNA ligase) is used to join the 5′ and 3′ ends of the cleavage flap to form a circular flap, such as... Figure 6B The figure on the right is schematically illustrated. By way of example and not limitation, the cleavage flap can be combined at 60°C for 2 hours in a ligation reaction mixture containing 50 mM HEPES, pH 8.0, 66 mM KOAc, 0.5 mM DTT, 1 M betaine, and 30 U CIRCLIGASE II™ (Epicentre Biotechnologies, Madison, WI). Subsequently, the ligase is heat-inactivated, for example, by incubating the reaction mixture at 80°C for 10 minutes. See, for example, U.S. Patent No. 9,938,568 to RC Heller et al., which is incorporated herein by reference in its entirety for all purposes.

[0525] In some embodiments, cleavage of the valve oligonucleotide is detected in a subsequent reaction (if it has occurred). Therefore, in some embodiments, after contacting the sample with a composition containing the valve oligonucleotide and a valve endonuclease under conditions that result in the formation of an invasive cleavage structure comprising the valve oligonucleotide and the target nucleic acid (e.g., if the target nucleic acid is present in the sample), cleavage of the valve sequence is detected by methods including the use of one or more of padlock probes (including, but not limited to, sequence library vectors), bridging oligonucleotides, splint oligonucleotides, ligases, DNA polymerases, and one or more primers. For example, in some embodiments, the cleavage valve is treated under suitable conditions such that the cleavage valve strand hybridizes with the internal segment of the bridging oligonucleotide, and the 5′ and 3′ ends of the bridging oligonucleotide hybridize with the first and second target ends of the padlock probe, respectively, thereby aligning the cleavage valve with the ends of the padlock probe to form two connectable cleavages, for example, as... Figure 5A This is illustrated schematically. Subsequent connections form a closed circular strand of nucleic acid containing the padlock probe sequence and the cleavage flap sequence.

[0526] In some embodiments, after contacting the sample with a composition containing a valve-like oligonucleotide and a valve-like endonuclease under conditions that result in the formation of an invasive cleavage structure comprising a valve-like oligonucleotide and a target nucleic acid (if the target nucleic acid is present in the sample), the cleavage of the valve is detected using a second composition containing a dumbbell probe and a ligase, and the reaction is carried out under suitable conditions that hybridize the cleavage valve sequence with the bridging sequence of the dumbbell probe. In some embodiments, the method includes using one or more dumbbell probes containing bridging sequences (including, but not limited to, sequence library vectors), a ligase, a DNA polymerase, and one or more primers. For example, in some embodiments, the cleavage valve is treated under suitable conditions such that the cleavage valve strand hybridizes with the inner segment of the bridging sequence of the dumbbell probe, thereby aligning the cleavage valve with the ends of the dumbbell probe to form two ligable cleavages, for example, as... Figure 5B As illustrated schematically. Subsequent connections form a closed circular chain of nucleic acid containing the dumbbell probe sequence and the cleavage lobe sequence (e.g., a closed loop-stem-loop structure).

[0527] In some embodiments, the sample is contacted with a composition containing a valve-like oligonucleotide and a valve-like endonuclease under conditions that allow the formation of an invasive cleavage structure comprising a valve-like oligonucleotide and a target nucleic acid (if the target nucleic acid is present in the sample), and the cleavage valves are treated under conditions in which the ends of the cleavage valves are joined together to form a circular valve sequence, such as... Figure 6B and Figure 6C This is shown schematically.

[0528] Enhance the connection of the cutting flap

[0529] Residual contamination from residual polymerases (e.g., GoTaq polymerase) in PCR-flap assays can reduce the ligation sensitivity and overall analytical sensitivity of the method, for example, by inhibiting hybridization of the cleft sequence with bridging oligonucleotides. Therefore, in an exemplary method, after forming the cleft sequence via an invasive cleft assay (e.g., PCR-flap assay, such as LQAS assay) in the presence of polymerase, the reaction mixture is treated with a phosphatase (e.g., calf intestinal alkaline phosphatase (CIP) or shrimp alkaline phosphatase (rSIP)) to dephosphorylate the dNTPs in the reaction mixture, thereby inhibiting the polymerase activity of residual polymerases used in PCR-flap assays. For information on the use of rSAP, see, for example, the New England Biolabs application note "Enzymatic PCR Cleanup using Exonuclease I and Shrimp Alkaline Phosphatase" (2019), NEB Cat. #M0371. (For example, 1 µL of rSAP is combined with 5 µL of PCR product, incubated at 37 °C for 15 min, heated to 80 °C for 15 min to inactivate, and then the PCR product is processed downstream).

[0530] Polymerase activity can also be limited by using a low ligation temperature, for example, below the temperature at which the polymerase is active; degrading the polymerase with a suitable protease (e.g., proteinase K) after PCR-valve assay; or heat-inactivating the polymerase (e.g., at 99°C for 10 min).

[0531] CIP treatment typically also removes the 5' phosphate from the oligonucleotide, for example, from the 5' end of the cleavage valve (if present). In a preferred embodiment, the cleavage valve is treated with a polynucleotide kinase (PNK), such as T4 polynucleotide kinase (T4 PNK). In some embodiments, under suitable conditions, the 5' end of the cleavage valve and / or the 5' end of a linkable molecule (e.g., the 5' linkable end of a cyclizable molecule or a linear carrier oligonucleotide) is phosphorylated using PNK to enable the cleavage valve to link with a cyclizable molecule or a carrier oligonucleotide.

[0532] Cleavage lobes and cyclizable molecules can hybridize with bridging or splinting oligonucleotides or sequences, for example, such as Figure 5A and Figure 5B The schematic illustration shows that the cleavage flap and vector pair can hybridize with bridging or splinting oligonucleotides, for example, as... Figure 14 As illustrated, this creates a ligation-compatible nick. A ligase (e.g., HiFi Taq DNA ligase) is then used to ligate the phosphorylated nick flap and the ligation-compatible molecule in the hybridization structure.

[0533] In an embodiment where the ligandable molecule is circularizable, the ligation forms a covalently closed circular nucleic acid. After ligation, residual non-circular molecules, such as uncircularized vectors, residual oligonucleotides, and other nucleic acid materials, are removed by digestion with an appropriate exonuclease (e.g., λ exonuclease, exonuclease III, exonuclease I, or exonuclease VIII), making the closed circular structure suitable for downstream applications such as amplification and sequencing.

[0534] The cleavage flaps were connected using RNA bridging sequences.

[0535] In some embodiments, the activity of residual DNA polymerase, such as that from a PCR-valve assay, is limited by using a bridging or splint chain containing RNA. For example, in some embodiments, the cleavage valve is linked to a ligation-compatible molecule (e.g., a circularizable valve-like vector or linear vector pair) using RNA-bridging oligonucleotides or chimeric bridging oligonucleotides, wherein at least a portion of the oligonucleotide chain contains RNA, e.g., as shown in the figure. Figure 14 This is shown schematically.

[0536] As discussed above, in some embodiments used to prepare ligable structures, the 3′ and 5′ ends of the flap vector are aligned with the 5′ and 3′ ends of the cleavage flap by bridging oligonucleotides, thereby forming a nick. A ligase suitable for joining DNA nicks to RNA or RNA analog bridge strands is used, such as SPLINTR ligase (also known as PBCV-1 DNA ligase or Chlorella virus DNA ligase, New England BioLabs; see also Krzywkowski, T. and M. Nilsson, Nucleic Acids Research, 2017, Vol. 45, No. 18: e161; and Shin, M et al., Nucleic Acids Therapeutics v32 (1): 66-72 (2022); these references are incorporated herein by reference in their entirety for all purposes), connecting the ends of the cleavage lobes to the lobular carrier to form cyclic molecules.

[0537] Bridging oligonucleotides can be entirely RNA or can be RNA in selected portions (e.g., where the 3′ end of a valve-like vector hybridizes). In some embodiments, the RNA portion of the bridging oligonucleotide comprises or is composed of a ribonucleotide analog, such as a 2′-modified ribonucleotide, such as 2′-O-methyl or 2′-O-methoxyethyl nucleotide. 2′-O-methyl oligonucleotides have the general structure of RNA oligonucleotides but are more stable than RNA oligonucleotides; for example, they are more resistant to nucleases.

[0538] In some implementations, the 5' end of the petal-like carrier is configured to be non-connectable unless activated, for example, by cutting away the 5' end (referred to as a "5'-activated petal-like carrier"). For example, as... Figure 14 As shown, in some embodiments, the 5′ end of the flap vector is not phosphorylated and / or engineered to have unpaired arms or flaps, such as a 2-nucleotide 5′ arm that is not complementary to the bridging oligonucleotide. Once the cleavage flap with the correct 3′ end hybridizes with the bridging oligonucleotide along with the flap vector, the flap endonuclease can cleave the 5′ arm from the flap vector, leaving a 5′ phosphate that can be attached to the 3′ end of the cleavage flap. In a preferred embodiment, the flap endonuclease comprises FEN-1 endonuclease left over from the PCR-flap assay step. Chimeric bridging oligonucleotides are used, for example, such as... Figure 14 As shown, a suitable DNA template strand is provided for use with the FEN-1 endonuclease, which exhibits reduced activity against the RNA strand. As noted above, while bacterial flap endonucleases (e.g., the 5′ nuclease of DNA polymerase) can cleave DNA probes within invasive cleavage structures formed on the RNA template strand, the strict FEN-1 endonuclease does not. See, for example, U.S. Patent No. 7,045,289 to Allavi et al.

[0539] enrichment of linker products

[0540] One approach may include enriching the ligation product prior to detection (e.g., enriching a ligation-closed circular structure comprising a cyclizable molecule and a cleavage lobe sequence, or a linear ligation product comprising a carrier oligonucleotide or carrier pair and a cleavage lobe sequence). The product may be enriched by amplification and / or by solid-phase chemistry. Circular nucleic acid products may be selectively enriched by treating the sample with an exonuclease (e.g., λ exonuclease, exonuclease III, exonuclease I, or exonuclease VIII) to digest linear nucleic acid products (e.g., target nucleic acid fragments, unligated padlock probes, etc.). Typically, exonuclease degradation may be used to enrich the ligation product when the ligation product is protected against exonuclease degradation. The exonuclease should then be inactivated (e.g., by heating) prior to any subsequent steps involving polymerization, such as before rolling circle amplification. See, for example, U.S. Patent No. 10,526,643 (ibid.) and U.S. Patent No. 11,186,863, both of which are incorporated herein by reference in their entirety for all purposes.

[0541] Exonucleases can be added to remove acyclic oligonucleotides. Suitable conditions include incubation at 37°C in the appropriate exonuclease buffer for 1 hour, followed by enzyme inactivation at 80°C for 20 minutes. When using a capture / detection method, the ligation product can be enriched by capturing the product on a solid phase via the capture portion. For example, as shown in Example 1 of U.S. Patent No. 10,526,643 (ibid.), a solution containing the ligation product can be mixed with 10 mL of M-280 streptavidin-coated magnetic beads (Invitrogen) in Tris-HCl (pH 7.5), 3.5 mM EDTA, and 0.07% Tween-20 to a final volume of 200 mL and incubated at room temperature for 15 minutes. After incubation, the magnetic beads are collected using a magnet, and the supernatant is removed. See also Example 3 of U.S. Patent No. 11,186,863. Other methods of enriching the ligation product include specific size screening of the ligation product.

[0542] As discussed above, using RNA or chimeric bridging oligonucleotides and 5′-activated valve vectors in the ligation step can reduce or eliminate the advantages of using this enrichment step.

[0543] Amplification of ligation products

[0544] In some implementations, the closed circular structure or linearly linked product is subsequently amplified and detected. A variety of different nucleic acid assays can be used, such as PCR (e.g., across the junction), ligase chain reaction, reporter probe hybridization, rolling circle amplification, rolling circle transcription (see, for example, SL Daubendiek et al. J Am Chem Soc. 1995;117:7818–7819, which is incorporated herein by reference in its entirety for all purposes). Suitable amplification techniques include, but are not limited to: rolling circle amplification (see below; also see JS Hartig et al., Nucleic Acids Res. 2004; 32(19): e152)), bridged PCR (Adessi C et al., Nucleic Acids Res. 2000 Oct 15;28(20):E87), emulsion PCR (digital PCR in emulsions described in Dressman et al., Proc Natl Acad Sci US A. July 22, 2003;100(15):8817-22. Electronic publication July 11, 2003), and digital PCR (Vogelstein and Kinzler, Proc Natl Acad Sci US A. August 3, 1999;96(16):9236-41), all of which are incorporated herein by reference in their entirety for all purposes. Local amplification of clones in gels is described in Mitra and Church, Nucleic Acids Res. 15 Dec 1999; 27(24): e34, which is incorporated herein by reference in its entirety for all purposes. Embodiments of this method may include amplifying ligation products and obtaining a cumulative signal, which is a combination of individual signals from the amplified products. Preferably, the ligation products are amplified across ligation nodes.

[0545] In some preferred embodiments, amplifying the ligation product is a step in preparing a library (e.g., a sequencing library). For example, in some embodiments, amplicons are generated by amplifying regions of the ligation molecule mixture using NGS index primers that initiate amplification from the adaptor sequence in the circularizable or vector molecule portion of the product mixture, such as... Figure 9A and Figure 14 This is an illustrative illustration. See also Bohmann K et al., "Strategies for sample labeling and library preparation in DNA metabarcoding studies." Mol Ecol Resour. U.S. Patent No. 2022;22:1231–1246; and U.S. Patent No. 10,526,643B2, which are incorporated herein by reference in their entirety for all purposes. In some embodiments, the linkable molecule (such as a vector used in NGS) includes, for example, an index sequence on the cyclizable molecule or the vector, such as Figure 8 , Figure 9B and Figure 14 The illustration is schematic. In some embodiments, ligand molecules containing index sequences may be used in addition to, in place of, or in combination with, the index sequence on the amplification primers. In some embodiments, the valve sequence used in valve assays or PCR-valve assays contains an index sequence, which may be used in addition to, in place of, or in combination with, the index sequence on the amplification primers and / or the circularizable molecule or vector. Using index sequences on vector and / or valve sequences provides additional indexing capabilities, for example, allowing for a higher level of multiplexing of samples analyzed, for example, in a single sequencing run, if such indexing is used in addition to indexing capabilities based on amplification primers. Index sequences in valve sequences and / or in circularizable molecules or vector oligonucleotides may be used, for example, for subject-specific (e.g., patient-specific) and / or sample-specific indexing of data. In some embodiments, multiple vectors containing different vector indexes may be used, for example, 2, 3, 4, 5, or more different index vectors. In other embodiments, ligand molecules containing index sequences are not used in combination with the index sequence on the amplification primers.

[0546] In a preferred embodiment, amplification may include providing conditions for rolling circle replication of a closed circular structure and detecting the products of rolling circle replication. Rolling circle replication is described, for example, in US 5,854,033 (Lizardi); Fire and Xu, ProcNatl Acad Sci US A. May 9, 1995;92(10):4641-5; and M. Ali et al., "Rollingcircle amplification: a versatile tool for chemical biology, materialsscience and medicine". Chemical Society Reviews 43 (10): 3324–3341, these references are incorporated into this paper in their entirety by reference for all purposes.

[0547] Rolling circle replication is the amplification of circular nucleic acid molecules using hybridization primers that use strand displacement DNA polymerase for extension, resulting in large DNA molecules containing dozens to hundreds of tandem repeats complementary to the circular template. ( See, for example, M. Ali et al., ibid.; also see U.S. Patent No. 10,526,643B2, which is incorporated herein by reference in its entirety for all purposes. The polymerase commonly used for RCA is... Phi 29 (Φ29), Bst and Vent exo - DNA polymerase, among which Phi 29 DNA polymerases are preferred for their superior continuous synthesis and strand substitution capabilities. Additional amplification can be obtained by cascades of strand substitution reactions. For example, rolling circle replication can be superbranched rolling circle replication, as described by Lizardi et al., Nat Genet. July 1998;19(3):225-32, which is incorporated herein by reference in its entirety for all purposes.

[0548] In some embodiments, the ligation reaction mixture is desalted or diluted prior to detection, for example, to provide buffer conditions more suitable for the selected polymerase. In some embodiments, the reaction conditions for cleavage and / or ligation are selected to be compatible with the selected polymerase, thereby allowing the ligated material to be used for amplification reactions without dilution or desalting.

[0549] As a non-limiting example, in some embodiments, primers hybridizing with the linked probe are used at 37°C in 50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 20 mM (NH4)SO4, 10 mM dithiothreitol, 0.2 µg / mL BSA, 0.25 mM dNTP, and 2 ng / mL Phi 29. The polymerization reaction takes place in a DNA polymerase. In some embodiments, primers for RCA are immobilized on a support (e.g., microbeads or other surface). See, for example, U.S. Patent Nos. 6,316,229 and 10,526,643B2 to Lizardi, which are incorporated herein by reference in their entirety for all purposes.

[0550] In some implementations, the RCA reaction can be monitored in real time during the extension reaction. For example, RCA can be performed in the presence of a 100 nM molecular beacon probe complementary to the multiply product and a 300 nM ROX dye. The molecular beacon probe typically contains a FRET system, which includes a fluorophore that is quenched in the unhybridized probe but not when the probe hybridizes to the target sequence. The reaction is tracked in real time by measuring fluorescence, for example, in an ABI 7700 real-time PCR instrument. The fluorescence value is typically given as a ratio between the fluorescence emitted by the molecular beacon (e.g., labeled with FAM, HEX, or other fluorophores) and the ROX reference dye.

[0551] Various methods have been developed for the detection of RCA products, such as in solution, in emulsion, and bound to microbeads or surfaces. See, for example, US 7,862,999, US Patent No. 6,316,229 to Lizardi et al.; and M. Ali et al., "Rolling circle amplification: a versatile tool for chemical biology, materials science and medicine". Chemical Society Reviews .43 (10): 3324–3341, and U.S. Patent No. 11,186,863, which are incorporated herein by reference in their entirety for all purposes.

[0552] In some embodiments, RCA is performed in the same buffer in which FEN cleavage and / or ligation occur. In some embodiments, the reaction mixture contains the target nucleic acid, a padlock probe, a FEN endonuclease, a ligase, and a DNA polymerase.

[0553] Identification of elements in connectable molecules

[0554] In some embodiments, cyclizable molecules (e.g., padlock probes, dumbbell probes, cleavage lobes) or linear vectors or vector pairs may contain sequence elements capable of downstream analysis of ligated molecules or amplification products. For example, in some embodiments, padlock probes, dumbbell probes, linear vector oligonucleotides or vector pairs, or the lobe portion of a lobe oligonucleotide contains coding elements, unique molecular identifier (UMI) sequences, one or more primer binding sites, etc., which are typically used to capture, identify, sequence, and / or count individual probes or lobes or families of probes or lobes that are ligated due to the presence of target nucleic acids (e.g., in the presence of cleavage lobes). For example, in some embodiments, detected coding is counted and the number of targets is estimated based on the detected coding count, for example, to estimate the number of targets based on the correlation between the detected coding count and the number of targets in the analyzed sample. See, for example, U.S. Patent No. 6,316,229 to Lizardi, which is incorporated herein by reference in its entirety for all purposes. In a particularly preferred embodiment, the linker molecule, such as a linked padlock probe, dumbbell probe, linear linker product of a carrier oligonucleotide or carrier pair, or a circularized cleavage flap, is used as a template for DNA polymerase. In some embodiments, the linker molecule is analyzed using sequencing, preferably in parallel analysis using next-generation sequencing, as discussed above.

[0555] Multiple detection of the cutting flap

[0556] This article presents a method that uses valve sequences generated by nucleases as target identifiers, which, when combined with next-generation sequencing, enables ultrasensitive and highly specific quantification in multiplex PCR assays. Figure 9A The document provides an overview of the exemplary methods described herein. Figure 9B Another implementation scheme is shown in the figure.

[0557] like Figure 9A As shown, the initial steps include an invasive cleavage assay (e.g., PCR-valve assay). The invasive cleavage assay is performed using a valve-shaped oligonucleotide containing a valve sequence and at least one target-specific region. The invasive cleavage assay can be performed on raw samples containing the target nucleic acid, or on sample material that has preferably been pre-amplified in a multiplex amplification reaction, for example, as described in PCT / US23 / 66278, filed April 27, 2023, and in U.S. Patent Nos. 10,648,025 and WO 2020 / 112869, each of which is incorporated herein by reference in its entirety.

[0558] This type of lobular oligonucleotide in Figure 9A The label is shown as "Sequence-Specific Probe 1", and the lobe sequence is shown in gray. Invasive cleavage assays typically use a forward primer (which also serves as an invasive oligonucleotide or invading oligonucleotide), a reverse primer, and a lobe endonuclease (e.g., FEN-1, preferably strict FEN-1) to induce cleavage of the lobe sequence.

[0559] The cleavage valve sequence is generated only when the valve-shaped oligonucleotide binds to the target, and is therefore target-specific. The cleavage valve in... Figure 9A The term "cutting marker-specific identification arm" is used in this context and may also be referred to herein as "cutting target-specific marker" or "cutting target-specific identifier." In addition to serving as a target-specific identifier, the cutting flap sequence may also contain subject-specific (e.g., patient-specific) and / or sample-specific identifiers, such as index sequences. In the absence of any specific target, a flap probe specific to that target is not cut, and no cutting flap is generated for that specific target.

[0560] like Figure 9A As shown, after generating the cleavage flap, the cleavage flap is ligated to the cyclizable vector. In this exemplary method, the cleavage flap is ligated to the cyclizable vector by hybridizing the ends of the cleavage flap and the cyclizable vector with bridging oligonucleotides to form a ligation-compatible cleavage structure. The bridging oligonucleotide is referred to as an "arm-specific bridging oligonucleotide" in the figure.

[0561] The ligation of the cleavage flap to a circularizable vector forms a closed circular nucleic acid molecule containing the cleavage flap sequence (e.g., a target-specific identifier containing the cleavage). Libraries containing multiple such closed circular structures can be created by amplifying a mixture of these closed circular structures, thereby generating a sequencing library. The cleavage flap sequence indicating the target can be detected using any suitable sequencing technology, including NGS (e.g., Illumina sequencing). The method described herein achieves a highly specific initial multiplex PCR reaction, followed by NGS counting of the generated target-specific identifiers (e.g., cleavage flap sequences), which significantly increases the number of simultaneously detected markers (e.g., simultaneously detected target-specific cleavage flap sequences) compared to conventional qPCR.

[0562] like Figure 9B As shown, the aforementioned circularizable vector may also contain unique sequences, in Figure 9B The term "vector index sequence" is used to increase the total index capacity of the system, enabling higher levels of multiplexing, for example, in individual sequencing runs. Using additional index sequences provides additional indexing capabilities (e.g., beyond the indexing capabilities based on amplification primer indexes), allowing for higher levels of multiplexing of samples analyzed, for example, in a single sequencing run. In some implementations, multiple vectors containing different vector indexes may be used, for example, two, three, four, five, or more different index vectors.

[0563] experiment

[0564] Example 1

[0565] Multilobular cutting, ligation and sequencing

[0566] Methods involving pre-amplification reactions (e.g., pre-amplification reactions of multiple genetic targets), secondary amplification reactions (e.g., LQAS, TELQAS) to generate cleavage lobes, ligating the cleavage lobes into cassettes, and indexing via PCR to generate sequencing libraries are referred to herein as lobular ligation and polymerase chaining (FLAP-seq). As discussed above, an exemplary method (e.g., an exemplary FLAP-seq method) for generating sequence libraries from highly multiplexed lobular cleavage assays based on PCR-lobular assay reactions (e.g., LQAS reactions) may include the following steps:

[0567] 1) Pre-amplification

[0568] 2) LQAS PCR-valve assay (preferably FRET-free oligonucleotides)

[0569] 3) Proteinase K treatment

[0570] 4) Ligation (e.g., HiFi Taq DNA ligase)

[0571] 5) Exonuclease treatment (e.g., Exo I and Exo III)

[0572] 6) Index PCR

[0573] 7) AMPure XP processing

[0574] 8) Illumina sequencing

[0575] 1) Pre-amplification

[0576] In a reaction mixture of 25 mM KCl, 7.5 mM MgCl2, 10 mM MOPS, 0.008% Tween 20, 0.008% IGEPAL, 0.025% LBSA, 0.25 mM dNTP, and pH 7.5, up to 50 µL of extracted nucleic acid (e.g., cfDNA from plasma) was combined with suitable primers / primer pairs and 1.875 units of GoTaq DNA polymerase. The reaction mixture was cycled: 95 °C for 5 min, followed by 95 °C for 30 sec and 64 °C for 1 min, repeated for 12 cycles. Aliquots were diluted 1:10 with 10 mM TrisHCl and 0.1 mM EDTA.

[0577] 2) LQAS PCR-valve assay

[0578] Aliquots of the diluted pre-amplification mixture were amplified in an LQAS reaction comprising 10 µL of the diluted pre-amplification mixture, suitable primer pairs and valve oligonucleotides, 0.05 units of GoTaq, and 14.6 ng / µL of FEN-1 endonuclease (Afu FEN-1, Hologic, Inc.) in a reaction mixture of 10 mM MOPS, 0.3 mM Tris HCl, 7.5 mM MgCl2, 0.8 mM KCl, 0.008% Tween 20, 0.008% IGEPAL, 0.1 µg / µL BSA, 1.25% glycerol, and 0.25 mM dNTPs. The reaction mixture was cycled: 95 °C for 3 min, followed by 92.5 °C for 10 s and 62.5 °C for 50 s, repeated 27 times. Aliquots of the LQAS reaction mixture were diluted 1:10 in 10 mM Tris-HCl, 0.1 mM EDTA, 10 mM MgCl2, and 10 mM KCl.

[0579] 3) Proteinase K treatment

[0580] 10 µL of diluted LQAS mixture was treated with 2.5 units of proteinase K at 37 °C for 45 min, followed by heat inactivation of the proteinase at 95 °C for 15 min.

[0581] 4) Connection

[0582] 13 µL of proteinase K-treated sample was combined with 6 µL of a premix containing 0.5 units of HiFi Taq DNA ligase dissolved in 50 mM Tris-HCl, 10 mM MgCl2, 10 mM DTT, 1 mM ATP, and 1 mM NAD at pH 7.5. The mixture was incubated at 55 °C for 1 min, followed by incubation at 37 °C for 6 min, for a total of 10 cycles.

[0583] 5) Exonuclease treatment

[0584] Combine 19 µL of the ligation mixture with 3 µL of an exonuclease mixture containing heat-sensitive exonuclease I (1.71 units) and exonuclease III (4.25 units). Incubate the mixture at 37 °C for 90 min, followed by incubation at 70 °C for 30 min.

[0585] 6) Index PCR

[0586] In a reaction mixture of 25 mM KCl, 7.5 mM MgCl2, 10 mM MOPS, 0.008% Tween 20, 0.008% IGEPAL, 0.025% L BSA, 0.25 mM dNTP, and pH 7.5, 22 µL of the reaction mixture from step 5 was combined with 53 µL of index PCR premix containing 1.875 units of GoTaq and index primers. The reaction was incubated at 95 °C for 5 min, followed by cycling at 95 °C for 30 s and at 64 °C for 1 min, repeated for 16 cycles.

[0587] 7) PCR purification

[0588] According to the manufacturer's instructions, such as using AMPure XP bead-based reagents (Beckman Coulter Life Sciences), the products of indexed PCR can be purified to remove unincorporated dNTPs, primers, primer dimers, etc.

[0589] 8) Illumina sequencing

[0590] The merged final library was analyzed using the Illumina miSEQ program.

[0591] Technicians will understand that, although this method is illustrated by reference to the Illumina NGS method, it is readily adaptable to other methods for characterizing slit lobes, including the use of other NGS methods.

[0592] Example 2

[0593] Methods to reduce the impact of residual DNA polymerase activity after PCR-flap assay

[0594] Further experiments were conducted to determine the effects of modifying various parameters measured by FLAP-seq.

[0595] The effects of pre-ligation dephosphorylation and phosphorylation (kinase treatment) of the flap assay products were tested, as well as the use of different types of ligases in preparing sequencing libraries from the flap sequences.

[0596] Effects of phosphatase and kinase treatment on LQAS cleavage products

[0597] PCR-valve assays were performed using LQAS conditions (FRET-free) and three different methylated DNA marker (MDM) target DNAs. The LQAS reaction conditions are further described in U.S. Patent No. 10,648,025 and WO 2020 / 112869. Experiments were performed using 200 strands (2e2) or 20 strands (2e1) of the calibrator as positive controls, or 200 ng of polyA as a template-free control (NTC). LQAS valve assay products were treated with calf intestinal alkaline phosphatase (CIP) to dephosphorylate residual dNTPs, followed by kinase treatment with polynucleotide kinase (PNK) to 5′ phosphorylate the cleaved valve molecules prior to the ligation step. This method was compared to the preparation of ligated molecules without CIP / PNK treatment.

[0598] Oligonucleotides and phosphorylated cyclizable molecules (sequencing library vectors) were specifically bridged using a 300pM arm and ligated as described above. After ligation, the reaction mixture was processed as described above, and 20 cycles of index PCR were performed, followed by sequencing of the products. Reads from the sequencing data are summarized in […]. Figure 10 These data show that, under all conditions, the use of CIP and PNK significantly improved assay sensitivity, as evidenced by the increased number of reads in all three MDMs.

[0599] In some implementations, other options for improving assay sensitivity include using 3′-terminal ribonucleotides on the bridging oligonucleotide, and mismatches between the bridging oligonucleotide and cyclizable molecules can also be used as an alternative or supplement to: lowering the ligation temperature, using bridging oligonucleotides modified with nucleotides that inhibit DNA polymerase activity, and / or using CIP and PNK or proteinase K treatment to improve assay sensitivity.

[0600] Nucleotide exonuclease treatment

[0601] Non-ligated acyclic materials (e.g., cyclizable molecules, residual primers, non-specific amplification products, etc.) can cause undesirable non-specific signals in downstream assays. For example, non-ligated acyclic materials can form primer dimers, which are undesirably amplified, generating non-specific signals. To remove these products from the reaction mixture, for example, before indexing PCR, treatment with one or more exonucleases can be performed after ligation.

[0602] The ligation reaction, comprising the LQAS reaction product and a cyclizable molecule, was performed as described above, but with an additional exonuclease treatment step prior to indexing PCR. The ligation reaction was carried out using different ratios and concentrations of bridging oligonucleotides:cyclizable molecules (e.g., splice:vector), covering a 10-fold dilution series of splice:vector concentrations. 200 ng of PolyA was used as a non-target control nucleic acid. The exonuclease was added to the ligation reaction mixture as described above, and the product was amplified and quantified; the results are shown below. Figure 11 In the middle. For example Figure 11 As shown, under all conditions, the addition of an exonuclease treatment step reduced nonspecific signaling, including at the highest concentration (4000 pM) of bridging oligonucleotides:cyclizable molecules. These data indicate that exonuclease treatment reduces nonspecific signaling when the target is not present.

[0603] ligase fidelity

[0604] Using high-fidelity ligases (with limited single-stranded DNA ligation activity) is another method to minimize or prevent unintended self-ligation of carrier molecules (cyclizable molecules or carrier pairs) in the absence of cleavage of flap oligonucleotides. Assays are performed as described above, and the ligation mixture is treated with T4 DNA ligase, HiFi Taq DNA ligase, Taq DNA ligase, or E. coli DNA ligase. Analysis of the ligation products is shown below. Figures 12A-12C middle.

[0605] Figure 12A This is a bioanalyzer gel image showing the final product after cleavage of the lobe oligonucleotide ligation and amplification enrichment by circularized probes. The top band (approximately 285 bp) represents the expected target-specific product (ligation on the target strand), while the bottom band rep...

Claims

1. A method comprising: a) A reaction mixture comprising or suspected to comprise a population of different target nucleic acids, multiple valve-shaped oligonucleotides, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises: i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion; In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed. In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to generate a population of cleavage flaps; b) Providing a population of linker molecules, wherein the linker molecules comprise sequences of the population of the cleavage lobes and / or sequences complementary to the population of the cleavage lobes, and c) Characterize the population of the linker molecules to characterize the population of the different target nucleic acids present in the reaction mixture.

2. The method of claim 1, wherein the connecting molecule is a cyclic molecule.

3. The method of claim 1, wherein the connecting molecule is a linear molecule.

4. The method of claim 1, wherein providing the population of linking molecules comprises treating the population of cleavage flaps with a linkable carrier molecule formulation, wherein the cleavage flaps are linked to the linkable carrier molecules to form the population of linking molecules, each linking molecule comprising a carrier molecule sequence and a cleavage flap sequence.

5. The method of any one of claims 1-4, wherein the reaction mixture is an amplification reaction mixture, and wherein the population of different target nucleic acids comprises amplified nucleic acids generated in the amplification reaction mixture.

6. The method of claim 5, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

7. The method of any one of claims 1-6, wherein characterizing the population of the linker molecules comprises counting the different cleavage sequences or their complementary sequences present in the population of the linker molecules.

8. The method of claim 7, wherein characterizing the population of linker molecules comprises sequencing the cleavage lobe sequence or its complementary sequence in the population of linker molecules.

9. The method according to any one of claims 1-8, wherein the valve endonuclease is a FEN-1 endonuclease.

10. The method of claim 9, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture contains Mg. ++ Lobe-shaped assay buffer.

11. The method of claim 10, wherein the strict FEN-1 endonuclease is selected from the group consisting of: *Afu* FEN-1, *Pfu* FEN-1, and *Ave* FEN-1 and its variants, and CLEAVASE 2.0 nuclease.

12. The method of any one of claims 1-11, wherein each linker in the group of linkers comprises one or more of the following: i) A first bridging oligonucleotide complementary arm located at the 3′ end of the first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of the second vector sequence, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm. ii) A pair of amplification primer binding sites configured to amplify a ligand molecule containing a cleavage lobe sequence; iii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification; iv) At least one unique molecular tag; v) Polynucleotide linkers; as well as vi) Polymerization terminator, preferably an internal spacer region.

13. The method of any one of claims 1-12, further comprising the step of amplifying the region of the linker molecule containing the cleavage lobe sequence to form a population of lobe amplicones.

14. The method of claim 13, further comprising sequencing the lobe amplicon to generate sequencing reads.

15. The method of any one of claims 4-14, wherein the connectable carrier molecule comprises one or more of a dumbbell probe, a padlock probe, a carrier pair, and / or a linear carrier molecule.

16. The method of any one of claims 4-15, wherein the connectable carrier molecule comprises a carrier pair, wherein each carrier pair comprises a first bridging oligonucleotide complementary arm located at the 3′ end of a first carrier sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of a second carrier sequence, and wherein step b) comprises contacting the population of the cleavage flaps with the connectable carrier molecule formulation and the population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 3′ end complementary to the 3′ end of the first carrier sequence, and a 5′ end complementary to the 5′ end of the second carrier sequence.

17. The method of claim 16, wherein each of the connectable carrier molecules at the 5′ end of the second carrier sequence comprises a 5′ blocking group.

18. The method of any one of claims 4-15, wherein the connectable carrier molecule is a dumbbell probe, wherein each dumbbell probe comprises a bridging sequence complementary to the cleavage lobe sequence.

19. The method of any one of claims 1-15 and 17-18, wherein the linker molecule comprises a cyclic molecule, and the method further comprises the step of using rolling circle replication to replicate the cyclic molecule comprising a cleavage lobe sequence to form a population of lobe replicons.

20. The method of any one of claims 1-19, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 10 unique valve oligonucleotides.

21. The method of claim 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 20 unique valve oligonucleotides.

22. The method of claim 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 30 unique valve oligonucleotides.

23. The method of claim 20, wherein the plurality of valve oligonucleotides in the reaction mixture comprises 40 to 1000 unique valve oligonucleotides.

24. The method of any one of claims 1-23, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

25. The method of claim 24, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is 13 to 100 nucleotides in length.

26. The method of any one of claims 1-25, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

27. The method of any one of claims 1-26, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

28. The method of claim 27, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid.

29. The method of claim 28, wherein the oligonucleotide comprises a flap oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

30. The method of claim 28 or claim 29, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

31. The method of claim 30, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

32. The method of any one of claims 28 to 31, wherein the reference nucleic acid comprises methylation. B3GALT6 DNA.

33. A composition comprising: i) One or both of the following: a) Groups of cleavage flaps; and b) A group of linker molecules, each linker molecule containing a cleavage valve sequence or its complementary sequence, wherein each cleavage valve sequence or its complementary sequence contains a nucleotide sequence indicating a target nucleic acid; as well as ii) Ligase.

34. The composition of claim 33, further comprising a population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

35. The composition of claim 33, further comprising a linker molecule, wherein each linker molecule comprises at least one linker molecule sequence.

36. The composition of claim 33 or claim 35, further comprising a group of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

37. The composition of claim 36, further comprising one or more of the following: i) A first linkable carrier molecule and a second linkable carrier molecule, the second linkable carrier molecule comprising a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the first linkable carrier molecule and the second linkable carrier molecule in a complex, the complex comprising: I) A first connectable slit, comprising the 3' end of the first connectable molecule and the 5' end of the cutting flap, and II) An invasive cleavage substrate comprising the 3' end of the cleavage flap and the 5' end of the second connectable carrier molecule comprising the 5' blocking group. The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups; ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions; iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications; iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease; v) Mg ++ Lobe-shaped assay buffer solution; vi) Amplification reagents; vii) Lobe-like assay reagent; and / or viii) Ligases, including the Chlorella virus PBCV-1 DNA ligase.

38. The composition of any one of claims 33-37, wherein each linker in the group of linker molecules comprises one or more of the following: i) A pair of amplification primer binding sites configured as part of a cleavage lobe sequence in an amplification ligand molecule; ii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification; iii) At least one unique molecular tag; iv) Polynucleotide linkers; and / or v) Polymerization terminator, preferably an internal spacer region.

39. The composition of any one of claims 33 to 38, further comprising a population of amplification products, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in a linker molecule of the population of linkers.

40. A method comprising: a) Providing in the mixture a bridging oligonucleotide, a valve sequence oligonucleotide, preferably a cleaved valve oligonucleotide, a first linker molecule, and a second linker molecule containing a 5′ blocking group, wherein the bridging oligonucleotide comprises: i) Internal segments complementary to the said lobe sequence oligonucleotides, ii) The 3' end complementary to the 3' end of the first connectable carrier molecule, and iii) The 5' end complementary to the 5' end of the second connectable carrier molecule. In the mixture, the bridging oligonucleotide hybridizes with the lobe sequence oligonucleotide, the first linker molecule, and the second linker molecule to form a complex, the complex comprising: I) A first connectable nick, comprising the 3′ end of the first connectable molecule and the 5′ end of the valve sequence oligonucleotide, and II) Invasive cleavage of a substrate comprising the 3′ end of the said lobe sequence oligonucleotide and the 5′ end of the second linkable vector molecule comprising the said 5′ blocking group; b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate cleaves the 5′ blocking group of the second linkable carrier molecule and creates a second linkable nick; and c) Treat the complex with a ligase, wherein the first ligase and the second ligase are joined to form a linker molecule.

41. The method of claim 40, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

42. The method of claim 40 or 41, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

43. The method of claim 42, wherein the RNA portion comprises one or more nucleotide modifications.

44. The method of claim 43, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modification.

45. The method of claim 43 or claim 44, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

46. ​​The method of claim 43, wherein the 3′ end and adjacent portion of the inner segment of the bridging oligonucleotide comprise DNA.

47. The method of any one of claims 40-46, further comprising detecting the linker molecule in a nucleic acid detection assay.

48. The method of any one of claims 40-47, wherein the ligase comprises Chlorella virus PBCV-1 DNA ligase.

49. A method comprising: a) A reaction mixture comprising or suspected to comprise a population of different target nucleic acids, multiple valve-shaped oligonucleotides, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises: i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion; In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed. In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to generate a population of cleavage flaps; b) Providing a population of cyclic molecules, wherein the cyclic molecules comprise sequences of the population of the cleavage lobes and / or sequences complementary to the population of the cleavage lobes, and c) Characterize the population of the cyclic molecules to characterize the population of the different target nucleic acids present in the reaction mixture.

50. The method of claim 49, wherein characterizing the population of the cyclic molecules includes detecting hybridization of the cleavage flap with the cyclic molecules.

51. The method of claim 49, wherein providing the population of the cyclic molecules comprises processing the population of the cleavage lobes, wherein the 5′ and 3′ ends of individual cleavage lobes are joined together to form cyclic molecules.

52. The method of claim 49, wherein providing the population of cyclic molecules comprises treating the population of cleavage flaps with a cyclizable molecular formulation, wherein the cleavage flaps are linked to the cyclizable molecules to form the population of cyclic molecules, each cyclic molecule comprising a cyclizable molecular sequence and a cleavage flap sequence.

53. The method of any one of claims 49-52, wherein the reaction mixture is an amplification reaction mixture, and wherein the population of different target nucleic acids comprises amplified nucleic acids generated in the amplification reaction mixture.

54. The method of claim 53, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

55. The method of any one of claims 49-54, wherein characterizing the population of the cyclic molecules comprises counting the different cleavage sequences or their complementary sequences present in the population of the cyclic molecules.

56. The method of claim 55, wherein characterizing the population of cyclic molecules comprises sequencing the cleavage sequence or its complementary sequence in the population of cyclic molecules.

57. The method according to any one of claims 49-56, wherein the valve endonuclease is a FEN-1 endonuclease.

58. The method of claim 57, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture comprises Mg ++ Lobe-shaped assay buffer.

59. The method of claim 58, wherein the strict FEN-1 endonuclease is selected from the group consisting of: *Afu* FEN-1, *Pfu* FEN-1, and *Ave* FEN-1 and its variants, and CLEAVASE 2.0 nuclease.

60. The method of any one of claims 49-59, wherein each cyclic molecule in the group of cyclic molecules comprises one or more of the following: i) A pair of bridging oligonucleotide complementary arms located at the 5′ and 3′ ends of the cyclizable molecule, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm; ii) A pair of amplification primer binding sites configured to amplify a portion of the cleavage lobe sequence of a circular molecule; iii) Primer binding sites configured to amplify the circular molecule via rolling circle amplification; iv) At least one unique molecular tag; v) Polynucleotide linkers; and / or vi) Polymerization terminator, preferably an internal spacer region.

61. The method of any one of claims 49-60, further comprising the step of amplifying the region of the cyclic molecule containing the cleavage lobe sequence to form a population of lobe amplicones.

62. The method of claim 61, further comprising sequencing the lobe amplicon to generate sequencing reads.

63. The method of any one of claims 52-62, wherein the cyclizable molecule comprises a dumbbell probe and / or a padlock probe.

64. The method of any one of claims 52-63, wherein each of the cyclizable molecules comprises a pair of bridging oligonucleotide complementary arms located at the 5′ end and the 3′ end of the cyclizable molecule, and wherein step b) comprises contacting a population of the cleavage flaps with the cyclizable molecule formulation and the population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 5′ end complementary to the 5′ end of the cyclizable molecule, and a 3′ end complementary to the 3′ end of the cyclizable molecule.

65. The method of claim 64, wherein each of the cyclizable molecules comprises a 5′ blocking group.

66. The method of any one of claims 52-65, wherein the cyclizable molecule is a dumbbell probe, wherein each dumbbell probe comprises a bridging sequence complementary to the cleavage lobe sequence.

67. The method of any one of claims 49-66, further comprising the step of using rolling ring replication to replicate the circular molecule containing the cleavage lobe sequence to form a population of lobe replicons.

68. The method of any one of claims 49-67, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 10 unique valve oligonucleotides.

69. The method of claim 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 20 unique valve oligonucleotides.

70. The method of claim 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises at least 30 unique valve oligonucleotides.

71. The method of claim 68, wherein the plurality of valve oligonucleotides in the reaction mixture comprises 40 to 1000 unique valve oligonucleotides.

72. The method of any one of claims 49-71, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

73. The method of claim 72, wherein the target-specific portion of the plurality of valve oligonucleotides in the reaction mixture is 13 to 100 nucleotides in length.

74. The method of any one of claims 49-73, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

75. The method of any one of claims 49-74, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

76. The method of claim 75, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid.

77. The method of claim 76, wherein the oligonucleotide comprises a lobular oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

78. The method of claim 76 or claim 77, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

79. The method of claim 78, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

80. The method of any one of claims 76 to 79, wherein the reference nucleic acid comprises methylation. B3GALT6 DNA.

81. A composition comprising: i) One or both of the following: a) Groups of cleavage flaps; and b) A group of circular molecules, each circular molecule containing a cleavage sequence or its complement, wherein each cleavage sequence or its complement contains a nucleotide sequence indicating a target nucleic acid; as well as ii) Ligase.

82. The composition of claim 81, further comprising a population of bridging oligonucleotides, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

83. The composition of claim 81, further comprising cyclizable molecules, wherein each cyclic molecule comprises a cyclizable molecular sequence.

84. The composition of claim 81 or claim 83, further comprising a group of bridging oligonucleotides, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the cyclizable molecule, and a 5′ end complementary to the 5′ end of the cyclizable molecule.

85. The composition of claim 84, further comprising one or more of the following: i) A cyclizable carrier molecule containing a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the cyclizable molecule to form a complex, the complex comprising: I) A first connectable slit, comprising the 3' end of the cyclizable molecule and the 5' end of the cleavage flap, and II) An invasive cleavage substrate comprising the 3′ end of the cleavage flap and the 5′ end of the cyclizable molecule comprising the 5′ blocking group; The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups; ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions; iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications; iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease; v) Mg ++ Lobe-shaped assay buffer solution; vi) Amplification reagents; vii) Lobe-like assay reagent; and / or viii) Ligases, including the Chlorella virus PBCV-1 DNA ligase.

86. The composition of any one of claims 81-85, wherein each cyclic molecule in the group of cyclic molecules comprises one or more of the following: i) A pair of amplification primer binding sites configured to amplify a circular molecule containing a cleavage lobe sequence; ii) Primer binding sites configured to amplify circular molecules via rolling circle amplification; iii) At least one unique molecular tag; iv) Polynucleotide linkers; and / or v) Polymerization terminator, preferably an internal spacer region.

87. The composition of any one of claims 81-86, further comprising a population of amplification products, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in a circular molecule within the population of circular molecules.

88. A method comprising: a) Providing a bridged oligonucleotide, a valve sequence oligonucleotide, preferably a valve-cleaving oligonucleotide, and a cyclizable molecule containing a 5′ blocking group in the mixture, wherein the bridged oligonucleotide comprises: i) Internal segments complementary to the said lobe sequence oligonucleotides, ii) The 3′ end complementary to the 3′ end of the cyclizable molecule, and iii) The 5' end complementary to the 5' end of the cyclizable molecule. In the mixture, the bridging oligonucleotide hybridizes with the valve sequence oligonucleotide and the cyclizable molecule to form a complex, the complex comprising: I) A first connectable nick, comprising the 3′ end of the cyclizable molecule and the 5′ end of the valve sequence oligonucleotide, and II) Invasive cleavage of a substrate comprising the 3′ end of the said valve sequence oligonucleotide and the 5′ end of the said cyclizable molecule comprising the said 5′ blocking group; b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate is cleaved to remove the 5′ blocking group and create a second connectable nick; and c) Treat the complex with a ligase such that the first ligase and the second ligase are joined to form a cyclic molecule.

89. The method of claim 88, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

90. The method of claim 88 or 89, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

91. The method of claim 90, wherein the RNA portion comprises one or more nucleotide modifications.

92. The method of claim 91, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modification.

93. The method of claim 91 or claim 92, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

94. The method of claim 93, wherein the 3′ end and the adjacent portion of the inner segment of the bridging oligonucleotide comprise DNA.

95. The method of any one of claims 93-94, further comprising detecting the cyclic molecule in a nucleic acid detection assay.

96. The method of any one of claims 88-95, wherein the ligase comprises Chlorella virus PBCV-1 DNA ligase.

97. A method comprising: a) A reaction mixture comprising or suspected to comprise at least one target nucleic acid, at least one valve-shaped oligonucleotide, and a valve-shaped endonuclease, wherein each valve-shaped oligonucleotide comprises: i) A target-specific portion, wherein if the target nucleic acid is present in the reaction mixture, the target-specific portion is annealed with the target nucleic acid, and ii) A 5′ petal-shaped portion containing a nucleotide sequence indicating annealing of the target nucleic acid with the target-specific portion; In this process, for the target nucleic acid present in the reaction mixture, an invasive cleavage structure comprising the target nucleic acid annealed with the petal oligonucleotide is formed. In the reaction mixture, the invasive cleavage structure is cleaved by the flap endonuclease to produce at least one cleavage flap. b) Provide at least one linker molecule, wherein the linker molecule comprises a sequence of the cleavage lobe and / or a sequence complementary to the cleavage lobe, and c) Characterize the at least one linker molecule to characterize the at least one target nucleic acid present in the reaction mixture.

98. The method of claim 97, wherein the connecting molecule is a cyclic molecule.

99. The method of claim 97, wherein the connecting molecule is a linear molecule.

100. The method of claim 97, wherein providing the linker molecule comprises treating the cleavage flap with a formulation comprising a linkable carrier molecule, wherein the cleavage flap is linked to the linkable carrier molecule to form a linker molecule, each linker molecule comprising a carrier molecule sequence and a cleavage flap sequence.

101. The method of any one of claims 97-100, wherein the reaction mixture is an amplification reaction mixture, and wherein the at least one target nucleic acid comprises at least one amplified nucleic acid generated in the amplification reaction mixture.

102. The method of claim 101, wherein the amplification reaction mixture is a PCR-valve assay reaction mixture.

103. The method of any one of claims 97-102, wherein characterizing the at least one linker molecule comprises counting different cleavage sequences or their complementary sequences if present in the reaction mixture.

104. The method of claim 103, wherein characterizing the at least one linker molecule comprises sequencing a cleavage lobe sequence or its complementary sequence if present in the reaction mixture.

105. The method according to any one of claims 97-104, wherein the valve endonuclease is a FEN-1 endonuclease.

106. The method of claim 105, wherein the FEN-1 endonuclease is a strictly FEN-1 endonuclease, and wherein the reaction mixture comprises Mg ++ Lobe-shaped assay buffer.

107. The method of claim 106, wherein the strict FEN-1 endonuclease is selected from the group consisting of: *Afu* FEN-1, *Pfu* FEN-1, and *Ave* FEN-1 and its variants, and CLEAVASE 2.0 nuclease.

108. The method of any one of claims 97-107, wherein each linker molecule comprises one or more of the following: i) A first bridging oligonucleotide complementary arm located at the 3′ end of the first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of the second vector sequence, wherein the nucleotide sequence of the first bridging oligonucleotide complementary arm is different from that of the second bridging oligonucleotide complementary arm. ii) A pair of amplification primer binding sites configured to amplify a ligand molecule containing a cleavage lobe sequence; iii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification; iv) At least one unique molecular tag; v) Polynucleotide linkers; and / or vi) Polymerization terminator, preferably an internal spacer region.

109. The method of any one of claims 97-108, further comprising the step of amplifying the region of the at least one linker molecule containing the cleavage lobe sequence to form at least one lobe amplicon.

110. The method of claim 109, further comprising sequencing the at least one lobe amplicon to generate sequencing reads.

111. The method of any one of claims 100-110, wherein the connectable carrier molecule is selected from dumbbell probes, padlock probes, carrier pairs and / or linear carrier molecules.

112. The method of any one of claims 100-111, wherein the connectable vector molecule comprises a vector pair, the vector pair comprising a first bridging oligonucleotide complementary arm located at the 3′ end of a first vector sequence and a second bridging oligonucleotide complementary arm located at the 5′ end of a second vector sequence, and wherein step b) comprises contacting the at least one cleavage flap with the connectable vector molecule and the bridging oligonucleotide, wherein the bridging oligonucleotide comprises an inner segment complementary to the cleavage flap sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

113. The method of claim 112, wherein the connectable carrier molecule comprising the 5′ end of the second carrier sequence contains a 5′ blocking group.

114. The method of any one of claims 100-111, wherein the connectable carrier molecule is a dumbbell probe comprising a bridging sequence complementary to the cleavage lobe sequence.

115. The method of any one of claims 97-111 and 114, wherein the linker molecule is a cyclic molecule, the method further comprising the step of using rolling circle replication to replicate the cyclic molecule containing the cleavage lobe sequence to form a lobe replicon.

116. The method of any one of claims 97-115, wherein the target-specific portion of the valve oligonucleotide in the reaction mixture is at least 6 nucleotides in length, preferably at least 7, preferably at least 8, preferably at least 9, preferably at least 10, preferably at least 11, preferably at least 12 nucleotides in length.

117. The method of claim 116, wherein the target-specific portion of the valve oligonucleotide in the reaction mixture is 13 to 100 nucleotides in length.

118. The method of any one of claims 97-117, wherein the target nucleic acid comprises one or more of cDNA, amplified DNA, genomic DNA, and circulating cell-free DNA.

119. The method of any one of claims 97-118, wherein the target nucleic acid comprises DNA treated with a methylation-specific reagent.

120. The method of claim 119, wherein the reaction mixture further comprises an oligonucleotide specific to a reference nucleic acid, the reference nucleic acid preferably comprising methylation. B3GALT6 DNA.

121. The method of claim 120, wherein the oligonucleotide comprises a lobular oligonucleotide, an invasive oligonucleotide, and optionally a non-invasive oligonucleotide.

122. The method of claim 120 or claim 121, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is changed relative to the concentration of a corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture, and / or wherein one or more of the oligonucleotides specific to the reference nucleic acid bind to the reference nucleic acid with one or more mismatched base pairs.

123. The method of claim 122, wherein the concentration of one or more of the oligonucleotides specific to the reference nucleic acid in the reaction mixture is reduced relative to the concentration of the corresponding oligonucleotide specific to the target nucleic acid in the reaction mixture.

124. A composition comprising: i) One or both of the following: a) at least one cutting flap; and b) At least one linker molecule comprising a cleavage lobe sequence or its complementary sequence, wherein the cleavage lobe sequence or its complementary sequence comprises a nucleotide sequence indicating a target nucleic acid; as well as ii) Ligase.

125. The composition of claim 124, further comprising at least one bridging oligonucleotide, wherein each bridging oligonucleotide comprises a 5′ end sequence complementary to the 5′ end of the cleavage sequence and a 3′ end sequence complementary to the 3′ end of the cleavage sequence, wherein the bridging oligonucleotide hybridizes with a cleavage if present in the composition to form a connectable cleavage.

126. The composition of claim 124, further comprising at least one linker molecule, wherein each linker molecule comprises at least one linker molecule sequence.

127. The composition of claim 124 or claim 126, further comprising at least one bridging oligonucleotide, wherein each bridging oligonucleotide comprises an inner segment complementary to the cleavage lobe sequence, a 3′ end complementary to the 3′ end of the first vector sequence, and a 5′ end complementary to the 5′ end of the second vector sequence.

128. The composition of claim 127, further comprising one or more of the following: i) A first linkable carrier molecule and a second linkable carrier molecule, the second linkable carrier molecule comprising a 5′ blocking group, wherein in the composition, the bridging oligonucleotide hybridizes with the cleavage flap and the first linkable carrier molecule and the second linkable carrier molecule in a complex, the complex comprising: I) A first connectable slit, comprising the 3' end of the first connectable molecule and the 5' end of the cutting flap, and II) An invasive cleavage substrate comprising the 3' end of the cleavage flap and the 5' end of the second connectable carrier molecule comprising the 5' blocking group. The 5′ blocking group preferably includes a subset selected from the following: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups; ii) Bridged oligonucleotides containing one or more of the RNA and DNA portions; iii) A bridging oligonucleotide comprising an RNA moiety comprising one or more nucleotide modifications, preferably selected from 2′-O-methyl and 2′-O-methoxyethyl nucleotide modifications; iv) A valve-shaped endonuclease, preferably a FEN-1 endonuclease, preferably a strict FEN-1 endonuclease, preferably a strict FEN-1 endonuclease selected from the group consisting of: *A. scintillans* FEN-1, *P. flamingococcus* FEN-1 and *A. ve* FEN-1 and their variants, and CLEAVASE 2.0 nuclease; v) Mg ++ Lobe-shaped assay buffer solution; vi) Amplification reagents; vii) Lobe-like assay reagent; and / or viii) Ligases, including the Chlorella virus PBCV-1 DNA ligase.

129. The composition of any one of claims 124-128, wherein the linker molecule comprises one or more of the following: i) A pair of amplification primer binding sites configured as part of a cleavage lobe sequence in an amplification ligand molecule; ii) Primer binding sites configured to amplify circular linker molecules via rolling circle amplification; iii) At least one unique molecular tag; iv) Polynucleotide linkers; and / or v) Polymerization terminator, preferably an internal spacer region.

130. The composition of any one of claims 124 to 129, further comprising at least one amplification product, each amplification product comprising a target nucleic acid sequence associated with a cleavage lobe sequence in the linker molecule.

131. A method comprising: a) Providing in the mixture a bridging oligonucleotide, a valve sequence oligonucleotide, preferably a cleaved valve oligonucleotide, a first linker molecule, and a second linker molecule containing a 5′ blocking group, wherein the bridging oligonucleotide comprises: i) Internal segments complementary to the said lobe sequence oligonucleotides, ii) The 3' end complementary to the 3' end of the first connectable carrier molecule, and iii) The 5' end complementary to the 5' end of the second connectable carrier molecule. In the mixture, the bridging oligonucleotide hybridizes with the lobe sequence oligonucleotide, the first linker molecule, and the second linker molecule to form a complex, the complex comprising: I) A first connectable nick, comprising the 3′ end of the first connectable molecule and the 5′ end of the valve sequence oligonucleotide, and II) Invasive cleavage of a substrate comprising the 3′ end of the said lobe sequence oligonucleotide and the 5′ end of the second linkable vector molecule comprising the said 5′ blocking group; b) Treat the complex with a valve-shaped endonuclease, wherein the invasive cleavage substrate cleaves the 5′ blocking group of the second linkable carrier molecule and creates a second linkable nick; and c) Treat the complex with a ligase, wherein the first ligase and the second ligase are joined to form a linker molecule.

132. The method of claim 131, wherein the 5′ blocking group comprises a portion selected from: hexane, non-standard nucleotides, baseless nucleotides, fluorophores, valve-shaped nucleic acid sequences, hairpin valve-shaped nucleic acid sequences, and hydroxyl groups.

133. The method of claim 131 or 132, wherein the bridging oligonucleotide comprises one or more of an RNA portion and a DNA portion.

134. The method of claim 133, wherein the RNA portion comprises one or more nucleotide modifications.

135. The method of claim 134, wherein the one or more nucleotide modifications include 2′-O-methyl or 2′-O-methoxyethyl modifications.

136. The method of claim 134 or claim 133, wherein the 5′ end and adjacent portion of the internal segment of the bridged oligonucleotide comprises RNA, 2′-O-methylRNA or 2′-O-methoxyethylRNA.

137. The method of claim 134, wherein the 3′ end and adjacent portion of the inner segment of the bridging oligonucleotide comprise DNA.

138. The method of any one of claims 131-137, further comprising detecting the linker molecule in a nucleic acid detection assay.

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