Compositions, kits, and methods for isolating target polynucleotides

Abstract

This document relates to compositions, kits, and methods for isolating target polynucleotides, and provides oligomers, compositions, kits, and methods for capturing target polynucleotides, for example, for downstream applications such as amplification, library preparation, or sequencing. In some embodiments, a capture oligomer comprising a capture sequence annealed to a complementary sequence is provided or used, the capture sequence preventing capture until the complementary sequence is substituted in a target-polynucleotide-dependent manner. In some embodiments, the amount of target polynucleotide captured is less than or equal to a predetermined amount.

Description

[0001] This patent application is a divisional application of the patent application with application number 2021800201009, application date January 15, 2021, entitled "Composition, kit and method for isolating target polynucleotides".

[0002] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 961,816, filed January 16, 2020; UK Patent Application No. 2000673.0, filed January 16, 2020; and UK Patent Application No. 2000672.2, filed January 16, 2020, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] The embodiments described herein relate to the isolation of target polynucleotides, such as polynucleotides and amplicones, from a composition. These embodiments can be used as part of, for example, a workflow to prepare target polynucleotides for sequencing or other analyses. Background Technology

[0004] Certain biochemical and molecular biology procedures benefit from or require a defined amount of input nucleic acid. For example, sequencing library preparation procedures may have a range of acceptable amounts of nucleic acid, where amounts below the minimum result in wasted bandwidth and low data output, while amounts above the maximum result in poor library quality. Furthermore, it is generally desirable to use a predetermined amount of library nucleic acid (often expressed as "number of molecules") for the cloning amplification step (if used) in sequencing workflows to avoid both large clonal populations and wasted bandwidth and low data output; and to use a predetermined amount of library nucleic acid for single-molecule sequencing workflows, where a defined number of molecules is expected to be input to the sequencing step for optimal results. For time-critical applications, such as rapid pathogen detection through sequencing of clinical samples, existing methods for providing samples with acceptable amounts of nucleic acid (e.g., involving quantification procedures and subsequent concentration or dilution steps) may undesirably slow down. In addition, various existing methods may suffer from other problems, such as excessive capture oligomers saturating solid vectors and the complexities associated with adding adaptors or other sequences. Summary of the Invention

[0005] Therefore, there is a need for compositions and methods that can provide improved nucleic acid capture, including (e.g.) rapid and accurate capture of nucleic acids in a controlled or restricted manner (e.g., at a predetermined amount less than or equal to); capture of oligomers that can be captured in a target-dependent manner; and efficient addition of adaptors or other sequences. This disclosure aims to provide compositions and methods that meet one or more of these needs, provide other beneficial effects, or at least provide useful options to the public. This document provides capture oligomers, combinations of capture oligomers with other oligomers, and related compositions, kits, and methods for capturing and / or controlling the amount of nucleic acids, such as wherein a predetermined amount of the capture oligomer or another restrictive agent (e.g., a second capture agent) or combinations thereof can control the output of the capture procedure, and the capture oligomer can be captured in a target-dependent manner; and / or efficiently add adaptors or other sequences.

[0006] More specifically, this document provides the following embodiments. Embodiment 1 is a trapping oligomer comprising, in the 5' to 3' direction:

[0007] Capture sequence,

[0008] Internal extension blocking element,

[0009] The complementary sequence of the captured sequence, and

[0010] Target hybridization sequence,

[0011] The complementary sequence of the captured sequence is configured to anneal with the captured sequence when there is no extended target sequence that anneals with the target hybridization sequence and the complementary sequence of the captured sequence.

[0012] Implementation scheme 2 is the capturing oligomer described in implementation scheme 1, wherein the capturing oligomer has the following formula:

[0013] 5'-A1-CLB-A2-C'-A3-RB-A4-THS-X-3',

[0014] Where A1 is the first additional sequence that exists arbitrarily;

[0015] C represents the capture sequence.

[0016] L is an optional connector.

[0017] B is an internally extended blocking element.

[0018] A2 is an arbitrarily existing second additional sequence.

[0019] C' is the complementary sequence to the captured sequence.

[0020] A3 is an arbitrarily existing third additional sequence.

[0021] RB is an arbitrarily existing reversible extension blocking element.

[0022] A4 is an arbitrarily existing fourth additional sequence.

[0023] THS is the target hybridization sequence; and

[0024] X represents any arbitrarily existing blocking component.

[0025] Implementation scheme 3 is the capture oligomer of any one of the preceding implementation schemes, wherein the capture sequence comprises a polyA or polyT sequence, and the complementary sequence of the capture sequence comprises a polyT or polyA sequence.

[0026] Implementation scheme 4 is the capture oligomer described in any of the preceding implementation schemes, wherein the capture oligomer comprises a first additional sequence located at the 5' of the capture sequence and a third additional sequence located at the 3' of the complementary sequence of the capture sequence, which respectively comprise a first stable sequence and a second stable sequence, optionally wherein the stable sequence is a GC clip sequence.

[0027] Implementation scheme 5 is the capture oligomer described in the previous implementation scheme, wherein the first stable sequence is located at the 5' of the remainder of the capture sequence and / or the second stable sequence is located at the 3' of the remainder of the complementary sequence of the capture sequence.

[0028] Implementation scheme 6 is the capture oligomer described in implementation scheme 4 or 5, wherein each of the GC clip sequences contains a (GC)3 sequence or a (CG)3 sequence.

[0029] Embodiment 7 is a capture oligomer as described in any of the preceding embodiments, comprising a linker located between the capture sequence and the internal extension blocker, the linker optionally being a nucleotide sequence or a non-nucleotide linker or a combination thereof.

[0030] Implementation scheme 8 is the capture oligomer described in any one of the preceding implementation schemes, wherein the internal extension blocking member comprises any one or more of the following: a non-nucleotide linker, or one or more debasement sites, a non-natural nucleotide, or a chemically modified natural nucleotide.

[0031] Implementation scheme 9 is a capture oligomer as described in any of the preceding implementation schemes, comprising a third additional sequence comprising a linker sequence located between the complementary sequence of the capture sequence and THS.

[0032] Implementation scheme 10 is the capture oligomer described in any of the preceding implementation schemes, wherein the capture oligomer contains a reversible extension blocking member located at the 5' of the target hybridization sequence.

[0033] Implementation scheme 11 is the capture oligomer described in the previous implementation scheme, wherein the reversible extension blocking member is located at the 3' of the complementary sequence of the capture sequence.

[0034] Implementation scheme 12 is the capture oligomer described in implementation scheme 10 or 11, wherein the capture oligomer comprises a third additional sequence located at the 3' of the complementary sequence of the capture sequence and the 5' of the target hybridization sequence, and a reversible extension blocking member is located at the 3' of the linker sequence, optionally wherein the third additional sequence comprises the linker sequence.

[0035] Implementation scheme 13 is a capture oligomer as described in any of the preceding implementation schemes, wherein the capture oligomer comprises a fourth additional sequence located at the 3' of the reversible blocking sequence and the 5' of the target hybridization sequence, optionally wherein the fourth additional sequence comprises a linker sequence.

[0036] Embodiment 14 is a capture oligomer as described in any of the preceding embodiments, comprising a second additional sequence located between the internal extension blocker and the complementary sequence of the capture sequence, optionally wherein the second additional sequence comprises a mixed nucleotide segment.

[0037] Implementation Scheme 15 is the capture oligomer described in the preceding embodiment, wherein the reversible extension blocking factor includes: Iso-dC or Iso-dG, xanthine or 5-(2,4-diaminopyrimidine), 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4-methylbenzimidazole or 2,4-difluorotoluene, 7-azaindole or isoquinolone, dMMO2 or d5SICS, dF or dQ; one or more chemically modified nucleotides, wherein the modification is by attachment via a reversible linker bond, and said linker bond can be reversed by providing any one or more of the following: chemicals, enzymes, temperature changes, reagent composition changes; reversible nucleic acid structural features; or molecules that reversibly bind to the capture oligomer, optionally wherein the reversibly binding molecule is a protein, enzyme, lipid, carbohydrate, or chemical moiety.

[0038] Implementation scheme 16 is a capture oligomer as described in any of the preceding implementation schemes, wherein the target hybridization sequence includes a blocking portion at its 3' end.

[0039] Implementation Scheme 17 is a capture oligomer as described in any of the preceding implementation schemes, comprising one or more affinity-enhancing modifications located in the target hybridization sequence (e.g., any one or more of 5-Me-C, 2-aminopurine, 2′-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2′-OMe, or restricted ethyl (cEt) substitution).

[0040] Implementation scheme 18 is a combination comprising the capturing oligomer and the second capturing agent as described in any of the preceding embodiments, the second capturing agent comprising a complementary sequence to the capturing sequence and (a) a binding partner (e.g., biotin) or (b) a solid carrier (e.g., beads or surface).

[0041] Implementation 19 is the combination described in the preceding implementation, wherein the combination further comprises a second capture oligomer and a second capture agent, wherein the second capture oligomer comprises a second target hybridization sequence different from the target hybridization sequence of the capture oligomer and a second capture sequence different from the capture sequence of the capture oligomer, and the second capture agent comprises a complementary sequence of the second capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid carrier (e.g., beads or surface).

[0042] Implementation 20 is the combination described in Implementation 18, wherein the second capture agent comprises a complementary sequence of the capture sequence and a binding partner (e.g., biotin), and the combination further comprises a solid carrier (e.g., beads) containing a second binding partner (e.g., a biotin binder, such as streptavidin) configured to bind to the binding partner of the second capture agent.

[0043] Implementation scheme 21 is a combination comprising any one of the preceding implementation schemes, wherein the capturing oligomer comprises a linker sequence located at the 5' of the target hybridization sequence, and the combination further comprises a non-extendable blocking oligomer comprising a linker sequence, optionally wherein the blocking oligomer is configured to bind a complementary sequence of the linker sequence with a greater affinity than the linker sequence of the capturing oligomer, or to form a complex with a complementary sequence of the linker sequence having a higher melting temperature than the complex of the linker sequence of the capturing oligomer and the complementary sequence of the linker sequence.

[0044] Implementation scheme 22 is a combination comprising the capture oligomer or combination of any of the preceding embodiments, wherein the capture oligomer comprises a linker sequence at the 5' of the target hybridization sequence, and the combination further comprises a second oligomer comprising a second target hybridization sequence at the 5' of a complementary sequence at at least a portion of the linker sequence, wherein, in the presence of a target polynucleotide having an accessible complementary sequence of the first and second target hybridization sequences, the capture oligomer and the second oligomer are configured to form a triplet link with the target polynucleotide.

[0045] Embodiment 23 is a reaction mixture comprising the capture oligomer or combination thereof as described in any of the preceding embodiments and the target polynucleotide.

[0046] Embodiment 24 is the reaction mixture described in the preceding embodiment, wherein the target is an amplicon, and optionally further comprises at least one amplification primer, and further optionally, wherein the target hybridization sequence that captures the oligomer has a higher affinity for the amplicon than for the primer (e.g., a longer THS or an affinity-enhancing modification).

[0047] Embodiment 25 is the reaction mixture of any one of Embodiments 23 or 24, further comprising a second capture oligomer, a second capture agent, and a second target polynucleotide, wherein the second capture oligomer comprises a second target hybridization sequence configured to anneal with the second target polynucleotide, and a second capture sequence different from the capture sequence of the capture oligomer, and the second capture agent comprises a complementary sequence of the second capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid carrier (e.g., beads or surface).

[0048] Implementation scheme 26 is the reaction mixture described in the previous implementation scheme, wherein the second target polynucleotide is present at a concentration lower than that of the target polynucleotide.

[0049] Embodiment 27 is the reaction mixture of any one of Embodiments 25 or 26, wherein the target polynucleotide and the second target polynucleotide are isolated from or generated from samples from organisms or environmental types, and the second target polynucleotide is not common in samples from organisms or environmental types.

[0050] Implementation scheme 28 is a combination comprising a capturing oligomer and a complementary oligomer, wherein:

[0051] (a) The captured oligomers contain: in the 5' to 3' direction:

[0052] The captured sequence contains a first part and a second part.

[0053] Internal extension blocking element,

[0054] An interval sequence, comprising a first part and a second part, and

[0055] Target hybridization sequence; and

[0056] (b) The complementary oligomers comprise, in the 3' to 5' direction:

[0057] The complementary sequence of the second part of the captured sequence, and

[0058] The complementary sequence of at least a first portion of the spacer sequence, wherein the complementary sequence of the second portion of the capture sequence and the complementary sequence of at least a first portion of the spacer sequence are configured to anneal with the capture oligomer when the complementary sequence of the spacer sequence is not present.

[0059] Implementation scheme 29 is the combination described in the previous implementation scheme, wherein the captured oligomer has the following formula:

[0060] 5′-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3′

[0061] Where A1 is the first additional sequence that exists arbitrarily.

[0062] C1 is the first part of the capture sequence.

[0063] C2 is the second part of the capture sequence.

[0064] B is an internally extended blocking element.

[0065] A2 is an arbitrarily existing second additional sequence.

[0066] S1 is the first part of the interval sequence.

[0067] S2 is the second part of the interval sequence.

[0068] A3 is an arbitrarily existing third additional sequence.

[0069] RB is an arbitrarily existing reversible extension blocking element.

[0070] A4 is an arbitrarily existing fourth additional sequence.

[0071] THS is the target hybridization sequence, and

[0072] X represents any arbitrarily existing blocking component.

[0073] Implementation scheme 30 is a combination of any one of implementation schemes 28 or 29, wherein the complementary oligomers have the following formula:

[0074] 5′-S1′-A2′-L-C2′-X-3′

[0075] Where S1' is the complementary sequence of at least the first part of the spacer sequence.

[0076] A2' is an optional complementary sequence of a second additional sequence that may be present in the captured oligomer;

[0077] L is an optional connector.

[0078] C2' is the complementary sequence to the second part of the captured sequence, and

[0079] X represents any arbitrarily existing blocking component.

[0080] Implementation scheme 31 is a combination comprising a capturing oligomer and a complementary oligomer, wherein:

[0081] (a) The captured oligomers contain: in the 5' to 3' direction:

[0082] The captured sequence contains a first part and a second part, and

[0083] The target hybridization sequence, comprising a second part and a first part; and

[0084] (b) The complementary oligomers comprise, in the 3' to 5' direction:

[0085] The complementary sequence of the second part of the captured sequence, and

[0086] The complementary sequence of the second part of the target hybridization sequence, wherein the complementary sequence of the second part of the capture sequence and the complementary sequence of the second part of the target hybridization sequence are configured to anneal with the capture oligomer when the complementary sequence of the target hybridization sequence is not present.

[0087] Implementation scheme 32 is the combination described in the previous implementation scheme, wherein the captured oligomer has the following formula:

[0088] 5′-A1-C1-C2-A2-S-A3-THS2-THS1-X-3′

[0089] Where A1 is the first additional sequence that exists arbitrarily.

[0090] C1 is the first part of the capture sequence.

[0091] C2 is the second part of the capture sequence.

[0092] A2 is an arbitrarily existing second additional sequence.

[0093] S is an arbitrarily existing interval sequence.

[0094] A3 is an arbitrarily existing third additional sequence.

[0095] THS2 is the second part of the target hybridization sequence.

[0096] THS1 is the first part of the target hybridization sequence, and

[0097] X represents any arbitrarily existing blocking component.

[0098] Implementation scheme 33 is a combination of any one of implementation schemes 31 or 32, wherein the complementary oligomer has the following formula:

[0099] 5′-THS2′-A3′-S'-A2′-C2′-X-3'

[0100] THS2′ is the complementary sequence to the second part of the target hybridization sequence.

[0101] A3' is an optional complementary sequence to the third additional sequence that may be present in the captured oligomer;

[0102] S' is a complementary sequence of spacers that are arbitrarily present in the captured oligomers.

[0103] A2' is an optional complementary sequence of a second additional sequence that may be present in the captured oligomer;

[0104] C2' is the complementary sequence of the second part of the captured sequence, and

[0105] X represents any arbitrarily existing blocking component.

[0106] Implementation scheme 34 is a combination of any one of implementation schemes 28 to 33, wherein the capturing oligomer and / or complementary oligomer includes a blocking portion at its 3' end.

[0107] Embodiment 35 is a combination of any one of Embodiments 28 to 34, wherein if the spacer sequence of the captured oligomer is occupied by a single complementary sequence, the complementary sequence of the second portion of the captured sequence is insufficient to stably anneal with the captured sequence of the captured oligomer at a temperature of 65°C or above.

[0108] Embodiment 36 is a capture oligomer, reaction mixture, or combination as described in any of the preceding embodiments, wherein the capture sequence comprises poly A or poly T, and the complementary sequence of the capture sequence or the complementary sequence of the second portion of the capture sequence comprises poly T or poly A.

[0109] Embodiment 37 is a capture oligomer, reaction mixture, or combination as described in any of the preceding embodiments, wherein the capture oligomer comprises a linker sequence as part or all of a spacer sequence, or a third additional sequence at the 3' of a spacer sequence or a fourth additional sequence at the 5' of a target hybridization sequence.

[0110] Embodiment 38 is a capture oligomer, reaction mixture, or combination thereof as described in any of the preceding embodiments, wherein, for example, the capture oligomer comprises an affinity-enhancing modification located in the target hybridization sequence (e.g., any one or more of 5-Me-C, 2-aminopurine, 2′-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2′-OMe, or restricted ethyl (cEt) substitution).

[0111] Embodiment 39 is a capture oligomer, reaction mixture, or combination thereof as described in any of the preceding embodiments, wherein the capture oligomer comprises a reversible extension blocking molecule located at the 5' of the target hybridization sequence.

[0112] Implementation scheme 40 is the capture oligomer, reaction mixture or combination described in the previous implementation scheme, wherein the reversibly extended blocking member is located at 3' of the second part of the spacer sequence.

[0113] Implementation Scheme 41 is the capture oligomer, reaction mixture, or combination thereof as described in any one of Implementation Schemes 39 or 40, wherein the reversible extension blocking factor includes: Iso-dC or Iso-dG, xanthine or 5-(2,4-diaminopyrimidine), 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4-methylbenzimidazole or 2,4-difluorotoluene, 7-azaindole or isoquinolone, dMMO2 or d5SICS, dF or dQ; one or more chemically modified nucleotides, wherein the modification is by attachment via a reversible linker bond, and the linker bond can be reversed by any one or any combination of two or more of the following: chemicals, enzymes, temperature changes, or reagent composition changes; reversible nucleic acid structural features; reversible binding to a nucleic acid template such as a protein, enzyme, lipid, carbohydrate, or chemical moiety.

[0114] Embodiment 42 is a capture oligomer, reaction mixture, or combination of any one of embodiments 28 to 41, comprising a second additional sequence located between the first portion of the internal extension blocker and the spacer sequence, wherein the second additional sequence comprises a mixed nucleotide segment.

[0115] Implementation scheme 43 is the capture oligomer, reaction mixture or combination described in the previous implementation scheme, wherein the mixed nucleotide segment contains 5 nucleotides, wherein each nucleotide is different from its immediate neighboring nucleotide.

[0116] Embodiment 44 is a capture oligomer, reaction mixture, or combination thereof as described in any one of Embodiments 42 or 43, wherein the mixed nucleotide segment does not contain repeating dinucleotides, repeating trinucleotides, and / or adjacent repeating sequences.

[0117] Embodiment 45 is the trapping oligomer, reaction mixture, or combination of any one of embodiments 42 to 44, further comprising a clamping oligomer comprising, in the 5′ to 3′ direction:

[0118] Complementary sequences to sequences not present in the captured oligomers;

[0119] Mixed nucleotide segments; and

[0120] The complementary sequence of the captured sequence.

[0121] Embodiment 46 is the capturing oligomer, reaction mixture, or combination described in the preceding embodiment, wherein the capturing oligomer comprises a third or fourth additional sequence, the third or fourth additional sequence comprising a linker sequence located between a second portion of the target hybridization sequence and the spacer sequence, or the target hybridization sequence of the capturing oligomer is a linker sequence; and

[0122] The splint oligomer also contains the complementary sequence of the 3' linker sequence located at the complementary sequence of the capture sequence.

[0123] Embodiment 47 is a capture oligomer, reaction mixture or combination of embodiments 28 to 46, further comprising a second capture agent comprising a complementary sequence to the capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid carrier (e.g., beads or surface).

[0124] Embodiment 48 is the capture oligomer, reaction mixture, or combination described in the preceding embodiment, wherein the second capture agent comprises a binding pair and the combination further comprises a solid carrier (e.g., one or more beads), the solid carrier comprising a second binding pair configured to bind to the binding pair of the second capture agent, optionally wherein the binding pair of the second capture agent is biotin and the second binding pair is a biotin binder (e.g., streptavidin).

[0125] Embodiment 49 is a capture oligomer, reaction mixture, or combination thereof as described in any one of embodiments 28 to 48, further comprising a substitution oligomer containing a substitution target hybridization sequence configured to bind a target polynucleotide in a certain orientation, wherein the 3' end of the substitution target hybridization sequence is oriented toward the site bound by the target hybridization sequence of the capture oligomer.

[0126] Embodiment 50 is the capture oligomer, reaction mixture, or combination described in the preceding embodiment, wherein the capture oligomer comprises a linker sequence located at the 5' of the target hybridization sequence and the 3' of the complementary sequence of the capture sequence.

[0127] Embodiment 51 is a capture oligomer, reaction mixture, or combination as described in any one of embodiments 49 or 50, further comprising an amplified oligomer containing a reverse target hybridization sequence configured to bind a target polynucleotide in an orientation opposite to that of the capture oligomer, and optionally also comprising a second linker sequence located at the 5' of the reverse target hybridization sequence.

[0128] Embodiment 52 is a reaction mixture comprising the capture oligomer or combination thereof as described in any of the preceding embodiments, and further comprising the target polynucleotide.

[0129] Embodiment 53 is the reaction mixture described in the preceding embodiment, wherein the target is an amplicon, and further comprises amplification primers including amplification primers that bind to the same strand as the capture oligomer, optionally wherein the THS of the capture oligomer has a higher affinity for the amplicon than for the primers that bind to the same strand as the capture oligomer (e.g., wherein the capture oligomer has a longer THS than the primers that bind to the same strand as the capture oligomer, or the capture oligomer contains modifications to enhance affinity).

[0130] Implementation scheme 54 is a combination comprising the capturing oligomer or combination thereof as described in any one of embodiments 1 to 51 and the amplifying oligomer, wherein:

[0131] The capture oligomer contains a first linker sequence located between the target hybridization sequence and the internal extension blocking sequence, and

[0132] The amplified oligomer comprises (i) a reverse target hybridization sequence that binds to the target polynucleotide in the reverse orientation relative to the capture oligomer and (ii) a second linker sequence located at the 5' of the reverse target hybridization sequence.

[0133] Implementation scheme 55 is the combination described in the previous implementation scheme, wherein the capturing oligomer includes a blocking portion at its 3′ end.

[0134] Implementation scheme 56 is a combination of implementation scheme 54, wherein the capture of oligomers is extensible.

[0135] Implementation scheme 57 is a combination of any one of implementation schemes 54 to 56, wherein the amplified oligomer comprises a reversible extension blocking member located between the second linker sequence and the reverse target hybridization sequence, and optionally, wherein the captured oligomer further comprises a reversible extension blocking member located between the target hybridization sequence and the first linker sequence.

[0136] Implementation Scheme 58 is a method for capturing target polynucleotides from a composition, the method comprising:

[0137] The target polynucleotide is contacted with the capture oligomer of any one of embodiments 1 to 22 or 36 to 51, wherein the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide at a site containing the 3′ end of the target polynucleotide;

[0138] The 3′ end of the target polynucleotide is extended using a DNA polymerase with strand substitution activity to form a complementary sequence to the complementary sequence of the capture sequence, which is then annealed with the capture oligomer so that the capture sequence of the capture oligomer can be used for binding.

[0139] The capture sequence of the capture oligomer is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding partner or (ii) a solid carrier to form a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0140] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0141] Implementation scheme 59 is a method for capturing target polynucleotides from a composition, the method comprising:

[0142] The composition is contacted with the composition of any one of embodiments 28 to 30 or 34 to 51, wherein at the site containing the 3' end of the target polynucleotide, the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide;

[0143] The 3′ end of the target polynucleotide is extended using a DNA polymerase with strand displacement activity to form a complementary sequence to the spacer sequence. This complementary sequence is annealed with the capture oligomer, such that the complementary oligomer is replaced to a degree sufficient to make the capture sequence of the capture oligomer available for binding.

[0144] The capture sequence of the capture oligomer is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding partner or (ii) a solid carrier to form a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0145] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0146] Implementation Scheme 60 is a method for capturing a target polynucleotide from a composition, the method comprising:

[0147] The composition is contacted with the composition of any one of embodiments 28 to 30 or 34 to 51, wherein at the site containing the 3' end of the target polynucleotide, the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide;

[0148] The 3′ end of the target polynucleotide is extended using DNA polymerase, which optionally has strand substitution activity, to form a complementary sequence to the spacer sequence, which anneals with the captured oligomer.

[0149] The free capturing oligomer is brought into contact with the complementary oligomer, wherein the complementary oligomer anneals with the free capturing oligomer and partially occupies its capturing sequence, wherein the complementary oligomer does not anneal with the complex of the capturing oligomer containing the complementary sequence with the spacer sequence.

[0150] The capture sequence of the capture oligomer complexed with the target polynucleotide is contacted with a complementary sequence comprising the capture sequence and a second capture agent comprising (i) a binding coupler or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent, wherein the complementary oligomer is introduced into the composition before, during, or after the 3' extension of the target polynucleotide; and

[0151] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0152] Implementation scheme 61 is the method of the previous implementation scheme, wherein the target hybridization sequence of the capture oligomer is extended to form an extended capture oligomer.

[0153] Implementation Scheme 62 is a method for capturing a target polynucleotide from a composition, the method comprising:

[0154] The composition is contacted with the combination of any one of embodiments 28 to 30 or 34 to 51, the combination further comprising an amplified oligomer comprising a reverse target hybridization sequence configured to bind a target polynucleotide in the opposite orientation to the capture oligomer, and optionally also comprising a second linker sequence located at the 5' of the reverse target hybridization sequence, wherein the target hybridization sequence of the capture oligomer is annealed with and extended to form an extended capture oligomer;

[0155] Anneal the amplified oligomers with the extended captured oligomers;

[0156] The 3' end of the oligomer is extended and amplified using a DNA polymerase, which optionally has strand substitution activity, to form a complementary sequence to at least the target hybridization sequence of the captured oligomer, which is then annealed with the captured oligomer.

[0157] The free capturing oligomer is brought into contact with the complementary oligomer, wherein the complementary oligomer anneals with the free capturing oligomer and partially occupies its capturing sequence, wherein the complementary oligomer does not anneal with a complex of the capturing oligomer containing a complementary sequence to the target hybridization sequence of the capturing oligomer;

[0158] The capture sequence of the capture oligomer complexed with the target polynucleotide is contacted with a complementary sequence comprising the capture sequence and a second capture agent comprising (i) a binding coupler or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent, wherein the complementary oligomer is introduced into the composition before, during, or after the 3' extension of the target polynucleotide; and

[0159] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0160] Implementation Scheme 63 is a method for capturing target polynucleotides from a composition, the method comprising:

[0161] Contact the target polynucleotide with a capture oligomer or combination thereof as described in any one of embodiments 1 to 22, 28 to 30 or 34 to 51, wherein the capture oligomer comprises a third or fourth additional sequence at the 3' of the second portion of the complementary sequence or spacer sequence of the target hybridization sequence at the 5' of the target hybridization sequence;

[0162] The target polynucleotide is contacted with a second oligomer, the second oligomer comprising a second target hybridization sequence at 5' of a complementary sequence located in at least a portion of a third or fourth additional sequence, wherein the capture oligomer and the second oligomer form a triple-stranded link with the target polynucleotide;

[0163] The 3′ end of the second oligomer is extended using a DNA polymerase with strand displacement activity, thereby forming a complementary sequence of the complementary sequence of the capture sequence or a complementary sequence of the spacer sequence, which is annealed with the capture oligomer so that the capture sequence of the capture oligomer can be used for binding.

[0164] The capture sequence of the capture oligomer is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding partner or (ii) a solid carrier to form a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0165] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0166] Implementation scheme 64 is the method of the preceding implementation scheme, wherein the capturing oligomer includes a blocking portion at its 3' end; and / or wherein the second oligomer includes a portion at its 5' end that blocks the substitution of polymerases with chain substitution activity.

[0167] Implementation Scheme 65 is a method for capturing a target polynucleotide from a composition, the method comprising:

[0168] The composition is contacted with the combination of any one of embodiments 31 to 51, wherein the target hybridization sequence that captures the oligomer is annealed with the target polynucleotide.

[0169] Before or after annealing the capture oligomer with the target polynucleotide, the capture oligomer is contacted with a complementary oligomer, wherein the complementary oligomer anneals with the free capture oligomer and partially occupies its capture sequence, wherein the complementary oligomer does not anneal with a complex of the capture oligomer containing a complementary sequence to the target hybridization sequence of the capture oligomer, and wherein if the contact between the capture oligomer and the complementary oligomer occurs before the capture oligomer is annealed with the target polynucleotide, the annealing of the target hybridization sequence with the target polynucleotide causes the complementary oligomer to dissociate from the capture oligomer;

[0170] The capture sequence of the capture oligomer complexed with the target polynucleotide is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding coupler or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0171] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0172] Embodiment 66 is the method of any one of embodiments 58 to 65, wherein the second capture reagent comprises a binding partner (e.g., biotin), and the separation comprises contacting the complex with a solid carrier (e.g., beads) comprising the second binding partner (e.g., streptavidin), the second binding partner being configured to bind to the binding partner of the second capture reagent.

[0173] Implementation scheme 67 is the method of any one of implementation schemes 58 to 66, wherein an excess of the capturing oligomer is provided relative to the second capturing agent.

[0174] Implementation scheme 68 is the method of any one of implementation schemes 58 to 67, wherein the target polynucleotide is obtained from a clinical sample.

[0175] Implementation scheme 69 is the method of any one of implementation schemes 58 to 68, wherein the target polynucleotide is derived from pathogens (bacteria, viruses, etc.).

[0176] Implementation scheme 70 is the method of any one of implementation schemes 58 to 69, wherein the target polynucleotide is the amplification product.

[0177] Implementation scheme 71 is the method of any one of implementation schemes 58 to 70, wherein the target polynucleotide is a member of the sequencing library.

[0178] Implementation scheme 72 is the method of any one of implementation schemes 58 to 71, wherein the captured oligomer comprises a third or fourth additional sequence located between the target hybridization sequence and the internal extension blocking sequence.

[0179] Embodiment 73 is the method of any one of Embodiments 63 to 72, wherein an extension product is formed by extending a capture oligomer along a target polynucleotide, and the method further includes contacting the extension product with a non-extendable blocking oligomer containing a third or fourth additional sequence, optionally wherein the blocking oligomer is configured to bind a complementary sequence of the third or fourth additional sequence with a higher affinity than the third or fourth additional sequence of the capture oligomer, or to form a complex with the complementary sequence of the third or fourth additional sequence having a higher melting temperature than the complex of the third or fourth additional sequence of the capture oligomer with the complementary sequence of the third or fourth additional sequence.

[0180] Implementation scheme 74 is the method described in any one of implementation schemes 58 to 73, wherein the method further includes:

[0181] The target polynucleotide is brought into contact with the amplified oligomer, which contains (i) a reverse target hybridization sequence that binds the target polynucleotide in a reverse orientation relative to the capturing oligomer and (ii) an additional sequence located at the 5' of the second target hybridization sequence.

[0182] And to extend oligomers along the target polynucleotide to form reverse extension products,

[0183] A portion of the captured oligomers is annealed with the reverse-stretching product.

[0184] Implementation scheme 75 is the method of the previous implementation scheme, wherein the captured oligomer includes a blocking portion at its 3′ end.

[0185] Implementation 76 is the method of the preceding implementation, wherein the method further includes separating a complex comprising a reverse extension product annealed with a capture oligomer, wherein the extension product is essentially a single strand located at the 5' of the target hybridization sequence of the capture oligomer.

[0186] Embodiment 77 is the method of any one of Embodiments 74 to 76, wherein the captured oligomer is extendable, and the method further includes extending a portion of the captured oligomer along the extension product to form a second extension product, the second extension product comprising a third or fourth additional sequence and a complementary sequence to the additional sequence of the amplified oligomer.

[0187] Embodiment 78 is the method of any one of Embodiments 58 to 77, wherein the target polynucleotide comprises a sequence from the DNA or RNA of the target organism and an additional sequence not present in the DNA or RNA of the target organism, and the target hybridization sequence for capturing oligomers is configured to anneal with the additional sequence of the target polynucleotide.

[0188] Implementation 79 is the method of implementation 78, wherein the composition comprises a plurality of target polynucleotides, the target polynucleotides comprising (i) an additional sequence and (ii) different sequences from DNA or RNA of a target organism and / or different samples, and the method includes capturing the plurality of target polynucleotides.

[0189] Embodiment 80 is the method of any one of embodiments 58 to 79, wherein the captured oligomer comprises a reversible extension blocking factor located at the 5' of the target hybridization sequence, and the method includes:

[0190] Copy or amplify the target polynucleotide before removing the blockade from the reversible extension blocker;

[0191] Unblocking of the reversible extension blocker; and

[0192] Perform another cycle of copying or amplifying the target polynucleotide.

[0193] Optionally, amplification is performed in only one cycle before the target polynucleotide is captured and after the reversible extension blocker is released.

[0194] Embodiment 81 is the method of any one of embodiments 58 to 64 or 66 to 80, wherein the capture oligomer comprises a mixed nucleotide segment located between the first portion of the internal extension blocker and the spacer sequence or the complementary sequence of the capture sequence, and

[0195] The method involves extending the target polynucleotide along the 3' end of the capture oligomer until the internal extension blocker, thereby forming an extension product containing a complementary sequence of mixed nucleotide segments at its 3' end;

[0196] Contact the elongation product with the splint oligonucleotide, which comprises, in the 5' to 3' direction:

[0197] The complementary sequence of the 5' terminal region of the extended product;

[0198] Mixed nucleotide segments;

[0199] The complementary sequence of the captured sequence; and

[0200] Optionally, a segment complementary to the 5' segment of the extended product immediately adjacent to the capture sequence; and

[0201] Connect the 5' end of the extended product to the 3' end of the extended product.

[0202] Implementation scheme 82 is the method described in the previous implementation scheme, wherein the 5′ end segment of the extended product is a linking sequence.

[0203] Implementation scheme 83 is the method described in implementation scheme 81 or 82, wherein the segment of the extended product immediately adjacent to the capture sequence 5' is a complementary sequence to the linker sequence.

[0204] Implementation scheme 84 is the method of any one of implementation schemes 81 to 83, wherein the mixed nucleotide segment comprises 5 nucleotides, wherein each nucleotide is different from its immediate neighboring nucleotide.

[0205] Implementation scheme 85 is the method of any one of implementation schemes 81 to 84, wherein the mixed nucleotide segment does not contain repeating dinucleotides, repeating trinucleotides and / or adjacent repeating sequences.

[0206] Implementation scheme 86 is the method of any one of implementation schemes 58 to 85, further comprising target polynucleotide sequencing.

[0207] Implementation scheme 87 is the method of any one of implementation schemes 58 to 86, further comprising cloning and amplifying the captured target polynucleotide.

[0208] Implementation scheme 88 is the method described in the previous implementation scheme, which further includes sequencing the target polynucleotide of the clone amplification.

[0209] Implementation scheme 89 is the method of any one of implementation schemes 86 or 88, wherein the sequencing is Sanger sequencing or next-generation sequencing, optionally wherein next-generation sequencing includes sequencing by synthesis, sequencing by ligation, sequencing by hybridization or single-molecule sequencing.

[0210] Implementation scheme 90 is a method for capturing target polynucleotides from a composition, the method comprising:

[0211] The target polynucleotide is contacted with a capture oligomer or combination of embodiments 1 to 22, 28 to 30 or 34 to 51, wherein the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide at a site upstream of the 3' end of the target polynucleotide;

[0212] The target polynucleotide is extended to capture the 3' end of the oligomer, thereby forming the first extended chain;

[0213] The target polynucleotide is brought into contact with the substitution oligomer, which contains the substitution target hybridization sequence. The substitution target hybridization sequence anneals with the target polynucleotide downstream of the target hybridization sequence that captures the oligomer.

[0214] The oligomer is replaced along the extension of the target polynucleotide, thereby replacing the first extension chain of the target polynucleotide.

[0215] Optionally, the capturing oligomer and the displacement oligomer are added to the composition simultaneously or sequentially.

[0216] Implementation scheme 91 is the method of the preceding implementation scheme, which further includes contacting the first extension strand with a reverse amplification oligomer containing a reverse target hybridization sequence, the reverse target hybridization sequence being configured to bind the first extension strand; and extending the reverse amplification oligomer to form a second extension strand.

[0217] Implementation scheme 92 is the method of the previous implementation scheme, wherein the reverse amplification oligomer includes an additional sequence located at the 5' of its target hybridization sequence, optionally wherein the 3' end of the first extension strand is further extended to form a complementary sequence to the additional sequence of the reverse amplification oligomer.

[0218] Embodiment 93 is the method of any one of Embodiments 90 to 92, wherein the capturing oligomer further comprises an additional sequence located at 5′ of the target hybridization sequence, optionally wherein, if there is an extension of the second extension chain, a complementary sequence to the additional sequence forming the capturing oligomer is formed.

[0219] Implementation scheme 94 is the method of the previous implementation scheme, wherein the second strand of the target polynucleotide contains a complementary sequence to the additional sequence that captures the oligomer.

[0220] Embodiment 95 is the method of Embodiment 90 or 91, wherein the target polynucleotide comprises a sequence of DNA or RNA from a target organism and an additional sequence not present in the DNA or RNA of the target organism, and the target hybridization sequence for capturing oligomers is configured to anneal with a first portion of the additional sequence of the target polynucleotide, and the substitution oligonucleotide is configured to anneal with a second portion of the additional sequence of the target polynucleotide, optionally wherein the additional sequence of the target polynucleotide further comprises one or more nucleotides located between the first and second portions.

[0221] Implementation scheme 96 is the method of implementation scheme 90 or 95, wherein the first extension strand comprises a second additional sequence located proximal to a sequence from the target organism's DNA or RNA and distal to a target hybridization sequence that captures oligomers, and

[0222] The method further includes: contacting a first extension strand with a reverse amplification oligomer, the reverse amplification oligomer including a reverse target hybridization sequence configured to bind to a second additional sequence; and extending the reverse amplification oligomer to form a second extension strand, optionally wherein the reverse amplification oligomer includes an additional sequence located at the 5' of its target hybridization sequence, and the method further includes extending the first extension strand further along the reverse amplification oligomer.

[0223] Implementation scheme 97 is a combination comprising a capturing oligomer and a second capturing agent, wherein the capturing oligomer comprises, in the 5' to 3' direction:

[0224] First self-complementary sequence,

[0225] Target hybridization sequence, and

[0226] Second self-complementary sequence,

[0227] The first self-complementary sequence and the second self-complementary sequence are configured such that when the target hybridization sequence is single-stranded, the first self-complementary sequence and the second self-complementary sequence anneal to each other, and when the target hybridization sequence anneales to its target, the first self-complementary sequence and the second self-complementary sequence do not anneal to each other.

[0228] The second capture agent comprises a complementary sequence of the first self-complementary sequence and the second self-complementary sequence, and a binding partner; and

[0229] The amount of the capturing oligomer in the combination is greater than that of the second capturing agent.

[0230] Implementation scheme 98 is the combination described in the previous implementation scheme, wherein the captured oligomer has the following formula:

[0231] 5′-SC1-THS2-THS1-L-THS2′-SC2-X-3'

[0232] or

[0233] 5′-SC2-THS2′-L-THS1-THS2-SC1-X-3′

[0234] SC1 is the first self-complementary sequence.

[0235] THS2 and THS1 are the second and first parts of the target hybridization sequence, respectively.

[0236] L is an optional connector.

[0237] THS2′ is an optional complementary sequence present in the second part of the target hybridization sequence.

[0238] SC2 is the second self-complementary sequence, and

[0239] X represents any arbitrarily existing blocking component.

[0240] Implementation scheme 99 is a combination of any one of implementation schemes 97 or 98, wherein the capture oligomer comprises a 3' linker located in the first portion of the target hybridization sequence and a 5' linker in the second self-complementary sequence.

[0241] Implementation scheme 100 is a combination of any one of implementation schemes 97 to 99, wherein the capturing oligomer comprises a complementary sequence of the second portion of the target hybridization sequence located at the 3' of the first portion of the target hybridization sequence and the 5' of the second self-complementary sequence.

[0242] Implementation 101 is a combination of any one of Implementations 97 to 100, wherein the capturing oligomer comprises a linker at the 3' of the first portion of the target hybridization sequence and a complementary sequence at the 3' of the linker and the 5' of the second portion of the target hybridization sequence.

[0243] Implementation scheme 102 is a combination of any one of implementation schemes 97 to 101, wherein the capture oligomer comprises a 5' linker located in the first part of the target hybridization sequence and a 3' linker in the second self-complementary sequence.

[0244] Implementation scheme 103 is a combination of any one of implementation schemes 97 to 102, wherein the capturing oligomer comprises the complementary sequence of the second part of the target hybridization sequence located at the 5' of the first portion of the target hybridization sequence and the 3' of the second self-complementary sequence.

[0245] Implementation scheme 104 is a combination of any one of implementation schemes 97 to 103, wherein the capture oligomer comprises a linker at the 5' of the first portion of the target hybridization sequence and a complementary sequence at the 5' of the linker and the 3' of the second portion of the target hybridization sequence.

[0246] Implementation scheme 105 is a combination of any one of implementation schemes 97 to 104, wherein the capturing oligomer and / or the second capturing agent are non-extendable.

[0247] Implementation scheme 106 is a combination of any one of implementation schemes 97 to 105, wherein the first self-complementary sequence or the second self-complementary sequence contains poly A or poly T, and the complementary sequence of the self-complementary sequence contains poly T or poly A.

[0248] Implementation 107 is a combination of any one of Implementations 97 to 106, wherein the capturing oligomer comprises one or more affinity-enhancing modifications located in the target hybridization sequence (e.g., any one or more of 5-Me-C, 2-aminopurine, 2′-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2′-OMe, or restricted ethyl (cEt) substitution).

[0249] Implementation scheme 108 is a kit comprising a combination or capture oligomer of any one of implementation schemes 1 to 22, 28 to 51, 54 to 57 or 97 to 107.

[0250] Embodiment 109 is a composition comprising any combination or capture oligomer of embodiments 1 to 22, 28 to 51, 54 to 57 or 97 to 107.

[0251] Implementation 110 is a combination of any one of Implementations 18 to 22, 28 to 51, 54 to 57 or 97 to 107, comprising (i) a second capture agent comprising a binding pair, and (ii) a solid carrier (e.g., one or more beads) comprising a second binding pair configured to bind to a binding pair of the second capture agent, optionally wherein the binding pair of the second capture agent is biotin and the second binding pair is a biotin binder (e.g., streptavidin).

[0252] Embodiment 111 is a reaction mixture comprising a combination of any one of embodiments 18 to 22, 28 to 51, 54 to 57, 97 to 107 or 110, and further comprising a target polynucleotide.

[0253] Embodiment 112 is the reaction mixture described in the preceding embodiment, wherein the target is in or obtained from an extract of cells, a sample, a virus, or a biological sample.

[0254] Implementation scheme 113 is a method for capturing target polynucleotides from a composition, the method comprising:

[0255] The target polynucleotide is contacted with the combination of any one of embodiments 97 to 107 or 110, wherein a capture oligomer and a second capture agent are added simultaneously or sequentially, and wherein the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide, and the second capture agent is annealed with the self-complementary sequence of the capture oligomer, thereby forming a complex;

[0256] The complex is brought into contact with a second binding pair, the second binding pair being configured to bind to a binding pair of a second capture reagent, wherein the second binding pair associates with a solid carrier, and wherein the second binding pair binds to a binding pair of a second capture reagent; and

[0257] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0258] Implementation scheme 114 is the method described in the previous implementation scheme, wherein the captured oligomer is in excess relative to the target polynucleotide.

[0259] Implementation scheme 115 is the method of any one of implementation schemes 113 or 114, wherein the target polynucleotide is in excess relative to the second capture reagent.

[0260] Implementation scheme 116 is the method of any one of implementation schemes 113 to 115, wherein only a portion of the target polynucleotide is captured.

[0261] Implementation scheme 117 is the method of any one of implementation schemes 113 to 116, wherein the target polynucleotide is obtained from a clinical sample.

[0262] Implementation scheme 118 is the method of any one of implementation schemes 113 to 117, wherein the target polynucleotide is derived from pathogens (bacteria, viruses, etc.).

[0263] Implementation scheme 119 is the method of any one of implementation schemes 113 to 118, wherein the target polynucleotide is the amplification product.

[0264] Implementation scheme 120 is the method of any one of implementation schemes 113 to 119, which further includes amplifying the target polynucleotide and / or attaching one or more additional sequences to the target polynucleotide.

[0265] Implementation scheme 121 is the method of any one of implementation schemes 113 to 120, further comprising preparing a sequencing library containing target polynucleotides.

[0266] Implementation scheme 122 is the method of any one of implementation schemes 113 to 121, further comprising one or both of cloning and amplifying members of a sequencing library and sequencing members of the sequencing library, optionally wherein the sequencing is Sanger sequencing or next-generation sequencing, optionally wherein next-generation sequencing includes sequencing by synthesis, ligation sequencing, hybridization sequencing or single-molecule sequencing.

[0267] Implementation scheme 123 is a method for capturing target polynucleotides from a composition, the method comprising:

[0268] The target polynucleotide is brought into contact with the capturing oligomer, which contains, in the 5' to 3' direction:

[0269] Capture sequence,

[0270] Optional internal extension blocking element,

[0271] Optional interval sequences, and

[0272] The target hybridization sequence is configured to anneal with the target polynucleotide.

[0273] Contact the capture oligomer with a first capture agent containing a complementary sequence of the capture sequence (before, during, or after contacting the target polynucleotide with the capture oligomer);

[0274] A second capture agent is provided, comprising a complementary sequence to a sequence in the capture oligomer other than the capture sequence, wherein if some or all of the capture oligomers do not anneal with the target polynucleotide, the second capture agent is contacted with the capture oligomers that do not anneal with the target polynucleotide.

[0275] Separate a first complex and a second complex from the composition, wherein the first complex comprises a target polynucleotide, and the second complex comprises a capture oligomer that has not been annealed to the target polynucleotide; and

[0276] Selective elution of target polynucleotides or subcomplexes containing target polynucleotides from the first complex;

[0277] Optionally, wherein (a) the first capture agent comprises (i) a binding coupler and (e.g., biotin) or (ii) a solid carrier (e.g., beads or surface) and / or (b) the second capture agent comprises (i) a binding coupler and (e.g., biotin) or (ii) a solid carrier (e.g., beads or surface).

[0278] Implementation scheme 124 is the method of implementation scheme 123, wherein the target polynucleotide is contacted with an excess of capture oligomers.

[0279] Implementation scheme 125 is the method of implementation scheme 123 or 124, wherein the first capture agent is provided in a limiting amount relative to the captured oligomer.

[0280] Implementation scheme 126 is the method of any one of implementation schemes 123 to 125, wherein the capture oligomer is provided in a limiting amount relative to the target polynucleotide.

[0281] Implementation scheme 127 is the method of any one of implementation schemes 123 to 126, wherein a portion of the capture oligomer in contact with the second capture reagent comprises a capture oligomer that has not been annealed with the target polynucleotide.

[0282] Implementation scheme 128 is the method of any one of implementation schemes 123 to 127, wherein the first capture reagent comprises a first solid carrier, the first solid carrier comprising a complementary sequence to the capture sequence.

[0283] Implementation scheme 129 is the method of any one of implementation schemes 123 to 128, wherein the second capture reagent comprises a second solid carrier, the second solid carrier comprising a complementary sequence to a sequence in the capture oligomer other than the capture sequence.

[0284] Embodiment 130 is the method of any one of Embodiments 123 to 129, wherein the capturing oligomer comprises a spacer sequence located between the target hybridization sequence and the internal extension blocker, and optionally, wherein the second capturing agent comprises a complementary sequence to the spacer sequence.

[0285] Implementation scheme 131 is the method of any one of implementation schemes 123 to 130, wherein at the 3′ end of the target polynucleotide, the target hybridization sequence is annealed with the target polynucleotide.

[0286] Implementation scheme 132 is the method described in the preceding implementation scheme, wherein the method includes extending the 3' end of the target polynucleotide.

[0287] Implementation scheme 133 is the method of any one of implementation schemes 123 to 132, wherein the second capturing agent has a greater affinity for the capturing oligomer than the first capturing agent has for the capturing oligomer, and / or the second capturing agent is configured to form a complex with the capturing oligomer, the melting temperature of which is higher than the melting temperature of the complex of the first capturing agent and the capturing oligomer.

[0288] Implementation scheme 134 is the method described in any one of implementation schemes 128 to 133, comprising:

[0289] The target polynucleotide is brought into contact with the capturing oligomer, which contains, in the 5' to 3' direction:

[0290] First capture sequence,

[0291] Internal extension blocking element,

[0292] The second capture sequence, and

[0293] The target hybridization sequence is configured to anneal to the 3' end of the target polynucleotide.

[0294] This causes the captured oligomers to anneal with the target polynucleotide;

[0295] The 3' end of the target polynucleotide is extended via a second capture sequence;

[0296] The first complex is brought into contact with a first solid carrier containing a complementary sequence of the first capture sequence, thereby forming the first complex;

[0297] Unbound capture oligomers are contacted with a second solid support containing a complementary sequence of a second capture sequence to form a second complex, wherein the melting temperature of the complementary sequence of the first capture sequence is lower than the melting temperature of the second complex formed by annealing the second capture sequence with the complementary sequence of the second capture sequence, and / or the affinity of the complementary sequence of the second capture sequence to the second capture sequence is greater than the affinity of the complementary sequence of the first capture sequence to the first capture sequence;

[0298] Separating the first and second complexes from substances not bound to the first or second solid support; and

[0299] Target polynucleotides are selectively eluted from the first complex.

[0300] Implementation 135 is the method of implementation 123, wherein the first capture agent further comprises a second capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method comprises: after contacting the annealed capture oligomer with the first capture agent, annealing the second capture sequence with a solid carrier comprising a complementary sequence of the second capture sequence.

[0301] Implementation 136 is the method of implementation 123 or 135, wherein the second capture agent further comprises a third capture sequence that is not complementary to the capture oligomer or target polynucleotide, and the method comprises: after contacting the unbound capture oligomer with the second capture agent, annealing the third capture sequence with a solid carrier containing a complementary sequence of the third capture sequence.

[0302] Implementation scheme 137 is the method of implementation scheme 123, wherein the first capture agent further comprises a second capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method comprises: after contacting the annealed capture oligomer with the first capture agent, annealing the second capture sequence with a solid support comprising a complementary sequence of the second capture sequence; and the complementary sequence of the third capture sequence has a greater affinity for the third capture sequence than the complementary sequence of the second capture sequence has for the second capture sequence, and / or the complementary sequence of the third capture sequence is configured to form a complex with the third capture sequence, the melting temperature of which is higher than the melting temperature of the complex of the complementary sequence of the second capture sequence and the second capture sequence.

[0303] Implementation scheme 138 is a combination comprising a capturing oligomer, a first solid carrier, and a second solid carrier, wherein:

[0304] The captured oligomers contain the following in the 5' to 3' direction:

[0305] First capture sequence,

[0306] Optional internal extension blocking element,

[0307] Optional second capture sequence, and

[0308] Target hybridization sequence;

[0309] The first solid carrier contains a complementary sequence to the first capture sequence;

[0310] The second solid carrier contains a complementary sequence to the second capture sequence; and

[0311] The melting temperature of the first complex formed by annealing the first capture sequence and its complementary sequence is lower than that of the second complex formed by annealing the second capture sequence and its complementary sequence, and / or the affinity of the complementary sequence of the second capture sequence to the second capture sequence is greater than the affinity of the complementary sequence of the first capture sequence to the first capture sequence.

[0312] Implementation scheme 139 is a combination comprising a capture oligomer, a first capture reagent, a second capture reagent, a first solid support, and a second solid support, wherein:

[0313] The captured oligomers contain the following in the 5' to 3' direction:

[0314] First capture sequence,

[0315] Optional internal extension blocking element, and

[0316] Target hybridization sequence;

[0317] The first capture reagent contains a second capture sequence and a complementary sequence to the first capture sequence, wherein the second capture sequence is not complementary to the capture oligomer;

[0318] The second capture reagent contains a third capture sequence and a complementary sequence to the capture oligomer sequence other than the first capture sequence, wherein the third capture sequence is not complementary to the capture oligomer.

[0319] The first solid carrier contains a complementary sequence to the second capture sequence;

[0320] The second solid carrier contains a complementary sequence to the third capture sequence; and

[0321] The melting temperature of the first complex formed by annealing the second capture sequence with its complementary sequence is lower than that of the second complex formed by annealing the third capture sequence with its complementary sequence, and / or the affinity of the complementary sequence of the third capture sequence to the third capture sequence is greater than the affinity of the complementary sequence of the second capture sequence to the second capture sequence.

[0322] Implementation scheme 140 is a combination comprising a capturing oligomer and a second capturing agent, wherein the capturing oligomer comprises:

[0323] The target hybridization sequence contains one or more affinity-enhancing nucleotides; and

[0324] Capture sequences; and

[0325] The second capture reagent contains a complementary sequence to the capture sequence and a binding partner.

[0326] Implementation scheme 141 is a combination of any one of implementation schemes 138 to 140, wherein the capture sequence is located at the 5' of the target hybridization sequence.

[0327] Implementation scheme 142 is a combination of any one of implementation schemes 138 to 141, wherein the target hybridization sequence is configured to anneal with an additional sequence.

[0328] Implementation scheme 143 is a combination of any one of implementation schemes 138 to 142, wherein the second capturing agent is present in the combination in a lower amount than the capturing oligomer.

[0329] Implementation scheme 144 is a combination of any one of implementation schemes 138 to 144, wherein, for example, the capturing oligomer comprises one or more affinity-enhancing modifications located in the target hybridization sequence (e.g., any one or more of 5-Me-C, 2-aminopurine, 2′-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2′-OMe, or restricted ethyl (cEt) substitution).

[0330] Implementation scheme 145 is a combination of any one of implementation schemes 138 to 145, wherein the capture sequence comprises a polyA or polyT sequence.

[0331] Implementation scheme 146 is a method for capturing target polynucleotides from a composition, the method comprising:

[0332] The target polynucleotide is contacted with the combination of any one of embodiments 138 to 145, wherein a capture oligomer and a second capture agent are added simultaneously or sequentially, and wherein the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide, and the second capture agent is annealed with the capture sequence of the capture oligomer, thereby forming a complex;

[0333] The complex is brought into contact with a second binding pair, the second binding pair being configured to bind to a binding pair of a second capture reagent, wherein the second binding pair associates with a solid carrier and binds to a binding pair of a second capture reagent; and

[0334] The complex is isolated from the composition, thereby capturing the target polynucleotide.

[0335] Implementation scheme 147 is the method of the preceding implementation scheme, wherein the second capture agent is provided in a lower amount than the capture oligomer and / or target polynucleotide.

[0336] Implementation scheme 148 is the method of implementation scheme 146 or 147, wherein the capture oligomer is provided in a lower amount than the target polynucleotide, and optionally, wherein a second capture agent is provided in a lower amount than the capture oligomer.

[0337] Implementation scheme 149 is the method of any one of implementation schemes 146 to 148, wherein the target polynucleotide includes an additional sequence, and the target hybridization sequence is annealed with the additional sequence.

[0338] Embodiment 150 is the method of any one of Embodiments 146 to 149, wherein the target hybridization sequence for capturing oligomers comprises an affinity-enhancing modification (e.g., any one or more of 5-Me-C, 2-aminopurine, 2′-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2′-OMe, or restricted ethyl (cEt) substitution).

[0339] Embodiment 151 is the method of any one of Embodiments 146 to 150, wherein the target polynucleotide comprises a sequence from the DNA or RNA of the target organism, and the additional sequence is not present in the DNA or RNA of the target organism.

[0340] Embodiment 152 is the method of any one of embodiments 58 to 96, 113 to 137, or 146 to 151, wherein the amount of target polynucleotide captured is less than or equal to a predetermined amount, optionally wherein the predetermined amount corresponds to the molar amount of the captured oligomer provided or the molar amount of the second captured reagent provided.

[0341] Implementation scheme 153 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 152, wherein the amount of captured oligomers present is 10. 7 1 molecule / reaction up to 10 13 molecule / reaction, 10 9 1 molecule / reaction up to 10 12 One molecule / reaction, or 10 10 1 molecule / reaction up to 10 12 Within the range of molecules / reactions.

[0342] Implementation scheme 154 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 153, wherein if a second capturing agent is present, the present second capturing agent is in 10 3 1 molecule / reaction up to 10 14 molecule / reaction, or 10 3 1 molecule / reaction up to 10 9 One molecule / reaction, or 10 5 1 molecule / reaction up to 10 13 One molecule / reaction, or 10 5 1 molecule / reaction up to 10 8 molecule / reaction, or 10 6 1 molecule / reaction up to 10 13 molecule / reaction, or 10 6 1 molecule / reaction up to 10 8 Within the range of molecules / reactions.

[0343] Implementation scheme 155 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 154, wherein if a blocking oligomer is present, the present blocking oligomer is in the range of 10 8 1 molecule / reaction up to 10 14 molecule / reaction, 10 10 1 molecule / reaction up to 10 13 molecule / reaction, or 1011 1 molecule / reaction up to 10 13 Within the range of molecules / reaction; and where, if a second oligomer is present, the presence of the second oligomer is within 10 7 1 molecule / reaction up to 10 14 One molecule / reaction or 10 8 1 molecule / reaction up to 10 13 Within the range of molecules / reactions.

[0344] Implementation scheme 156 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 155, wherein if complementary oligomers are present, the present complementary oligomers are in the range of approximately 1.5 × 10⁻⁶. 7 1 molecule / reaction up to 10 14 1 molecule / reaction or approximately 1.5 × 10⁻⁶ 9 1 molecule / reaction up to 10 13 Within the range of molecules / reaction; and where, if sandwich oligomers are present, the presence of sandwich oligomers is within 10 3 1 molecule / reaction up to 10 14 molecule / reaction, or 10 4 1 molecule / reaction up to 10 10 molecule / reaction, or 2 × 10 5 1 molecule / reaction up to 10 14 molecule / reaction, or 10 6 1 molecule / reaction up to 10 9 molecule / reaction, or 2 × 10 6 1 molecule / reaction up to 10 14 molecule / reaction, or 2 × 10 6 1 molecule / reaction up to 10 9 Within the range of molecules / reactions.

[0345] Implementation scheme 157 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 156, wherein if an amplified oligomer is present, the present amplified oligomer is present in about 10 7 1 molecule / reaction up to 10 14 One molecule / reaction or approximately 10 8 1 molecule / reaction up to 10 13 Within the range of molecules / reaction; and where, if a blocking oligomer is present, the presence of the blocking oligomer is within 10 8 1 molecule / reaction up to 10 14 molecule / reaction, 10 10 1 molecule / reaction up to 10 13 molecule / reaction, or 10 11 1 molecule / reaction up to 10 13Within the range of molecules / reactions.

[0346] Implementation scheme 158 is the method of any one of implementation schemes 58 to 96, 113 to 137, or 146 to 157, wherein if a substitution oligomer is present, the present substitution oligomer is in the range of about 10 7 1 molecule / reaction up to 10 14 One molecule / reaction or approximately 10 8 1 molecule / reaction up to 10 13 Within the range of molecules / reactions.

[0347] Embodiment 159 is a capture oligomer, combination, reaction mixture, kit, composition or method as described in any of the preceding embodiments, wherein if a first additional sequence is present in the capture oligomer, the length of the first additional sequence (optionally a stable sequence) is about 2 to 15 nucleotides or about 3 to 10 nucleotides.

[0348] Embodiment 160 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein the length of the capture sequence is about 10 to 35 nucleotides or about 10 to 25 nucleotides.

[0349] Embodiment 161 is a capture oligomer, combination, reaction mixture, kit, composition or method as described in any of the preceding embodiments, wherein if a linker is present, the linker is about 10 to 20 nucleotides long, or if the linker is non-nucleotide-based, the linker is about 3 to 20 or more atoms long, or about 2 to 15 or more repeating units long.

[0350] Embodiment 162 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein if an internal extension blocker is present, the length of the internal extension blocker is about 1 to 20 nucleotides or about 1 to 8 nucleotides, or if the internal extension blocker is non-nucleotide-based, the length of the internal extension blocker is about 3 to 20 or more atoms or about 1 to 8 or more repeating units.

[0351] Embodiment 163 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein if a second additional sequence (optionally a mixture of nucleotide sequences) is present, the length of the second additional sequence is about 2 to 10 nucleotides or about 4 to 8 nucleotides.

[0352] Embodiment 164 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein the length of the complementary sequence of the capture sequence is about 10 to 35 nucleotides or about 10 to 25 nucleotides.

[0353] Embodiment 165 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions, or methods described above, wherein if a third additional sequence (optionally a stabilizing sequence and / or a linker sequence) is present, the length of the third additional sequence is about 2 to 50 nucleotides or about 4 to 35 nucleotides, and the length of the reversible extension blocker is about 1 to 20 nucleotides or about 1 to 8 nucleotides (natural or non-natural).

[0354] Embodiment 166 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein if a fourth additional sequence (optionally a linker sequence) is present, the length of the fourth additional sequence is about 4 to 40 nucleotides or about 6 to 25 nucleotides.

[0355] Embodiment 167 is any one of the preceding embodiments for capturing oligomers, combinations, reaction mixtures, kits, compositions or methods, wherein the length of the target hybridization sequence is about 10 to 60 nucleotides or about 12 to 25 nucleotides.

[0356] Embodiment 168 is any one of the capture oligomers, combinations, reaction mixtures, kits, compositions or methods described above, wherein if a blocking portion is present, the length of the blocking portion is about 1 to 10 or about 1 to 5 nucleotides, or if the blocking portion is non-nucleotide-based, the length of the blocking portion is about 3 to 20 atoms or about 1 to 5 repeating units.

[0357] Embodiment 169 is a capture oligomer, combination, reaction mixture, kit, composition or method as described in any of the preceding embodiments, wherein the total length of the capture oligomer is about 40 to 200 nucleotides, or about 40 to 150 nucleotides, or about 60 to 140 nucleotides, or about 60 to 130 nucleotides, and optionally, wherein the capture oligomer further comprises a nonnucleotide element of about 3 to 60 atoms or 5 to 28 repeating units.

[0358] Embodiment 170 is a combination, reaction mixture, kit, composition or method of any one of Embodiments 18 to 169, wherein if a second capture agent is present, the second capture agent comprises a complementary sequence to the capture sequence, the complementary sequence being about 10 to 35 nucleotides or about 10 to 20 nucleotides in length.

[0359] Embodiment 171 is a combination, reaction mixture, kit, composition or method of any one of Embodiments 18 to 170, wherein if a blocking oligomer is present, the length of the blocking oligomer is about 10 to 35 nucleotides or about 10 to 20 nucleotides, optionally, wherein the blocking oligomer further comprises a nonnucleotide element of about 3 to 20 atoms or 1 to 5 repeating units.

[0360] Embodiment 172 is a combination, reaction mixture, kit, composition or method of any one of Embodiments 18 to 171, wherein the length of the second oligomer is about 15 to 50 nucleotides or about 20 to 40 nucleotides.

[0361] Embodiment 173 is a combination, reaction mixture, kit, composition or method as described in any one of Embodiments 18 to 172, wherein if a complementary oligomer is present, the length of the complementary oligomer is about 10 to 50 nucleotides or about 15 to 35 nucleotides.

[0362] Implementation scheme 174 is a combination, reaction mixture, kit, composition or method of any one of implementation schemes 18 to 173, wherein if a splint oligomer is present, the length of the splint oligomer is about 15 to 60 nucleotides or about 20 to 50 nucleotides.

[0363] Implementation scheme 175 is a combination, reaction mixture, kit, composition or method of any one of implementation schemes 18 to 174, wherein if a substitution oligomer is present, the length of the substitution oligomer is about 10 to 50 nucleotides or about 20 to 40 nucleotides.

[0364] Implementation scheme 176 is a combination, reaction mixture, kit, composition or method of any one of implementation schemes 18 to 175, wherein if an amplifying oligomer is present, the length of the amplifying oligomer is about 10 to 80 nucleotides or about 20 to 60 nucleotides. Attached Figure Description

[0365] Figures 1A to 1C An exemplary capture oligomer according to this disclosure is shown, comprising a capture sequence, a blocking portion, a complementary sequence (C') of the capture sequence, an additional sequence (e.g., a third or fourth additional sequence), a target hybridization sequence, and other molecules. Figure 1A In the process, the captured oligomer anneals to the target polynucleotide (target), and the 3' end of the target polynucleotide anneals to the 5' end of the target hybridization sequence. This allows the captured sequence to anneal to the C' end. Figure 1B In the process, the 3' end of the target has been extended to the blocking portion, and the resulting target extension sequence is annealed with the additional sequence and C', while the capture sequence has been replaced and has become single-stranded. The 3' end of the capture oligomer also extends along the target polynucleotide. Figure 1C middle, Figure 1B The complex is annealed with a second capture agent containing a complementary sequence to the capture sequence and a binding partner or solid matrix. Simultaneously, excess capture oligomers, along with the capture sequence annealed with the complementary sequence to the capture sequence, are retained and do not interact with the second capture agent.

[0366] Figure 2A One embodiment of this disclosure is shown, wherein, as Figure 1B The complex in the mixture is annealed with a second capture agent that contains a complementary sequence of the capture sequence and is associated with a solid matrix (in this case, streptavidin-coated magnetic beads). The solid matrix may be part of the second capture agent or may associate with the second capture agent through interaction with a binding partner of the second capture agent (e.g., biotin).

[0367] Figure 2B One embodiment of this disclosure is shown, wherein the [implementation details] are derived from Figure 2A The extended capture oligomer and target complex have been eluted from the second capture reagent.

[0368] Figure 3 An embodiment of a capture oligomer according to the present disclosure is shown, comprising a stable (clamp) sequence as a first additional sequence, a capture sequence, a linker, an internal extension blocker, a complementary sequence to the capture sequence, a stable (clamp) sequence as a third additional sequence, a fourth additional sequence, and a target hybridization sequence.

[0369] Figure 4AExemplary molecules and exemplary reaction schemes according to this disclosure are shown. A target molecule is provided, wherein a first strand contains the sequence Sf at its 5' end and Sr′ at its 3' end, and a second strand contains the sequence Sf' at its 3' end and Sr at its 5' end. Herein and throughout, sequence names with a ' indicate complementarity to sequences without a '. The target molecule may be, for example, an amplicon from a previous reaction using primers having sequences Sf and Sr. A first cycle extension (cycle 1) is performed, wherein a capture oligomer according to this disclosure is annealed with a first target strand 1 (+), the capture oligomer comprising a capture sequence C, an internal extension blocking unit (solid circle), a complementary sequence C′ of the capture sequence, a fourth additional sequence A4, and a target hybridization sequence THS complementary to Sr'. A reverse amplification oligomer is annealed with a second target strand 1 (-), the reverse amplification oligomer comprising an additional sequence A2 and a target hybridization sequence Sf* complementary to at least Sf'. Sf* may contain affinity-enhancing modifications and / or additional nucleotides complementary to the second target strand to enhance its affinity for the target and promote competition for binding with primers from previous reactions having the sequence Sf (if present). The extension of the capture oligomer and the reverse amplification oligomer produces products 2(-) and 2(+), respectively, while the first strand extends along the capture oligomer to produce product 1(+)e, and the second strand extends along the reverse amplification oligomer to produce product 1(-)e. Essentially as follows... Figure 1B The capture sequence in the extended capture oligomer 2(-) is replaced. A second cycle (cycle 2) is performed, in which 2(-) is annealed with the reverse amplification oligomer, causing extension to produce products 2(-)e and 3.1(+). Simultaneously, 1(+)e is annealed with the capture oligomer, and the latter extends to produce product 3.1(-). Other instances of 1(-)e and 2(+), and 2(-)e and 3.1(+), are also produced by appropriate hybridization and extension events. This reaction protocol illustrates the inclusion of additional sequences at each end of the target, and the capture of the target by introducing C in a form that can be used for binding, for example, to bind with a second capture agent.

[0370] Figure 4BExemplary molecules and exemplary reaction schemes according to this disclosure are illustrated. A target molecule is provided, wherein a first strand contains the sequence Sf at its 5' end and Sr′ and A4′ at its 3' end, and a second strand contains the sequence Sf′ at its 3' end and Sr and A4 at its 5' end. The target molecule may be, for example, an amplicon from a previous reaction using primers having the sequences Sf and A4-Sr, e.g., where A4 is an additional sequence not initially present in the template. A first extension cycle (cycle 1) is performed, wherein a capture oligomer according to this disclosure is annealed with a first target strand (not shown), the capture oligomer comprising a capture sequence C, an internal extension blocking unit (solid circle), a complementary sequence C' of the capture sequence, a fourth additional sequence A4, and a target hybridization sequence THS complementary to A4'. A reverse amplification oligomer is annealed with a second target strand (not shown), the reverse amplification oligomer comprising an additional sequence A2 and a target hybridization sequence Sf* complementary to at least Sf'. Sf* may contain affinity-enhancing modifications and / or additional nucleotides complementary to the second target strand to enhance its affinity for the target and promote competition for binding with primers having the sequence Sf from previous reactions (if present). The extension of these complexes produces an extended capture oligomer 2(-) and an extended first target strand 1(+)e, as well as an extended second target strand 1(-)e and an extended reverse amplification oligomer 2(+). Essentially as... Figure 1B The capture sequence in the extended capture oligomer 2(-) is replaced. A second reaction cycle (cycle 2) is performed, in which 2(-) is annealed with the reverse amplification oligomer, causing extension to produce products 2(-)e and 3.1(+). Simultaneously, 1(+)e is annealed with the capture oligomer, and the latter extends to produce product 3.1(-). Other instances of 1(-)e and 2(+) and 2(-)e and 3.1(+) are also produced by appropriate hybridization and extension events. This reaction protocol illustrates the inclusion of an additional sequence at the target end, remote from the capture oligomer binding site, and the introduction of C (in a form usable for binding) using a capture oligomer capable of being attached to the target in the previous step (e.g., by amplification or ligation) and having a universal THS (i.e., binding additional sequence A4'), thereby making the target captureable.

[0371] Figure 5Exemplary molecules and exemplary reaction schemes according to this disclosure are shown. A capture oligomer is provided, comprising a 3' blocking portion and a target hybridization sequence (THS) that binds to sequence A1' in a target strand. A1' may be an additional sequence attached to the target in a previous step (e.g., by amplification or ligation). The capture oligomer also comprises a sequence x containing a complementary sequence to the capture sequence of the capture oligomer, and may also contain a third or fourth additional sequence between the complementary sequence of the capture sequence and the THS. As discussed elsewhere, the target strand may extend along the capture oligomer to replace the capture sequence from the complementary sequence of the capture sequence. The target strand may be positioned relative to the target (e.g., 10...). 14 The limitation quantity (e.g., 10 copies) 12 (1 copy) provides capture of oligomers. Also provides capture of more than the target (e.g., 10). 15 A primer (one copy) is used, and the target contains the sequences A2 and Sf. Extension of this primer produces a strand containing A2 at its 5' end and A1' at its 3' end. The target strand is also extended along the primer to include the sequence A2'. If a second extension cycle is performed (downward arrow), a mixture of products is formed, including the aforementioned substances as well as a complex of the target strand and the trapping oligomer, wherein the target strand contains A2 at its 5' end and A1' near its 3' end. This reaction protocol illustrates the production of single-stranded trapping products, including (during the second extension cycle) single-stranded trapping products where the target strand already includes the additional sequence.

[0372] Figure 6 The diagram (above the dashed line) illustrates how hybridization of a capture oligomer with an extendable 3' end to another capture oligomer produces a dimer after extension, where the capture sequence is substituted from C′. This dimer becomes captureable and can potentially interfere with downstream processes, such as competing for capture with the desired target by occupying a second capture reagent (not shown), and subsequent analysis of the interference (e.g., the dimer becoming part of the sequencing library, thus reducing the output and quality of subsequent sequencing runs). Sx′ is a complementary sequence to a portion of the target hybridization sequence, and other elements are as shown in the previous figure. Below the dashed line, a capture oligomer (circled x) with a blocking portion at its 3' end is shown, which prevents the formation of the dimer extension product, ensuring that no C substitution occurs in any dimer.

[0373] Figure 7AOne embodiment is shown, in which a capture oligomer comprising a capture sequence, various intermediate elements (indicated by “…”), a reversible extension blocking unit (solid circle), and a target hybridization sequence (THS) is used. The capture sequence and various intermediate elements (if present) are not templates for extension (e.g., extension of the target strand or amplification oligomer) until the blocking is released from the reversible extension blocking unit. This facilitates more efficient and specific extension or amplification by avoiding the introduction of additional sequences complementary to the capture sequence and various intermediate elements (if present) into the product (e.g., into any mis-initiated products that may form) throughout the extension or amplification process until the blocking is released from the reversible extension blocking unit; after the blocking is released, the capture sequence and various intermediate elements (if present) can be introduced.

[0374] Figure 7B An embodiment is illustrated, in which a first amplifying oligomer is used, comprising from 3' to 5': a target hybridization sequence Sr, a reversible extension blocking unit (solid square), an additional sequence A1, and optional additional elements (indicated by "..."), such as an optional capture sequence. Optionally, a second amplifying oligomer is used, comprising from 3' to 5': a target hybridization sequence Sf, a reversible extension blocking unit (hollow square; this sequence may be the same as or different from the reversible extension blocking unit in the first amplifying oligomer), an additional sequence A2, and optional additional elements (indicated by "...", these additional elements may be the same as or different from those in the first amplifying oligomer) (as shown in the figure). The additional sequence and optional additional elements (if present) are not templates for extension (e.g., extension of the target strand or the amplifying oligomer) until one or more reversible extension blocking units release the blocking. This facilitates more efficient and specific extension or amplification by avoiding the introduction of sequences complementary to the additional sequence and various other elements (if present) into the product (e.g., into any error-induced product that may form) throughout the initial extension or amplification process. The reversible extension blocker is unblocked (if two reversible extension blockers are present, unblocking can occur simultaneously or separately), and the additional sequence and any other elements present can be introduced in later stages of the method (e.g., in a subsequent cycle of extension).

[0375] Figure 8A Exemplary molecules and exemplary reaction schemes according to this disclosure are shown. Initial target chains 1(+) and 1(-) are... Figure 4A The same applies below the vertical arrow. A trapping oligomer is provided, containing the target hybridization sequence THS, and as shown below. Figure 4AThe oligomer contains additional elements A4, C′, an internally extended blocking agent, and C. THS binds to the internal site of the target chain and is extended to produce the product 2N(-). A substitution oligomer containing Sr is provided and extended to replace 2N(-) with 1(+) and produce 2(-). As provided... Figure 4A The reverse amplification oligomer, extending along 1(-), produces 2.1(+), while 1(-) extending along the reverse amplification oligomer produces 1(-)e. Once 2N(-) is replaced (curved arrow on the left), the reverse amplification oligomer anneals with 2N(-) and extends independently, producing products 2N(-)e and 2.2(+), which now contain A2' and in which C' is replaced by C. This reaction scheme illustrates the use of substitution oligomers to facilitate the production of capture products containing additional sequences (e.g., adaptors) at both ends of the target sequence, even in cycle 1 only. Furthermore, this reaction scheme shows an embodiment in which the capture oligomer does not bind to sites including the 3' end of the target strand.

[0376] Figure 8B Further exemplary molecules and another exemplary reaction scheme according to this disclosure are shown. The reaction scheme is substantially similar to... Figure 4A The reaction protocol described herein differs from the following 1) and 2). 1) The initial target strands 1(+) and 1(-) contain additional sequences, which include the target hybridization sequence THS, an optional spacer sequence S, and a substitution oligomer binding site D. These additional sequences are arbitrary user-defined sequences and can be introduced into the target, for example, by using an amplification reaction with an amplification oligomer containing Sr and a sequence tag containing THS, S, and D, and an amplification oligomer containing Sf. 2) The THS of the captured oligomer binds to the user-defined THS site. Otherwise, as Figure 8A The reaction was carried out as shown, and the resulting product is shown in... Figure 8B Optional spacing sequences can be used to improve the extension of displaced oligomers and subsequent displacement of trapped oligomers. Figure 4A Similar to the scheme illustrated, this reaction scheme demonstrates the use of substitution oligomers to facilitate (e.g., only in cycle 1) the generation of a capture product containing additional sequences (e.g., adaptors) at both ends of the target sequence. Furthermore, this scheme illustrates the use of additional, user-defined sequences that can serve as binding sites for both the capture and substitution oligomers. This design allows for the generalization of the method and enables simpler and more cost-effective means of designing capture and substitution oligomers for different targets, including in multiple forms.

[0377] Figure 9This illustrates the general principle by which a blocking oligomer prevents hybridization between an additional sequence in the oligomer and its complementary sequence in the extension product. An amplification reaction is performed using a forward primer containing sequence f that hybridizes to the target strand T(-), and a reverse primer containing sequence A (an additional sequence absent in the target) and sequence r that hybridizes to the target strand T(+). Extension produces products 1(-) and 1(+). A blocking oligomer containing sequence A and a 3' blocking portion is provided. In cycle 2, the forward primer extends along 1(-) to produce 2(+), and the reverse primer extends along 1(+) to produce 2(-). In cycle 3 and subsequent cycles, the blocking oligomer is annealed to 2(+), meaning that hybridization of r with r' is necessary for the reverse primer to initiate extension along 2(+). This is advantageous in the event of any mis-initiation event that produces a small amount of incompletely complementary sequence with r but extending to include A' as a byproduct. Without the blocking oligomer, the interaction between A and A' of the reverse amplification oligomer will more favor the binding of the reverse amplification oligomer to the mis-initiated byproducts, resulting in more byproduct amplification than with the blocking oligomer. (Simultaneously, the forward primer is annealed with 2(-) and extended).

[0378] Figure 10A Exemplary molecules and exemplary reaction schemes according to this disclosure are illustrated. The following combinations are provided: (i) a capture oligomer comprising a first and second portion (C1 and C2) of a capture sequence, an internal extension blocking member (solid circle), a first and second portion (S1 and S2) of a spacer sequence, and a target hybridization sequence (THS) binding to a site in the target strand containing its 3' end; and (ii) a complementary oligomer comprising S1' and C2'. The complementary oligomer is substituted when the capture oligomer hybridizes with the target, and the target extends along the capture oligomer to the internal extension blocking member, thereby introducing S' into the target strand. The capture oligomer also extends along the target (note that in other embodiments described herein, the capture oligomer may be blocked, and thus this extension does not occur). A second capture agent is provided comprising a binding partner or solid carrier (circled B) connected to the complementary sequence C' of the capture sequence via a linker (zigzag curve). The second capture reagent anneals with the capture oligomer bound to the extended target, but not with the capture oligomer bound to the complementary oligomer, because the latter occupies C2, which is a sufficient amount of the capture sequence to substantially prevent the second capture reagent from annealing with the capture oligomer.

[0379] Figure 10BOne embodiment is shown, wherein, if desired, a combination of oligomers can be used to capture target polynucleotides from a composition comprising an amount (e.g., a limiting amount, or an amount less than or equal to a predetermined amount) of the target polynucleotide. The combination comprises: a capture oligomer comprising, from 5' to 3', a first portion C1 of the capture sequence, a second portion C2 of the capture sequence, an optional spacer sequence S, a second portion THS2 of the target hybridization sequence, a first portion THS1 of the target hybridization sequence, and an optional blocking portion (circled X); a separate complementary oligomer comprising, from 5' to 3', THS2′, S′ (optional; may or may not be used when S is present in the capture oligomer), and C2′ (where the complementary sequence of the element is indicated by “′”) and an optional blocking portion (circled X) at the 3' end; and a second capture agent comprising the complementary sequence of the capture sequence comprising, from 5' to 3', C2′, C1′ (C1′ or C2′ may be complementary to or not complementary to the full length of C1 and C2) and a binding partner (illustrated in this diagram by way of a biotin molecule, indicated by a circled B). In the absence of the target polynucleotide, the complementary oligomer binds to the capture oligomer and blocks the accessibility of the intact capture sequence to a degree sufficient to block the binding of the complementary sequence of the capture sequence in the second capture reagent (see the complex of the complementary oligomer and the capture oligomer at the top of the figure). When the target is present, the THS1 region of the capture oligomer binds to the target, followed by the THS2 region (which is energy-advantageous), thus displacing the THS2' region of the individual complementary oligomer from the capture oligomer. When this occurs, the C2' region of the individual complementary sequence is no longer stable enough to bind the capture oligomer and thus becomes unbound, resulting in the intact capture sequence available for binding, as shown below the first arrow. Then, as shown below the second arrow, the complementary sequence of the capture sequence in the second capture reagent binds to the capture sequence of the capture oligomer. The complex can then be isolated from the mixture, for example, by streptavidin-coated magnetic microspheres (as described elsewhere in this disclosure), thereby capturing and purifying the target polynucleotide. Optionally, the capture oligomer may be present in the combination in a larger amount than the second capture reagent. Such oligomers and combinations can be used to capture a certain amount (e.g., a limiting amount or an amount less than or equal to a predetermined amount) of target polynucleotides from the composition.

[0380] Figures 11A to 11B Exemplary molecules and exemplary reaction schemes according to this disclosure are shown. Figure 11A In this context, a method for capturing oligomers is provided, which comprises essentially as follows: Figure 4AThe element described in the capture oligomer differs in that THS binds to a site in a target chain (which may be circular or linear as shown) that does not contain a 3' end. A complementary oligomer is provided comprising (i) a target hybridization sequence annealed to an adjacent sequence of THS of the capture oligomer and (ii) a complementary sequence of at least a portion of A4. In the absence of a target chain, the complementary sequence of at least a portion of A4 is insufficient to anneal to the capture oligomer. Figure 11B In this process, the complementary oligomer is extended, which replaces C and makes it suitable for capture using a second capturing agent (not shown). This scheme can be used to capture cyclic molecules and / or represents an alternative method using capturing oligomers that do not bind at the 3' end of the target chain.

[0381] Figure 12 Exemplary molecules and exemplary reaction schemes according to this disclosure are shown. Containing, for example... Figure 4A A capture oligomer containing elements of the capture oligomer and having a second additional sequence A2 is annealed with the target strand at a site including its 3' end. The second additional sequence may contain a mixed nucleotide segment between C' and the internal extension blocking member. The target strand also includes a sequence A5 at its 5' end, which may be any sequence, a primer binding site used in a previous amplification reaction, or a sequence added in a previous step (e.g., amplification or ligation). Sequences A4', C', and A2' are added to the 3' end of the target strand along the extension of the capture oligomer. The presence of A4 in the capture oligomer and A4' in the extended target strand is optional. The extended target strand can then be annealed with a splice oligomer containing sequences A5', A2, C', and A4, wherein the 5' and 3' ends of the target strand are adjacent when the extended target strand is annealed with the splice oligomer. The extended target strand can then be circularized by ligation. The A2 and A2' sequences are used to ensure the proper juxtaposition of the 5' and 3' ends of the extended target strand. This can be helpful when C and C' are repetitive sequences (e.g., poly-A and poly-T or vice versa), which may otherwise tend to slip, inhibiting the formation of substrates for ligation. This scheme can be used to capture and subsequently cyclize target molecules, for example, in rolling circle amplification processes.

[0382] Figure 13Exemplary molecules and exemplary reaction schemes according to this disclosure are illustrated. A capture oligomer is provided comprising a capture sequence C (including a first portion C1 and a second portion C2; not shown), an internal extension blocking agent (solid circle), a spacer sequence S (including a first portion S1 and a second portion S2; not shown), and a target hybridization sequence THS, and a reverse amplification oligomer comprising sequence S2 is provided. THS and S2 are used to generate an amplified target (e.g., by PCR). A complementary oligomer is added comprising a complementary sequence S1′ of the first portion of the spacer sequence and a complementary sequence C2′ of the second portion of the capture sequence. When S′ anneals with the other strand S of the amplified target, C2′ is insufficient to anneal with C of the amplified target. The complementary oligomer anneals to capture the oligomer that has not annealed with the amplified strand. To capture the amplified target, a second capture agent comprising C' and a binding coupler or solid carrier (circled B) is added. The second capture agent binds the amplified target but does not bind the capture oligomer that has not annealed with the amplified strand, wherein C2′ of the complementary oligomer blocks C to a sufficient extent.

[0383] Figure 14 The fold difference in output is shown using methods for capturing oligomers with or without clamp sequences. Detailed Implementation

[0384] A. Definition

[0385] Before describing this teaching in detail, it should be understood that this disclosure is not limited to specific compositions or method steps, as these can vary. It should be noted that, as used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural indicators unless the context explicitly indicates otherwise, and expressions such as “one or more items” include singular indicators. Thus, for example, reference to “an oligomer” includes multiple oligomers, etc. The conjunction “or” should be interpreted in an inclusive sense, i.e., equivalent to “and / or,” unless such inclusive meaning is unreasonable in the context. When there is “at least one” member of a category (e.g., oligomer), reference to “the” member (e.g., oligomer) means either the member present (if only one) or at least one of the members present (e.g., multiple oligomers) (if more than one).

[0386] It should be understood that the terms "about" are implied before the temperatures, concentrations, amounts, times, etc., discussed in this disclosure, making slight and non-substantial deviations within the scope of the teachings herein. Generally, the term "about" indicates a non-substantial change in the amount of a component of the composition that has no significant effect on the activity or stability of the composition, for example, a variation within 10%, 5%, 2%, or 1%. Therefore, unless stated to the contrary, the numerical parameters set forth in the following description and the appended claims are approximate values ​​that may vary depending on the desired properties sought. At least, and without attempting to limit the application of the doctrine of equivalence to the scope of the claims, each numerical parameter should be interpreted by taking into account at least the number of significant figures and by applying conventional rounding techniques. Where there is no exclusionary expression such as "excluding endpoints," all ranges should be interpreted to include endpoints; thus, for example, "within 10 to 15" includes the values ​​10 and 15, as well as all intermediate integers and (where appropriate) non-integer values. Furthermore, the use of “comprise,” “comprises,” “comprising,” “contain,” and “include,” “includes,” “including” is not intended to be restrictive. It should be understood that the foregoing general and detailed descriptions are merely illustrative and explanatory, and not intended to limit the teachings. The paragraph headings provided are for the convenience of the reader only and are not intended to limit this disclosure. In the event of any discrepancy between any material incorporated by reference and the expression of this disclosure, the expression of this disclosure shall prevail.

[0387] Unless otherwise specified, embodiments described in the specification as “comprising” various components are also considered to “consist of the described components” or “substantially consist of the described components.” “Substantially consist of” means that the compositions or methods described herein may include one or more additional components, ingredients, or method steps that do not substantially alter the essential and novel characteristics of those compositions and methods. Depending on the specific circumstances, these characteristics include, for example, the ability to hybridize with target polynucleotides and undergo further binding and / or extension reactions as described herein.

[0388] "Sample" refers to a substance that may contain target polynucleotides, including but not limited to biological samples, clinical samples, environmental samples, and food samples. Environmental samples include environmental materials such as surface material, soil, water, air, and industrial samples, as well as samples obtained from food and dairy processing instruments, equipment, apparatus, utensils, disposable and non-disposable items. "Biological" or "clinical" samples refer to tissues or substances from living or dead human, animal, or other organisms that may contain target polynucleotides, including, for example, swabs, washes, aspirates, exudates, biopsy tissue, or bodily fluids such as blood or urine. Samples may be processed to physically or mechanically destroy tissue or cellular structures, thereby releasing intracellular nucleic acids into solutions that may contain enzymes, buffers, salts, detergents, etc., to prepare samples for analysis. These examples should not be construed as limiting the types of samples that may be applied to this disclosure.

[0389] “Nucleic acid” and “polynucleotide” refer to polymeric compounds containing nucleosides or nucleoside analogs, having nitrogen-containing heterocyclic bases or base analogs linked together to form polynucleotides, including conventional RNA, DNA, mixed RNA-DNA, and polymers as their analogs. The nucleic acid “backbone” can consist of a variety of bonds, including sugar-phosphodiester bonds, peptide-nucleic acid bonds (“peptide nucleic acid” or PNA; PCT No. WO 95 / 32305), thiophosphate bonds, methylphosphonate bonds, or combinations thereof, or more. The sugar moiety of a nucleic acid can be ribose, deoxyribose, or similar compounds with substituents (e.g., 2′methoxy or 2′halogen substitution). The nitrogenous bases can be conventional bases (A, G, C, T, U), their analogs (e.g., inosine or others; see The Biochemistry of the Nucleic Acids 5-36, Adams et al., 11th ed., 1992), or derivatives of purines or pyrimidines (e.g., N...). 4 -Methyldeoxyguanosine, denitropurine or azapurine, denitropyrimidine or azapyrimidine, pyrimidine bases with substituents at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with substituents at the 2, 6 or 8 position, 2-amino-6-methylaminopurine, O 6 -Methylguanine, 4-thiopyrimidine, 4-aminopyrimidine, 4-dimethylhydrazine-pyrimidine and O 4-alkyl-pyrimidine; U.S. Patent No. 5,378,825 and PCT No. WO 93 / 13121). Nucleic acids may include one or more "base-free" residues, wherein the backbone includes a base-free site of the polymer (U.S. Patent No. 5,585,481). Nucleic acids may contain only conventional RNA or DNA sugars, bases, and linkages, or may include conventional components and substitutions (e.g., conventional bases having a 2' methoxy linkage, or polymers containing conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), which are analogs containing one or more LNA nucleotide monomers having bicyclic furanose units locked in RNA in a sugar-mimicking conformation, which enhances hybridization affinity for complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Implementations of oligomers capable of affecting the stability of hybridization complexes include PNA oligomers, oligomers containing 2′-methoxy or 2′-fluorine-substituted RNA, or oligomers affecting the overall charge, charge density, or spatial relationships of the hybridization complex, including oligomers containing charged linkages (e.g., phosphate thioides) or neutral groups (e.g., methylphosphonates). Unless otherwise specified, methylated cytosine such as 5-methylcytosine can be used in combination with any of the aforementioned backbone / sugar / linkages, including RNA or DNA backbones (or mixtures thereof). RNA and DNA equivalents have different sugar moieties (i.e., ribose versus deoxyribose) and can differ due to the presence of uracil in RNA and thymine in DNA. Differences between RNA and DNA equivalents do not result in homology differences because the equivalents have the same degree of complementarity to a particular sequence. It should be understood that when referring to the length range of oligonucleotides, amplicones, or other nucleic acids, the range includes all integers (e.g., a length of 19 to 25 linked nucleotides includes 19, 20, 21, 22, 23, 24, and 25). Unless otherwise stated, “SEQ ID NO: X” refers to the base sequence of the corresponding sequence listing entry and does not require identity with the main strand (e.g., RNA, 2′-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues). Furthermore, unless otherwise stated, T residues are understood to be interchangeable with U residues and vice versa.

[0390] "Target polynucleotide" refers to a polynucleotide for which the composition or method described herein is sought to capture, isolate, amplify, detect, and / or sequence. In some embodiments, the target polynucleotide comprises a sequence of DNA or RNA from an organism (e.g., any virus, prokaryote, eukaryote, protist, plant, fungus, animal, mammal, or other biological entity, which may be alive or formerly alive). Exemplary DNA includes genomic DNA, free or plasmid DNA, and mitochondrial DNA. Exemplary RNA includes mRNA, more generally transcribed RNA, ribosomal RNA, miRNA, non-coding RNA, etc. (and, where applicable, genomic RNA, such as in the case of certain viruses). The target polynucleotide also includes an amplicon containing the aforementioned nucleic acid, wherein additional sequences (e.g., any additional sequences described herein) may be added. In some embodiments, the target polynucleotide comprises a sequence that is not naturally occurring, such as a sequence generated by in vitro synthesis, ligation, site-directed mutagenesis, recombination, etc.

[0391] "Oligomer" or "oligonucleotide" refers to nucleic acids typically less than 1,000 nucleotides (nt), including those in a size range of about 2 nt to 5 nt (lower limit) and about 500 nt to 900 nt (upper limit). Some specific embodiments are oligomers in a size range of about 5 nt to 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, or 20 nt (lower limit) and about 50 nt to 600 nt (upper limit), and other specific embodiments are oligomers in a size range of about 10 nt to 20 nt (lower limit) and about 30 nt to 100 nt (upper limit). Oligomers can be purified from naturally occurring sources but can be synthesized using any well-known enzymatic or chemical method. Oligomers may be referred to by functional names (e.g., capture probes, primers, or promoter primers), but those skilled in the art will understand that such terms refer to oligomers. Oligomers can form secondary and tertiary structures through self-hybridization or hybridization with other polynucleotides. Such structures can include, but are not limited to, double strands, hairpins, cross-shaped structures, curved structures, and triple strands. Oligomers can be generated by any means, including chemical synthesis, DNA replication, reverse transcription, PCR, or combinations thereof. In some embodiments, oligomers forming invasive cleavage structures are generated during a reaction (e.g., by primer extension in an enzymatic extension reaction).

[0392] "Arbitrary sequence" refers to any sequence selected, chosen, determined, designed, etc., by the user, typically used to provide the desired function or purpose in a downstream process. In a preferred mode, the arbitrary sequence is designed to be non-complementary to or non-reactive with the target sequence under given method conditions. In some embodiments, the arbitrary sequence may be a randomly generated sequence or a collection of sequences, for example, used as a unique molecular identifier.

[0393] "Capture oligomer", "capture oligonucleotide", "capture probe", "target capture oligomer" and "capture probe oligomer" are used interchangeably and refer to a nucleic acid oligomer containing: (i) a target hybridization sequence capable of specifically hybridizing to a target sequence in a target nucleic acid and (ii) a capture sequence capable of hybridizing to a second oligomer, for example, immobilized on a solid support or linked to a binding coupler to facilitate the separation of a complex containing the capture oligomer, the target, and the second oligomer from other molecules in the composition.

[0394] An "amplifier" or "amplification product" is a nucleic acid molecule generated in a nucleic acid amplification reaction, derived from a template nucleic acid. An amplifier or amplification product contains an amplified nucleic acid sequence (e.g., a target nucleic acid), which may be in the same or opposite direction as the template nucleic acid. In some embodiments, the length of an amplifier is approximately 100 to 30,000 nucleotides, approximately 100 to 10,000 nucleotides, approximately 100 to 5,000 nucleotides, 100 to 2,000 nucleotides, approximately 100 to 1,500 nucleotides, approximately 100 to 1,000 nucleotides, approximately 100 to 800 nucleotides, approximately 100 to 700 nucleotides, approximately 100 to 600 nucleotides, or approximately 100 to 500 nucleotides.

[0395] "Amplifying oligonucleotide" or "amplifying oligomer" refers to an oligonucleotide that hybridizes with a target nucleic acid or its complementary sequence and participates in a nucleic acid extension or amplification reaction, such as an oligonucleotide acting as a primer and / or promoter-primer. The amplifying oligomer also includes a promoter provider containing a promoter capable of initiating transcription but not necessarily extended by DNA polymerase, and may contain a 3' blocking portion. A particular amplifying oligomer contains a target hybridization sequence of at least about 10 consecutive bases, and optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases, which is complementary to a region of the target nucleic acid sequence or its complementary strand. Other exemplary lengths or length ranges of the target hybridization sequence are described elsewhere herein and may be applied to amplifying oligomers. The consecutive bases may be at least about 70%, at least about 80%, at least about 90%, or completely complementary to the target sequence binding the amplifying oligomer. In some embodiments, the amplification oligomer comprises an intermediate linker or a non-complementary sequence located between two segments of a complementary sequence. For example, the two complementary segments of the oligomer together comprise at least about 10 complementary bases, and optionally together at least 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 complementary bases. In some embodiments, the amplification oligomer is about 10 to about 60 bases long and may optionally include modified nucleotides. The amplification oligomer may be optionally modified, for example by including a 5' region that is non-complementary to the target sequence. Such modification may include functional additions, such as tags, promoters, or other sequences for manipulating or amplifying the primer or target oligonucleotide.

[0396] A primer is an oligomer that hybridizes to a template nucleic acid and has a 3' end that extends through polymerization. Primers can optionally be modified, for example by including a 5' region that is not complementary to the target sequence. Such modifications may include functional additions, such as tags, promoters, or other sequences for manipulating or amplifying the primer or the target oligonucleotide.

[0397] The first sequence is a “complementary sequence” (or equivalent to “complementary to” the second sequence) of the second sequence, wherein the first sequence has sufficient length and content to anneal with the second sequence under reasonable binding conditions, which may be, but need not be, the stringent hybridization conditions described herein, and also include, for example, the annealing conditions used in standard PCR and other techniques involving primer or probe binding and extension.

[0398] A “tag” is any additional sequence that may be included in an oligomer, other than the target hybridization sequence. Any sequence that exists besides the target hybridization sequence can be used as a tag. Tags include, but are not limited to, adaptors (see below). Other examples of tags are promoters, mixed nucleotide elements described elsewhere herein, and stable sequences including clips.

[0399] An "adaptor" is a sequence that modifies a molecule to provide a binding site for another molecule, such as a target hybridization sequence (THS) of a sequencing primer or a capture oligomer. The binding site can be a universal binding site (e.g., binding multiple capture oligomers, all having the same THS, in multiple forms, or binding a universal primer). Other examples of binding sites include binding sites for substitution oligomers, probes, capture oligomers, or nucleic acid-modifying enzymes (e.g., RNA polymerases, primases, ligases, RNases (e.g., RNase H), or restriction enzymes), or binding sites for attachment to a solid phase (including attachment via solid-phase primers or capture oligomers), including those for clonal amplification, or other functional elements for downstream applications (e.g., enrichment, library preparation, clonal amplification, or sequencing). Therefore, sample barcodes or index sequences, key sequences or calibration sequences, molecular barcodes (including unique molecular identifiers), sites for downstream cloning, and sites for target molecule circularization are other examples of elements that can be included in an adaptor.

[0400] A "connector" is a sequence or non-sequence element, or a combination thereof, that links one part of an oligomer to another. In some embodiments, the sequence adapter comprises a sequence that does not hybridize with the target polynucleotide and / or other oligomers in the combination or composition. In some embodiments, the non-sequence adapter comprises an alkyl, alkenyl, amide, or polyethylene glycol group [(-CH2CH2O-]]. n ].

[0401] "Stable sequences" are clamps, mixed nucleotide regions, or other sequences that function to increase the stability of double-stranded regions and / or control hybridization (e.g., when located near sequences that tend to slide, such as sequences containing repeating nucleotides, such as poly-dA or poly-dT sequences). "Alignment sequences" are stable sequences that control hybridization. In addition to clamps and mixed nucleotide regions described elsewhere herein, stable sequences include GC-rich sequences and sequences containing affinity-enhancing modifications.

[0402] An "internal extension blocker" is an element located within or bound to a nucleic acid sequence that prevents the complementary strand from extending along the nucleic acid. Examples include non-nucleotide linkers, one or more debasement sites, non-natural nucleotides or chemically modified natural nucleotides, and the reversible extension blockers discussed below.

[0403] A “reversible extension blocker” is an internal extension blocker whose blocking function can be reversed, i.e., it allows the complementary strand to extend. Exemplary reversible extension blockers are non-natural nucleotides with complementary nucleotides that are suitable for polymerases and exhibit specificity relative to the natural nucleotide (i.e., the polymerase does not add a natural base across the reversible extension blocker). Providing a complementary nucleotide reverses the blocking function. Examples of non-natural base pairs are Iso-dC or Iso-dG; xanthine or 5-(2,4-diaminopyrimidine); 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one; 4-methylbenzimidazole or 2,4-difluorotoluene; 7-azaindole or isoquinolone; dMMO2 or d5SICS; or dF or dQ, wherein any member of these non-natural base pairs can serve as a reversible extension blocker. Other examples of reversible extension blocking molecules are chemically modified nucleotides, wherein the modification is performed by attachment via a reversible linker bond, and said linker bond can be reversed by providing one or more of the following: chemicals, enzymes, temperature changes, reagent composition changes, etc.; reversible nucleic acid structural features; or molecules that reversibly bind to the capture oligomer, optionally wherein said reversibly binding molecule is a protein, enzyme, lipid, carbohydrate, or chemical moiety.

[0404] "Nucleic acid amplification" refers to any in vitro method that produces multiple copies of a target nucleic acid sequence or its complementary sequence or fragment thereof (i.e., an amplified sequence containing fewer copies than the complete target nucleic acid). Examples of nucleic acid amplification methods include transcription-related methods, such as transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), and other methods (e.g., U.S. Patent Nos. 5,399,491, 5,554,516, 5,437,990, 5,130,238, 4,868,105, and 5,124,246), replicase-mediated amplification (e.g., U.S. Patent No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Patent No. 4,786,600), and polymerase chain reaction (PCR). Examples of replication-mediated amplification include: U.S. Patent Nos. 4,683,195, 4,683,202, and 4,800,159; rolling circle amplification (RCA) (e.g., U.S. Patent Nos. 5,854,033 and 6,143,495); recombinase polymerase amplification (RPA) (e.g., U.S. Patent No. 7,666,598); ligase chain reaction (LCR) (e.g., European Patent Application 0320308); and strand displacement amplification (SDA) (e.g., U.S. Patent No. 5,422,252). Replicaase-mediated amplification uses self-replicating RNA molecules and replicases such as QB replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of the two complementary strands of DNA or cDNA. LCR amplification uses at least four individual oligonucleotides to amplify the target and its complementary strand through multiple hybridization, ligation, and denaturation cycles. SDA uses primers containing a restriction endonuclease recognition site, which creates a nick on one strand of a semi-modified DNA duplex containing the target sequence, followed by amplification through a series of primer extension and strand displacement steps. A specific embodiment uses PCR, but it will be apparent to those skilled in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods.

[0405] "Hybridization" or "hybridize" refers to the ability of two completely or partially complementary nucleic acid chains to come together in parallel or antiparallel directions under specific hybridization test conditions to form a stable structure with double-stranded regions. "Hybridization" is synonymous with "annealing" or "anneal." The two component chains of this double-stranded structure (sometimes called a hybrid) are held together by hydrogen bonds. Although these hydrogen bonds most commonly form between nucleotides containing the bases adenine and thymine or uracil (A and T or U) or cytosine and guanine (C and G) on a single nucleic acid chain, base pairing can also form between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, for example, RLPAdams et al., The Biochemistry of the Nucleic Acids (11th edition, 1992))

[0406] As used herein, the term "specific hybridization" refers to a probe, primer, or other oligomer (e.g., a capture oligomer) that, under given hybridization conditions, detectably hybridizes essentially only with one or more target sequences in a sample containing one or more target sequences (i.e., exhibits little or no detectable hybridization with non-target sequences). It is noteworthy that oligomers can be configured to specifically hybridize with any one of a set of targets (e.g., sequences from organisms of a particular taxonomic group (e.g., genus)). In some embodiments, the probe, primer, or other oligomer (e.g., a capture oligomer) may hybridize with its target nucleic acid to form stable oligomers: target heterozygotes, but not a sufficient number of stable oligomers: non-target heterozygotes, for amplification or capture, depending on the circumstances. Amplifying oligomers and capture oligomers that specifically hybridize with target nucleic acids can be used to amplify and capture target nucleic acids, but not for amplifying and capturing non-target nucleic acids, especially those from closely related organisms. Therefore, oligomers hybridize to a much greater extent with target nucleic acids than with non-target nucleic acids, enabling those skilled in the art to accurately capture, amplify, and / or detect the presence (or absence) of nucleic acids derived from a specific target (e.g., a specific pathogen). Typically, reducing the complementarity between an oligonucleotide sequence and its target sequence will decrease the degree or rate of hybridization between the oligonucleotide and its target region. However, the inclusion of one or more non-complementary nucleosides or bases can enhance the oligonucleotide's ability to distinguish non-target nucleic acid sequences.

[0407] "Strong hybridization conditions" or "strict conditions" refer to conditions that: (1) allow oligomers to preferentially hybridize with the target nucleic acid rather than with different nucleic acids (e.g., nucleic acids that differ from the target nucleic acid by as little as one nucleotide in identity) or (2) allow only oligomers with a target hybridization sequence of higher affinity (as opposed to oligomers with a target hybridization sequence of lower affinity) to hybridize with the target, for example, where the higher affinity target hybridization sequence is longer than the lower affinity target hybridization sequence and / or the higher affinity target hybridization sequence contains an affinity-enhancing modification while the lower affinity target hybridization sequence does not contain that affinity-enhancing modification. While the definition of strict hybridization conditions remains unchanged, the actual reaction environment that can be used for strict hybridization can vary depending on factors including GC content and oligomer length, and the degree of similarity between the oligomer sequence and the target and non-target nucleic acid sequences that may be present in the test sample. Hybridization conditions include temperature and the composition of the hybridization reagent or solution. When the concentration of the monovalent cation is in the range of about 0.4 M to 1 M, the concentration of the divalent cation is in the range of about 0 to 10 mM, and the pH is in the range of about 5 to 9, the exemplary stringent hybridization conditions using the oligomers of this disclosure correspond to temperatures of about 40°C to 75°C, for example, 40°C to 50°C, 50°C to 60°C, or 60°C to 75°C. Further details of the hybridization conditions will be set forth in the Examples section. Other acceptable stringent hybridization conditions can be readily determined by those skilled in the art.

[0408] A “label” or “detectable label” refers to a portion or compound that is directly or indirectly attached to a probe that is being detected or that generates a detectable signal. Any detectable portion can be used, such as a radionuclide, a ligand such as biotin or avidin, an enzyme, an enzyme substrate, a reactive group, a chromophore such as a dye, or particles that impart a detectable color (e.g., latex or metal beads), a luminescent compound (e.g., a bioluminescent, phosphorescent, or chemiluminescent compound), and a fluorescent compound (i.e., a fluorophore). Embodiments of fluorophores include those that absorb (e.g., have a peak absorption wavelength) light in the range of about 495 nm to 690 nm and emit (e.g., have a peak emission wavelength) light in the range of about 520 nm to 710 nm, including those referred to as FAM. TM TET TM HEX, CAL FLUOR TM (orange or red), CY and QUASAR TM The fluorophore of the compound. The fluorophore can be used in combination with a quencher molecule; when in close proximity to the fluorophore, the quencher molecule absorbs light to attenuate background fluorescence. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHAER. TM (or BHQ) TM Blackberry (or BBQ) ), Or TAMRA TM Compounds.

[0409] "Non-extending" oligomers, or oligomers comprising a "blocking portion at its 3' end," include a blocking portion sufficiently close to its 3' end (also referred to as the 3' end) to prevent extension. For the purposes of this disclosure, any blocking portion sufficiently close to the 3' end to prevent extension is considered to be "located" at the 3' end, even if it is not bound to or present in place of a 3' hydroxyl or oxygen. In some embodiments, the blocking portion near the 3' end is within five residues of the 3' end and is large enough to limit the binding of the polymerase to the oligomer, and other embodiments include a blocking portion covalently linked to the 3' end. Many different chemical groups can be used to block the 3' end, such as alkyl groups, non-nucleotide linkers, alkane-diol dideoxynucleotide residues (e.g., 3'-hexanediol residues), and cordycepin. Other examples of blocking portions include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other markers that interfere with elongation; reverse nucleotides (e.g., linked to the preceding nucleotide via a 3′-to-3′ phosphodiester, optionally having an exposed 5'-OH or phosphate ester); or proteins or peptides linked to oligonucleotides to prevent further elongation of the initial nucleic acid chain by polymerase. The non-elongating oligonucleotides of this disclosure can be at least 10 bases in length and can be up to 15, 20, 25, 30, 35, 40, 50, or more nucleotides in length. Non-elongating oligonucleotides containing detectable markers can be used as probes.

[0410] "Conjugation couple" refers to a member of a pair of parts that can be used to form a non-covalent bond. Exemplary groups of conjugation couples are biotin and biotin-binding agents. Other examples of conjugation couples include, but are not limited to, digoxigenin / anti-digoxigenin, and more generally, antibodies and their targets.

[0411] "Biotin binders" are reagents (e.g., peptides) capable of specifically binding biotin. Streptavidin, avidin, and neutral avidin represent examples of biotin binders. Anti-biotin antibodies are also considered biotin binders.

[0412] The term "antibody" includes any polypeptide containing a functional antigen-binding region with a complementarity-determining region and a framework region (e.g., VH and VL domains), including but not limited to scFv, Fab, and full-length antibodies (e.g., IgA, IgG, IgD, IgE, or IgM antibodies).

[0413] As used herein, a “kit” is a package of reagents that includes, for example, one or more oligonucleotides disclosed herein. For example, a kit may include a package of one or more vials, tubes, or cartridges having multiple chambers containing reagents for isolating target polynucleotides. Reagents may include capturing oligonucleotides, primer oligonucleotides, and probe oligonucleotides (such as those described herein), and nucleotide polymerases (e.g., DNA polymerase, reverse transcriptase, RNA polymerase, etc.). In some embodiments, the reagents may be in liquid, solid (e.g., lyophilized), or semi-solid (e.g., glass). In some embodiments, oligonucleotide reagents and enzyme reagents are present in the kit as components of a single lyophilized composition (e.g., pellets). In this case, primers, probes, and one or more enzymes (e.g., DNA polymerase) may be lyophilized in the same reaction chamber or container, the lyophilized form being reconstituted with an aqueous reagent, wherein the same kit includes separate vials or tubes containing the aqueous reagent. The kit may further include a number of optional components, such as other oligomers. Other reagents that may be present in the kit include those suitable for in vitro amplification, such as buffers, salt solutions, and / or appropriate triphosphates (e.g., dATP, dCTP, dGTP, dTTP; and / or ATP, CTP, GTP, and UTP). The kit may further include a solid support material (e.g., magnetically attractable particles, such as magnetic beads) for directly or indirectly immobilizing the oligomers during sample preparation procedures. In some embodiments, the kit further includes a set of instructions for carrying out the methods according to this disclosure, wherein the instructions may relate to packaging inserts and / or the packaging of the kit or its components.

[0414] As used herein, a “combination” of oligomers means any plurality of oligomers that are close to each other, such as in different or the same containers in a kit, or in a combination or group of combinations placed side by side, such as in a plate, rack or other container.

[0415] Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by those skilled in the art. General definitions can be found in technical books related to the field of molecular biology, such as the 2nd edition of the Dictionary of Microbiology and Molecular Biology (Singleton et al., 1994, John Wiley & Sons, New York, NY) or The Harper Collins Dictionary of Biology (Hale & Marham, 1991, Harper Perennial, New York, NY).

[0416] B. Exemplary compositions, kits, methods, and uses

[0417] This disclosure provides oligomers, compositions, and kits for isolating target polynucleotides and / or attachment tags (such as adaptors). Isolation involves separation in limited quantities (limited capture) and specific quantities (copy control). For example, in some workflows, it is desirable to capture (or amplify and capture) no more than a predetermined quantity (e.g., the maximum expected value for downstream applications such as sequencing library preparation), but otherwise, it is desirable to capture as high a quantity of target polynucleotides as possible. Similarly, in some workflows, it is desirable to capture (or amplify and capture) a predetermined specific quantity (e.g., a specific number of molecules or molecular copies) of target polynucleotides (which may be, for example, natural DNA or RNA, amplicon, or sequencing library) for downstream applications (e.g., cloning amplification, including in next-generation sequencing workflows). Furthermore, in some workflows, it is desirable to introduce additional sequences into the target polynucleotides, such as introducing adaptors into sequencing libraries. Oligomers comprising various elements are described herein. Unless otherwise stated, other elements may exist before, between, or after the elements of the oligomer, as long as these other elements do not interfere with the functionality of the elements.

[0418] In some embodiments, for example, the oligomer is provided in a kit or composition. The oligomer typically contains a target hybridization region, which is configured to specifically hybridize to a target polynucleotide. While oligomers of varying lengths and base compositions can be used, in some embodiments, the length of the target hybridization region of the oligomer in this disclosure is described in a paragraph discussing the target hybridization sequence. In some embodiments, the oligomer includes an additional sequence region, as detailed elsewhere herein, which may be located at the 5' of the target hybridization region. In some embodiments, the oligomer does not contain a second sequence region. In some embodiments, the additional sequence region includes a capture sequence.

[0419] 1. Capture oligomers and combinations for copy control and other applications

[0420] a. Capture oligomers containing the capture sequence and its complementary sequence

[0421] In some embodiments, a capture oligomer is provided, comprising a capture sequence, an internal extension blocking group, a complementary sequence of the capture sequence, and a target hybridization sequence, wherein the complementary sequence of the capture sequence is configured to anneal with the capture sequence when no extended target sequence annealed to the target hybridization sequence and the complementary sequence of the capture sequence is present. Figures 1A to 1C As illustrated in the example of the capture oligomer, such capture oligomers can be captured in a target-dependent manner because the capture sequence is initially annealed with the complementary sequence (C') of the capture sequence. Figure 1A However, after the target polynucleotide is extended, it becomes able to bind via C'. Figure 1B The internal extension blocker prevents the target from extending along the capture sequence itself. Therefore, the target-capture oligomer extension product readily binds to a second capture agent containing a complementary sequence to the capture sequence and a binding pair or solid matrix. Figure 1C Simultaneously, any unbound capture oligomers do not serve as extension templates, allowing C' to remain annealed with the capture sequence, and the unbound capture oligomers substantially do not interact with the second capture reagent. Therefore, after separating the complex of the target-capture oligomer extension product, the unbound capture oligomers will remain substantially in the original solution, and separation can be accomplished using appropriate standard techniques based on the properties of the binding coupler (e.g., biotin) or solid matrix (e.g., magnetic beads). Figure 2A The diagram schematically illustrates the complex of the target bound to the beads and the trapping oligomers, and Figure 2B The elution of the target from the beads is shown.

[0422] Such capture oligomers and their combinations with suitable second capture reagents can be used in any application requiring capture, restriction capture, or copy control, as well as any application requiring the introduction of additional sequences. The capture oligomers described herein with an extendable 3' end can be used as amplification oligomers (e.g., primers), for example, together with amplification primers, target strands, or amplicon strands having the reverse orientation of the capture oligomer, to produce an extension product or amplicon, which can then be separated by contacting the formed complex with a second capture reagent and performing an appropriate separation step. In other embodiments, the capture oligomer is not used as an amplification oligomer, or not used for multiple cycles of extension, and the amplicon or native DNA or RNA can be captured simply by annealing with the capture oligomer (and optionally, or where appropriate, by other oligomers discussed herein, such as complementary or substitutional oligomers plus a reverse amplification oligomer, which may facilitate target-dependent substitution of the complementary sequence of the capture sequence, as discussed in detail elsewhere herein), extension with a polymerase, followed by contacting the extended complex with a second capture reagent and performing an appropriate separation step. In either case, the amount of the second capture reagent can be selected to set a specific amount or maximum amount required for capture. Furthermore, the amounts of both the capture oligomer and the second capture reagent can be selected to set a specific amount or maximum amount required for capture; for example, the amount of the capture oligomer can be less than the amount of the target polynucleotide, and the amount of the second capture reagent can be less than the amount of the capture oligomer.

[0423] According to the exemplary formula for capturing oligomers disclosed herein,

[0424] 5′-A1-CLB-A2-C'-A3-RB-A4-THS-X-3'

[0425] Where A1 is the first additional sequence that exists arbitrarily;

[0426] C represents the capture sequence.

[0427] L is an optional connector.

[0428] B is an internally extended blocking element.

[0429] A2 is an arbitrarily existing second additional sequence.

[0430] C′ is the complementary sequence of the captured sequence.

[0431] A3 is an arbitrarily existing third additional sequence.

[0432] RB is an arbitrarily existing reversible extension blocking element.

[0433] A4 is an arbitrarily existing fourth additional sequence.

[0434] THS is the target hybridization sequence; and

[0435] X represents any arbitrarily existing blocking component.

[0436] In the embodiments described herein, including but not limited to capture oligomers according to the above formula, the ordinal numbers (first, second, third, and fourth) preceding each additional sequence are used only to allow specific reference to each possible individual additional sequence and do not necessarily imply the presence of any other additional sequence. Therefore, a capture oligomer may contain any one additional sequence, or any combination of two or more additional sequences, such as only the fourth additional sequence; the third and fourth additional sequences; the first, third, and fourth additional sequences; and so on. Furthermore, any additional sequence may contain multiple elements (e.g., barcodes and primer binding sites, or either or both of these plus an additional sequence) or consist of a single element.

[0437] When a first additional sequence is present, the first additional sequence may contain any sequence or one or more bases that may be used, for example, to protect the 5' end of the capture sequence or as an enzyme cleavage site, enzyme recognition site (e.g., for restriction endonucleases) or as a stabilizing sequence (e.g., a clamp) for stabilizing the duplex and / or aligning the duplex regions. Figure 3 The captured oligomer containing the clip sequence is shown.

[0438] The capture sequence may include any sequence suitable for use as a capture sequence, such as any capture sequence implementation described herein, including the implementations set forth in the oligomeric element paragraphs above and below.

[0439] When a linker is present, it can be a sequence or non-sequence element, or a combination thereof. The linker can provide flexibility to facilitate self-hybridization between the complementary sequence of the capturing sequence and the capturing sequence, and can adopt a cyclic conformation (e.g., essentially single-stranded when the linker is a sequence element). Exemplary non-sequence linkers include alkyl, alkenyl, amide, and polyethylene glycol groups [(-CH2CH2O-]]. n The exemplary length of the connector is 3, 6, 12, or 18 or more atoms. Other connector implementations are provided elsewhere in this document.

[0440] Internal extension blockers are elements that DNA polymerase cannot penetrate, thus preventing the extension of the strand complementary to the one to which the internal extension block sequence is part. In some embodiments, internal extension blockers are abase sites, chemically modified nucleotides, non-natural nucleotides (e.g., isoC or isoG or other non-natural nucleotides described elsewhere herein; for the blocker to be effective, the reaction mixture must not contain complementary non-natural nucleotides; for example, if isoC is the blocker, isoG cannot be present, and vice versa), or non-sequence elements (e.g., any of the non-sequence adapters described above). Non-sequence adapters can function as internal extension blockers, or adapters and internal extension blockers can exist separately. When non-natural bases such as isoC or isoG are used as internal extension blockers, there are no complementary bases, thus preventing DNA polymerase from penetrating the modified base site.

[0441] When a second additional sequence is present, the second additional sequence may contain a tag, such as a mixed nucleotide element as described below. In some embodiments, for example, when used in combination with a corresponding complementary sequence in another additional sequence, the second additional sequence contains an enzyme cleavage site or a sequence (e.g., a clip sequence) for stabilizing and / or aligning the overlapping of double-stranded regions.

[0442] The complementary sequence of the capture sequence may comprise a complementary sequence of any sequence suitable for use as a capture sequence, such as the complementary sequence of any capture sequence embodiment described herein, including the embodiments set forth in the oligomeric element paragraphs above and below. In some embodiments, the complementarity level of the complementary sequence of the capture sequence to the capture sequence is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. Furthermore, the complementary sequence of the capture sequence may be complementary to only a portion of the capture sequence, such as 50%, 60%, 70%, 80%, or 90% of the capture sequence.

[0443] When a third additional sequence is present, such as when used in combination with a corresponding complementary sequence in the first additional sequence, the third additional sequence may contain a tag or restriction site or a sequence for stabilizing and / or aligning the superposition of duplex regions (e.g., a clip sequence). Complementary clip sequences can make self-hybridization more energy-favorable and / or help complementary regions C and C' maintain the desired superposition (alignment), and can improve the performance of capturing oligomers, as demonstrated in the examples. Figure 3The diagram illustrates a capture oligomer containing a clip sequence. In some embodiments, the third additional sequence comprises an adaptor or tag, such as a sequence that can be used as a binding site for primers (including sequencing primers), an anchoring site for oligomers or probes in subsequent reactions or steps, or a binding site for enzyme cleavage or enzymes (e.g., restriction endonucleases), or as a barcode or other tag, for example, to identify the origin of a sample or molecule and facilitate methods involving pooling, such as highly parallel sequencing. Such a sequence can be part of the third additional sequence, as well as a stable (e.g., clip) sequence.

[0444] When a reversible extension blocker is present, it can be used to restrict the extension of the complementary strand along the capture oligomer for a first time period, (e.g.) to maintain the specificity of the binding of the target hybridization sequence (THS) to the target polynucleotide when the capture oligomer is used as the amplification oligomer. Exemplary reversible extension blockers include IsoG and IsoC, as well as other reversible extension blockers described elsewhere herein, which can be unblocked by adding a complementary nucleotide (e.g., IsoC for unblocking IsoG, and vice versa). When using a reversible extension blocker, an element different from the reversible extension blocker should be used as an internal extension blocker at the 5' of the complementary sequence of the capture sequence. The reversible extension blocker can also be used for amplification oligomers (e.g., in extension / amplification reactions, reverse amplification primers other than the capture oligomer), positioned at the 5' of the THS and the 3' of any other sequence present. As described above, this can maintain the binding specificity of the THS of the amplification oligomer to the target polynucleotide during amplification, among other advantages. The reversible extension blocking factor can be reversed after amplification, and a complementary sequence to the additional sequence can be generated in a single extension step, thereby introducing the additional sequence into the amplicon. If the capture oligomer also contains a reversible extension blocking factor, it can also optionally be reversed, and a complementary sequence of the capture oligomer up to the inner extension blocking factor (as described above) is generated concurrently with a similar process in the amplification oligomer. This single extension step can optionally be carried out in the same reaction mixture as the amplification reaction. This represents a rapid and simple method to maintain specificity while also introducing the additional sequence into the product (e.g., it can be used to add adaptors to sequencing libraries).

[0445] When a fourth additional sequence is present, it may contain an adaptor or tag, which may serve as a binding site sequence for primers, probes, anchored oligonucleotides, etc., or as a barcode or other tag, for example, to identify the origin of a sample or molecule and facilitate hybrid sequencing methods such as highly parallel sequencing. Such a sequence may be part of the fourth additional sequence, as well as a stable sequence, alignment sequence, restriction enzyme site, or enzyme binding site. In some embodiments, the fourth additional sequence contains a stable sequence or alignment sequence.

[0446] In any of the foregoing embodiments, any of the additional sequences (or their complementary sequences formed by extending along the additional sequences) contain tags useful for downstream processes (e.g., adding adaptors useful for library preparation, clonal amplification, sequencing, or data analysis), and combining tag addition with target capture can make the overall workflow more efficient.

[0447] The target hybridization sequence can be any sequence with sufficient length and complementarity to hybridize with a given target, such as any target hybridization sequence implementation described herein, including the implementations set forth in the oligomeric element paragraphs above and below.

[0448] When a blocking moiety is present, it can be any portion that prevents the polymerase from extending its 3' end. Exemplary blocking moieties are described in detail elsewhere herein. The blocking moiety can prevent the extension of the dimer of the capture oligomer and subsequent capture, which would otherwise occur, for example, when the concentration of the capture oligomer is high and / or the target hybridization sequence has a certain dimerization potential. See also Figure 6 .

[0449] i. Combination

[0450] In some embodiments, a combination is provided comprising a capturing oligomer according to the present disclosure and one or more additional oligomers. The additional oligomers may comprise any one of, or any combination of two or more of, the following: complementary oligomers, which are used to capture target molecules (which may be cyclic) without binding the capturing oligomer to a site containing the 3′ end of the target (see [link to documentation] for an explanation of this combination and its use in capturing cyclic target molecules). Figures 11A to 11B The combination may include: an amplifying oligomer, for example, binding a target polynucleotide in the reverse direction relative to the capturing oligomer; a pair of amplifying oligomers, for example, configured to amplify the target; a second capturing agent, for example, containing a complementary sequence of the capturing sequence and a binding partner or solid carrier; a splint oligomer; a blocking oligomer; or a displacement oligomer. These additional oligomers are described in detail elsewhere herein. The combination may also include one or more additional capturing oligomers, for example, for multiple capture of multiple target polynucleotides. Additional capturing oligomers may be accompanied by other additional oligomers, such as suitable reverse amplifying oligomers, displacement oligomers, blocking oligomers, etc. When multiple capturing oligomers are used, these capturing oligomers may have the same or sufficiently similar capturing sequences so that a single second capturing agent can be used. Alternatively, these capturing oligomers may have different capturing sequences, for example, such that if a target is present in high abundance, the resulting complex with extended capturing oligomers will not saturate the second capturing agent to exclude target complexes of lower abundance.

[0451] In some embodiments, the combination comprises a capture oligomer and a complementary oligomer as described herein. The capture oligomer includes a complementary sequence to the capture sequence, at least one second, third, or fourth additional sequence, and other elements. The complementary oligomer includes a complementary sequence to at least a portion of the target hybridization sequence and the second, third, or fourth additional sequence in the 5' to 3' direction. See also Figure 11A The target hybridization sequence of the complementary oligomer should bind to a portion of the target near the target (3' side, depending on the target orientation) of the target hybridization sequence of the capture oligomer. At least a portion of the complementary sequence of the second or third additional sequence should be configured such that it anneals to the capture oligomer in a target-dependent manner, i.e., it should not anneal in the absence of a target. The ternary complex of the target, the capture oligomer, and the complementary oligomer is a substrate used to extend the 3' end of the complementary oligomer via polymerase, and by this extension, to replace the capture sequence with the complementary sequence of the capture sequence, making it usable for capture using a second capture reagent.

[0452] b. Combinations containing capturing oligomers and complementary oligomers

[0453] In some embodiments, a combination comprising a trapping oligomer and a complementary oligomer is provided, wherein: (a) the trapping oligomer comprises, in the 5' to 3' direction: a trapping sequence comprising a first portion and a second portion; an internal extension blocking member; a spacer sequence comprising a first portion and a second portion; and a target hybridization sequence; and (b) the complementary oligomer comprises, in the 3' to 5' direction: a complementary sequence of the second portion of the trapping sequence and a complementary sequence of at least the first portion of the spacer sequence, wherein the complementary sequence of the second portion of the trapping sequence and the complementary sequence of the first portion of the spacer sequence are configured to anneal simultaneously with the trapping oligomer when the complementary sequence of the spacer sequence is not present. See also Figure 10A This demonstrates this combination and its use.

[0454] Such captured oligomers can have the following formula:

[0455] 5′-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3'

[0456] Where A1 is the first additional sequence that exists arbitrarily.

[0457] C1 is the first part of the capture sequence.

[0458] C2 is the second part of the capture sequence.

[0459] B is an internally extended blocking element.

[0460] A2 is an arbitrarily existing second additional sequence.

[0461] S1 is the first part of the interval sequence.

[0462] S2 is the second part of the interval sequence.

[0463] A3 is an arbitrarily existing third additional sequence.

[0464] RB is an arbitrarily existing reversible extension blocking element.

[0465] A4 is an arbitrarily existing fourth additional sequence.

[0466] THS is the target hybridization sequence, and

[0467] X represents any arbitrarily existing blocking component.

[0468] Complementary oligomers can have the following formula:

[0469] 5′-S1'-A2'-L-C2′-X-3'

[0470] Where S1' is the complementary sequence of the first part of the spacer sequence.

[0471] A2' is an optional complementary sequence of a second additional sequence that may be present in the captured oligomer;

[0472] L is an optional connector.

[0473] C2' is the complementary sequence to the second part of the captured sequence, and

[0474] X represents any arbitrarily existing blocking component.

[0475] Figure 10A An exemplary combination of capture oligomers according to the description above is shown. In the absence of a target, the complementary oligomer is annealed with the capture oligomer. After hybridization with the target and subsequent 3' end extension, the complementary oligomer is substituted. Due to the extension, substitution, and generation of a double strand in the "S" region (which blocks the re-annealing of the complementary oligomer), the capture sequence remains essentially single-stranded and can therefore contact the complementary sequence containing the capture sequence and a second capture agent that binds to the partner or bead, thereby facilitating subsequent separation steps.

[0476] Figure 13 Different exemplary workflows using combinations of capturing oligomers as described above are shown, wherein complementary oligomers are provided after the target extends along the capturing oligomers, rather than before annealing the capturing oligomers with the target. Furthermore, in Figure 13In the described exemplary workflow, a reverse amplification oligomer is provided, which is used in conjunction with a capture oligomer in an amplification reaction (e.g., PCR) to produce a product that introduces a spacer region into the amplicon. This spacer region is an arbitrary sequence. In some embodiments of this workflow, a complementary oligomer is provided during the amplification reaction. In this mode, the specificity of the amplification reaction is improved by blocking at least a portion of the spacer and capture sequences during the annealing phase of the reaction, and by annealing with the capture oligomer after the amplification reaction is complete, even in the absence of a complementary sequence to the spacer sequence.

[0477] When a first additional sequence is present, the first additional sequence may contain a tag. In some embodiments, the first additional sequence contains one or more bases that can be used, for example, to protect the 5' end of the capture sequence or as a cleavage site or enzyme recognition site (e.g., restriction endonuclease).

[0478] The first and second portions of the capture sequence form the capture sequence, for example, any sequence suitable for use as a capture sequence, such as any capture sequence implementation described herein, including the implementations set forth in the oligomeric element paragraphs above and below.

[0479] Internal extension blocking elements are those that DNA polymerase cannot pass through. In some embodiments, internal extension blocking elements are debase sites, chemically modified nucleotides, non-natural nucleotides (e.g., isoC or isoG or any other non-natural nucleotide added without using a natural nucleotide as a template), or non-sequence elements (e.g., any non-sequence adapter described above). Non-sequence adapters can function as internal extension blocking elements. When modified bases such as isoC or isoG are used as internal extension blocking elements, they lack complementary bases, thus preventing DNA polymerase from passing through the modified base site.

[0480] When a second additional sequence is present, it contains an adaptor or tag, which may serve as a binding site sequence for primers, probes, anchored oligonucleotides, etc., or as a barcode or other tag, for example, to identify the origin of a sample or molecule and facilitate hybrid sequencing methods such as highly parallel sequencing. Such a sequence may be part of the second additional sequence, as well as a stable sequence, alignment sequence, restriction enzyme site, or enzyme binding site. In some embodiments, the second additional sequence contains a stable sequence or alignment sequence.

[0481] The first and second portions of the spacer sequence form the spacer sequence. At least the first portion of the spacer sequence is responsible for binding the complementary oligomer. When the target polynucleotide, including its 3' end, anneals with the capture oligomer, the spacer sequence is configured to act as a template for extension, wherein such extension causes substitution of the complementary oligomer, thereby making the capture sequence available to a second capture agent. The spacer sequence is distinct from the target hybridization sequence, the capture sequence, and their complementary sequences; that is, the spacer sequence should not substantially hybridize with any of the aforementioned sequences. The spacer sequence, or its first or second portion, may also contain any of the elements described above with respect to the second additional sequence.

[0482] When a third additional sequence is present, it may comprise an adaptor or tag, such as any sequence or a sequence that acts as a target binding site, to be used in subsequent reactions or steps as a primer or probe, anchor oligonucleotide, or similar binding site (e.g., an adaptor), or as a barcode or other tag, for example, to identify the origin of a sample or molecule and to facilitate hybrid sequencing methods involving highly parallel sequencing, such as sequencing in parallel. Such a target sequence may be part of the third additional sequence, as well as a clamping stable sequence, alignment sequence, restriction enzyme site, or enzyme binding site sequence. In some embodiments, the third additional sequence comprises a stable or alignment clamp sequence.

[0483] When a reversible extension blocker is present, it can be used to restrict the extension of the complementary strand along the capture oligomer for a first time period, (e.g.) to maintain the specificity of the binding of the target hybridization sequence to the target polynucleotide when the capture oligomer is used as the amplification oligomer. Exemplary reversible extension blockers include IsoG and IsoC, as well as other members of non-natural nucleotide pairs described elsewhere herein, which can be unblocked by adding a complementary nucleotide (e.g., IsoC is used to unblock IsoG, and vice versa). When using a reversible extension blocker, elements different from the reversible extension blocker should be used as internal extension blockers at the 5' of the complementary sequence of the capture sequence.

[0484] When a fourth additional sequence is present, it may contain an adaptor or tag, which may serve as a binding site sequence for primers, probes, anchored oligonucleotides, etc., or as a barcode or other tag, for example, to identify the origin of a sample or molecule and facilitate hybrid sequencing methods such as highly parallel sequencing. Such a sequence may be part of the fourth additional sequence, as well as a stable sequence, alignment sequence, restriction enzyme site, or enzyme binding site. In some embodiments, the fourth additional sequence contains a stable sequence or alignment sequence.

[0485] In any of the foregoing embodiments, any of the additional sequences (or their complementary sequences formed by extending along the additional sequences) contain tags useful for downstream processes (e.g., adding adaptors useful for library preparation, clonal amplification, sequencing, or data analysis), and combining tag addition with target capture can make the overall workflow more efficient.

[0486] The target hybridization sequence can be any sequence with sufficient length and complementarity to hybridize with a given target, such as any target hybridization sequence implementation described herein, including the implementations set forth in the oligomeric element paragraphs above and below.

[0487] When a blocking moiety is present, it can be any portion that prevents the polymerase from extending its 3' end. Exemplary blocking moieties are described in detail elsewhere herein. The blocking moiety can prevent the extension of the dimer of the capture oligomer and subsequent capture, which would otherwise occur, for example, when the concentration of the capture oligomer is high and / or the target hybridization sequence has a certain dimerization potential. See also Figure 6 .

[0488] The complementary oligomer is then used, with the complementary sequence of the first portion of the spacer sequence serving to stabilize the complex of the complementary oligomer and the trapping oligomer, which does not act as a template for the extension of the target polynucleotide. Although not required, the complementary oligomer may contain the complementary sequence of the entire spacer sequence.

[0489] When complementary oligomers are used in conjunction with capture oligomers containing a second additional sequence, the complementary sequence in the complementary oligomer containing the second additional sequence may be useful in promoting the binding of the complementary oligomer to the capture oligomer.

[0490] When a connector is present, the connector can be a sequential or non-sequential element as discussed above regarding connectors. Depending on the size and properties of the internal extension blocking molecule in the captured oligomer, the connector can provide a suitable spacer to facilitate the simultaneous annealing of the complementary sequence of the first part of the spacer sequence and the complementary sequence of the second part of the captured sequence with the corresponding part of the captured oligomer.

[0491] The length and quantity of the complementary sequence of the second part of the capture oligomer should be sufficient to anneal the capture oligomer together with the complementary sequence of the first part of the spacer sequence (and, if applicable, other parts of the complementary oligomer that are complementary to the sequence in the capture oligomer), but not sufficient to anneal independently of the complementary sequence of at least the first part of the spacer sequence. In other words, when the complementary sequence of at least the first part of the spacer sequence is replaced, for example, by extending the target polynucleotide along the spacer sequence, the complementary sequence of the second part of the capture sequence should dissociate because, without the energy contribution of other hybridizations of the other parts of the oligomer, the complementary sequence of the second part of the capture sequence lacks sufficient affinity to bind the second part of the capture sequence.

[0492] When a blocking moiety is present, it can be any portion that prevents the polymerase from extending its 3' end. Exemplary blocking moieties are described in detail elsewhere in this document. A blocking moiety can prevent the complementary oligomer from extending along any sequence it binds to, which could result in undesirable byproducts, primer dimers, etc.

[0493] i. Other combinations; kits and reaction mixtures

[0494] In some embodiments, additional combinations comprising one or more additional oligomers are provided. These additional oligomers may include any one of, or any combination of two or more of, the following: complementary oligomers for capturing target molecules (which may be cyclic) without binding the capturing oligomer to a site containing the 3' end of the target; amplifying oligomers, for example, binding target polynucleotides in the reverse direction relative to the capturing oligomer; a pair of amplifying oligomers, for example, configured to amplify the target; a second capturing agent, such as comprising a complementary sequence of the capturing sequence and a binding coupler or solid carrier; a splint oligomer; a blocking oligomer; or a displacement oligomer. These additional oligomers are described in detail elsewhere herein. Combinations may also include one or more additional capturing oligomers, for example for multiple capture of multiple target polynucleotides, which may be accompanied by one or more additional suitable complementary oligomers (and / or complementary oligomers that bind two or more, or all, of the capturing oligomers). Additional capture oligomers may be accompanied by other oligomers, such as appropriate reverse amplification oligomers, substitution oligomers, blocking oligomers, etc. When multiple capture oligomers are used, these capture oligomers may have the same or sufficiently similar capture sequences so that a single secondary capture agent can be used. Alternatively, these capture oligomers may have different capture sequences, (for example) such that if a target is present in high abundance, the resulting complex with extended capture oligomers will not saturate the secondary capture agent to exclude target complexes with lower abundance.

[0495] 2. Detailed description of oligomeric elements and other reagents and components

[0496] a. Oligomeric elements

[0497] i. Capture sequence

[0498] Any capture sequence that can be bound by a suitable complementary sequence can be used. In some embodiments, the capture sequence comprises a poly-A or poly-T sequence (e.g., at least 10, 12, 15, 20, 25, or 30 consecutive A residues, or at least 10, 12, 15, 20, 25, or 30 consecutive T residues). In some embodiments, the poly-A sequence comprises 25 or 30 consecutive A residues. In some embodiments, the poly-T sequence comprises 25 or 30 consecutive T residues. Unless otherwise specified, T residues include U residues for a suitable poly-T sequence. In some embodiments, the capture sequence is any sequence not present in the target polynucleotide. For a given method, any sequence can be selected or designed to produce desired properties, such as selected to produce desired thermostability, affinity, and kinetic properties. In some embodiments, when the target polynucleotide is a sequence derived from a gene or contains a sequence derived from a gene, the capture sequence is any sequence not present in the genome of the gene. In some embodiments, when the target polynucleotide is a sequence derived from an organism or contains a sequence derived from an organism, the capture sequence is any sequence not present in the genome of the organism. In some embodiments, the capture sequence comprises 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 nucleotides. In some embodiments, the capture sequence consists of 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 nucleotides.

[0499] ii. Capture the complementary sequence of the sequence

[0500] Any sequence sufficiently complementary to the capture sequence to bind it with appropriate levels of specificity and stability can be used as the complementary sequence to the capture sequence. In some embodiments, the complementary sequence to the capture sequence comprises residues that are at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary to the capture sequence. In some embodiments, the complementary sequence to the capture sequence is configured to form a complex with the capture sequence at a melting temperature in the range of 40°C to 75°C, for example, 40°C to 50°C, 50°C to 60°C, or 60°C to 75°C, under hybridization conditions such as 0.4M to 1M monovalent cation, 0 to 10mM divalent cation (e.g., magnesium), 10mM to 100mM buffer (e.g., citrate, phosphate, Tris, borate) pH 5 to 9, and 0 to 1 mg / mL BSA.

[0501] iii. Additional sequences

[0502] As described herein, the capture oligomer may contain one or more additional sequences, for example, which provide additional functionality for the capture oligomer and / or related extended products.

[0503] The capture oligomer may include, for example, a stable (e.g., clip) sequence located at the 5' of the capture sequence and the 3' of the complementary sequence of the capture sequence, which makes self-hybridization more energy-favorable and / or helps align the sequence of the capture oligomer with the complementary sequence of the capture oligomer as desired. In some embodiments, the clip sequence contains G and / or C residues, for example, alternating G and C residues. Exemplary clip sequences are 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the clip sequence contains one or more affinity-enhancing modifications as described elsewhere herein.

[0504] In some implementations, the additional sequence is a label. Labels can be provided to spatially separate other elements or to impart other properties.

[0505] In some implementations, the additional sequence provides a binding site for an adaptor, such as a probe or primer. For example, the additional sequence (or its complementary sequence formed by extending along the additional sequence) can serve as a binding site for a universal primer or a sequencing primer. Therefore, including the additional sequence can make some processes more efficient, such as sequencing library preparation where an adaptor sequence needs to be added by combining the adaptor addition with target capture.

[0506] In some implementations, the additional sequence provides a tag sequence, such as a barcode sequence that helps identify molecules from a specific source or that have been processed in a specific manner.

[0507] In some implementations, the additional sequence provides one or more enzyme recognition sites, such as restriction sites. Therefore, the additional sequence facilitates enzyme digestion and subsequent ligation or molecular cloning processes. Another example of an enzyme recognition site is a site recognized by a site-specific recombinase.

[0508] In some embodiments, the additional sequence comprises an alignment sequence, such as a mixed nucleotide segment. The mixed nucleotide segment is not a continuous series of identical nucleotides. In some embodiments, the alignment sequence or mixed nucleotide segment is adjacent to the 3' end of an internal extension blocker, such that the extension product formed using an amplifying oligomer containing a capture oligomer bound in the opposite direction will have a complementary sequence to the mixed nucleotide segment at its 3' end. In some embodiments, the alignment sequence or mixed nucleotide segment comprises 4, 5, 6, 7, or 8 nucleotides. In some embodiments, each nucleotide in the mixed nucleotide segment is different from each of its directly adjacent nucleotides (e.g., having different base pairing specificities). The alignment sequence or mixed nucleotide segment can be used to prepare substrates for cyclization reactions as described elsewhere herein.

[0509] iv. Connector

[0510] In some embodiments, the connector includes a sequence. The connector sequence can be arbitrary or have any of the characteristics discussed regarding the additional sequence. In some embodiments, the connector comprises a non-sequence element. Exemplary non-sequence connector elements include alkyl, alkenyl, amide, and polyethylene glycol groups with a chain length of 3 to 20 atoms or more (or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more atoms) or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 repeating units, such as the -CH2CH2O- unit.

[0511] v. Internal extension blocking element

[0512] The various capture oligomers described herein contain internal extension blockers. Any part that prevents the polymerase from continuing the extension reaction along the template when present in the template can serve as an internal extension blocker. Exemplary internal extension blockers, depending on the polymerase, may include a base-degrading site, a chemically modified nucleotide, a non-natural nucleotide such as IsoG or IsoC (for the blocker to be effective, complementary non-natural nucleotides cannot be present in the reaction mixture; for example, if isoC is the blocker, isoG cannot be present, and vice versa), a non-sequence linker element as described above, or a combination thereof.

[0513] In some implementations, the internal extension blocking factor includes a debasement site. A debasement site is a location in a nucleic acid where a nucleobase is missing from its normal position.

[0514] In some implementations, the internal elongation blocker comprises a chemically modified nucleotide. A variety of chemically modified nucleotides are known to block elongation by DNA polymerase, such as alkylated nucleotides and modified nucleotides formed by the reaction of nucleotides with certain DNA-damaging agents (e.g., chemotherapeutic drugs). Other modified nucleotides used as internal elongation blockers include those with backbone modifications, sugar modifications, base modifications, and combinations thereof.

[0515] In some implementations, the internal elongation blocker contains a non-natural nucleotide such as IsoG (6-amino-2-ketopurine) or IsoC (2-amino-4-ketopyrimidine). IsoG and IsoC are nucleotides that do not form Watson-Crick base pairs with any of the four natural DNA nucleotides but pair with each other. See, for example, Hirao et al., Proc Jpn Acad Ser BPhys Biol Sci. 2012; 88(7):345–367. Therefore, if (i) the template contains IsoG and (ii) the IsoC nucleotide is unavailable, or vice versa, elongation will be blocked because the polymerase cannot introduce any substance relative to IsoG or IsoC.

[0516] In some embodiments, the internal extension blocking element comprises a non-sequence linker element, such as an alkyl, alkenyl, amide, or polyethylene glycol group, with a chain length of 3 to 20 atoms or more (or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more atoms) or 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 repeating units, such as the -CH2CH2O- unit.

[0517] vi. Reversible extension blocking element

[0518] In some implementations, the capture oligomer contains a reversible extension blocker. A reversible extension blocker is a component that, when present in the template, prevents the polymerase from continuing the extension reaction along the template until the blocker is reversed. Examples of reversible extension blockers include members of non-natural base pairs, such as IsoG and IsoC discussed above. See, for example, Hirao et al., above. IsoG-based blocking can be reversed by providing IsoC nucleoside triphosphate, allowing extension to continue, and vice versa. Figure 7AThe document provides general instructions for using reversible extension blocking molecules. In some embodiments, the amplifying oligomer contains a reversible extension blocking molecule. This amplifying oligomer can be used alone in the extension or amplification reaction, or in combination with a trapping oligomer without a reversible extension blocking molecule, or in combination with a trapping oligomer having a reversible extension blocking molecule. If amplifying oligomers and trapping oligomers both containing reversible extension blocking molecules are used in the same method, their respective reverse steps can be performed simultaneously or separately.

[0519] vii. Target hybridization sequence

[0520] Various capture oligomers according to this disclosure include a target hybridization sequence. The target hybridization sequence may hybridize with, for example, a naturally occurring sequence in the nucleic acids (e.g., DNA or RNA) of an organism, or it may hybridize with a binding site or, for example, using an amplifying oligomer containing an adapter sequence or using an adapter sequence added to an amplicon by ligation. In some embodiments, the length of the target hybridization sequence is in the range of about 10 to 60 bases, about 12 to 50 nucleotides, about 12 to 40 nucleotides, about 12 to 35 nucleotides, about 12 to 30 nucleotides, about 15 to 30 nucleotides, or about 17 to 25 nucleotides. Various algorithms for selecting a suitable sequence for use as a target hybridization sequence are available to those skilled in the art.

[0521] In some implementations, the target hybridization sequence is configured to bind to a site containing the 3' end of the target. This binding produces a substrate for 3' end extension, which, for the various oligomers and combinations described herein, causes a chain substitution that allows the capture sequence to be used to bind a second capture agent, as discussed in more detail elsewhere herein.

[0522] In some implementations, the target hybridization sequence is configured to bind to an internal site within the target. This binding can be used in combination with a substitution sequence or other additional oligomers, or more generally in capture methods described elsewhere herein that do not involve the 3′ end of an extended target.

[0523] viii. Blocking part

[0524] In some embodiments, the capturing oligomer or other oligomer described herein includes a blocking portion at its 3′ end. The blocking portion prevents the oligomer from extending along the template, wherein the blocking portion is part of the oligomer. Exemplary blocking portions include alkyl groups, non-nucleotide linkers, alkane-diols (e.g., 3′-hexanediol residues), and cordycepin. Other examples of blocking portions include 3′-deoxynucleotides (e.g., 2′,3′-dideoxynucleotides); 3′-phosphorylated nucleotides; fluorophores, quenchers, or other markers that interfere with extension; reverse nucleotides (e.g., linked to the preceding nucleotide via a 3′-to-3′ phosphodiester, optionally having an exposed 5′-OH or phosphate ester); or proteins or peptides linked to oligonucleotides to prevent extension. Figure 5 The use of an exemplary capture oligomer having a blocking portion at its 3' end is illustrated schematically. Such a capture oligomer can be used, for example, in combination with a suitable reverse amplification oligomer (which may also provide additional sequences to be introduced into the target) to isolate a single-stranded target. A capture oligomer having a blocking portion at its 3' end can also provide the benefit of preventing dimerization of the capture oligomer extension and subsequent capture. See also Figure 6 An exemplary illustration.

[0525] ix. Modifications to enhance affinity

[0526] In some embodiments, the capture oligomer, or other oligomers described herein, includes, for example, one or more affinity-enhancing modifications located in the portion of the sequence responsible for binding another molecule, such as the target polynucleotide or a second capture agent. Even when competing oligomers containing sequences that bind the same target are present, affinity-enhancing modifications can be used to ensure effective competition for the target by the oligomer, such as from unintroduced primers in the amplification reaction. Competitive oligomers can be present in excess relative to the capture oligomer and / or the target, meaning that effective competition with relatively high affinity for the target will have a significant effect on how many capture oligomers bind to their target without eliminating the capture oligomer from the pool.

[0527] Exemplary modifications that enhance affinity include 5-methylation of cytosine; use of 2-aminopurine; 2'-fluorine modification; 2′-methoxy modification; C-5-propyne; and restricted ethyl (cEt) substitution. Embodiments of oligomers that affect the stability of the hybridization complex include PNA oligomers, LNAs (locked nucleic acids, which stabilize nucleotides in certain conformations and reduce the degree of entropy reduction in hybridization free energy), or oligomers that affect the total charge, charge density, or spatial association of the hybridization complex, including ZNAs (Zip nucleic acids), oligomers containing charged linkages (e.g., phosphate thioesters) or neutral groups (e.g., methylphosphonates).

[0528] b. Second capture reagent

[0529] In some embodiments, a second capture agent is present in the combination or used in conjunction with the capture oligomers described herein. The second capture agent may comprise a complementary sequence to the capture sequence in the capture oligomer and (i) a binding partner or (ii) a solid carrier, such as beads (e.g., magnetic beads), pores, planes, packed columns, fibers, resins, or gels, to facilitate the separation of a complex containing the second capture agent and the capture oligomer, said complex being hybridized to a target polynucleotide and / or having undergone extension to include a copy of the target polynucleotide sequence.

[0530] Any suitable binding coupler can be used, including any binding coupler described in the definition paragraphs herein (e.g.). In some embodiments, the binding coupler is biotin.

[0531] c. Blocking oligomers

[0532] In some implementations, the blocking oligomer is present in the combination or used in conjunction with the capturing oligomer described herein. The blocking oligomer typically contains a complementary sequence to an additional sequence, or a complementary sequence to at least a portion of the additional sequence, and can be used to promote specific hybridization of the target hybridization sequence of the capturing oligomer (or the amplifying oligomer used to prepare an amplicon captured by the capturing oligomer) with its target by competing for hybridization with the complementary sequence of the additional sequence (i.e., blocking the additional sequence in the capturing oligomer from hybridizing with its complementary sequence, or at least reducing its hybridization ability). See also Figure 9 This illustrates the general principle of using blocking oligomers in amplification reactions. For example, when it is undesirable to block oligomer extension, the blocking oligomer can contain a blocking portion at its 3′ end. Using blocking oligomers can reduce the extent to which any misguided products undergo further amplification during the amplification reaction, because essentially only targets containing the complementary sequence to the target hybridization sequence are good substrates for hybridization with either the capture oligomer or the amplification oligomer, regardless of the presence of the complementary sequence of the additional sequence.

[0533] d. Splice oligomers

[0534] In some implementations, the splint oligomer is present in the composition or used in conjunction with the trapping oligomers described herein. The splint oligomer can be used to facilitate the linking of extended products to achieve cyclization. See also Figure 12 The sandwich oligomer may comprise: a complementary sequence to a sequence not present in the capture oligomer; a second additional sequence present in the capture oligomer; and a complementary sequence to the capture sequence of the capture oligomer. Optionally, the sandwich oligomer may further comprise a fourth additional sequence present in the capture oligomer.

[0535] The splint oligomer is configured to anneal with the target polynucleotide, which has been extended from its 3' end along the capture oligomer. The capture oligomer contains, in the 3' to 5' direction, an optional fourth additional sequence, a complementary sequence to the capture sequence, and a second additional sequence, followed by an internal extension blocker. Thus, the complementary sequence to the second additional sequence is located at the 3' end of the target polynucleotide.

[0536] The complementary sequence of the sequence in the splint oligomer that is not present in the capture oligomer is complementary to the target polynucleotide sequence at the 5' end (i.e., distal to the site where the capture oligomer binds). This may be a linker sequence added during amplification or may correspond to some or all of the amplification oligomer used to amplify the target polynucleotide.

[0537] The second additional sequence is configured to anneal to the complementary sequence of the second additional sequence located at the 3' end of the target polynucleotide. The complementary sequence of the capture sequence and the fourth additional sequence (if present) are also annealed to the corresponding sequence near the 3' end of the target polynucleotide. Upon annealing with the splint oligomer, the target polynucleotide becomes a substrate for cyclization via ligation. See also Figure 12 This illustrates exemplary clip-on oligonucleotides according to this specification and their use for cyclization. Cycling target molecules can be useful, for example, for preparing targets used in rolling circle amplification procedures.

[0538] e. Displacement oligomers

[0539] In some implementations, the substitution oligomer (e.g., substitution primer) is present in the combination or used in conjunction with the capture oligomer described herein. The substitution oligomer can be used to replace the template strand of the target polynucleotide with a capture oligomer. See, for example, [link to relevant documentation]. Figure 8A and Figure 8B In some embodiments, a substitution oligomer and a capture oligomer having a target hybridization sequence are provided in combination, the target hybridization sequence binding to an internal site within the target (e.g., a site within the target polynucleotide, an amplicon, or an additional sequence introduced during a previous extension or amplification step), which is positioned relative to the binding site of the substitution oligomer to facilitate substitution by extension of the substitution oligomer.

[0540] In some embodiments, the combination comprising a substitution oligomer (e.g., a substitution primer) and a capture oligomer further comprises an amplification oligomer (e.g., a primer), the amplification oligomer being configured to bind the extended capture oligomer (and thus having a binding orientation opposite to that of the capture oligomer and the substitution oligomer). Depending on the configuration of the capture oligomer and / or the combination, the extension of the amplification oligomer can cause a substitution of the complementary oligomer or the capture sequence, thereby making the capture sequence of the extended capture oligomer available for interaction with a second capture reagent. As described elsewhere, the amplification oligomer can also be used to introduce an additional sequence (e.g., a tag, such as a linker sequence) at the end of the extended capture oligomer, the end being remote from the initial capture oligomer sequence (e.g., see [link]). Figure 8A The process involves further extension of the captured oligomer through extension and substitution to generate a complementary sequence to the additional sequence of the amplified oligomer. Therefore, a product containing both additional sequences and an accessible captured sequence can be prepared in a simple and rapid one-step, one-pot process.

[0541] f. Amplifying oligomers

[0542] In some implementations, the amplifying oligomer (e.g., a primer) is present in the combination or used in conjunction with the capturing oligomer described herein. For example, the amplifying oligomer may be configured to anneal with the target hybridization sequence of the capturing oligomer in opposite orientations. See, for example, [link to relevant documentation]. Figure 4A The amplified oligomer may contain a target hybridization sequence and, optionally, an additional sequence located at the 5′ end of the target hybridization sequence. Using the additional sequence of the amplified oligomer as a template, the additional sequence is introduced as the chain containing the extended capture oligomer is further extended, thereby providing one or more tags that may contain one or more linker sequences. In some embodiments, the amplified oligomer contains a reversible internal extension blocking member, for example, located at the 3′ end of one or more elements other than THS, for example, located at the 5′ end of the target hybridization sequence, between the target hybridization sequence and the additional sequence. For example, as described elsewhere herein, combinations containing amplified oligomers may further contain one or more other oligomers, such as substitutional oligomers, splice oligomers, etc.

[0543] g. Reagent kit

[0544] In some embodiments, a kit is provided comprising a capture oligomer or combination thereof, such as any capture oligomer or combination described herein, and further comprising one or more additional elements, such as reagents, buffers, or other substances used with the capture oligomer or combination thereof, and / or instructions for use of the capture oligomer or combination thereof. Exemplary reagents include dNTPs, DNA polymerases, sodium salts, magnesium salts, and beads, said beads comprising, for example, a complementary sequence of a capture sequence or a second binding pair bound to a first binding pair present together with a complementary sequence of a capture sequence in a second capture reagent.

[0545] In some implementations, the content of the capture oligomers in the kit is 10 7 1 molecule / reaction up to 10 13 molecule / reaction, 10 9 1 molecule / reaction up to 10 12 molecule / reaction, or 10 10 1 molecule / reaction up to 10 12 Within the range of molecules / reactions.

[0546] In some embodiments, the kit contains a second capture agent comprising a complementary sequence of the capture oligomer in a concentration of 10. 3 1 molecule / reaction up to 10 14 molecule / reaction, or 10 3 1 molecule / reaction up to 10 9 molecule / reaction, or 10 5 1 molecule / reaction up to 10 13 molecule / reaction, or 10 5 1 molecule / reaction up to 10 8 molecule / reaction, or 10 6 1 molecule / reaction up to 10 13 molecule / reaction, or 10 6 1 molecule / reaction up to 10 8 Within the range of molecules / reactions.

[0547] In some implementations, the kit contains capture oligomers in a liquid, frozen, or lyophilized state.

[0548] In some embodiments, the kit comprises at least a first container and a second container, each containing a capture oligomer and one or more other oligomers as described herein (e.g., complementary oligomers, a second capture reagent, or amplifying oligomers, clip-on oligomers, blocking oligomers, second or replacement oligomers). Alternatively, a combination of such oligomers may be provided in a single container.

[0549] In some implementations, the kit includes a solid carrier / bead that includes a second binding partner (e.g., streptavidin) configured to bind to a binding partner of a second capture reagent.

[0550] h. Composition

[0551] Compositions comprising the capture oligomers or combinations described herein are also provided. Any oligomers or combinations described herein may be present in the composition. In some embodiments, the composition is in the form of a lyophilized product or a solution.

[0552] In some embodiments, a reaction mixture comprising a capture oligomer or a combination thereof (e.g., any capture oligomer or combination described herein). The reaction mixture may further comprise one or more target polynucleotides and / or one or more reagents (e.g., any reagents discussed herein in conjunction with a kit or method).

[0553] The reaction mixture may further comprise one or more target polynucleotides (e.g., any target discussed herein in conjunction with the method). In some embodiments, the target polynucleotide comprises a sequence from the DNA or RNA (e.g., genomic DNA or mRNA) of the target organism and an additional sequence, and the target hybridization sequence of the capture oligomer is configured to anneal with the additional sequence of the target polynucleotide. The additional sequence may be added in a prior reaction, such as an extension, ligation, or amplification reaction. For example, the additional sequence may be a sequence not present in the DNA or RNA of the target organism. Such an additional sequence may be added in an early extension reaction using a primer containing the additional sequence. This approach facilitates the design of capture oligomers for multiple capture or reuse of different targets with the same additional sequence attached. In some embodiments, the composition comprises multiple target polynucleotides comprising (i) an additional sequence and (ii) different sequences from the DNA or RNA of the target organism, and the method includes capturing multiple target polynucleotides.

[0554] 3. Detailed description of methods for capturing polynucleotides

[0555] This article provides a method for capturing target polynucleotides from a composition, the method comprising:

[0556] The target polynucleotide is contacted with a capture oligomer comprising a capture sequence and a complementary sequence thereof, as described herein, wherein at a site comprising the 3' end of the target polynucleotide, the target hybridization sequence of the capture oligomer anneals with the target polynucleotide. The method further comprises extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity to form a complementary sequence to the complementary sequence of the capture sequence, which anneals with the capture oligomer such that the capture sequence of the capture oligomer is available for binding.

[0557] Contact the capture sequence of the capture oligomer with a complementary sequence containing the capture sequence and a second capture agent (i) a binding partner or (ii) a solid carrier to form a complex containing the target polynucleotide, the capture oligomer and the second capture agent; and isolate the complex from the composition to capture the target polynucleotide. Figures 4A to 4B An exemplary scheme illustrating the contact and extension steps is shown. Figure 4A In this process, the 3′ end sequence of the THS-binding target of the oligomer is captured. Figure 4B In this process, an additional sequence at the 3′ end of the THS-binding target of the oligomer is captured, for example, by adding this additional sequence in an earlier reaction.

[0558] This article also provides a method for capturing target polynucleotides from a composition, the method comprising:

[0559] The composition is contacted with the combination comprising a trapping oligomer and a complementary oligomer as described herein, wherein the target hybridization sequence of the trapping oligomer is annealed with the target polynucleotide or an additional sequence at a site containing the 3′ end of the target polynucleotide;

[0560] The 3′ end of the target polynucleotide is extended using a DNA polymerase with strand displacement activity to form a complementary sequence to the spacer sequence. This complementary sequence is annealed with the capture oligomer, such that the complementary oligomer is replaced to a degree sufficient to make the capture sequence of the capture oligomer available for binding.

[0561] The capture sequence of the capture oligomer is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding partner or (ii) a solid carrier to form a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0562] The complex is isolated from the composition, thereby capturing the target polynucleotide. The complementary oligomer of the combination can be provided to the composition at any point before the capture sequence of the capturing oligomer comes into contact with the second capturing agent, for example, provided together with the capturing oligomer, or provided after the target extends along the capturing oligomer. See Figure 10 and... Figure 13 This is for illustrating exemplary methods according to this specification.

[0563] This article also provides a method for capturing target polynucleotides from a composition, the method comprising:

[0564] Contact the target polynucleotide with the capture oligomer or combination described herein, wherein the capture oligomer contains a third or fourth additional sequence at the 3' of the second part of the complementary sequence or spacer sequence of the target hybridization sequence at the 5' of the target hybridization sequence;

[0565] The target polynucleotide is contacted with a second oligomer, the second oligomer comprising a second target hybridization sequence at 5' of a complementary sequence located in at least a portion of a third or fourth additional sequence, wherein the capture oligomer and the second oligomer form a triple-stranded link with the target polynucleotide;

[0566] The 3' end of the second oligomer is extended using a DNA polymerase with strand displacement activity to form a complementary sequence to the complementary sequence of the capture sequence or a complementary sequence to the spacer sequence. This sequence is annealed with the capture oligomer so that the capture sequence of the capture oligomer can be used for binding.

[0567] Contact the capture sequence of the capture oligomer with a complementary sequence comprising the capture sequence and (i) a binding partner or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0568] The complex is isolated from the composition, thereby capturing the target polynucleotide. In some embodiments of this method, the target polynucleotide is cyclic. For an explanation of the exemplary method according to this specification, see [link to documentation]. Figures 11A to 11B In some embodiments, the target polynucleotide is not cyclic. For example, the target polynucleotide may include a translocation link (e.g., BCR-ABL, or any other translocation) that brings the binding sites of the trapping oligomer and the second oligomer closer together. In some embodiments, the second oligomer is provided to the reaction mixture at a concentration higher than that of the trapping oligomer. In some embodiments, the second oligomer is provided to the reaction mixture at a concentration lower than that of the trapping oligomer.

[0569] The following further embodiments are applicable to any of the foregoing methods. In some embodiments, the second capture agent comprises a binding partner (e.g., biotin), and separation comprises contacting the complex with a solid carrier (e.g., beads) comprising a second binding partner (e.g., streptavidin), the second binding partner being configured to bind to the binding partner of the second capture agent. An excess of the capture oligomer may be provided relative to the second capture agent.

[0570] Where applicable, exemplary concentration and quantity ranges for capturing oligomers, complementary oligomers, and / or second oligomers include: for capturing oligomers, 10 7 1 molecule / reaction up to 10 13 molecule / reaction, 10 9 1 molecule / reaction up to 10 12 molecule / reaction, or 10 10 1 molecule / reaction up to 10 12 One molecule per reaction; for complementary oligomers, approximately 1.5 × 10⁻⁶. 7 1 molecule / reaction up to 10 14 1 molecule / reaction, or approximately 1.5 × 10⁻⁶9 1 molecule / reaction up to 10 13 One molecule / reaction; and for the second oligomer, it is 10. 6 1 molecule / reaction up to 10 14 molecule / reaction, or 10 8 1 molecule / reaction up to 10 13 One molecule / reaction. An exemplary range of second capture reagents is provided above.

[0571] The method can be used to capture target polynucleotides from any source. In some embodiments, the source of the target polynucleotide is a clinical sample, pathogen, or environmental sample. In some embodiments, the clinical sample is a sample from a subject who has or is suspected of having sepsis (e.g., a blood, serum, or plasma sample), and in some embodiments, the clinical sample is a sample from a patient who has or is suspected of having cancer. In some embodiments, the target polynucleotide is an extension or amplification product.

[0572] In some implementations, the target polynucleotide is a member of the sequencing library.

[0573] In some embodiments, an extension product (extended capture oligomer) is formed by extending a capture oligomer along a target polynucleotide, and the capture oligomer contains a third or fourth additional sequence. In some embodiments, the method further includes contacting the extension product with a blocking oligomer containing a non-extendable third or fourth additional sequence, optionally wherein the blocking oligomer is configured to bind a complementary sequence of the third or fourth additional sequence with a greater affinity than the third or fourth additional sequence of the capture oligomer, or the melting temperature of the complex formed with the complementary sequence of the third or fourth additional sequence of the capture oligomer is higher than the melting temperature of the complex formed with the complementary sequence of the third or fourth additional sequence of the capture oligomer. This method can be used to reduce or avoid nonspecific extension, for example, in cases where the product of a false triggering event of the capture oligomer can generate additional cyclic amplification through hybridization of the third or fourth additional sequence with the reverse extension product, where the reverse extension product would be generated if the blocking oligomer were not present.

[0574] In some implementations, the method further includes:

[0575] The target polynucleotide is contacted with an amplified oligomer comprising (i) a reverse target hybridization sequence that binds the target polynucleotide in an orientation opposite to that of the capture oligomer, and (ii) an additional sequence located at the 5' of a second target hybridization sequence. The method further includes extending the amplified oligomer along the target polynucleotide to form a reverse extension product, wherein a portion of the capture oligomer is annealed with the reverse extension product. Such embodiments can be used to introduce an additional sequence (e.g., a tag, such as an adaptor) at the end of the target, which is away from the end that binds to or introduces the capture oligomer. As discussed elsewhere, extension along the capture oligomer (e.g., an extended capture oligomer) can replace the complementary sequence of the capture sequence with the capture sequence. The amplified oligomer can also be used to initiate such extension.

[0576] In some embodiments, for example, wherein the capturing oligomer includes a blocking portion at its 3' end, the method further includes separating a complex comprising a reverse extension product annealed with the capturing oligomer (e.g., as described above or a reverse extension product generated using an amplifying oligomer that does not necessarily contain an additional sequence), wherein the extension product is essentially a single strand of the 5' end of the target hybridization sequence of the capturing oligomer, and the capturing element of the capturing oligomer has been replaced by the extension of the reverse extension product, for example, as... Figure 5 As shown in the diagram. If at least half of the molecule does not hybridize with the other strand, the extended product is considered to be substantially single-stranded. Therefore, for example, a short extension of a self-hybridized nucleic acid cannot prevent the molecule or a portion thereof from being substantially single-stranded.

[0577] In some embodiments where a reverse extension product is generated, the capture oligomer is extendable, and the method further includes extending the capture oligomer along the reverse extension product to form a second extension product containing a complementary sequence of the amplified oligomer. The second extension product will contain a complementary sequence of any additional sequence present in the capture oligomer and any additional sequence present in the reverse amplified oligomer. In this way, products containing additional sequences (e.g., tags, such as one or more adapters) near or at one or both ends (e.g., both ends) can be generated.

[0578] In some embodiments, the target polynucleotide comprises a sequence from the DNA or RNA of the target organism and an additional sequence, and the target hybridization sequence of the capture oligomer is configured to anneal with the additional sequence of the target polynucleotide. The additional sequence can be added in a prior reaction, such as an extension, ligation, or amplification reaction. For example, the additional sequence can be a sequence not present in the DNA or RNA of the target organism. This additional sequence can be added in an early extension reaction using primers containing the additional sequence. This approach facilitates the design of capture oligomers for multiple capture or reuse of different targets with the same additional sequence attached. In some embodiments, the composition comprises multiple target polynucleotides, each containing (i) an additional sequence and (ii) a different sequence from the DNA or RNA of the target organism, and the method includes capturing multiple target polynucleotides.

[0579] In some embodiments, the capture oligomer contains a reversible extension blocker located at the 5' of the target hybridization sequence, and the method includes: copying or amplifying the target polynucleotide before unblocking the reversible extension blocker; unblocking the reversible extension blocker; and performing another cycle or multiple cycles of copying or amplifying the target polynucleotide. In some embodiments, only one cycle of amplification / extension is performed before capturing the target polynucleotide and after unblocking the reversible extension blocker. In some embodiments, the amplification oligomer contains a reversible extension blocker and serves a similar function as described above. In some embodiments, amplification oligomers and capture oligomers, both having reversible extension blockers, are used together in the same process. In this case, the reversible extension blocker of the amplification capture oligomer can be reversed simultaneously or in separate steps. In some embodiments, the reversible extension blocker is a member of a non-natural nucleotide pair (e.g., IsoC or IsoG), and unblocking the reversible extension blocker includes providing a complementary member of the non-natural nucleotide pair.

[0580] In some embodiments, the method includes cyclizing an extension product of the target polynucleotide. This is, for example, when the capture oligomer contains a mixed nucleotide segment located between the first portion of the extension blocker and the spacer sequence or the complementary sequence of the capture sequence (e.g., immediately following the 3' of the inner extension blocker). The method includes extending the 3' end of the target polynucleotide along the capture oligomer to the inner extension blocker, thereby forming an extension product containing the complementary sequence of the mixed nucleotide segment at its 3' end. The method further includes contacting the extension product with a splint oligonucleotide comprising, in the 5' to 3' direction: the complementary sequence of the 5' terminal segment of the extension product; the mixed nucleotide segment; the complementary sequence of the capture sequence; and optionally, a segment complementary to the 5' segment of the extension product immediately adjacent to the 5' of the capture sequence. The method further includes attaching the 5' end of the extension product to the 3' end of the extension product. In some embodiments, the 5' terminal region of the extension product includes at least a portion of the sequence for generating an amplified oligomer of the target polynucleotide, for example, at least a portion of the target hybridization sequence of the amplified oligomer, or (where applicable) at least a portion of an additional sequence of the amplified oligomer, wherein the additional sequence is the 5' of the target hybridization sequence of the amplified oligomer. See also Figure 12 This illustrates an exemplary method according to this specification.

[0581] In some embodiments, any of the foregoing methods includes adding additional sequences at one or both ends of the target polynucleotide. For example, such a method may include contacting the target polynucleotide with the capture oligomer described herein, wherein the target hybridization sequence of the capture oligomer is annealed at a site upstream of the 3' end of the target polynucleotide;

[0582] The target polynucleotide is extended to capture the 3' end of the oligomer, thereby forming the first extended chain;

[0583] The target polynucleotide is brought into contact with a substitution oligomer containing a substitution target hybridization sequence, which is annealed with the target polynucleotide downstream of the target hybridization sequence of the capture oligomer.

[0584] The substitution oligomer extends along the target polynucleotide, thereby replacing the first extension chain.

[0585] Optionally, the capturing oligomer and the substitution oligomer are added to the composition simultaneously or sequentially (e.g., such that the extension of the capturing oligomer begins before the substitution caused by the extension of the substitution oligomer). The capturing oligomer may further comprise an additional sequence (e.g., a third or fourth additional sequence) located at the 5' of the target hybridization sequence. In some embodiments, the THS of the capturing oligomer has a higher Tm, or the binding of the THS of the capturing oligomer and its complementary sequence has a higher affinity than the binding of the THS of the substitution oligomer and its complementary sequence, or the capturing oligomer is provided at a higher concentration than the substitution oligomer. This can promote the binding and extension (or at least the initiation of extension) of the capturing oligomer before the extension of the substitution oligomer. When the Tm and / or affinity of the capturing oligomer is high, this effect can be further promoted by first incubating the reaction mixture at a higher temperature that allows the binding and extension of the capturing oligomer to take precedence over the binding and extension of the substitution oligomer, and then at a lower temperature that allows the binding and extension of the substitution oligomer.

[0586] In some embodiments, such a method further includes: contacting a first extended strand of the target polynucleotide with a reverse amplification oligomer containing a reverse target hybridization sequence configured to bind the first extended strand; and extending the reverse amplification oligomer to form a second extended strand. The reverse amplification oligomer may contain an additional 5′ sequence of its target hybridization sequence.

[0587] In some embodiments, such a method further includes: contacting a first extension strand with a reverse amplification oligomer containing a reverse target hybridization sequence configured to bind to the 3' end of the first extension strand; and extending the reverse amplification oligomer to form a second extension strand. The reverse amplification oligomer may contain an additional sequence at the 5' end of its target hybridization sequence, in which case the additional sequence is used as a template to further extend the first extension strand, thereby producing a complementary sequence to the additional sequence. See also Figure 8A .

[0588] In some embodiments, the target polynucleotide comprises a sequence from the DNA or RNA of the target organism and an additional sequence not present in the DNA or RNA of the target organism, and the target hybridization sequence of the capture oligomer is configured to anneal with the additional sequence of the precursor of the target polynucleotide. The substitution oligomer is also configured to anneal with the additional sequence of the precursor of the target polynucleotide, wherein, in the additional sequence, the binding site of the substitution oligomer is downstream of the binding site of the target hybridization sequence of the capture oligomer (e.g., see...). Figure 8B ).

[0589] In some embodiments, the first strand of the target polynucleotide includes a second linker sequence located proximal to the DNA or RNA sequence of the target organism and distal to the target hybridization sequence that captures the oligomer.

[0590] The method further includes: contacting the first strand of the target polynucleotide with a reverse amplification oligomer containing a reverse target hybridization sequence configured to bind a second linker sequence; and extending the reverse amplification oligomer to form the second strand of the target polynucleotide.

[0591] In some embodiments, any of the foregoing methods further includes cloning and amplifying the captured target polynucleotides. In some embodiments, such methods further include sequencing the cloned and amplified target polynucleotides. In some embodiments, any of the foregoing methods further includes sequencing the captured target polynucleotides. In some embodiments, the sequencing is Sanger sequencing or next-generation sequencing, optionally wherein next-generation sequencing includes sequencing by synthesis, ligation sequencing, hybridization sequencing, or single-molecule sequencing. Compared to Sanger sequencing, next-generation sequencing includes any form of sequencing that generates reads in a significantly parallel manner.

[0592] 4. Capture oligomers and combinations for limited capture and other applications

[0593] a. Capture oligomers containing self-complementary sequences

[0594] In some embodiments, a capture oligomer is provided, comprising a first self-complementary sequence, a target hybridization sequence, and a second self-complementary sequence.

[0595] The first and second self-complementary sequences are configured such that when the target hybridization sequence is single-stranded, the first and second self-complementary sequences anneal to each other, and when the target hybridization sequence anneales to its target, the first and second self-complementary sequences do not anneal to each other. Such a capturing oligomer can be combined with a complementary sequence containing either the first or second self-complementary sequence and a second capturing agent that binds to a partner. The capturing oligomer can be present in the combination in a larger amount than the second capturing agent. Such oligomers and combinations can be used to capture a certain amount (e.g., a limited amount, or less than or equal to a predetermined amount) of target polynucleotides from the composition.

[0596] In some implementations, such captured oligomers have the following formula:

[0597] 5′-SC1-THS2-THS1-L-THS2′-SC2-3′.

[0598] In some implementations, such captured oligomers have the following formula:

[0599] 5′-SC2-THS2′-L-THS1-THS2-SC1-3′.

[0600] In each of the above formulas, SC1 is the first self-complementary sequence; THS2 and THS1 are the second and first parts of the target hybridization sequence, respectively; L is an optional adapter; THS2′ is an optional complementary sequence of the second part of the target hybridization sequence; and SC2 is the second self-complementary sequence.

[0601] Either the first self-complementary sequence or the second self-complementary sequence can be used as the capturing sequence. When the first self-complementary sequence is the capturing sequence, the second self-complementary sequence is the complement of the capturing sequence. When the second self-complementary sequence is the capturing sequence, the first self-complementary sequence is the complement of the capturing sequence. Capturing sequences and their complements are discussed in detail elsewhere in this document. Any suitable capturing sequence and its complement can be used.

[0602] When a linker is present, it can be any suitable linker, such as any linker described elsewhere herein, including sequence and non-sequence linkers. The linker can serve as a flexible element facilitating intramolecular hybridization of a first self-complementary sequence and a second self-complementary sequence. When the linker contains a sequence, in some embodiments, the sequence is a sequence not present in sources such as organisms, genomes, genes, or other nucleic acids or combinations thereof, from which the target hybridization sequence originates or is complementary. The linker should generally not be too long so that it enables intramolecular hybridization of the first and second self-complementary sequences even when the target hybridization sequence anneals to its target (see discussion below).

[0603] Target hybridization sequences are discussed in detail elsewhere in this article. Any suitable target hybridization sequence (and a complementary sequence of a portion of the target hybridization sequence, if available) can be used.

[0604] The second capture reagent is discussed in detail elsewhere in this paper, and it contains a complementary sequence to the capture sequence. Any suitable second capture reagent can be used.

[0605] In the combination, when the amount of the capturing oligomer is greater than that of the second capturing agent, the ratio can be approximately 10,000:1, 5,000:1, 2,000:1, 1,000:1, 500:1, 200:1, 100:1, 50:1, 20:1, 10:1, 5:1 or 2:1.

[0606] This capturing oligomer can be used in methods for capturing target polynucleotides. When the target polynucleotide is absent, the capturing oligomer can adopt a self-hybridization conformation. This self-hybridization conformation involves the first and second self-complementary sequences annealing to each other, thus making the capturing oligomer essentially unable to interact with the complementary sequence of the capturing sequence of the second capturing reagent. However, the target hybridization sequence is at least partially single-stranded. When the target polynucleotide is present, the target hybridization sequence can hybridize with it. When the target hybridization sequence is therefore essentially double-stranded, intramolecular hybridization of the first and second self-complementary sequences cannot occur because, among other potential factors, they are too far apart. Therefore, the capturing sequence (i.e., one of the first and second self-complementary sequences) is essentially single-stranded and can hybridize with the second capturing reagent. It may be advantageous that hybridization of the target hybridization sequence is energy-preferential than intramolecular hybridization of the first and second self-complementary sequences, thus causing the capturing oligomer to preferentially hybridize with the available target polynucleotide. Excess capturing oligomers can also be used to drive the formation of complexes with the target polynucleotide.

[0607] Then, for example, standard techniques suitable for binding couplers or solid carriers of the second capture reagent can be used to separate the complex of the second capture reagent, the capture oligomer, and the target. The amount of captured polynucleotides can be limited, or determined by the amount of the second capture reagent or by a combination of the amount of the capture oligomer and the amount of the second capture reagent.

[0608] b. A combination containing a capturing oligomer and a complementary oligomer that is complementary to a portion of the capturing sequence.

[0609] In some implementations, a combination is provided comprising a trapping oligomer and a complementary oligomer, wherein:

[0610] (a) The captured oligomers contain: in the 5' to 3' direction:

[0611] The captured sequence contains a first part and a second part, and

[0612] The target hybridization sequence, comprising a second part and a first part; and

[0613] (b) The complementary oligomers comprise, in the 3' to 5' direction:

[0614] The complementary sequence of the second part of the captured sequence, and

[0615] The complementary sequence of the second part of the target hybridization sequence, wherein the complementary sequence of the second part of the capture sequence and the complementary sequence of the second part of the target hybridization sequence are configured to anneal with the capture oligomer when the complementary sequence of the target hybridization sequence is not present. Figure 10B Illustrations of exemplary oligomers according to these embodiments are provided. They may be as described in other embodiments listed above and / or as... Figure 10B (For example, Figure 10B As shown in any individual element or any combination thereof, there are optional additional elements.

[0616] Combinations can be used for limited capture because complementary oligomers can be configured to bind free capture oligomers but not capture oligomers bound to the target polynucleotide. For example, binding of the target hybrid sequence of the capture oligomer to the target polynucleotide can be energy-advantageous compared to binding of the complementary sequence of the second part of the capture sequence to the second part of the target hybrid sequence. When the target polynucleotide is absent, the complementary oligomer binds to the capture oligomer and blocks the capture sequence ( Figure 10B The accessibility of C1+C2 in the second capture reagent is sufficient to block the binding of the capture sequence to the complementary sequence of the capture sequence in the second capture reagent, which can be any second capture reagent described elsewhere herein.

[0617] Accordingly, a method for capturing target polynucleotides from a composition is also provided, the method comprising:

[0618] Contact the composition with the above combination or any other embodiment thereof described herein, wherein the target hybridization sequence that captures the oligomer is annealed with the target polynucleotide;

[0619] Before or after annealing the capture oligomer with the target polynucleotide, the capture oligomer is contacted with a complementary oligomer, wherein the complementary oligomer anneals with the free capture oligomer and partially occupies its capture sequence, wherein the complementary oligomer does not anneal with a complex containing the capture oligomer annealed with the target polynucleotide, and wherein if the contact between the capture oligomer and the complementary oligomer occurs before the capture oligomer annealed with the target polynucleotide, the annealing of the target hybridization sequence with the target polynucleotide causes the complementary oligomer to dissociate from the capture oligomer;

[0620] The capture sequence of the capture oligomer complexed with the target polynucleotide is contacted with a complementary sequence containing the capture sequence and a second capture agent (i) a binding coupler or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the second capture agent; and

[0621] The complex is isolated from the composition, thereby capturing the target polynucleotide. This can be done as described in other embodiments listed above and / or as... Figure 10B (For example, Figure 10B As shown in any individual element or any combination thereof, there are optional additional elements.

[0622] 5. Differential capture methods and oligomer combinations

[0623] This document also provides methods and combinations of oligomers for differentially capturing oligomers (e.g., utilizing different affinities) based on whether the capturing oligomer is in a complex with the target polynucleotide. Such methods allow for the elution of the target polynucleotide while substantially not eluting the capturing oligomers that do not associate with the target polynucleotide. The capturing sequence, target hybridization sequence, and other elements of the oligomer in such combinations and methods can be, for example, any capturing sequence, target hybridization sequence, etc., consistent with the characteristics of the oligomers, combinations, and methods described herein.

[0624] In some embodiments, such a method includes contacting a target polynucleotide with a capture oligomer comprising, in the 5' to 3' direction: a capture sequence; an optional internal extension blocker; an optional spacer sequence; and a target hybridization sequence configured to anneal with the target polynucleotide. This contact may thereby anneal at least some of the capture oligomers (i.e., a portion of a group of capture oligomers) with the target polynucleotide. The method further includes contacting the capture oligomers with a first capture agent containing a complementary sequence to the capture sequence (before, simultaneously with, or after contacting the target polynucleotide with the capture oligomers); and providing a second capture agent containing a complementary sequence to a sequence in the capture oligomers other than the capture sequence, wherein if some or all of the capture oligomers do not anneal with the target polynucleotide, the second capture agent contacts the capture oligomers that do not anneal with the target polynucleotide. The sequence in the capture oligomer other than the capture sequence may be some or all of the target hybridization sequence, or a sequence overlapping the target hybridization sequence, or a sequence that otherwise renders the second capture agent inaccessible when the capture oligomer anneals with the target polynucleotide. The method further includes: separating a first complex and a second complex from the composition, wherein the first complex contains a target polynucleotide and the second complex contains a capture oligomer that has not been annealed with the target polynucleotide; and selectively eluting the target polynucleotide or a subcomplex containing the target polynucleotide from the first complex.

[0625] In some embodiments, the target polynucleotide is contacted with an excess of the capture oligomer. In some embodiments, a first capture agent is provided in a limited amount relative to the capture oligomer. In some embodiments, the capture oligomer is provided in a limited amount relative to the target polynucleotide. In some embodiments, a portion of the capture oligomer contacted with a second capture agent comprises unbound capture oligomers.

[0626] In some embodiments, the first capture agent comprises a first solid carrier containing a complementary sequence to the capture sequence. Further aspects of these embodiments are set forth elsewhere herein, including those described in the overview above.

[0627] In some embodiments, the first capture agent further comprises a second capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method includes, after contacting the annealed capture oligomer with the first capture agent, annealing the second capture sequence with a solid support containing a complementary sequence of the second capture sequence. Further aspects of these embodiments are set forth elsewhere herein, including those set forth in the foregoing overview.

[0628] This paper also provides an assembly comprising a trapping oligomer, a first solid support, and a second solid support. The trapping oligomer comprises, in the 5' to 3' direction:

[0629] The sequence consists of a first capture sequence, an internal extension blocking molecule, a second capture sequence, and a target hybridization sequence.

[0630] The first solid support contains a complementary sequence to the first capture sequence. The second solid support contains a complementary sequence to the second capture sequence. The first complex formed by annealing the first capture sequence and its complementary sequence has a lower melting temperature than the second complex formed by annealing the second capture sequence and its complementary sequence, and / or the complementary sequence of the second capture sequence has a higher affinity for the second capture sequence than the complementary sequence of the first capture sequence has for the first capture sequence.

[0631] This article also provides a combination comprising a capture oligomer, a first capture reagent, a second capture reagent, a first solid support, and a second solid support.

[0632] The captured oligomers contain the following in the 5' to 3' direction:

[0633] The first capture sequence, the internal extension blocking sequence, and the target hybridization sequence.

[0634] The first capture agent comprises a second capture sequence and a complementary sequence to the first capture sequence, wherein the second capture sequence is not complementary to the capture oligomer. The second capture agent comprises a third capture sequence and a complementary sequence to a capture oligomer sequence different from the first capture sequence, wherein the third capture sequence is not complementary to the capture oligomer. The first solid support comprises a complementary sequence to the second capture sequence. The second solid support comprises a complementary sequence to the third capture sequence. The first complex formed by annealing the second capture sequence with its complementary sequence has a lower melting temperature than the second complex formed by annealing the third capture sequence with its complementary sequence, and / or the affinity of the complementary sequence of the third capture sequence to the third capture sequence is greater than the affinity of the complementary sequence of the second capture sequence to the second capture sequence.

[0635] This document also provides a combination comprising a capture oligomer and a second capture agent. The capture oligomer comprises a target hybridization sequence containing one or more affinity-enhancing nucleotides and a capture sequence. The second capture agent comprises a complementary sequence to the capture sequence and a binding partner. In some embodiments, the capture sequence is located at the 5' of the target hybridization sequence. In some embodiments, the target hybridization sequence is configured to anneal with an adapter sequence. In some embodiments, the second capture agent is present in the combination in a lower amount than the capture oligomer.

[0636] This document also provides a method for capturing a target polynucleotide from a composition, the method comprising contacting the target polynucleotide with such a composition as described above. A capturing oligomer and a second capturing agent are added simultaneously or sequentially. The target hybridization sequence of the capturing oligomer is annealed with the target polynucleotide, and the second capturing agent is annealed with the capturing sequence of the capturing oligomer, thereby forming a complex.

[0637] The method further includes: contacting the complex with a second binding partner configured to bind to a binding partner of a second capture reagent, wherein the second binding partner is associated with a solid carrier and the second binding partner binds to a binding partner of the second capture reagent; and separating the complex from the composition to capture the target polynucleotide.

[0638] In some embodiments, the second capture agent is present in the combination in a lower amount than the capture oligomer and / or the target polynucleotide. In some embodiments, the capture oligomer is present in the combination in a lower amount than the target polynucleotide, and the second capture agent is present in the combination in a lower amount than the capture oligomer. In some embodiments, the target polynucleotide comprises a linker sequence, and the target hybridization sequence anneales with the linker sequence. In some embodiments, the target polynucleotide comprises a sequence from the DNA or RNA of the target organism, and the linker sequence is not present in the DNA or RNA of the target organism.

[0639] IV. Examples

[0640] The following examples are provided to illustrate certain disclosed implementations, but should not be construed as limiting the scope of this disclosure in any way.

[0641] A. Capturing a predetermined amount of amplicon using a capture oligomer containing the capture sequence and its complementary sequence.

[0642] Oligomer.

[0643] The following primers were used for PCR to amplify a fragment of the uidA gene from Escherichia coli (E. coli):

[0644] Ec_uidA_F:GTATCAGCGCGAAGTCTTTATACC(SEQ ID NO:1)

[0645] Ec_uidA_R:GGCAATAACATACGGAGTGACATC(SEQ ID NO:2)

[0646] Design primers to generate amplicons with the following sequences:

[0647] GTATCAGCGCGAAGTCTTTATAACCGAAAGGTTGGGCGGCCAG

[0648] CGTATTGTACTGCGTTTCGATGCGGTCACTCATTACGGCAAAGT

[0649] GTGGGTAAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACTCCGTATGTTATTGCC(SEQ ID NO:3)

[0650] A capture oligomer named uidA_PA_1.2 is provided, which has the following sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTCTA / iSp18 / TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGACGCAAGCTACTGGTGATTTGGCAATAACATACGGAGTGACATCGGCTTC (SEQ ID NO:4) (iSp18 = hexaethylene glycol (HEG) internal spacer (IDT))

[0651] In this oligomer, the 5′ poly-A sequence is the capture sequence. CCTCTA is the adapter sequence. iSp18 is the internal extension blocker. The poly-T sequence following iSp18 is the complementary sequence to the capture sequence. AGACGCAAGCTACTGGTGATTT (SEQ ID NO:5) is the fourth additional sequence. The target hybridization sequence (THS) is GGCAATAACATACGGAGTGACATCGGCTTC (SEQ ID NO:6), which hybridizes specifically with the uidA gene sequence in the target amplicon. In this embodiment, THS is longer than the inverse PCR primer that overlaps with it (see the sequence above) to increase the Tm of THS and give it a competitive advantage over the inverse primer in hybridization with the target.

[0652] Use a second capture reagent with the following sequence:

[0653] dT 30-Biotin: TTTTTTTTTTTTTTTTTTTT / 3'Biotin (SEQ ID NO:7)

[0654] The following primers and probes are used for quantitative PCR (qPCR) analysis of copy control products:

[0655] Ec_uidA_F GTATCAGCGCGAAGTCTTTATACC(SEQ ID NO:1)

[0656] uidA_probe

[0657] 5′FAM / TAGCCGCCCTGATGCTCCATCACTTCCTG / 3′IowaBlac k(SEQ ID NO:8)

[0658] TQ_R AGACGCAAGCTACTGGTGAT(SEQ ID NO:9)

[0659] Protocol / Reaction Conditions.

[0660] (1) Use the above primers to generate PCR amplicon of target uidA; the amplicon is purified by AMPure XP (Beckman Coulter) according to the manufacturer’s recommended protocol, and quantified by qPCR using uidA forward and reverse primers and uidA probe.

[0661] (2) Capture oligomer annealing and amplicon chain extension – Dilute the purified uidA amplicon 2-fold, 10-fold, or 100-fold. Add 20 μL of each dilution of the amplicon to a solution of 0.07 U / μL SDPol (Bioron), 1×SDPol reaction buffer, 0.17 mM dNTPs, 3 mM MgCl2, 1 mg / ml BSA, and 5×10⁻⁶ dNTPs. 10 The final volume of the capture oligomers, consisting of one copy, was 30 μL in an annealing / extension reaction. The capture oligomers were annealed to the 3' end of the complementary strand of the uidA amplicon, and the amplicon strand was extended using a thermal cycler according to the following temperature control: 92°C for 2 minutes, 54°C for 2 minutes, 68°C for 10 minutes, 54°C for 2 minutes, followed by controlled cooling (0.3°C / second) to 20°C. In this embodiment, the 3' end of the capture oligomers was also extended.

[0662] (3) Hybridization of the complementary sequences of the capture oligomers – The entire capture oligomer / amplifier extension reaction mixture was added to 10 μL of a second capture reagent at a concentration of 4X, resulting in a final concentration of 125 mM NaCl, 0.25 mg / ml BSA, and 10 710 8 One or 10 9 The complementary sequence of the captured sequence was obtained from 3 copies (i.e., 3 different amounts were tested). Hybridization was performed by incubating the reaction mixture at room temperature (20°C to 24°C) for 15 minutes.

[0663] (4) Capture of amplicon and capture oligomer extension product / capture oligomer complex - Add 5 μL aliquots (50 μg) of MyOneC1 streptavidin beads (ThermoFisherScientific) in 250 mM NaCl and 1 mg / ml BSA to the hybridization mixture (step 3 above) to capture the capture oligomer / amplicon extension product / capture sequence complementary sequence complex on the beads and wash the beads according to the manufacturer's recommendations.

[0664] (5) Elution - After the final washing and removal of the washing buffer, add 10 μL of water to the bead cluster, resuspend the beads, and incubate at 70°C for 2 minutes. Use a magnet to clump the beads together and remove the eluent.

[0665] (6) Quantification - Using primers targeting the uidA_F primer site and the TQ primer-adaptor site, as well as the uidA_ probe, the amount of eluted products and captured oligomer extension products (see step 2 above) is quantified by qPCR.

[0666] Results and conclusions:

[0667] Use 10 7 10 8 One or 10 9 Each copy of the capture oligomer was used to control the copying of the amplicon produced in the targeted enrichment step (see Step 1 above) at 2-fold, 10-fold, and 100-fold dilutions (see Steps 2 through 5 above). As shown in Table 1, the amount of recovered amplicon was proportional to the amount of capture oligomer added to each PCR dilution (approximately at the expected ratio; see the “Ratio” column in the table). The differences between replicates at each data point were small (see the “Standard Deviation” column).

[0668] Table 1

[0669]

[0670] *Average of 3 repeated data

[0671] **Set the value of the 2x dilution to equal 1.**

[0672] Furthermore, despite a 50-fold difference in amplicon input amount, for 10 7 10 8 109 The number of captured oligomers varied by 2.36, 3.11, and 3.44 times, respectively (Table 1). As mentioned above, the differences between repeated data were small.

[0673] These data demonstrate that the capture oligomers described in this paper can be used to generate a predetermined amount of target output from a range of different target inputs.

[0674] Essentially as described above, but additional experiments were conducted using capture oligomers in multiple forms, where the THS region was designed to target the uidA gene of *E. coli*, the nuc gene of *Staphylococcus aureus*, the vanA gene of *Enterococcus faecallis*, or the rpb7 gene of *Candida albicans* (i.e., four capture oligomers per reaction). Using primers for uidA as shown above, and other primers designed for nuc and vanA genes, PCR was performed separately using the methods described above to amplify the uidA, nuc, and vanA genes. The resulting amplicon was diluted 10-fold or 100-fold, and 20 μL aliquots of each individual target were added to different capture oligomer annealing and amplicon chain extension reactions containing 5 × 10⁻⁶ ppm. 10 Each of the four trapping oligomers described above was copied (i.e., a quadruple trapping oligomer, but with only one target). The reaction conditions were the same as described, except for the following temperature control: 92°C for 2 minutes, 64°C for 2 minutes, 68°C for 10 minutes, 98°C for 2 minutes, 57°C for 2 minutes, followed by controlled cooling (0.3°C / second) to 20°C. After the reaction was complete, 5 × 10⁻⁶ copies were... 8 The complementary sequence of the capture sequence of each copy of the capture oligomer is added to each reaction. The capture, washing, elution, and quantification steps described above are then performed.

[0675] Table 2 shows the output levels after capture for amplicon inputs that differed by a factor of 10. The results indicate that for each of the three individual target amplicones tested in multiplex form, after control copying, the 10-fold difference in amplicon input levels was reduced to no more than a factor of 1.4 in the average output levels. Furthermore, for all three target amplicons, the average output level range after control copying spanned approximately 1.6-fold, while their input level range exceeded 270-fold.

[0676] Table 2

[0677]

[0678]

[0679] *Average of 3 repeated data

[0680] Essentially as described above, additional experiments are performed in singlet form, but using a capture oligomer in which a universal binding site in the tag sequence of the target is introduced during the PCR amplification step via THS annealing. Primers are designed to target the bacterial 23S rRNA gene, and PCR amplicons are generated from bacterial genomic DNA. The reverse primer contains a universal sequence tag introduced into the amplicons during PCR. The capture oligomer annealing and amplicon strand extension are performed as described above, except that the reaction mixture contains 0.02 U / μL SD polymerase (Bioron), 0.4x SD polymerase reaction buffer (Bioron), 0.012 mM 4dNTPs, 1.8 mM MgCl2, 0.6 mg / mL BSA, and 5 × 10⁻⁶ ppm of 4 dNTPs. 10 The final reaction volume for capturing one copy of the oligomer was 100 μL of the reaction mixture, with 40 μL of pure aliquot (i.e., undiluted; approximately 9 × 10⁻⁶) added. 12 One copy) or 50-fold dilution (approximately 2 × 10⁻⁶ copies) 11 (One copy). The temperature control method used was 92°C for 2 minutes, 54°C for 2 minutes, 68°C for 10 minutes, 54°C for 2 minutes, and then gradually decreasing to 20°C (0.3°C / second). The entire volume of the above reaction was added to 50 μL containing 1 mg / ml BSA, 125 mM NaCl, and 5 × 10⁻⁶ ppm BSA. 8 A copy of the second capture reagent was added to the 3X annealing mixture. Annealing was performed by incubating the reaction mixture on a thermal block at 25°C for 10 minutes. 50 μL (200 μg in this experiment) of MyOneC1 streptavidin beads (ThermoFisherScientific) in 4X wash buffer (composed of 4M NaCl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.20% Tween 20, and 2 mg / mL BSA) was added to the 150 μL reaction mixture described above. The resulting complex, including the capture oligomer, amplicon extension product, and the complementary sequence of the capture sequence, was captured onto the beads, and the beads were washed according to the manufacturer's instructions (in this case, using 1X wash buffer; see the 4X formulation above). Elution was performed using 20 μL of water (other aspects of the protocol are the same as described above). qPCR was performed using specific forward and reverse primers targeting a universal tag.

[0681] Despite a 50-fold difference in amplicon input levels, the output changed only 2.5-fold after the capture oligomer annealing and amplicon chain extension steps, and only 1-fold after contact with the second capture reagent and separation of the resulting complex (i.e., fully normalized) (see Table 3). Furthermore, these results demonstrate the embodiments of this disclosure, wherein THS

[0682] Combined into a general tag sequence.

[0683] Table 3

[0684]

[0685] *Average of number of repetitions** Pure / 50x dilution output

[0686] Essentially as described above (single, universal THS), but using capture oligomers in multiplex form for additional experiments, the THS in the capture oligomers is introduced into the universal tag sequence of the target during the annealing to PCR amplification step. In eight separate singlet reactions, eight different amplicons targeting regions in the bacterial 16S rRNA gene, 23S rRNA gene, and antibiotic resistance marker KPC were generated from Klebsiella pneumoniae genomic DNA. Targeted synthesis of internal controls (ICs) was also generated.

[0687] One type of DNA amplicon, nine separate amplicones in total. All nine amplicones were combined in equal volumes, and 54 μL of pure aliquots (i.e., undiluted; approximately 1 × 10⁻⁶) were added. 13 (one copy) or a 10-fold dilution (approximately 1 × 10⁻⁶ copies) 12 (One copy) to separate the oligomer annealing and amplicon chain extension reaction mixtures (each a final volume of 100 μL). The remainder of the workflow is performed essentially the same as described above, except that 5 × 10⁻⁶ copies are used. 11 One copy of the captured oligomer and 5 × 10 9 A second capture reagent was used, with 9 copies. qPCR was performed using specific forward and reverse primers targeting the universal tag to quantify each target (9 separate PCRs). The recoveries of each individual target were summed to determine the overall recovery rate of the capture process.

[0688] Despite a 10-fold difference in amplicon input levels, the total output changed by only 2.5 after the capture oligomer annealing and amplicon chain extension steps, and by only 1.6 after contacting with the second capture reagent and separating the resulting complex (see table below). Furthermore, these results demonstrate embodiments of the invention in which THS binds to a universal tag sequence in multiple forms, thereby capturing all target amplicon present in the mixture.

[0689] Table 4

[0690]

[0691] B. Capture a predetermined amount of amplicon using a capture oligomer containing a capture sequence, its complementary sequence, and a sandwich sequence.

[0692] Basically, the target amplicon was prepared as described above regarding uidA and used in experiments with trap oligomers that have no or have clip sequences (GCGCGC) embedded as the first and third additional sequences (see [link to documentation]). Figure 3 Capture was performed using undiluted, 10X diluted, and 100X diluted amplicon, essentially as described above. The amount of captured product was quantified, essentially as described above. Results are shown in... Figure 14 The use of capture oligomers containing clip sequences improves the ability to normalize output across different amplicon dilution fold spans.

[0693] C. Capturing a predetermined amount of amplicon using trapping oligomers and complementary oligomers.

[0694] Oligomer.

[0695] The following primers were used to perform PCR to amplify a segment of the vanA gene from Enterococcus faecalis:

[0696] Efm_vanA_F:GGCTGCGATATTCAAAGCTCAG(SEQ ID NO:10)Efm_vanA_R:CTGAACGCGCCGGCTTAAC(SEQ ID NO:11)

[0697] Design primers to generate amplicons with the following sequences:

[0698] GGCTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTT

[0699] GACATACATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCT

[0700] TTTGGGTTATTAATAAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAG (SEQ ID NO: 12)

[0701] A capture oligomer named CC_Blo_vanA_001 is provided, which has the following sequence:

[0702] AAAAAAAAAAAAAAAAAAAAAAA / iSp18 / CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTCCTGAACGCGCCGGCTTAAC(SEQ ID NO:13)(iSp18 = Hexaethylene glycol (HEG) internal spacer (IDT))

[0703] The oligomer includes Figure 10A The exemplary capture oligomer illustrates the elements shown. In this oligomer, the 5' poly-A sequence is a capture sequence having a first part and a second part. iSp18 is an internal extension blocker. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC (SEQ ID NO:14) is a spacer sequence having a first part and a second part. The target hybridization sequence (THS) is CTGAACGCGCCGGCTTAAC (SEQ ID NO:15), which hybridizes specifically with the vanA gene sequence in the target amplicon.

[0704] A complementary oligomer named blocking agent_vanA_001 is provided, which contains Figure 10A The exemplary complementary oligomer shown has the following sequence: CGGTGCCAGAGGAGTTTTTTTTTT / invdt / (SEQ ID NO:16), where invdt is the reverse T nucleotide, which serves as the blocking portion. In this oligomer, CGGTGCCAGAGGAG (SEQ ID NO:17) is the complementary sequence to the first portion of the spacer sequence of the capturing oligomer, and TTTTTTTTTT (SEQ ID NO:18) is the complementary sequence to the second portion of the capturing sequence.

[0705] Use a second capture reagent with the following sequence:

[0706] dT 20 -Biotin:TTTTTTTTTTTTTTTTTTTT / 3'Biotin (SEQ ID NO:19)

[0707] The following primers and probes are used for quantitative PCR (qPCR) analysis of copy control products:

[0708] vanA_PCR2_Fwd TTGTATGGACAAATCGTTGACATACA(SEQ ID NO:20)

[0709] Efm probe FAM

[0710] 5′FAM / TGCTGGGATAGCTACTCCCGCCTTTTGG / 3’IowaBlack(SEQ ID NO:21)

[0711] CC_Univ_Inner_Rev ACCGTGCTGCCTTGGCTTC(SEQ ID NO:22)

[0712] Protocol / Reaction Conditions.

[0713] (1) PCR amplicon targeting vanA was generated using the Efm_vanA_F and Efm_vanA_R primers shown above; the amplicon was quantified by chip-based capillary electrophoresis using Agilent BioAnalyzer.

[0714] (2) Capture oligomer annealing and amplicon chain extension - in undiluted (pure) or diluted 10-fold (approximately 2 × 10⁻⁶ respectively) 13 One copy and 2×10 12 Using vanA amplicon in the case of (one copy) . 80 μL of each amplicon amount (pure and 10-fold diluted) was combined with 20 μL of the capture oligomer annealing / extension reaction mixture to produce a solution of 0.02 U / μL Deep Vent (exo-)Pol (NEB), 0.4x Deep Vent Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, 1×10 11 One copy of the captured oligomer (CC_Blo_vanA_001) and 1×10 12 The final mixture, consisting of one copy of the complementary oligomer (blocker_vanA_001), had a final volume of 100 μL. The capturing oligomer was annealed to the 3' end of the complementary strand of the vanA amplicon, and the amplicon strand was extended using a thermal cycler according to the following temperature control: 92°C for 2 minutes, 64°C for 2 minutes, and 68°C for 10 minutes. In this embodiment, the 3' end of the capturing oligomer was also extended.

[0715] (3) Hybridization of the complementary sequences of the capture oligomers – 50 μL of a second capture reagent at a concentration of 3X was added to the entire capture oligomer / amplifier extension reaction mixture to obtain a final concentration of 42 mM NaCl, 0.33 mg / ml BSA, and 10 9 The complementary sequence of the captured sequence (dT) of each copy 20 -Biotin). Hybridization was carried out by incubating the reaction mixture at 30°C for 10 minutes.

[0716] (4) Capture of Amplicon and Capture Oligomeric Extension Products / Capture Oligomeric Complexes – 50 μL aliquots (200 μg) of streptavidin-coated magnetic beads were added to the entire hybridization mixture (150 μL) to produce products with final concentrations of 1 M NaCl, 5 mM TrisHCl (pH 7.5), 0.5 mM EDTA, 0.05% Tween 20, and 0.5 mg / ml BSA. The complexes were captured on the beads at 25 °C, and the beads were washed with a washing reagent having the same composition as just detailed above.

[0717] (5) Elution - After the final washing and removal of the washing buffer, add 30 μL of water to the bead clump, resuspend the beads, and incubate at 70°C for 2 minutes. Use a magnet to clump the beads together and remove the eluent.

[0718] (6) Quantification - Using primers targeting the vanA_PCR2_Fwd primer site and the universal primer-adaptor site (CC_Univ_Inner_Rev) and the Efm_probe_FAM, the amount of eluted products and captured oligomer extension products (see step 2 above) is quantified by qPCR.

[0719] Results and conclusions:

[0720] Use 10 9 The second capture oligomer, consisting of one copy, was used to perform copy control on the 0-fold (pure) and 10-fold dilutions of the amplicon generated in step 1 (PCR) above (see steps 2 to 5 above). As shown in Table 5, despite a 10-fold difference in target input, the output levels were essentially the same.

[0721] Table 5

[0722] PCR dilution Output (#copy) Multiple difference pure 3.24E+08 - 10 times 3.14E+08 1.03

[0723] These data demonstrate that the capture oligomers and complementary oligomers described in this paper can be used to generate a predetermined normalized target output when the target input varies by a factor of 10.

[0724] Essentially, as described above, another experiment was conducted, which differed in the following ways:

[0725] (1) The PCR amplicon was purified using the QIAGEN QIAquick PCR Purification Kit according to the manufacturer’s instructions and then quantified by chip-based capillary electrophoresis using the Agilent BioAnalyzer.

[0726] (2) Capture oligomer annealing and amplicon chain extension - at undiluted (pure), 10-fold dilution and 100-fold dilution (approximately 8 × 10⁻⁶ each). 11One copy, 8×10 10 One copy and 6×10 9 Using vanA amplicon in cases of (one copy) . At 1×10 12 The capture oligomer for each copy / reaction uses the capture oligomer (CC_Blo_vanA_001) and is zero or 1×10 13 Each copy / reaction uses a complementary oligomer (blocker_vanA_001). The captured oligomer is annealed to the 3' end of the complementary strand of the vanA amplicon, and the amplicon strand is extended using a thermal cycler according to the following temperature control: 95°C for 2 minutes and 64°C for 15 minutes. All other conditions in this step are the same as in step 2 above.

[0727] (3 to 6) Steps 3 to 6 are performed as described above, except that in step 4, the complex is captured on the beads at 30°C instead of 25°C.

[0728] Results and conclusions:

[0729] Copy control was performed on the 0-fold (pure), 10-fold, and 100-fold dilutions of the amplicon generated in step 1 (PCR) above (see steps 2 to 5 above). In this experiment, the amount of each nucleic acid component used was approximately 8 × 10⁻⁶. 11 8×10 10 6 × 10 9 One copy of the target, 1×10 12 One copy of the captured oligomer, 0 or 1×10 13 One copy of complementary oligomers and 1×10 9 A second captured oligomer of one copy. Table 6 shows the results with or without complementary oligomers.

[0730] Table 6

[0731]

[0732] *Target amplicon generated in step 1

[0733] When complementary oligomers are lacking, the second trapping oligomer (dT) will not bind to the target, regardless of whether the trapping oligomer binds to it. 20 Biotin can bind to any capturing oligomer molecule. In this experiment, 1 × 10⁻⁶ ppm was used. 12 One copy of the captured oligomer and 1×10 9The second capture oligomer has a copy number, meaning there is a 1000-fold difference in these quantities. At the highest target level (pure), most of the capture oligomers will bind to the target, therefore most of the second capture oligomers will bind to the capture oligomer associated with the target, and the output copy number after capture and elution will be relatively high. This is confirmed in data with pure target input (no complementary oligomers), where a relatively high output (9.3 × 10⁻⁶) was observed. 7 (One copy). However, for a 10-fold dilution of the target, there will be an excess of capture oligomers, so not all capture oligomers will bind to the target. Some secondary capture oligomers will bind to the capture oligomers associated with the target, but some secondary capture oligomers will capture the oligomers that have not bound to the target. Therefore, as actually observed, the output will decrease (output = 2.6 × 10⁻⁶). 7 (1 copy). For a 100-fold dilution of the target, most of the trapping oligomers will not bind to the target, and therefore, similarly, most of the second oligomers will bind to the trapping oligomers that do not associate with the target. Therefore, the expected outcome under these conditions is a significant reduction in output, which was actually observed (output = 5.0 × 10⁻⁶). 5 One copy.

[0734] In the presence of complementary oligomers, capture oligomers not bound to the target will bind to the complementary oligomer, which in turn will block the binding of the second capture agent. Conversely, capture oligomers already bound to the target will not bind to the complementary oligomer (which has been replaced), which in turn will allow the second capture agent to bind. Therefore, at all target input levels tested in this experiment, the output in the presence of complementary oligomers was expected to be higher than the output in the absence of complementary oligomers (as stated above). This is exactly what was observed (see Table 6). Furthermore, the data demonstrate that normalization occurred, with only about a 2-fold difference between the output at the pure target level and the output at the 10-fold diluted target level, and only slightly more than a 14-fold difference between the output at the pure target level and the output at the 100-fold diluted target level. The output of the 100-fold target dilution was slightly lower than the theoretical value because the binding kinetics were slower due to the low target level. If the incubation time is longer, the output will increase, and the normalization factor will be improved.

[0735] These data demonstrate that the capture oligomers and complementary oligomers described in this paper can be used to generate a predetermined normalized target output when the target input varies by a factor of 100.

[0736] D. Using trapping oligomers, complementary oligomers, substitution oligomers, and forward primers, trappable products containing additional sequences (e.g., adaptors) at both ends of the target sequence are generated.

[0737] Oligomer.

[0738] The following primers were used to perform PCR to amplify a fragment of the vanA gene from Enterococcus faecalis:

[0739] Efm_vanA_F:GGCTGCGATATTCAAAGCTCAG(SEQ ID NO:23)

[0740] Efm_vanA_R:CTGAACGCGCCGGCTTAAC(SEQ ID NO:24)

[0741] Design primers to generate amplicons with the following sequences:

[0742] GGCTGCGATATTCAAAGCTCAGCAATTTGTATGGACAAATCGTT

[0743] GACATACATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCT

[0744] TTTGGGTTATTAATAAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAG (SEQ ID NO: 25)

[0745] A capture oligomer named PCR2R_adaptor_CC is provided, which has the following sequence:

[0746] AAAAAAAAAAAAAAAAAAAA / iSp18 / CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTCGTAGCTGCCACCGGCCTAT(SEQ ID NO:26)

[0747] (iSp18 = Hexaethylene glycol (HEG) internal spacer (IDT))

[0748] The oligomer contains Figure 10A The exemplary capture oligomer illustrates the elements shown. In this oligomer, the 5' poly-A sequence is a capture sequence having a first part and a second part. iSp18 is an internal extension blocker. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC (SEQ ID NO:27) is a spacer sequence having a first part and a second part. The target hybridization sequence (THS) is GTAGCTGCCACCGGCCTAT (SEQ ID NO:28), which hybridizes specifically with the vanA gene sequence in the target amplicon.

[0749] A complementary oligomer named blocking agent_vanA_001 is provided, which contains Figure 10A The exemplary complementary oligomer shown has the following sequence: CGGTGCCAGAGGAGTTTTTTTTTT / invdt / (SEQ ID NO:29), where invdt is the reverse T nucleotide, which serves as the blocking portion. In this oligomer, CGGTGCCAGAGGAG (SEQ ID NO:30) is the complementary sequence to the first portion of the spacer sequence of the capturing oligomer, and TTTTTTTTTT (SEQ ID NO:31) is the complementary sequence to the second portion of the capturing sequence.

[0750] Efm_vanA_R (the above sequence), which contains Figure 8A The exemplary substitution oligomers shown are examples of the elements. An oligomer named PCR1F_adaptor is also provided, which contains... Figure 8A The element shown in the exemplary forward primer with an adaptor, the PCR1F_adaptor, has the following sequence:

[0751] AAAACGAGACATGCCGAGCATCCGCGGCTGCGATATTCAAAGCTCAG (SEQ ID NO: 32).

[0752] Use a second capture reagent with the following sequence:

[0753] dT 20 -Biotin:TTTTTTTTTTTTTTTTTTTT / 3'Biotin (SEQ ID NO:33)

[0754] The following primers and probes are used for quantitative PCR (qPCR) analysis of copy control products:

[0755] CCRPA_uni_FAAAACGAGACATGCCGAGCATC(SEQ ID NO:34)

[0756] Efm_probe_FAM5'FAM / TGCTGGGATAGCTACTCCCGCCTTTTGG / 3'IowaBlack(SEQ ID NO:35)

[0757] CC_Univ_Inner_Rev ACCGTGCTGCCTTGGCTTC(SEQ ID NO:36)

[0758] Scheme / Reaction Conditions (1).

[0759] (1) Use the Efm_vanA_F and Efm_vanA_R primers shown above to generate PCR amplicon of target vanA; purify the amplicon using the QIAGEN QIAquick PCR purification kit according to the manufacturer’s instructions, and then quantify it by chip-based capillary electrophoresis using the Agilent BioAnalyzer.

[0760] (2) Annealing and extension of the capturing oligomer, the displacement oligomer, and the forward primer with the linker - containing approximately 1 × 10 12 Aliquots of one copy of the vanA amplicon were combined with the annealing / extension reaction mixture to produce a solution of 0.02 U / μL Deep Vent (exo-)Pol (NEB), 0.4x Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, and 5 × 10⁻⁶ ppm dNTPs. 13 One copy of the capture oligomer (PCR2R_adaptor_CC), ±1×10 13 One copy of the substitution oligomer (Efm_vanA_R) and 5×10 13 The final mixture consisted of one copy of the forward primer with the adaptor (PCR1F_adaptor), with a final volume of 100 μL. Annealing and extension of the capture oligomer and the substitution oligomer with the input amplicon, as well as annealing and extension of the extension product of the forward primer with the adaptor and the capture oligomer, all occurred in the same annealing / extension reaction, performed using a thermal cycler according to the following temperature control: 95 °C for 5 minutes, followed by 64 °C for 20 minutes.

[0761] (3) Quantification - Dilute each aliquot of the annealing extension reaction 100 times and use primers CCRPA_uni_F and CC_Univ_Inner_Rev (targeting the universal adaptor [ZW1] region in the forward and reverse directions, respectively) and Efm_probe_FAM to quantify the amount of product contained in each sample by qPCR.

[0762] Results and conclusions:

[0763] A single-cycle annealing and extension reaction was performed using the target and oligomer described above (a single cycle is defined as only one denaturation step, such as incubation at 95°C; another cycle would begin with another thermal denaturation step). As shown in Table 7, products containing universal linkers at both ends of the molecule were formed, as demonstrated by amplification using universal primers.

[0764] Table 7

[0765] Replacement oligomers Output (#copy) + 1.3E+11 - 2.8E+11

[0766] These data prove Figure 8A The embodiments of the invention described herein can be used to generate products with adaptors (or other desired sequences) at both ends of a molecule using single-cycle annealing / extension. Furthermore, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2R_adaptor_CC) can bind to an internal site in the target, not just to the ends. These data also demonstrate that the desired product can be generated without a substitution oligomer. Without wishing to be bound by any particular theory, different mechanisms may be at work in the disclosed embodiments to produce the desired product. In the presence of a substitution oligomer, multiple mechanisms may be at play to produce the observed results.

[0767] Essentially, as described above, another experiment was conducted, which differed in the following ways:

[0768] (2) Annealing and extension of the capturing oligomer, the displacement oligomer, and the forward primer with the linker - containing approximately 1 × 10 13 Aliquots of one copy of the vanA amplicon were mixed with an annealing / extension reaction mixture to produce a solution consisting of 0.02 U / μL Deep Vent(exo-)Pol(NEB), 0.4x Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, and 5 × 10⁻⁶ ppm dNTPs. 14 One copy of the capture oligomer (PCR2R_adaptor_CC) and 5 × 10 14 The final mixture, consisting of 1 copy of the forward primer with the adaptor (PCR1F_adaptor), was prepared in a final volume of 100 μL. Annealing and extension of this mixture were performed using a thermal cycler at the following temperature control: 95 °C for 5 minutes, followed by 64 °C for 15 minutes. At this point, 5 × 10⁻⁶ copies of the forward primer with the adaptor were added. 14 One copy of the substitution oligomer (Efm_vanA_R) was added to some of the duplicate samples of the reaction mixture, and for some samples, only buffer was added, and annealing and extension were continued using the following temperature control: 64°C for 5 minutes, 75°C for 5 minutes and 72°C for 15 minutes.

[0769] Results and conclusions:

[0770] A single-cycle annealing and extension reaction was performed using the target and oligomer described above (a single cycle is defined as only one denaturation step, e.g., incubation at 95°C; another cycle would begin with another thermal denaturation step). Midway through the process, a substitution oligomer was added to the annealing and extension reactions to further optimize performance. As shown in Table 8, products containing universal linkers at both ends of the molecule were formed, as demonstrated by amplification using universal primers.

[0771] Table 7

[0772] Replacement oligomers Output (#copy) + 3.2E+12 - 1.9E+12

[0773] As stated above, these data indicate that Figure 8A The embodiments of the invention described herein can be used to generate products with adaptors (or other desired sequences) at both ends of a molecule using a single-cycle annealing / extension process. As also stated above, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2R_adaptor_CC) can bind to an internal site in the target, not just to the ends. Furthermore, these data show that overall performance can be improved by adjusting the annealing and extension temperature control, and in this case, by adding a substitution oligomer midway through the process. Particularly noteworthy is that under these conditions, the amount of desired product generated is greater when the substitution oligomer is present than when it is absent, thus demonstrating that the substitution scheme, as described above, yields a significantly higher yield. Figure 8A The operation is as shown. Similarly, it is not intended to be bound by any particular theory; different mechanisms can also function in the disclosed implementation to produce the desired product.

[0774] Scheme / Reaction Conditions (2).

[0775] (1) Use the Efm_vanA_F and Efm_vanA_R primers shown above to generate PCR amplicon of target vanA; purify the amplicon using the QIAGEN QIAquick PCR purification kit according to the manufacturer’s instructions, and then quantify it by chip-based capillary electrophoresis using the Agilent BioAnalyzer.

[0776] (2) Annealing and extension of the capturing oligomer, the displacement oligomer, and the forward primer with the linker - containing approximately 1 × 10 12 Aliquots of one copy of the vanA amplicon were combined with the annealing / extension reaction mixture to produce a solution of 0.02 U / μL Deep Vent (exo-)Pol (NEB), 0.4x Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, and 5 × 10⁻⁶ ppm dNTPs. 13 One copy of the capture oligomer (PCR2R_adaptor_CC), ±5×10 12 One copy of the substitution oligomer (Efm_vanA_R) and 5×10 13The final mixture consisted of one copy of the forward primer with the adaptor (PCR1F_adaptor), with a final volume of 100 μL. Annealing and extension of the capture oligomer and the substitution oligomer with the input amplicon, as well as annealing and extension of the extension product of the forward primer with the adaptor and the capture oligomer, all occurred in the same annealing / extension reaction, performed using a thermal cycler according to the following temperature control: 95 °C for 5 minutes, followed by 64 °C for 20 minutes.

[0777] (3) Hybridization of the complementary sequences of the capture sequences of the capture oligomers - 50 μL of a second capture reagent at a concentration of 3X was added to the entire extension reaction mixture to obtain a final concentration of 42 mM NaCl, 0.33 mg / ml BSA, and 5 × 10⁻⁶ ppm. 14 The complementary sequence of the captured sequence (dT) copies 20 -Biotin). Hybridization was carried out by incubating the reaction mixture at 30°C for 10 minutes.

[0778] (4) Capture of Amplicon and Capture Oligomeric Extension Products / Capture Oligomeric Complexes – 50 μL aliquots (200 μg) of streptavidin-coated magnetic beads were added to the entire hybridization mixture (150 μL) to produce a final concentration of 1 M NaCl, 5 mM TrisHCl (pH 7.5), 0.5 mM EDTA, 0.05% Tween 20, and 0.5 mg / ml BSA. The complexes were captured on the beads at 30 °C, and the beads were washed with a washing reagent having the same composition as just detailed above.

[0779] (5) Elution - After the final washing and removal of the washing buffer, add 30 μL of water to the bead clump, resuspend the beads, and incubate at 70°C for 2 minutes. Use a magnet to clump the beads together and remove the eluent.

[0780] (6) Quantification - The eluted products were quantified by qPCR using primers CCRPA_uni_F and CC_Univ_Inner_Rev (targeting the universal adaptor region in the forward and reverse directions, respectively) and Efm_probe_FAM.

[0781] Results and conclusions:

[0782] Using the target and oligomers described above, a single cycle of annealing and extension reactions was performed to capture, wash, and elute the products, which were then quantified using qPCR. As shown in Table 8, products containing universal adaptors at both ends of the captured and eluted molecules were formed, as demonstrated by amplification using universal primers.

[0783] Table 8

[0784] Replacement oligomers Output (#copy) + 1.4E+09 - 1.5E+10

[0785] These data indicate that Figure 8A The embodiments of the invention described herein can be used to generate products with adaptors (or other desired sequences) at both ends of a molecule using single-cycle annealing / extension, and these products can be separated by capture onto beads, washing, and elution. Furthermore, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2R_adaptor_CC) can bind to an internal site in the target, not just to the ends. These data also show that the desired product can be generated in the absence of a substitution oligomer. Without wishing to be bound by any particular theory, different mechanisms may be at work in the disclosed embodiments to produce the desired product. In the presence of a substitution oligomer, multiple mechanisms may be at play to produce the observed results.

[0786] Essentially, as described above, another experiment was conducted, which differed in the following ways:

[0787] (2) The annealing and extension-annealing / extension reaction mixtures for capturing oligomers, displacing oligomers, and forward primers with linkers are the same as those described above, except that 5 × 10⁻⁶ ppm are added. 14 A new sample containing one copy of the complementary oligomer (blocker_vanA_001). Annealing and extension of the trapping and substitution oligomers with the input amplicon, annealing of the complementary oligomer with the trapping oligomer, and annealing and extension of the extension product of the forward primer with the trapping oligomer all occurred in the same annealing / extension reaction, which was performed using a thermal cycler according to the temperature control method shown in Table 9 below.

[0788] Table 9

[0789]

[0790]

[0791] (3) Hybridization of complementary sequences of the capture sequence of the capture oligomer - The conditions are the same as above, except that 1×10⁻⁶ is used. 9 The complementary sequence of the captured sequence (dT) of each copy 20 -Biotin).

[0792] Results and conclusions:

[0793] A single-cycle annealing and extension reaction was performed using the target and oligomer described above, with a predetermined amount of the complementary sequence (dT) of the capture sequence. 20 The biotin-captured product was washed, eluted, and then quantified using qPCR. The results are shown in Table 10.

[0794] Table 10

[0795] PCR dilution Output (#copy) Multiple difference pure 2.0E+06 - 10 times 4.3E+05 4.7

[0796] These data indicate that Figure 8A The embodiments of the invention described herein can be used to generate products with adaptors (or other desired sequences) at both ends of a molecule using single-cycle annealing / extension, and these products can be separated by capture onto beads, washing, and elution. Furthermore, a 10-fold difference at the input target level was normalized to a 4.7-fold difference, which exceeds a 2-fold normalization factor. Moreover, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2R_adaptor_CC) can bind to an internal site in the target, not just to the ends.

[0797] Essentially, as described above, another experiment was conducted, which differed in the following ways:

[0798] (2) The annealing and extension-annealing / extension reaction mixtures for capturing oligomers, displacing oligomers, and forward primers with linkers are the same as described above, except that 1E+13 and 1E+12 (10-fold dilution) copies / reaction of the target input are used; and 5×10⁻⁶ copies of the target input are added. 14 A new sample containing one copy of the complementary oligomer (blocker_vanA_001). Annealing and extension of the trapping oligomer with the input amplicon, annealing of the complementary oligomer with the trapping oligomer, and annealing and extension of the extension product of the forward primer with the trapping oligomer all occurred in the same annealing / extension reaction, which was performed using a thermal cycler according to the following temperature control: 95°C for 5 minutes, followed by 64°C for 15 minutes.

[0799] (3) Hybridization of complementary sequences of the capture sequence of the capture oligomer - The conditions are the same as above, except that 1×10⁻⁶ is used. 9 The complementary sequence of the captured sequence (dT) of each copy 20 -Biotin). Hybridize at 30°C for 30 minutes.

[0800] Results and conclusions:

[0801] A single-cycle annealing and extension reaction was performed using the target and oligomer described above, with a predetermined amount of the complementary sequence (dT) of the capture sequence. 20 The biotin-captured product was washed, eluted, and then quantified using qPCR. The results are shown in Table 10.

[0802] Table 11

[0803]

[0804] These data indicate that Figure 8A The embodiments of the invention described herein can be used to generate products with adaptors (or other desired sequences) at both ends of a molecule using single-cycle annealing / extension, and these products can be separated by capture onto beads, washing, and elution. Furthermore, when complementary oligomers are present, a 10-fold difference in the input target level is normalized to a 0.67-fold difference (~1). Without complementary oligomers, the product recovery decreases by more than 10-fold, as expected, while normalization is similar. Moreover, these data demonstrate that at least one primer-adaptor oligomer (in this case, PCR2R_adaptor_CC) can bind to an internal site in the target, not just the ends.

Claims

1. A composition comprising a trapping oligomer and a complementary oligomer, wherein: (a) The captured oligomer comprises, in the 5' to 3' direction: The captured sequence contains a first part and a second part. Internal extension blocking element, An interval sequence, comprising a first part and a second part, and Target hybridization sequence; and (b) The complementary oligomer comprises, in the 3' to 5' direction: The complementary sequence of the second part of the captured sequence, and The complementary sequence of at least a first portion of the spacer sequence, wherein the complementary sequence of the second portion of the capture sequence and the complementary sequence of at least a first portion of the spacer sequence are configured to anneal with the capture oligomer when the complementary sequence of the spacer sequence is not present.

2. The composition according to claim 1, wherein the trapping oligomer has the following formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3' Where A1 is the first additional sequence that exists arbitrarily. C1 is the first part of the capture sequence. C2 is the second part of the captured sequence. B is the internally extended blocking element. A2 is an arbitrarily existing second additional sequence. S1 is the first part of the interval sequence. S2 is the second part of the interval sequence. A3 is an arbitrarily existing third additional sequence. RB is an arbitrarily existing reversible extension blocking element. A4 is an arbitrarily existing fourth additional sequence. THS is the target hybridization sequence, and X represents any arbitrarily existing blocking component.

3. The composition according to claim 1, wherein the complementary oligomer has the following formula: 5'-S1'-A2'-L-C2'-X-3' Where S1' is the complementary sequence of at least the first part of the interval sequence. A2' is an optional complementary sequence of a second additional sequence that may be present in the captured oligomer; L is an optional connector. C2' is the complementary sequence of the second part of the captured sequence, and X represents any arbitrarily existing blocking component.

4. The composition according to any one of claims 1 to 3, wherein the capturing oligomer and / or complementary oligomer comprises a blocking portion at its 3' end.

5. The composition according to any one of claims 1 to 3, wherein if the spacer sequence of the trapping oligomer is occupied by a single complementary sequence, the complementary sequence of the second portion of the trapping sequence is insufficient to stably anneal with the trapping sequence of the trapping oligomer at a temperature of 65°C or above.

6. The composition according to any one of claims 1 to 3, wherein the capture sequence comprises poly A or poly T, and the complementary sequence of the capture sequence or the complementary sequence of the second portion of the capture sequence comprises poly T or poly A.

7. The composition according to any one of claims 1 to 3, wherein the capturing oligomer comprises a linker sequence as part or all of the spacer sequence, or as a third additional sequence at the 3' of part or all of the spacer sequence or a fourth additional sequence at the 5' of the target hybridization sequence.

8. The composition according to any one of claims 1 to 3, wherein, The capture oligomer contains an affinity-enhancing modification located in the target hybridization sequence.

9. The composition according to claim 8, wherein, The affinity-enhancing modification is any one or more of 5-Me-C, 2-aminopurine, 2'-fluoro, C-5-propyne, LNA, PNA, ZNA, thiophosphate, 2'-OMe, or restricted ethyl (cEt) substitution.

10. The composition according to any one of claims 1 to 3, wherein the capturing oligomer comprises a reversible extension blocking member located at the 5' of the target hybridization sequence.

11. A method for capturing a target polynucleotide from a composition, the method comprising: The composition is contacted with the composition of any one of claims 1 to 10, wherein the target hybridization sequence of the capture oligomer is annealed with the target polynucleotide at a site comprising the 3' end of the target polynucleotide; The 3' end of the target polynucleotide is extended using a DNA polymerase with strand displacement activity to form a complementary sequence to the spacer sequence. This complementary sequence is annealed with the capture oligomer such that the complementary oligomer is replaced to a degree sufficient to make the capture sequence of the capture oligomer available for binding. The capture sequence of the capture oligomer is contacted with a complementary sequence comprising the capture sequence and a second capture agent comprising (i) a binding partner or (ii) a solid carrier, thereby forming a complex comprising the target polynucleotide, the capture oligomer and the second capture agent; as well as The complex is isolated from the composition to capture the target polynucleotide.

12. The method of claim 11, wherein the second capturing agent comprises a binding partner, and separation comprises contacting the complex with a solid carrier comprising the second binding partner, the second binding partner being configured to bind to the binding partner of the second capturing agent.

13. The method of claim 12, wherein the binding partner is biotin.

14. The method according to claim 12, wherein the solid carrier is a bead.

15. The method of claim 12, wherein the second binding partner is streptavidin.

16. A method for capturing a target polynucleotide from a composition, the method comprising: The target polynucleotide is contacted with the composition according to any one of claims 1 to 10, wherein the target hybridization sequence of the capturing oligomer is annealed with the target polynucleotide at a site upstream of the 3' end of the target polynucleotide; The 3' end of the capture oligomer is extended along the target polynucleotide to form a first extended chain; The target polynucleotide is contacted with a substitution oligomer, the substitution oligomer containing a substitution target hybridization sequence, the substitution target hybridization sequence being annealed downstream of the target hybridization sequence of the capturing oligomer with the target polynucleotide; The substitution oligomer extends along the target polynucleotide, thereby replacing the first extended chain of the target polynucleotide. Optionally, the capturing oligomer and the displacement oligomer are added to the composition simultaneously or sequentially.

17. The method of claim 16, further comprising: The first extended strand is brought into contact with a reverse amplification oligomer containing a reverse target hybridization sequence configured to bind the first extended strand; and the reverse amplification oligomer is extended to form a second extended strand.

18. The method of claim 17, wherein the reverse amplification oligomer comprises an additional sequence at the 5' of its target hybridization sequence, optionally wherein the 3' end of the first extension strand is further extended to form a complementary sequence to the additional sequence of the reverse amplification oligomer.

19. The method of any one of claims 16 to 18, wherein the capturing oligomer further comprises an additional sequence located at the 5' of the target hybridization sequence, optionally wherein, if an extension of the second extension chain is present, the additional sequence forming the capturing oligomer is a complementary sequence.

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