Compositions, kits and methods for isolating target polynucleotides

Abstract

To provide improved compositions and methods for capturing nucleic acid, including, e.g., rapid and accurate capture of nucleic acid in a controlled or limited way, e.g., an amount less than or equal to a predetermined amount; capture oligomers that can capture in a target-dependent manner; and streamlined addition of adaptors or other sequences.SOLUTION: The disclosure provides a combination comprising a capture oligomer and a complementary oligomer, where (a) the capture oligomer comprises, in the 5' to 3' direction, a capture sequence comprising first and second portions, an internal extension blocker, a spacer sequence comprising first and second portions, and a target-hybridizing sequence; and (b) the complementary oligomer comprises, in the 3' to 5' direction, a complement of the second portion of the capture sequence, and a complement of at least the first portion of the spacer sequence, where the complement of the second portion of the capture sequence and the complement of the at least first portion of the spacer sequence are configured to anneal simultaneously to the capture oligomer in the absence of a complement of the spacer sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] 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, each of which is incorporated by reference in its entirety for all purposes.

[0002] I. Introduction and Overview Embodiments herein relate to isolating target polynucleotides, such as polynucleotides and amplicons, from a composition derived from an organism of interest. The embodiments are useful, for example, as part of a workflow for preparing target polynucleotides for sequencing or other analysis.

[0003] Certain biochemical and molecular biology procedures benefit from or require a defined amount of input nucleic acid. For example, a sequencing library preparation procedure may have a range of acceptable amounts of nucleic acid, with amounts below a minimum amount resulting in wasted throughput and low data output, and amounts above a maximum amount resulting in poor library quality. Furthermore, a defined amount (often expressed as a "number of molecules") of library nucleic acid is generally desirable for use in the clonal amplification step (if utilized) of a sequencing workflow to avoid, on the one hand, polyclonal molecular populations and, on the other hand, wasted throughput and low data output, as well as for use in single molecule sequencing workflows where a defined number of molecules input to the sequencing step is desirable for optimal results. For time-critical applications, such as rapid detection of pathogens by sequencing clinical samples, existing approaches to provide samples with acceptable amounts of nucleic acid, including, for example, quantification procedures and subsequent concentration or dilution steps, can be unnecessarily slow. In addition, various existing methods may suffer from other problems, such as saturation of solid supports with excess capture oligomers and complications associated with the addition of adapters or other sequences.

[0004] Thus, there is a need for compositions and methods that can provide improved nucleic acid capture, including, for example, rapid and accurate capture of nucleic acids in a controlled or limited manner, e.g., in amounts not exceeding a predetermined amount; capture oligomers capable of being captured in a target-dependent manner; and streamlined addition of adaptors or other sequences. The present disclosure aims to provide compositions and methods that meet one or more of these needs, provide other benefits, or at least provide a useful option to the public. Provided herein are capture oligomers, combinations of capture oligomers and other oligomers, and related compositions, kits, and methods for capturing and / or controlling the amount of nucleic acid (e.g., a defined amount of capture oligomer or another limiting reagent (e.g., a secondary capture reagent) or a combination thereof can control the output of the capture procedure, such that the capture oligomer is capable of being captured in a target-dependent manner); and / or for adding adaptors or other sequences in a streamlined manner. Summary of the Invention

[0005] More specifically, the following embodiments are provided herein: Embodiment 1 comprises, in the 5' to 3' direction: Capture sequence, Internal extension blockers, the complement of the capture sequence, and Target Hybridizing Sequence A capture oligomer comprising: The complement of the capture sequence is a capture oligomer that is configured to anneal to the capture sequence in the absence of the target hybridizing sequence and the extended target sequence that anneals to the complement of the capture sequence.

[0006] In a second embodiment, the capture oligomer has the formula 5'-A1-CLB-A2-C'-A3-RB-A4-THS-X-3' (wherein A1 is a first additional sequence that is optionally present; C is a capture sequence, L is an optional linker, B is an internal extension blocker, A2 is an optional second additional sequence, C' is the complement of the capture sequence, A3 is an optional third additional sequence, RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence, THS is the target hybridizing sequence; X is an optional blocking moiety. The capture oligomer of embodiment 1 has the following structure:

[0007] Embodiment 3 is a capture oligomer of any one of the preceding embodiments, wherein the capture sequence comprises a polyA or polyT sequence and the complement of the capture sequence comprises a polyT or polyA sequence.

[0008] Embodiment 4 is a capture oligomer of any one of the preceding embodiments, wherein the capture oligomer comprises a first additional sequence 5' of the capture sequence and a third additional sequence 3' of the complement of the capture sequence, which comprise a first and second stabilizing sequence, respectively, and optionally the stabilizing sequence is a GC clamp sequence.

[0009] Embodiment 5 is a capture oligomer according to the immediately preceding embodiment, wherein a first stabilizing sequence is located 5' to the remainder of the capture sequence and / or a second stabilizing sequence is located 3' to the remainder of the complement of the capture sequence.

[0010] Embodiment 6 is the capture oligomer of embodiment 4 or 5, wherein the GC clamp sequence comprises a (GC)3 sequence or a (CG)3 sequence, respectively.

[0011] Embodiment 7 is a capture oligomer according to any one of the preceding embodiments, comprising a linker between the capture sequence and the internal extension blocker, which linker is optionally a nucleotide sequence or a non-nucleotide linker, or a combination thereof.

[0012] Embodiment 8 is a capture oligomer of any one of the preceding embodiments, wherein the internal extension blocker comprises a non-nucleotidic linker, or one or more of abasic sites, non-natural nucleotides, or chemically modified natural nucleotides.

[0013] Embodiment 9 is a capture oligomer of any one of the preceding embodiments, comprising a third additional sequence comprising an adapter sequence between the complement of the capture sequence and the THS.

[0014] Embodiment 10 is the capture oligomer of any one of the preceding embodiments, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridizing sequence.

[0015] Embodiment 11 is a capture oligomer according to the immediately preceding embodiment, wherein the reversible extension blocker is located 3' to the complement of the capture sequence.

[0016] Embodiment 12 is the capture oligomer of embodiment 10 or 11, wherein the capture oligomer comprises a third additional sequence located 3' of the complement of the capture sequence and 5' of the target hybridizing sequence, and the reversible extension blocker is located 3' of the adapter sequence, and optionally the third additional sequence comprises an adapter sequence.

[0017] Embodiment 13 is a capture oligomer of any one of the preceding embodiments, wherein the capture oligomer comprises a fourth additional sequence located 3' of the reversible blocker and 5' of the target hybridizing sequence, optionally wherein the fourth additional sequence comprises an adapter sequence.

[0018] Embodiment 14 is a capture oligomer of any one of the preceding embodiments, comprising a second additional sequence between the internal extension blocker and the complement of the capture sequence, optionally wherein the second additional sequence comprises a mixed nucleotide segment.

[0019] Embodiment 15 is the capture oligomer of the immediately preceding embodiment, wherein the reversible extension blocker comprises Iso-dC or Iso-dG, Xanthine or 5-(2,4 diaminopyrimidine), 2-Amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4-Methylbenzimidizole or 2,4-difluorotoluene, 7-azaindole or isocarbostyril, dMMO2 or d5SICS, dF or dQ; one or more chemically modified nucleotides, wherein the modification is attached via a reversible linkage, which linkage can be reversed by providing one or more of a chemical, an enzyme, a temperature change, or a change in reagent composition; a reversible nucleic acid structural feature; or a molecule reversibly bound to the capture oligomer, optionally wherein the reversibly bound molecule is a protein, an enzyme, a lipid, a carbohydrate, or a chemical moiety.

[0020] Embodiment 16 is a capture oligomer of any one of the preceding embodiments, wherein the target hybridizing sequence comprises a blocking moiety at its 3' end.

[0021] Embodiment 17 is a capture oligomer according to any one of the preceding embodiments, e.g., comprising one or more affinity enhancing modifications (e.g., any one or more of 5-Me-C, 2-aminopurine, 2'-fluoro, C-5-propyne, LNA, PNA, ZNA, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions) in the target hybridizing sequence.

[0022] Embodiment 18 is a combination comprising a capture oligomer as described in any one of the preceding embodiments and a secondary capture reagent comprising a complement of the capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid support (e.g., a bead or surface).

[0023] Embodiment 19 is the combination of the immediately preceding embodiment, wherein the combination further comprises a second capture oligomer and a second secondary capture reagent, wherein the second capture oligomer comprises a second target hybridizing sequence that is different from the target hybridizing sequence of the capture oligomer and a second capture sequence that is different from the capture sequence of the capture oligomer, and the second secondary capture reagent comprises a complement of the second capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid support (e.g., a bead or surface).

[0024] Embodiment 20 is the combination of embodiment 18, wherein the secondary capture reagent comprises a complement of the capture sequence and a binding partner (e.g., biotin), and the combination further comprises a solid support (e.g., a bead) comprising a second binding partner (e.g., a biotin binder, such as streptavidin) configured to bind to the binding partner of the secondary capture reagent.

[0025] Embodiment 21 is a combination comprising a capture oligomer or combination according to any one of the preceding embodiments, wherein the capture oligomer comprises an adapter sequence 5' of the target hybridizing sequence, and the combination further comprises a blocker oligomer comprising a non-extendable adapter sequence, optionally configured such that the blocker oligomer binds to the complement of the adapter sequence with a higher affinity than the adapter sequence of the capture oligomer, or forms a complex with the complement of the adapter sequence that has a higher melting temperature than a complex between the adapter sequence of the capture oligomer and the complement of the adapter sequence.

[0026] Embodiment 22 is a combination comprising a capture oligomer or combination according to any one of the preceding embodiments, wherein the capture oligomer comprises an adapter sequence 5' of the target hybridizing sequence, and the combination further comprises a second oligomer comprising a second target hybridizing sequence 5' that is a complement of at least a portion of the adapter sequence, and wherein in the presence of a target polynucleotide having an accessible complement of the first and second target hybridizing sequences, the capture oligomer and the second oligomer are configured to form a triple-stranded junction with the target polynucleotide.

[0027] Embodiment 23 is a reaction mixture comprising a capture oligomer or combination according to any one of the preceding embodiments and a target polynucleotide.

[0028] Embodiment 24 is a reaction mixture of the immediately preceding embodiment, wherein the target is an amplicon, optionally further comprising at least one amplification primer, and optionally wherein the target-hybridizing sequence of the capture oligomer has a greater affinity (e.g., a longer THS or an affinity-enhancing modification) for the amplicon than the primer.

[0029] Embodiment 25 is a reaction mixture of any one of embodiments 23 or 24, further comprising a second capture oligomer, a second secondary capture reagent, and a second target polynucleotide, wherein the second capture oligomer comprises a second target hybridizing sequence configured to anneal to the second target polynucleotide and a second capture sequence that is different from the capture sequence of the capture oligomer, and the second secondary capture reagent comprises a complement of the second capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid support (e.g., a bead or surface).

[0030] Embodiment 26 is the reaction mixture of the immediately preceding embodiment, wherein the second target polynucleotide is present at a lower concentration than the target polynucleotide.

[0031] Embodiment 27 is a reaction mixture of any one of embodiments 25 or 26, wherein the target polynucleotide and the second target polynucleotide are isolated or generated from a sample from an environmental organism or type, and the second target polynucleotide is less commonly observed in samples from the environmental organism or type.

[0032] Embodiment 28 is (a) The capture oligomer is arranged in the 5' to 3' direction as follows: a capture sequence comprising a first and a second portion; Internal extension blockers, a spacer sequence comprising a first and a second portion, and Target Hybridizing Sequence Including, (b) A complementary oligomer having, in the 3' to 5' direction: the complement of a second portion of the capture sequence, and The complement of at least a first portion of the spacer sequence wherein the complement of the second portion of the capture sequence and the complement of at least the first portion of the spacer sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the spacer sequence. The combination comprises a capture oligomer and a complementary oligomer.

[0033] Embodiment 29 is a method for preparing a capture oligomer having the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3' (wherein A1 is a first additional optional sequence; C1 is a first portion of the capture sequence, C2 is a second portion of the capture sequence, B is an internal extension blocker, A2 is an optional second additional sequence, S1 is a first portion of the spacer sequence, S2 is a second portion of the spacer sequence, A3 is an optional third additional sequence, RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence, THS is the target hybridizing sequence; X is an optional blocking moiety. The combination according to the immediately preceding embodiment,

[0034] Embodiment 30 is an embodiment in which the complementary oligomer has the formula: 5'-S1'-A2'-L-C2'-X-3' where S1' is the complement of at least a first portion of the spacer sequence, A2' is the optional complement of a second additional sequence optionally present in the capture oligomer; L is an optional linker, C2' is the complement of the second portion of the capture sequence; X is an optional blocking moiety. 30. The combination according to any one of embodiments 28 or 29, wherein

[0035] Embodiment 31 is (a) The capture oligomer is arranged in the 5' to 3' direction as follows: a capture sequence comprising a first and a second portion; a target hybridizing sequence comprising a second and a first portion; Including, (b) A complementary oligomer having, in the 3' to 5' direction: the complement of a second portion of the capture sequence; and the complement of the second portion of the target hybridizing sequence; wherein the complement of the second portion of the capture sequence and the complement of the second portion of the target hybridizing sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the target hybridizing sequence; The combination comprises a capture oligomer and a complementary oligomer.

[0036] Embodiment 32 is a method for preparing a capture oligomer having the formula: 5'-A1-C1-C2-A2-S-A3-THS2-THS1-X-3' where A1 is a first additional optional sequence, C1 is a first portion of the capture sequence, C2 is a second portion of the capture sequence, A2 is an optional second additional sequence, S is an optional spacer sequence, A3 is an optional third additional sequence, THS2 is a second portion of the target hybridizing sequence, THS1 is the first portion of the target hybridizing sequence, X is an optional blocking moiety. The combination according to the immediately preceding embodiment,

[0037] Embodiment 33 is an embodiment in which the complementary oligomer has the formula: 5'-THS2'-A3'-S'-A2'-C2'-X-3' where THS2' is the complement of a second portion of the target hybridizing sequence, A3' is the optional complement of a third additional sequence optionally present in the capture oligomer; S' is the optional complement of a spacer optionally present in the capture oligomer; A2' is the optional complement of a second additional sequence optionally present in the capture oligomer; C2' is the complement of the second portion of the capture sequence; X is an optional blocking moiety. 33. The combination according to any one of embodiments 31 or 32, wherein

[0038] Embodiment 34 is a combination according to any one of embodiments 28 to 33, wherein the capture oligomer and / or the complementary oligomer comprises a blocking moiety at its 3' end.

[0039] Embodiment 35 is a combination according to any one of embodiments 28 to 34, wherein when the spacer sequence of the capture oligomer is occupied by a distinct complement, the complement of the second portion of the capture sequence is insufficient to stably anneal to the capture sequence of the capture oligomer at a temperature of 65° C. or greater.

[0040] Embodiment 36 is a capture oligomer, reaction mixture, or combination of any one of the preceding embodiments, wherein the capture sequence comprises polyA or polyT, and the complement of the capture sequence or the complement of the second portion of the capture sequence comprises polyT or polyA.

[0041] Embodiment 37 is a capture oligomer, reaction mixture, or combination of any one of the preceding embodiments, wherein the capture oligomer comprises an adapter sequence as part or all of the spacer sequence, or as part or all of the third additional sequence 3' of the spacer sequence or the fourth additional sequence 5' of the target hybridizing sequence.

[0042] Embodiment 38 is a capture oligomer, reaction mixture, or combination according to any one of the preceding embodiments, wherein the capture oligomer 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, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions), e.g., in the target hybridizing sequence.

[0043] Embodiment 39 is a capture oligomer, reaction mixture, or combination of any one of the preceding embodiments, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridizing sequence.

[0044] Embodiment 40 is a capture oligomer, reaction mixture, or combination according to the immediately preceding embodiment, wherein the reversible extension blocker is located 3' to the second portion of the spacer sequence.

[0045] Embodiment 41 is the capture oligomer, reaction mixture, or combination of any one of embodiments 39 or 40, wherein the reversible extension blocker comprises Iso-dC or Iso-dG, Xanthine or 5-(2,4 diaminopyrimidine), 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4-Methylbenzimidizole or 2,4-difluorotoluene, 7-azaindole or isocarbostyril, dMMO2 or d5SICS, dF or dQ, chemically modified nucleotide or nucleotides, wherein the modification is attached via a reversible linkage, optionally the linkage is reversible by any one or more of a chemical, an enzyme, a temperature change, a change in reagent composition, in any combination of two or more, a reversible nucleic acid structural feature, a protein, an enzyme, a lipid, a carbohydrate, or a reversible binding of a chemical moiety to the nucleic acid template.

[0046] Embodiment 42 is a capture oligomer, reaction mixture, or combination of any one of embodiments 28-41, comprising a second additional sequence between the internal extension blocker and the first portion of the spacer sequence, wherein the second additional sequence comprises a mixed nucleotide segment.

[0047] Embodiment 43 is a capture oligomer, reaction mixture, or combination of the immediately preceding embodiment, wherein the mixed nucleotide segment comprises five nucleotides, each nucleotide different from its nearest neighbor.

[0048] Embodiment 44 is a capture oligomer, reaction mixture, or combination of any one of embodiments 42 or 43, wherein the mixed nucleotide segments do not include repeated dinucleotides, repeated trinucleotides, and / or adjacent repeats.

[0049] Embodiment 45 relates to a 5' to 3' arrangement of: the complement of a sequence not present in the capture oligomer; mixed nucleotide segments; and Complement of the capture sequence 45. The capture oligomer, reaction mixture, or combination of any one of embodiments 42-44, further comprising a splint oligomer comprising:

[0050] Embodiment 46 is an embodiment in which the capture oligomer comprises a third or fourth additional sequence comprising an adapter sequence between the target hybridizing sequence and the second portion of the spacer sequence, or the target hybridizing sequence of the capture oligomer is an adapter sequence; the splint oligomer further comprises a complement of an adapter sequence 3' of its complement of the capture sequence; A capture oligomer, reaction mixture, or combination as described in the immediately preceding embodiment.

[0051] Embodiment 47 is a capture oligomer, reaction mixture, or combination according to any one of embodiments 28-46, further comprising a secondary capture reagent comprising a complement of the capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid support (e.g., a bead or surface).

[0052] Embodiment 48 is a capture oligomer, reaction mixture, or combination described in the immediately preceding embodiment, wherein the secondary capture reagent comprises a binding partner, and the combination further comprises a solid support (e.g., one or more beads) comprising a second binding partner configured to bind to the binding partner of the secondary capture reagent, and optionally, the binding partner of the secondary capture reagent is biotin and the second binding partner is a biotin binder (e.g., streptavidin).

[0053] Embodiment 49 is a capture oligomer, reaction mixture, or combination described in any one of embodiments 28 to 48, further comprising a displacer oligomer comprising a displacer target hybridizing sequence configured to bind to a target polynucleotide, wherein the 3' end of the displacer target hybridizing sequence is oriented toward the site bound by the target hybridizing sequence of the capture oligomer.

[0054] Embodiment 50 is a capture oligomer, reaction mixture, or combination according to the immediately preceding embodiment, wherein the capture oligomer comprises an adapter sequence located 5' of the target hybridizing sequence and 3' of the complement of the capture sequence.

[0055] Embodiment 51 is a capture oligomer, reaction mixture, or combination described in any one of embodiments 49 or 50, further comprising an amplification oligomer that comprises a reverse target hybridizing sequence configured to bind a target polynucleotide in an opposite orientation to the capture oligomer, and optionally further comprises a second adapter sequence located 5' to the reverse target hybridizing sequence.

[0056] Embodiment 52 is a reaction mixture comprising a capture oligomer or combination according to any one of the preceding embodiments and further comprising a target polynucleotide.

[0057] Embodiment 53 is the reaction mixture of the immediately preceding embodiment, wherein the target is an amplicon and further comprises an amplification primer comprising an amplification primer that binds to the same strand as the capture oligomer, and optionally wherein the THS of the capture oligomer has a greater affinity for the amplicon than the primer that binds to the same strand as the capture oligomer (e.g., the capture oligomer has a longer THS than the primer that binds to the same strand as the capture oligomer, or the capture oligomer comprises an affinity-enhancing modification).

[0058] Embodiment 54 is a combination comprising a capture oligomer or combination according to any one of embodiments 1 to 51 and an amplification oligomer, the capture oligomer comprises a first adapter sequence between the target hybridizing sequence and the internal extension blocker; the amplification oligomer comprises (i) a reverse target hybridizing sequence that binds to the target polynucleotide in a reverse orientation relative to the capture oligomer, and (ii) a second adapter sequence located 5' to the reverse target hybridizing sequence; It is a combination.

[0059] Embodiment 55 is a combination according to the immediately preceding embodiment, wherein the capture oligomer comprises a blocking moiety at its 3' end.

[0060] Embodiment 56 is the combination of embodiment 54, wherein the capture oligomer is extendable.

[0061] Embodiment 57 is a combination according to any one of embodiments 54 to 56, wherein the amplification oligomer comprises a reversible extension blocker positioned between the second adapter sequence and the reverse target hybridizing sequence, and optionally the capture oligomer further comprises a reversible extension blocker positioned between the target hybridizing sequence and the first adapter sequence.

[0062] Embodiment 58 is a method comprising: contacting a target polynucleotide with a capture oligomer of any one of embodiments 1-22 or 36-51, wherein a target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site that includes the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence that is annealed to the capture oligomer, such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0063] Embodiment 59 is a method comprising: contacting the composition with the combination of any one of embodiments 28-30 or 34-51, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site that includes the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming the complement of the spacer sequence that is annealed to the capture oligomer such that the complementary oligomer is displaced to an extent sufficient that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0064] Embodiment 60 is a method comprising: contacting the composition with the combination of any one of embodiments 28-30 or 34-51, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site that includes the 3' end of the target polynucleotide; optionally extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming the complement of the spacer sequence annealed to the capture oligomer; contacting the free capture oligomer with a complementary oligomer, where the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, and where the complementary oligomer does not anneal to a complex comprising the capture oligomer annealed to the complement of the spacer sequence; contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent, wherein the complementary oligomer is introduced into the composition before, during, or after extension of the 3' end of the target polynucleotide; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0065] Embodiment 61 is a method according to the immediately preceding embodiment, wherein the target-hybridizing sequence of the capture oligomer undergoes extension, thereby forming an extended capture oligomer.

[0066] Embodiment 62 is a method comprising: contacting the composition with the combination of any one of embodiments 28-30 or 34-51, further comprising an amplification oligomer comprising a reverse target hybridizing sequence configured to bind to the target polynucleotide in an opposite orientation relative to the capture oligomer, and optionally further comprising a second adapter sequence located 5' to the reverse target hybridizing sequence, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide and undergoes extension, thereby forming an extended capture oligomer; annealing the amplification oligomer to the extended capture oligomer; extending the 3' end of the amplification oligomer, optionally with a DNA polymerase having strand displacement activity, thereby forming a complement of at least the target hybridizing sequence of the capture oligomer that is annealed to the capture oligomer; contacting the free capture oligomer with a complementary oligomer, where the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, and where the complementary oligomer does not anneal to a complex comprising the capture oligomer annealed to the complement of the target hybridizing sequence of the capture oligomer; contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent, wherein the complementary oligomer is introduced into the composition before, during, or after extension of the 3' end of the target polynucleotide; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0067] Embodiment 63 is a method comprising: contacting the target polynucleotide with a capture oligomer or combination of any one of embodiments 1-22, 28-30, or 34-51, wherein the capture oligomer comprises a third or fourth additional sequence 5' of the target hybridizing sequence and a second portion 3' of the complement of the capture sequence or spacer sequence; contacting the target polynucleotide with a second oligomer comprising a second target hybridizing sequence 5' that is the complement of at least a portion of a third or fourth additional sequence, wherein the capture oligomer and the second oligomer form a triple-stranded junction with the target polynucleotide; extending the 3' end of the second oligomer with a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence or a complement of the spacer sequence that is annealed to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0068] Embodiment 64 is the method of the immediately preceding embodiment, wherein the capture oligomer comprises a blocking moiety at its 3' end; and / or the second oligomer comprises a portion at its 5' end that blocks displacement by a polymerase having strand displacement activity.

[0069] Embodiment 65 is a method comprising: contacting the composition with the combination of any one of embodiments 31-51, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide; contacting the capture oligomer with a complementary oligomer before or after the capture oligomer anneals to the target polynucleotide, where the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, where the complementary oligomer does not anneal to a complex that includes the capture oligomer annealed to the complement of the capture oligomer's target hybridizing sequence, and where if the contacting of the capture oligomer with the complementary oligomer occurs before the capture oligomer anneals to the target polynucleotide, annealing of the target hybridizing sequence to the target polynucleotide results in dissociation of the complementary oligomer from the capture oligomer; contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a secondary capture reagent comprising a complement of the capture sequence and (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0070] Embodiment 66 is a method according to any one of embodiments 58 to 65, wherein the secondary capture reagent comprises a binding partner (e.g., biotin) and the isolating comprises contacting the complex with a solid support (e.g., a bead) comprising a second binding partner (e.g., streptavidin) configured to bind to the binding partner of the secondary capture reagent.

[0071] Embodiment 67 is the method of any one of embodiments 58 to 66, wherein the capture oligomer is provided in excess relative to the secondary capture reagent.

[0072] Embodiment 68 is the method according to any one of embodiments 58 to 67, wherein the target polynucleotide is obtained from a clinical specimen.

[0073] Embodiment 69 is the method of any one of embodiments 58 to 68, wherein the target polynucleotide is derived from a pathogen (bacteria, viruses, etc.).

[0074] Embodiment 70 is the method according to any one of embodiments 58 to 69, wherein the target polynucleotide is an amplification product.

[0075] Embodiment 71 is the method of any one of embodiments 58 to 70, wherein the target polynucleotide is a member of a sequencing library.

[0076] Embodiment 72 is the method of any one of embodiments 58 to 71, wherein the capture oligomer comprises a third or fourth additional sequence between the target hybridizing sequence and the internal extension blocker.

[0077] Embodiment 73 is the method of any one of embodiments 63-72, wherein the extension product is formed by extending the capture oligomer along the target polynucleotide, and the method further comprises contacting the extension product with a blocker oligomer comprising a non-extendable third or fourth additional sequence, optionally configured to bind to the complement 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 complement of the third or fourth additional sequence that has a higher melting temperature than a complex of the third or fourth additional sequence of the capture oligomer and the complement of the third or fourth additional sequence.

[0078] Embodiment 74 is a method comprising: contacting the target polynucleotide with an amplification oligomer, the amplification oligomer comprising (i) a reverse target hybridizing sequence that binds to the target polynucleotide in a reverse orientation relative to the capture oligomer, and (ii) an additional sequence located 5' to the second target hybridizing sequence; and extending the amplification oligomer along the target polynucleotide to form a reverse extension product. Further comprising: 74. The method of any one of embodiments 58-73, wherein a portion of the capture oligomer anneals to the reverse extension product.

[0079] Embodiment 75 is the method of the immediately preceding embodiment, wherein the capture oligomer comprises a blocking moiety at its 3' end.

[0080] Embodiment 76 is the method of the immediately preceding embodiment, wherein the method further comprises isolating the complex comprising the reverse extension product annealed to the capture oligomer, wherein the extension product is substantially single-stranded 5' to the target hybridizing sequence of the capture oligomer.

[0081] Embodiment 77 is the method of any one of embodiments 74 to 76, wherein the capture oligomer is extendable, and the method further comprises extending a portion of the capture oligomer along the extension product, thereby forming a second extension product comprising a third or fourth additional sequence and the complement of the additional sequence of the amplification oligomer.

[0082] Embodiment 78 is a method according to any one of embodiments 58 to 77, wherein the target polynucleotide comprises a sequence derived 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 hybridizing sequence of the capture oligomer is configured to anneal to the additional sequence of the target polynucleotide.

[0083] Embodiment 79 is the method of embodiment 78, wherein the composition comprises a plurality of target polynucleotides comprising (i) additional sequences and (ii) sequences different from the DNA or RNA of the target organism and / or different samples, and the method comprises capturing the plurality of target polynucleotides.

[0084] Embodiment 80 is a method according to the present invention, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridizing sequence, and the method comprises: copying or amplifying the target polynucleotide prior to unblocking the reversible extension blocker; unblocking the reversible elongation blocker; and performing further rounds of copying or amplification of the target polynucleotide, Optionally, after unblocking the reversible extension blocker prior to capturing the target polynucleotide, further rounds of copying or amplification are performed, where only a single cycle of amplification is performed. The method according to any one of embodiments 58 to 79, comprising:

[0085] Embodiment 81 is an embodiment in which the capture oligomer comprises a mixed nucleotide segment between the internal extension blocker and the first portion of the spacer sequence or the complement of the capture sequence; the method extending a 3' end of the target polynucleotide along the capture oligomer to the internal extension blocker, thereby forming an extension product that includes the complement of the mixed nucleotide segment at its 3' end; The extension products are synthesized in the 5' to 3' direction as follows: the complement of the 5'-terminal segment of the extension product; mixed nucleotide segments; the complement of the capture sequence; and Optionally, a segment complementary to a segment in the extension product immediately 5' of the capture sequence. contacting said splint oligonucleotide with a splint oligonucleotide comprising: Ligating the 5' end of the extension product to the 3' end of the extension product The method according to any one of embodiments 58 to 64 or 66 to 80, comprising:

[0086] Embodiment 82 is the method of the immediately preceding embodiment, wherein the 5' terminal segment of the extension product is an adapter sequence.

[0087] Embodiment 83 is the method of embodiment 81 or 82, wherein the segment in the extension product immediately 5' to the capture sequence is the complement of the adapter sequence.

[0088] Embodiment 84 is the method of any one of embodiments 81 to 83, wherein the mixed nucleotide segment comprises five nucleotides, each nucleotide different from its nearest neighbor.

[0089] Embodiment 85 is the method of any one of embodiments 81 to 84, wherein the mixed nucleotide segment does not contain repeated dinucleotides, repeated trinucleotides, and / or adjacent repeats.

[0090] Embodiment 86 is the method of any one of embodiments 58 to 85, further comprising sequencing the target polynucleotide.

[0091] Embodiment 87 is the method of any one of embodiments 58 to 86, further comprising performing clonal amplification of the captured target polynucleotides.

[0092] Embodiment 88 is the method of the immediately preceding embodiment, further comprising sequencing the clonally amplified target polynucleotide.

[0093] Embodiment 89 is a method according to any one of embodiments 86 or 88, wherein the sequencing is Sanger sequencing or next-generation sequencing, optionally wherein next-generation sequencing comprises sequencing by synthesis, sequencing by ligation, sequencing by hybridization or single molecule sequencing.

[0094] Embodiment 90 is a method comprising: contacting a target polynucleotide with a capture oligomer or combination of any one of embodiments 1-22, 28-30, or 34-51, wherein a target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site upstream of the 3' end of the target polynucleotide; extending the 3' end of the capture oligomer along the target polynucleotide, thereby forming a first extended strand; contacting the target polynucleotide with a displacer oligomer that includes a displacer target hybridizing sequence that anneals to the target polynucleotide downstream of the target hybridizing sequence of the capture oligomer; extending the displacer oligomer along the target polynucleotide, thereby displacing the first extended strand of the target polynucleotide. Including, Optionally, the capture oligomer and the displacer are added to the composition simultaneously or sequentially. A method for capturing a target polynucleotide from a composition.

[0095] Embodiment 91 is the method of the immediately preceding embodiment, further comprising contacting the first extension strand with a reverse amplification oligomer comprising a reverse target hybridizing sequence configured to bind to the first extension strand and extending the reverse amplification oligomer, thereby forming a second extension strand.

[0096] Embodiment 92 is the method of the immediately preceding embodiment, wherein the reverse amplification oligomer comprises an additional sequence 5' of its target hybridizing sequence, and optionally, the 3' end of the first extended strand is further extended, thereby forming the complement of the additional sequence of the reverse amplification oligomer.

[0097] Embodiment 93 is a method according to any one of embodiments 90 to 92, wherein the capture oligomer further comprises an additional sequence located 5' of the target hybridizing sequence, and optionally an extension of the second extension strand, if present, forms the complement of the additional sequence of the capture oligomer.

[0098] Embodiment 94 is the method of the immediately preceding embodiment, wherein the second strand of the target polynucleotide comprises the complement of an additional sequence of the capture oligomer.

[0099] Embodiment 95 is the method of embodiment 90 or 91, wherein the target polynucleotide comprises a sequence derived from the DNA or RNA of the target organism and an additional sequence not present in the DNA and RNA of the target organism, the target hybridizing sequence of the capture oligomer is configured to anneal to a first portion of the additional sequence of the target polynucleotide, and the displacer oligonucleotide is configured to anneal to a second portion of the additional sequence of the target polynucleotide, and optionally the additional sequence of the target polynucleotide further comprises one or more nucleotides between the first portion and the second portion.

[0100] Embodiment 96 is an embodiment of the present invention, wherein the first extension comprises a second additional sequence located proximal to the sequence derived from the DNA or RNA of the target organism and distal to the target hybridizing sequence of the capture oligomer; 96. The method of embodiment 90 or 95, wherein the method further comprises contacting the first extended strand with a reverse amplification oligomer comprising a reverse target hybridizing sequence configured to bind to a second additional sequence and extending the reverse amplification oligomer, thereby forming a second extended strand, optionally the reverse amplification oligomer comprising additional sequence 5' of its target hybridizing sequence, and the method further comprises extending the first extended strand further along the reverse amplification oligomer.

[0101] Embodiment 97 is a method for producing a capture oligomer comprising the steps of: a first self-complementary sequence, a target hybridizing sequence, and a second self-complementary sequence, Including, the first and second self-complementary sequences are configured to anneal to one another when the target hybridizing sequence is single stranded, but not to anneal when the target hybridizing sequence is annealed to its target; the secondary capture reagent comprises a complement of the first or second self-complementary sequence and a binding partner; the capture oligomer is present in the combination in an amount greater than the secondary capture reagent; A combination of oligomers comprising a capture oligomer and a secondary capture reagent.

[0102] Embodiment 98 is a method for preparing a capture oligomer having the following formula: 5'-SC1-THS2-THS1-L-THS2'-SC2-X-3' or 5'-SC2-THS2'-L-THS1-THS2-SC1-X-3' where SC1 is the first self-complementary sequence, THS2 and THS1 are the second and first portions of the target hybridizing sequence, respectively; L is an optional linker, THS2' is the optional complement of a second portion of the target hybridizing sequence; SC2 is a second self-complementary sequence, X is an optional blocking moiety. The combination according to the immediately preceding embodiment,

[0103] Embodiment 99 is a combination of any one of embodiments 97 or 98, wherein the capture oligomer comprises a linker located 3' to the first portion of the target hybridizing sequence and 5' to the second self-complementary sequence.

[0104] Embodiment 100 is a combination described in any one of embodiments 97 to 99, wherein the capture oligomer comprises a complement of a second portion of the target hybridizing sequence located 3' of the first portion of the target hybridizing sequence and 5' of a second self-complementary sequence.

[0105] Embodiment 101 is a combination of any one of embodiments 97-100, wherein the capture oligomer comprises a linker located 3' of a first portion of the target hybridizing sequence and a complement of a second portion of the target hybridizing sequence located 3' of the linker and 5' of a second self-complementary sequence.

[0106] Embodiment 102 is a combination according to any one of embodiments 97 to 101, wherein the capture oligomer comprises a linker located 5' of the first portion of the target hybridizing sequence and 3' of the second self-complementary sequence.

[0107] Embodiment 103 is a combination according to any one of embodiments 97 to 102, wherein the capture oligomer comprises a complement of a second portion of the target hybridizing sequence located 5' of the first portion of the target hybridizing sequence and 3' of a second self-complementary sequence.

[0108] Embodiment 104 is a combination described in any one of embodiments 97 to 103, wherein the capture oligomer comprises a linker located 5' of a first portion of the target hybridizing sequence and a complement of a second portion of the target hybridizing sequence located 5' of the linker and 3' of a second self-complementary sequence.

[0109] Embodiment 105 is a combination according to any one of embodiments 97 to 104, wherein the capture oligomer and / or the secondary capture reagent are non-extendable.

[0110] Embodiment 106 is a combination according to any one of embodiments 97 to 105, wherein the first or the second self-complementary sequence comprises polyA or polyT, and the complement of the self-complementary sequence comprises polyT or polyA.

[0111] Embodiment 107 is a combination according to any one of embodiments 97-106, wherein the capture oligomer comprises, e.g., in the target hybridizing sequence, one or more affinity enhancing modifications (e.g., any one or more of 5-Me-C, 2-aminopurine, 2'-fluoro, C-5-propyne, LNA, PNA, ZNA, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions).

[0112] Embodiment 108 is a kit comprising a combination or capture oligomer according to any one of embodiments 1-22, 28-51, 54-57, or 97-107.

[0113] Embodiment 109 is a composition comprising a combination or capture oligomer according to any one of embodiments 1-22, 28-51, 54-57, or 97-107.

[0114] Embodiment 110 is a combination of any one of embodiments 18-22, 28-51, 54-57, or 97-107, comprising (i) a secondary capture reagent comprising a binding partner and (ii) a solid support (e.g., one or more beads) comprising a second binding partner configured to bind to the binding partner of the secondary capture reagent, optionally wherein the binding partner of the secondary capture reagent is biotin and the second binding partner is a biotin binder (e.g., streptavidin).

[0115] Embodiment 111 is a reaction mixture comprising the combination of any one of embodiments 18-22, 28-51, 54-57, 97-107, or 110, further comprising a target polynucleotide.

[0116] Embodiment 112 is a reaction mixture of the immediately preceding embodiment, wherein the target is in or obtained from a cell, specimen, virus, or extract from a biological sample.

[0117] Embodiment 113 is a method comprising: contacting a target polynucleotide with the combination of any one of embodiments 97-107 or 110, wherein a capture oligomer and a secondary capture reagent are added simultaneously or sequentially, such that the target-hybridizing sequence of the capture oligomer anneals to the target polynucleotide and the secondary capture reagent anneals to the self-complementary sequence of the capture oligomer, thereby forming a complex; contacting the complex with a second binding partner configured to bind to the binding partner of the secondary capture reagent, where the second binding partner is associated with a solid support and the second binding partner binds to the binding partner of the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0118] Embodiment 114 is the method of the immediately preceding embodiment, wherein the capture oligomers are in excess of the target polynucleotide.

[0119] Embodiment 115 is the method of any one of embodiments 113 or 114, wherein the target polynucleotide is in excess of the secondary capture reagent.

[0120] Embodiment 116 is the method according to any one of embodiments 113 to 115, wherein only a fraction of the target polynucleotides is captured.

[0121] Embodiment 117 is the method according to any one of embodiments 113 to 116, wherein the target polynucleotide is obtained from a clinical specimen.

[0122] Embodiment 118 is the method of any one of embodiments 113 to 117, wherein the target polynucleotide is derived from a pathogen (bacteria, viruses, etc.).

[0123] Embodiment 119 is the method according to any one of embodiments 113 to 118, wherein the target polynucleotide is an amplification product.

[0124] Embodiment 120 is the method of any one of embodiments 113 to 119, further comprising amplifying the target polynucleotide and / or attaching one or more additional sequences to the target polynucleotide.

[0125] Embodiment 121 is the method of any one of embodiments 113 to 120, further comprising preparing a sequencing library comprising the target polynucleotide.

[0126] Embodiment 122 is a method according to any one of embodiments 113 to 121, further comprising one or both of a clonal amplification member of a sequencing library and a sequencing member of a sequencing library, and optionally wherein the sequencing is Sanger sequencing or next-generation sequencing, and optionally wherein the next-generation sequencing comprises sequencing by synthesis, sequencing by ligation, sequencing by hybridization or single molecule sequencing.

[0127] Embodiment 123 is a method comprising: The target polynucleotide is sequenced in the 5' to 3' direction as follows: Capture sequence, any internal extension blocker, an optional spacer sequence, and A target hybridizing sequence configured to anneal to a target polynucleotide contacting said oligomer with a capture oligomer comprising: contacting the capture oligomer with a first capture reagent that comprises the complement of the capture sequence (before, during, or after contacting the target polynucleotide with the capture oligomer); providing a second capture reagent comprising a complement of a sequence in the capture oligomer other than the capture sequence, where if some or all of the capture oligomer is not annealed to the target polynucleotide, the second capture reagent contacts the capture oligomer that is not annealed to the target polynucleotide; isolating the first and second complexes from the composition, where the first complex comprises the target polynucleotide and the second complex comprises a capture oligomer that is not annealed to the target polynucleotide; and Selectively eluting the target polynucleotide or a subcomplex containing the target polynucleotide from the first complex. Including, Optionally, a method for capturing a target polynucleotide from a composition, wherein (a) a first capture reagent comprises (i) a binding partner and (e.g., biotin) or (ii) a solid support (e.g., a bead or a surface), and / or (b) a second capture reagent comprises (i) a binding partner and (e.g., biotin) or (ii) a solid support (e.g., a bead or a surface).

[0128] Embodiment 124 is the method of embodiment 123, wherein the target polynucleotide is contacted with an excess of capture oligomers.

[0129] Embodiment 125 is the method of embodiment 123 or 124, wherein the first capture reagent is provided in a limiting amount relative to the capture oligomer.

[0130] Embodiment 126 is the method of any one of embodiments 123 to 125, wherein the capture oligomer is provided in a limiting amount relative to the target polynucleotide.

[0131] Embodiment 127 is the method of any one of embodiments 123 to 126, wherein the fraction of capture oligomers contacted with the second capture reagent comprises capture oligomers that are not annealed to the target polynucleotide.

[0132] Embodiment 128 is the method of any one of embodiments 123 to 127, wherein the first capture reagent comprises a first solid support comprising the complement of the capture sequence.

[0133] Embodiment 129 is the method of any one of embodiments 123 to 128, wherein the second capture reagent comprises a second solid support comprising the complement of a sequence in the capture oligomer other than the capture sequence.

[0134] Embodiment 130 is a method according to any one of embodiments 123 to 129, wherein the capture oligomer comprises a spacer sequence between the target hybridizing sequence and the internal extension blocker, and optionally the second capture reagent comprises the complement of the spacer sequence.

[0135] Embodiment 131 is the method according to any one of embodiments 123 to 130, wherein the target hybridizing sequence anneals to the target polynucleotide at the 3' end of the target polynucleotide.

[0136] Embodiment 132 is the method of the immediately preceding embodiment, wherein the method comprises extending the 3' end of the target polynucleotide.

[0137] Embodiment 133 is a method according to any one of embodiments 123 to 132, wherein the second capture reagent has a greater affinity for the capture oligomer than the affinity of the first capture reagent for the capture oligomer and / or the second capture reagent is configured to form a complex with the capture oligomer that has a higher melting temperature than the complex between the first capture reagent and the capture oligomer.

[0138] Embodiment 134 is The target polynucleotide is sequenced in the 5' to 3' direction as follows: First acquisition sequence, Internal extension blockers, A second acquisition sequence, and a target hybridizing sequence configured to anneal to the 3' end of a target polynucleotide and contacting the capture oligomer with a capture oligomer comprising thereby annealing the capture oligomer to the target polynucleotide; extending the 3' end of the target polynucleotide through a second capture sequence; contacting the first complex with a first solid support that contains the complement of the first capture sequence, thereby forming a first complex; contacting the unbound capture oligomer with a second solid support comprising the complement of a second capture sequence, thereby forming a second complex, wherein the complement of the first capture sequence has a lower melting temperature than a second complex formed by annealing of the second capture sequence with the complement of the second capture sequence and / or the complement of the second capture sequence has an affinity for the second capture sequence that is greater than the affinity of the complement of the first capture sequence for the first capture sequence; isolating the first and second complexes from material not bound to the first or second solid support; and Selectively eluting the target polynucleotide from the first complex. The method according to any one of embodiments 128 to 133, comprising:

[0139] Embodiment 135 is the method of embodiment 123, wherein the first capture reagent 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 reagent, annealing the second capture sequence to a solid support that comprises the complement of the second capture sequence.

[0140] Embodiment 136 is the method of embodiment 123 or 135, wherein the second capture reagent further comprises a third capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method comprises, after contacting the unbound capture oligomer with the second capture reagent, annealing the third capture sequence to a solid support that comprises the complement of the third capture sequence.

[0141] Embodiment 137 is the method of embodiment 123, wherein the first capture reagent further comprises a second capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method is configured to, after contacting the annealed capture oligomer with the first capture reagent, anneal the second capture sequence to a solid support comprising a complement of the second capture sequence; such that the affinity of the complement of the third capture sequence for the third capture sequence is greater than the affinity of the complement of the second capture sequence for the second capture sequence and / or the complement of the third capture sequence forms a complex with the third capture sequence that has a higher melting temperature than the complex of the complement of the second capture sequence and the second capture sequence.

[0142] Embodiment 138 is an embodiment in which the capture oligomer has, in a 5' to 3' direction: A first capture sequence, any internal extension blocker, an optional second capture sequence, and Target Hybridizing Sequence Includes; a first solid support comprising the complement of the first capture sequence; a second solid support comprising the complement of the second capture sequence; a first complex formed by annealing a first capture sequence with its complement has a lower melting temperature than a second complex formed by annealing a second capture sequence with its complement, and / or the complement of the second capture sequence has an affinity for the second capture sequence that is greater than the affinity of the complement of the first capture sequence for the first capture sequence; The combination includes a capture oligomer, a first solid support, and a second solid support.

[0143] Embodiment 139 is an embodiment in which the capture oligomer has, in a 5' to 3' direction: A first capture sequence, any internal extension blocker, and Target Hybridizing Sequence Includes; the first capture reagent comprises a second capture sequence (wherein the second capture sequence is not complementary to the capture oligomer) and the complement of the first capture sequence; the second capture reagent comprises a third capture sequence (wherein the third capture sequence is not complementary to the capture oligomer) and a complement of a sequence of the capture oligomer other than the first capture sequence; the first solid support comprises the complement of the second capture sequence; the second solid support comprises the complement of the third capture sequence; a first complex formed by annealing the second capture sequence with its complement has a lower melting temperature than a second complex formed by annealing the third capture sequence with its complement, and / or the complement of the third capture sequence has an affinity for the third capture sequence that is greater than the affinity of the complement of the second capture sequence for the second capture sequence; The combination includes a capture oligomer, a first capture reagent, a second capture reagent, a first solid support, and a second solid support.

[0144] Embodiment 140 is an embodiment in which the capture oligomer comprises: a target hybridizing sequence comprising one or more affinity enhancing nucleotides; and Capture Sequence Includes; The secondary capture reagent is a combination that includes a capture oligomer and a secondary capture reagent, where the secondary capture reagent includes the complement and binding partner of the capture sequence.

[0145] Embodiment 141 is a combination according to any one of embodiments 138 to 140, wherein the capture sequence is located 5' to the target hybridizing sequence.

[0146] Embodiment 142 is a combination according to any one of embodiments 138 to 141, wherein the target hybridizing sequence is configured to anneal to an additional sequence.

[0147] Embodiment 143 is a combination according to any one of embodiments 138 to 142, wherein the secondary capture reagent is present in the combination in an amount less than the capture oligomer.

[0148] Embodiment 144 is a combination according to any one of embodiments 138 to 144, wherein the capture oligomer comprises, e.g., in the target hybridizing sequence, one or more affinity enhancing modifications (e.g., any one or more of 5-Me-C, 2-aminopurine, 2'-fluoro, C-5-propyne, LNA, PNA, ZNA, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions).

[0149] Embodiment 145 is a combination according to any one of embodiments 138 to 145, wherein the capture sequence comprises a polyA or polyT sequence.

[0150] Embodiment 146 is a method comprising: contacting a target polynucleotide with the combination of any one of embodiments 138-145, wherein a capture oligomer and a secondary capture reagent are added simultaneously or sequentially, such that the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide and the secondary capture reagent anneals to the capture sequence of the capture oligomer, thereby forming a complex; contacting the complex with a second binding partner configured to bind to the binding partner of the secondary capture reagent, where the second binding partner is associated with a solid support and the second binding partner binds to the binding partner of the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. A method for capturing a target polynucleotide from a composition, comprising:

[0151] Embodiment 147 is the method of the immediately preceding embodiment, wherein the secondary capture reagent is provided in an amount less than the capture oligomer and / or the target polynucleotide.

[0152] Embodiment 148 is the method of embodiment 146 or 147, wherein the capture oligomer is provided in a smaller amount than the target polynucleotide, and optionally the secondary capture reagent is provided in a smaller amount than the capture oligomer.

[0153] Embodiment 149 is the method according to any one of embodiments 146 to 148, wherein the target polynucleotide comprises an additional sequence and the target hybridizing sequence anneals to the additional sequence.

[0154] Embodiment 150 is a method according to any one of embodiments 146 to 149, wherein the target-hybridizing sequence of the capture oligomer 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, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions).

[0155] Embodiment 151 is a method according to any one of embodiments 146 to 150, wherein the target polynucleotide comprises a sequence derived from the DNA or RNA of the target organism, and the additional sequence is not present in the DNA and RNA of the target organism.

[0156] Embodiment 152 is a method according to any one of embodiments 58 to 96, 113 to 137, or 146 to 151, wherein an amount of target polynucleotide is captured that is equal to or less than a predetermined amount, and optionally the predetermined amount corresponds to a molar amount of capture oligomer provided or a molar amount of secondary capture reagent provided.

[0157] Embodiment 153 is an embodiment in which the capture oligomer comprises 10 7 ~10 13 Molecules / Reactions, 10 9 ~10 12 molecules / reaction, or 10 10 ~1012 The method of any one of embodiments 58-96, 113-137, or 146-152, wherein the hydroxyl group is present in an amount ranging from 0.1 to 1.0 mole / reaction.

[0158] Embodiment 154 relates to a method for preparing a secondary capture reagent, when present, comprising: 3 ~10 14 molecules / reaction, or 10 3 ~10 9 molecules / reaction, or 10 5 ~10 13 molecules / reaction, or 10 5 ~10 8 molecules / reaction, or 10 6 ~10 13 molecules / reaction, or 10 6 ~10 8 The method of any one of embodiments 58-96, 113-137, or 146-153, wherein the range of molecules / reactions is present.

[0159] Embodiment 155 is a method for preparing a blocker oligomer, when present, comprising administering to a patient a 8 ~10 14 molecules / reaction, or 10 10 ~10 13 molecules / reaction, or 10 11 ~10 13 A second oligomer, if present, is present in the range of 10 7 ~10 14 molecules / reaction or 10 8 ~10 13 The method of any one of embodiments 58-96, 113-137, or 146-154, wherein the range of molecules / reactions is present.

[0160] Embodiment 156 relates to a method for preparing a method for preparing a complementary oligomer, when present, comprising the steps of: 7 ~10 14 molecules / reaction or approximately 1.5 x 10 9 ~10 13 Sprint oligomers, if present, are present in the range of 10 3 ~10 14 molecules / reaction, or 10 4 ~1010 molecules / reaction, or 2×10 5 ~10 14 molecules / reaction, or 10 6 ~10 9 molecules / reaction, or 2×10 6 ~10 14 molecules / reaction, or 2×10 6 ~10 9 The method of any one of embodiments 58-96, 113-137, or 146-155, wherein the range of molecules / reactions is present.

[0161] Embodiment 157 relates to a method for preparing an amplification oligomer, the amplification oligomer being, when present, about 10 7 ~10 14 molecules / reaction or about 10 8 ~10 13 in the range of molecules / reaction; blocker oligomers, if present, are present in the range of 10 8 ~10 14 Molecules / Reactions, 10 10 ~10 13 molecules / reaction, or 10 11 ~10 13 The method of any one of embodiments 58-96, 113-137, or 146-156, wherein the range of molecules / reactions is present.

[0162] Embodiment 158 ​​is a method for preparing a displacer oligomer, when present, comprising the steps of: 7 ~10 14 molecules / reaction or about 10 8 ~10 13 The method of any one of embodiments 58-96, 113-137, or 146-157, wherein the range of molecules / reactions is present.

[0163] Embodiment 159 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the first additional sequence (optionally a stabilizing sequence), if present in the capture oligomer, is about 2-15 nucleotides or about 3-10 nucleotides in length.

[0164] Embodiment 160 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the capture sequence is about 10-35 nucleotides or about 10-25 nucleotides in length.

[0165] Embodiment 161 is a capture oligomer, combination, reaction mixture, kit, composition, or method of any one of the preceding embodiments, wherein the linker, if present, is about 10-20 nucleotides in length, or, if non-nucleotide based, about 3-20 or more atoms or about 2-15 or more repeat units.

[0166] Embodiment 162 is a capture oligomer, combination, reaction mixture, kit, composition, or method of any one of the preceding embodiments, wherein the length of the internal extension blocker, if present, is about 1-20 nucleotides or about 1-8 nucleotides, or, if non-nucleotide based, about 3-20 or more atoms, or 1-8 or more repeat units.

[0167] Embodiment 163 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the length of the second additional sequence (optionally a mixed nucleotide sequence), if present, is about 2-10 nucleotides or about 4-8 nucleotides.

[0168] Embodiment 164 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the length of the complement of the capture sequence is about 10-35 nucleotides or about 10-25 nucleotides.

[0169] Embodiment 165 is a capture oligomer, combination, reaction mixture, kit, composition, or method of any one of the preceding embodiments, wherein the third additional sequence, if present (optionally a stabilizing sequence and / or an adapter sequence), is about 2-50 nucleotides or about 4-35 nucleotides in length. The reversible extension blocker is about 1-20 or about 1-8 nucleotides (natural or non-natural).

[0170] Embodiment 166 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the length of the fourth additional sequence (optionally an adapter sequence), if present, is about 4-40 nucleotides or about 6-25 nucleotides.

[0171] Embodiment 167 is a capture oligomer, combination, reaction mixture, kit, composition, or method according to any one of the preceding embodiments, wherein the length of the target hybridizing sequence is from about 10 to 60 nucleotides or from about 12 to 25 nucleotides.

[0172] Embodiment 168 is a capture oligomer, combination, reaction mixture, kit, composition, or method of any one of the preceding embodiments, wherein the length of the blocking moiety, if present, is about 1-10 or about 1-5 nucleotides, or, if non-nucleotide based, about 3-20 atoms or about 1-5 repeat units.

[0173] Embodiment 169 is a capture oligomer, combination, reaction mixture, kit, composition, or method of any one of the preceding embodiments, wherein the total length of the capture oligomer is about 40-200 nucleotides, or about 40-150 nucleotides, or about 60-140 nucleotides, or about 60-130 nucleotides, and optionally the capture oligomer further comprises a non-nucleotide element having a length of about 3-60 atoms or 5-28 repeat units.

[0174] Embodiment 170 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 169, wherein the secondary capture reagent, if present, comprises a complement of the capture sequence having a length of about 10 to 35 nucleotides or about 10 to 20 nucleotides.

[0175] Embodiment 171 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 170, wherein the length of the blocker oligomer, if present, is about 10-35 nucleotides or about 10-20 nucleotides, and optionally the blocker oligomer further comprises a non-nucleotide element having a length of about 3-20 atoms or 1-5 repeat units.

[0176] Embodiment 172 is a combination, reaction mixture, kit, composition, or method according to 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.

[0177] Embodiment 173 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 172, wherein the length of the complementary oligomer, if present, is about 10 to 50 nucleotides or about 15 to 35 nucleotides.

[0178] Embodiment 174 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 173, wherein the length of the splint oligomer, if present, is about 15 to 60 nucleotides or about 20 to 50 nucleotides.

[0179] Embodiment 175 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 174, wherein the length of the displacer oligomer, if present, is about 10 to 50 nucleotides or about 20 to 40 nucleotides.

[0180] Embodiment 176 is a combination, reaction mixture, kit, composition, or method according to any one of embodiments 18 to 175, wherein the length of the amplification oligomer, if present, is about 10 to 80 nucleotides or about 20 to 60 nucleotides. [Brief description of the drawings]

[0181] II. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] 1 shows an exemplary capture oligomer according to the present disclosure, which includes, among other molecules, a capture sequence, a blocking moiety, a complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence. In FIG. 1A, the capture oligomer is annealed to a target polynucleotide (target), and the 3' end of the target polynucleotide is annealed to the 5' end of the target hybridizing sequence. The capture sequence is annealed to C'. [Figure 1B] 1 shows an exemplary capture oligomer according to the present disclosure, which includes, among other molecules, a capture sequence, a blocking moiety, the complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence. In FIG. 1B, the 3' end of the target is extended to the blocking moiety, and the resulting target extension is annealed to the additional sequence and C', but the capture sequence is displaced and becomes single-stranded. The 3' end of the capture oligomer is also extended along the target polynucleotide. [Figure 1C] FIG. 1C shows an exemplary capture oligomer according to the present disclosure, which includes a capture sequence, a blocking moiety, a complement of the capture sequence (C'), an additional sequence (e.g., a third or fourth additional sequence), and a target hybridizing sequence, along with other molecules. In FIG. 1C, the complex of FIG. 1B is annealed to a secondary capture reagent that includes a complement of the capture sequence and a binding partner or solid substrate. Meanwhile, excess capture oligomers have their capture sequences annealed to the complement of the capture sequence and do not interact with the secondary capture reagent.

[0182] [Figure 2A]FIG. 1B shows an embodiment of the disclosure in which a complex such as that in FIG. 1B is annealed to a secondary capture reagent that contains the complement of the capture sequence and associated with a solid substrate (in this case, a streptavidin-coated magnetic bead). The solid substrate may be part of the secondary capture reagent or may be associated with the secondary capture reagent through an interaction with a binding partner of the secondary capture reagent (e.g., biotin).

[0183] [Figure 2B] FIG. 2B shows an embodiment of the present disclosure in which the extended capture oligomer-target complex of FIG. 2A has been eluted from the secondary capture reagent.

[0184] [Diagram 3] 1 shows an embodiment of a capture oligomer according to the present disclosure, comprising a stabilizing (clamp) sequence as a first additional sequence, a capture sequence, a linker, an internal extension blocker, a complement of the capture sequence, a stabilizing (clamp) sequence as a third additional sequence, a fourth additional sequence, and a target hybridizing sequence.

[0185] [Figure 4A]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided in which the first strand comprises the sequence Sf at its 5' end and Sr' at its 3' end, and the second strand comprises the sequence Sf' at its 3' end and Sr at its 5' end. Here and throughout, a sequence designation with a' indicates complementarity to the sequence having the designation without '. The target molecule can be, for example, an amplicon from a reaction previously performed using primers with sequences Sf and Sr. A first extension cycle (cycle 1) is performed in which a capture oligomer according to the present disclosure, comprising a capture sequence C, an internal extension blocker (black circle), a complement of the capture sequence C', a fourth additional sequence A4, and a target hybridizing sequence THS complementary to Sr', anneals to the first target strand 1 (+). A reverse amplification oligomer, comprising an additional sequence A2 and a target hybridizing sequence Sf* complementary to at least Sf', anneals to the second target strand 1 (-). Sf* may contain affinity enhancing modifications and / or additional complementary nucleotides to the second target strand to enhance affinity to the target and, if present, promote competition for binding with a primer having sequence Sf from a previous reaction. Extension of the capture oligomer and reverse amplification oligomer produces products 2(-) and 2(+), respectively, with the first strand being extended along the capture oligomer to produce product 1(+)e and the second strand being extended along the reverse amplification oligomer to produce product 1(-)e. The capture sequence in the extended capture oligomer 2(-) is essentially displaced as described for FIG. 1B. A second reaction cycle (cycle 2) is performed in which 2(-) anneals to the reverse amplification oligomer, resulting in extension to produce products 2(-)e and 3.1(+). Meanwhile, 1(+)e anneals to the capture oligomer, and extension of the capture oligomer produces product 3.1(-). Additional examples of 1(-)e and 2(+) and 2(-)e and 3.1(+) are also generated from appropriate hybridization and extension events. This reaction scheme illustrates the inclusion of additional sequences at each end of the target, as well as rendering the target captureable, for example, by incorporation of C in a form available for binding to a second capture reagent.

[0186] [Figure 4B]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A target molecule is provided in which the first strand comprises sequence Sf at its 5' end and Sr' and A4' at its 3' end, and the second strand comprises sequence Sf' at its 3' end and Sr and A4 at its 5' end. The target molecule can be, for example, an amplicon from a reaction previously performed using a primer with sequence Sf and A4-Sr, where A4 is an additional sequence not originally present in the template. A first extension cycle (cycle 1) is performed in which a capture oligomer according to the present disclosure, comprising capture sequence C, an internal extension blocker (black circle), a complement of the capture sequence C', a fourth additional sequence A4, and a target hybridizing sequence THS complementary to A4', anneals to the first target strand (not shown). A reverse amplification oligomer, comprising additional sequence A2, and a target hybridizing sequence Sf* complementary to at least Sf', anneals to the second target strand (not shown). Sf* may contain affinity enhancing modifications and / or additional complementary nucleotides to the second target strand to enhance its affinity to the target and promote competition for binding with a primer having sequence Sf from a previous reaction that exists. Extension of these complexes produces extended capture oligomer 2(-) and extended first target strand 1(+)e, as well as extended second target strand 1(-)e and extended reverse amplification oligomer 2(+). The capture sequence in extended capture oligomer 2(-) is essentially displaced as described for FIG. 1B. A second reaction cycle (cycle 2) is performed in which 2(-) anneals to the reverse amplification oligomer, resulting in extension to produce products 2(-)e and 3.1(+). Meanwhile, 1(+)e anneals to the capture oligomer, and extension of the capture oligomer produces product 3.1(-). Additional examples of 1(-)e and 2(+) and 2(-)e and 3.1(+) are also generated from appropriate hybridization and extension events.This reaction scheme shows that a universal THS (i.e., a capture oligomer that may have, in an earlier step, bound (e.g., via amplification or ligation) an additional sequence A4' that can bind to the target, along with the inclusion of an additional sequence at the end of the target distal from the capture oligomer binding site, can be used to enable capture of the target by incorporating C (in a form available for binding).

[0187] [Diagram 5] 1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. Among other things, a capture oligomer is provided that includes a 3' blocking portion and a target hybridizing sequence (THS) that binds to sequence A1' in the target strand. A1' can be an additional sequence that was attached to the target in a previous step (e.g., via amplification or ligation). The capture oligomer further includes a sequence x that includes the complement of the capture sequence of the capture oligomer, and can also include a third or fourth additional sequence between the complement of the capture sequence and the THS. The target strand can be extended along the capture oligomer to displace the capture sequence from the complement of the capture sequence, as discussed elsewhere. The capture oligomer can be provided in a limited amount (e.g., 1012 copies) relative to the target (e.g., 1014 copies). A primer is also provided in excess over the target (e.g., 1015 copies) that includes sequences A2 and Sf. This extension of the primer results in a strand that includes 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 those discussed above and a complex of the target strand and capture oligomer in which the target strand includes A2 at its 5' end and A1' near its 3' end. This reaction scheme illustrates the production of single-stranded capturable products, including those in which additional sequences are included in the target strand (if a second extension cycle is performed).

[0188] [Figure 6]It is shown how hybridization of a capture oligomer with an extendable 3' end to another capture oligomer can create a dimer in which the capture sequence is displaced from C' upon extension (above the dashed line). This dimer is now captureable and may interfere with downstream processes, such as competing with capture of the desired target by occupying a secondary capture reagent (not shown), interference with subsequent analysis (e.g., the dimer becomes part of the sequencing library, thereby reducing the output and quality of the subsequent sequencing run), etc. Sx' is the complement of a portion of the target hybridizing sequence, with other elements as in the previous figures. Below the dashed line is shown a capture oligomer with a blocking moiety (circled x) at its 3' end, which prevents the formation of a dimeric extension product so that no dimer undergoes C displacement.

[0189] [Figure 7A] 1 shows an embodiment in which a capture oligomer is used that includes a capture sequence, various intermediate elements (indicated by "..."), a reversible extension blocker (filled circle), and a target hybridizing sequence (THS). Prior to unblocking the reversible extension blocker, the capture sequence and various intermediate elements (if present) are not templates for extension (e.g., of the target strand or the amplification oligomer). This can promote more efficient and more specific extension or amplification by avoiding the incorporation of additional sequences complementary to the capture sequence and various intermediate elements (if present) in the product (e.g., in any mispriming products that may be formed) throughout the extension or amplification process until the reversible extension blocker is unblocked; following unblocking, the capture sequence and various intermediate elements (if present) can be incorporated.

[0190] [Figure 7B]The embodiment shows that a first amplification oligomer is used, which includes from 3' to 5' a target hybridization sequence Sr, a reversible extension blocker (black square), an additional sequence A1, and any additional elements such as an optional capture sequence (indicated by "..."). A second amplification oligomer is optionally used (as shown in the figure), which includes from 3' to 5' a target hybridization sequence Sf, a reversible extension blocker (white square; this may be the same or different from the reversible extension blocker in the first amplification oligomer), an additional sequence A2, and any additional elements (indicated by "...": these may be the same or different from those in the first amplification oligomer). Before unblocking one or more reversible extension blockers, the additional sequence and any additional elements (if present) are not templates for extension (e.g., of the target strand or the amplification oligomer). This can promote more efficient and more specific extension or amplification by avoiding the incorporation of sequences complementary to additional sequences and various other elements (if present) in the products (e.g., in any mispriming products that may be formed) throughout the initial extension or amplification process. One or more reversible extension blockers are unblocked (if two are present, unblocking can occur simultaneously or separately) and the additional sequences and any other elements present can be incorporated at a later stage in the process, such as in a later extension round.

[0191] [Figure 8A]4A shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. The initial target strands 1(+) and 1(-) are as in FIG. 4A. Below the vertical straight arrow, a capture oligomer is provided that contains the target hybridizing sequence THS and additional elements A4, C', an internal extension blocker, and C as described for the oligomer in FIG. 4A. THS binds at an internal site on the target strand and undergoes extension to produce product 2N(-). A displacer oligomer containing Sr is provided, the extension of which displaces 2N(-) from 1(+) to produce 2(-). A reverse amplification oligomer as in FIG. 4A is provided, and extension along 1(-) produces 2.1(+), and extension of 1(-) along the reverse amplification oligomer produces 1(-)e. When 2N(-) is displaced (left curved arrow), the reverse amplification oligomer anneals to 2N(-) and is extended, respectively, resulting in products 2N(-)e and 2.2(+), which now contain A2' and where C has been displaced from C'. This reaction scheme illustrates the use of a displacer oligomer to facilitate the generation of a capturable product containing additional sequences (e.g., adapters) on both ends of the target sequence in only one cycle. Additionally, this reaction scheme illustrates an embodiment in which the capture oligomer does not bind to a site that contains the 3' end of the target strand.

[0192] [Figure 8B]8A and 8B show additional exemplary molecules and additional exemplary reaction schemes according to the present disclosure. The reaction schemes are substantially similar to those shown in FIG. 4A, except for 1) and 2) below. 1) The initial target strands 1(+) and 1(-) contain additional sequences including the target hybridization sequence THS, an optional spacer sequence S, and a displacer oligomer binding site D. These additional sequences are user-defined arbitrary sequences and can be incorporated into the target by an amplification reaction using, for example, 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 capture oligomer binds to the user-defined THS site. Otherwise, the reaction proceeds as shown in FIG. 8A, and the resulting products are shown in FIG. 8B. The optional spacer can be useful to improve the extension of the displacer oligomer and the subsequent displacement of the capture oligomer. Similar to the scheme shown in Figure 4A, this reaction scheme illustrates the use of a displacer oligomer to facilitate the generation (e.g., in only one cycle) of a capturable product that contains additional sequences (e.g., adapters) on both ends of the target sequence. Additionally, this scheme illustrates the use of an additional user-defined sequence that can serve as a binding site for both the capture and displacer oligomers. This design can generalize the approach, allowing for a simpler and much more cost-effective means of designing capture and displacer oligomers for use with a variety of targets, including multiplex formats.

[0193] [Figure 9]The general principle of how a blocker oligomer can prevent hybridization between an additional sequence in an oligomer and its complement in the extension product is shown. An amplification reaction is performed with a forward primer with sequence f that hybridizes to the target strand T(-) and a reverse primer that contains sequence A (an additional sequence not present in the target) and sequence r that hybridizes to the target strand T(+). Extension produces products 1(-) and 1(+). A blocker oligomer is provided that contains sequence A and a 3' blocking portion. In cycle 2, the forward primer is extended along 1(-) to produce 2(+) and the reverse primer is extended along 1(+) to produce 2(-). From cycle 3 onwards, the blocker oligomer anneals to 2(+), which means that hybridization of r to r' is required for the reverse primer to prime extension along 2(+). This can be beneficial if a mispriming event occurs that creates a small amount of by-product that has an incomplete complement of r but is extended to include A'. Without the blocker oligomer, the binding of the reverse amplification oligomer to the misprimed by-product becomes more favorable due to the interaction between A and A' of the reverse amplification oligomer, resulting in the amplification of more by-products than if the blocker oligomer was provided. (Meanwhile, the forward primer anneals to 2(-) and undergoes extension.)

[0194] [Figure 10A]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. (i) A capture oligomer is provided that includes a first and second portion of a capture sequence (C1 and C2), an internal extension blocker (black circle), a first and second portion of a spacer sequence (S1 and S2), and a target hybridizing sequence (THS) that binds to a site in a target strand including its 3' end, and (ii) a complementary oligomer that includes S1' and C2'. Upon hybridization of the capture oligomer to the target and extension along the capture oligomer to the internal extension blocker of the target, incorporation of S' into the target strand displaces the complementary oligomer. The capture oligomer is also extended along the target (note that in other embodiments described herein, the capture oligomer may be blocked and no such extension occurs). A secondary capture reagent is provided that includes a binding partner or solid support (circled B) linked by a linker (zigzag line) to the complement C' of the capture sequence. The secondary capture reagent anneals to capture oligomers bound to the extended target, but not to capture oligomers bound to complementary oligomers, since the latter occupy a sufficient amount of the capture sequence, C2, to substantially prevent annealing of the secondary capture reagent to the capture oligomer.

[0195] [Figure 10B]Embodiments are provided in which the oligomer combinations are useful, if desired, to capture a target polynucleotide from a composition that contains an amount (eg, a limited amount or an amount not greater than a predetermined amount) of the target polynucleotide. The combination includes a capture oligomer comprising, from 5' to 3', a first portion of the capture sequence C1, a second portion of the capture sequence C2, an optional spacer sequence S, a second portion of the target hybridizing sequence THS2, a first portion of the target hybridizing sequence THS1, and an optional blocking moiety (circled X); a separate complementary oligo comprising, from 5' to 3', THS2', S' (optional; may or may not be used if S is present in the capture oligomer), and C2' (where the complement of the element is indicated with a "") and an optional blocking moiety (circled X) at the 3' end; and a secondary capture reagent comprising, from 5' to 3', the complement of the capture sequence, including C2', C1' (C1' or C2' may or may not be complementary to the entire length of C1 and C2), and a binding partner (exemplified in this figure with a biotin molecule represented as a circled B). In the absence of target polynucleotide, the complementary oligomer binds to the capture oligomer, blocking access to the complete capture sequence to an extent sufficient to block binding of the complement of the capture sequence in the secondary capture reagent (see the complex of the complementary oligomer to the capture oligomer at the top of the figure). In the presence of target, the THS1 region of the capture oligomer binds to the target, followed by the energetically favorable THS2 region, thereby displacing the THS2' region of the separate complementary oligo from the capture oligomer. When this occurs, the C2' region of the separate complementary oligo is no longer stable enough to bind to the capture oligomer, and therefore does not bind, thus leaving the complete capture sequence available for binding, as shown under the first arrow. The complement of the capture sequence in the secondary capture reagent then binds to the capture sequence of the capture oligomer, as shown under the second arrow. This complex can then be isolated from the mixture, for example, by streptavidin-coated magnetic microspheres (described elsewhere in this disclosure), thus capturing and purifying the target polynucleotide. Optionally, the capture oligomer may be present in the combination in greater amounts than the secondary capture reagent.Such oligomers and combinations are useful for capturing an amount (eg, a limited amount or up to a predetermined amount) of a target polynucleotide from a composition.

[0196] [Figure 11A] 11A shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. In FIG. 11A, a capture oligomer is provided that essentially contains the elements described for the capture oligomer of FIG. 4A, except that the THS binds to a site on the target strand that does not include the 3' end (which may be circular as shown or linear). A complementary oligomer is provided that contains (i) a target hybridizing sequence that anneals adjacent to the THS of the capture oligomer and (ii) at least a partial complement of A4. The at least a partial complement of A4 is insufficient to anneal to the capture oligomer in the absence of the target strand. [Figure 11B] 11A-11C show exemplary molecules and exemplary reaction schemes according to the present disclosure. In FIG. 11B, the complementary oligomer has undergone extension, which displaces C and renders it available for capture using a secondary capture reagent (not shown). This scheme is useful for capturing circular molecules and / or represents an alternative approach for using capture oligomers that do not bind to the 3' end of the target strand.

[0197] [Figure 12]4A shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A capture oligomer containing elements such as the capture oligomer of FIG. 4A, with a second additional sequence A2, which may contain a mixed nucleotide segment between C' and the internal extension blocker, anneals to a target strand at a site containing its 3' end. The target strand also contains 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 during a previous step (e.g., amplification or ligation). Extension of the target strand along the capture oligomer adds sequences A4', C, and A2' to the 3' end of the target strand. 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 to a splint oligomer containing sequences A5', A2, C', and A4, and when the extended target strand is annealed to the splint oligomer, the target strand 5' and 3' ends are immediately adjacent. The extended target strand can then be circularized by ligation. The A2 and A2' sequences serve to ensure proper juxtaposition of the extended target strand 5' and 3' ends. This can be useful when C and C' are slippery repeat sequences (e.g., polyA and polyT, or vice versa) that would otherwise inhibit the formation of a substrate for ligation. This scheme is useful for capturing and then circularizing a target molecule, for example for use in rolling circle amplification procedures.

[0198] [Figure 13]1 shows an exemplary molecule and an exemplary reaction scheme according to the present disclosure. A capture oligomer is provided that includes a capture sequence C (including a first and second portion, C1 and C2; not shown), an internal extension blocker (black circle), a spacer sequence S (including a first and second portion, S1 and S2; not shown), and a target hybridization sequence THS, together with a reverse amplification oligomer that includes sequence S2. THS and S2 serve to create an amplified target (e.g., by PCR). A complementary oligomer is added that includes a complement of the first portion of the spacer sequence, S1', and a complement of the second portion of the capture sequence, C2'. C2' is insufficient to anneal to C of the amplified target if S' is annealed to S of the other strand of the amplified target. The complementary oligomer anneals to the capture oligomer that is not annealed to the amplified strand. A secondary capture reagent is added that includes C' and a binding partner or solid support (circled B) to capture the amplified target. The secondary capture reagent binds to the amplified target but not to capture oligomers that are not annealed to the amplified strand and C is blocked to a sufficient extent by C2' of the complementary oligomer.

[0199] [Figure 14] The fold difference in output of methods using capture oligomers with and without clamp sequences is shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0200] III. DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS A.Definition Before describing the present teachings in detail, it should be understood that the present disclosure is not limited to specific compositions or process steps, which may vary. It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents, and expressions such as "one or more items" include singular referents unless the context clearly dictates otherwise. Thus, for example, reference to "an oligomer" includes a plurality of oligomers, etc. The conjunction "or" should be interpreted in an inclusive sense, i.e., equivalent to "and / or," unless a more inclusive sense is undue in the context. When "at least one" member of a class (e.g., oligomer) is present, a reference to "the" member (e.g., oligomer) refers to at least one of the members (e.g., oligomers) present (if only one).

[0201] It will be understood that there is an implicit "about" before temperatures, concentrations, amounts, times, etc. discussed in this disclosure, such that minor and very minor deviations are within the scope of the teachings herein. In general, the term "about" refers to minor variations in the amounts of the components of the composition that do not have any significant effect on the activity or stability of the composition, e.g., within 10%, 5%, 2%, or 1%. Thus, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximations that may vary depending on the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed at least by considering the number of reported significant digits and applying ordinary rounding techniques. All ranges should be construed to include the endpoints unless there is an express exclusion, such as "not including the endpoints". Thus, for example, "within 10 to 15" includes the values ​​10 and 15 and all intervening integer and (where appropriate) non-integer values. Additionally, the use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not intended to be limiting of the teachings. Section headings are provided merely for the convenience of the reader and are not intended to limit the disclosure. To the extent that anything incorporated by reference is inconsistent with the express content of this disclosure, the express content shall control.

[0202] Unless otherwise indicated, embodiments herein described as "comprising" various components are also contemplated as "consisting of" or "consisting essentially of" the described components. By "consisting essentially of," it is meant that additional component(s), composition(s) or method step(s) may be included in the compositions or methods that do not substantially alter the basic and novel characteristics of the compositions and methods described herein. Such characteristics may include, in some cases, for example, the ability to hybridize to a target polynucleotide and undergo further binding and / or extension reactions as described herein.

[0203] "Sample" refers to materials that may contain target polynucleotides, including but not limited to biological, clinical, environmental and food samples. Environmental samples include environmental materials such as surface materials, soil, water, air and industrial samples, as well as samples obtained from food and dairy processing equipment, devices, facilities, instruments, disposable and non-disposable items. "Biological" or "clinical" samples refer to tissues or materials derived from living or dead humans, animals, or other organisms that may contain target polynucleotides, including, for example, swabs, lavage fluids, aspirates, exudates, biopsy tissues, or bodily fluids such as blood or urine. Samples can be treated to physically or mechanically disrupt tissue or cellular structures and release intracellular nucleic acids into solutions that may include enzymes, buffers, salts, detergents, etc. to prepare the sample for analysis. These examples should not be construed as limiting the types of samples applicable to this disclosure.

[0204] "Nucleic acid" and "polynucleotide" refer to polymeric compounds that include nucleosides or nucleoside analogs having nitrogenous heterocyclic bases or base analogs linked together to form polynucleotides, including polymers that are conventional RNA, DNA, mixed RNA-DNA, and analogs thereof. The nucleic acid "backbone" can be composed of a variety of linkages, including sugar-phosphodiester linkages, peptide-nucleic acid linkages ("peptide nucleic acids" or PNA; WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. The sugar portion of the nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, such as 2' methoxy or 2' halide substitutions. The nitrogenous bases can be the conventional bases (A, G, C, T, U), their analogs (e.g., inosine, or others; The Biochemistry of the Nucleic Acids 5-36, eds. Adams et al., 11th ed., 1992), derivatives of purines or pyrimidines (e.g., N 4 -methyldeoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, 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-thio-pyrimidine, 4-amino-pyrimidine, 4-dimethylhydrazine-pyrimidine, and O 4-alkyl-pyrimidines; U.S. Pat. No. 5,378,825 and WO 93 / 13121). Nucleic acids can include one or more "abasic" residues, where the backbone does not include a nitrogenous base for the position(s) of the polymer (U.S. Pat. No. 5,585,481). Nucleic acids can include only conventional RNA or DNA sugars, bases and linkages, or can include both conventional building blocks and substitutions (e.g., conventional bases with 2' methoxy linkages, or polymers containing both conventional bases and one or more base analogs). Nucleic acids include "locked nucleic acids" (LNAs), analogs that include one or more LNA nucleotide monomers with bicyclic furanose units locked to RNA that mimic the sugar conformation, enhancing hybridization affinity to complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Examples of oligomers that may affect the stability of the hybridization complex include PNA oligomers, oligomers containing 2'-methoxy or 2'-fluoro substituted RNA, or oligomers that affect the overall charge, charge density, or steric association of the hybridization complex, including oligomers containing charged bonds (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates). Methylated cytosines, such as 5-methylcytosine, may be used in conjunction with any of the above backbones / sugars / linkages, including RNA or DNA backbones (or mixtures thereof), unless otherwise indicated. RNA and DNA equivalents have different sugar moieties (i.e., ribose vs. deoxyribose) and may differ by the presence of uracil in RNA and thymine in DNA. Differences between RNA and DNA equivalents do not contribute to differences in homology, since the equivalents have the same degree of complementarity to a particular sequence. When referring to a range of lengths of oligonucleotides, amplicons, or other nucleic acids, it is understood that the range includes all integers (e.g., a length of 19 to 25 contiguous nucleotides includes 19, 20, 21, 22, 23, 24, and 25).Reference to a "sequence of SEQ ID NO:X" refers to the base sequence of the corresponding sequence listing entry and does not require identity of the backbone (e.g., RNA, 2'-O-Me RNA, or DNA) or base modifications (e.g., methylation of cytosine residues) unless otherwise indicated. Further, unless otherwise indicated, it is understood that T residues are interchangeable with U residues and vice versa.

[0205] "Target polynucleotide" refers to a polynucleotide that is sought to be captured, isolated, amplified, detected, and / or sequenced using the compositions or methods described herein. 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, whether living or previously living). Exemplary DNA includes genomic DNA, episomal or plasmid DNA, and mitochondrial DNA. Exemplary RNA includes mRNA, more generally transcribed RNA, ribosomal RNA, miRNA, non-coding RNA, etc. (and, where applicable, e.g., in the case of certain viruses, genomic RNA). Target polynucleotides also include amplicons that include the nucleic acids discussed above to which additional sequences (such as any additional sequences described herein) may be added. In some embodiments, the target polynucleotide comprises a non-naturally occurring sequence resulting, for example, from in vitro synthesis, ligation, site-directed mutagenesis, recombination, etc.

[0206] "Oligomer" or "oligonucleotide" refers to a nucleic acid generally less than 1,000 nt, including those in a size range having a lower limit of about 2-5 nucleotides (nt) and an upper limit of about 500-900 nt. Some particular embodiments are oligomers in a size range having a lower limit of about 5-15, 16, 17, 18, 19, or 20 nt and an upper limit of about 50-600 nt, and other particular embodiments are in a size range having a lower limit of about 10-20 nt and an upper limit of about 30-100 nt. Oligomers can be purified from natural sources, but can be synthesized by using any well-known enzymatic or chemical method. Oligomers may be referred to by their functional name (e.g., capture probe, primer, or promoter primer), but one of skill in the art will understand that such terms refer to oligomers. Oligomers can form secondary and tertiary structures by self-hybridizing or by hybridizing to other polynucleotides. Such structures may include, but are not limited to, duplex, hairpin, cruciform, bent, and triplex. The oligomer may be generated in any manner, including chemical synthesis, DNA replication, reverse transcription, PCR, or a combination thereof. In some embodiments, the oligomer that forms the invasive cleavage structure is generated in a reaction (e.g., by extension of a primer in an enzymatic extension reaction).

[0207] "Arbitrary sequence" refers to any sequence that is chosen, selected, determined, designed, etc., by a user, typically to perform a desired function or purpose in a downstream process. In a preferred mode, the arbitrary sequence is designed to be non-complementary or otherwise non-reactive to one or more target sequences under given conditions of the process. In some embodiments, the arbitrary sequence may be a randomly generated sequence or set of sequences, for example, for use as a unique molecular identifier.

[0208] "Capture oligomer," "capture oligonucleotide," "capture probe," "target capture oligomer," and "capture probe oligomer" are used interchangeably to refer to a nucleic acid oligomer that comprises (i) a target hybridizing sequence capable of specifically hybridizing to a target sequence in a target nucleic acid, and (ii) a capture sequence capable of hybridizing to a secondary oligomer (e.g., immobilized on a solid support or linked to a binding partner to facilitate isolation of a complex comprising the capture oligomer, target and secondary oligomer from other molecules in the composition).

[0209] "Amplicon" or "amplification product" refers to a nucleic acid molecule produced in a nucleic acid amplification reaction and derived from a template nucleic acid. An amplicon or amplification product includes an amplified nucleic acid sequence (e.g., a target nucleic acid) that can be of the same or opposite sense as the template nucleic acid. In some embodiments, an amplicon has a length of about 100-30,000 nucleotides, about 100-10,000 nucleotides, about 100-5000 nucleotides, 100-2000 nucleotides, about 100-1500 nucleotides, about 100-1000 nucleotides, about 100-800 nucleotides, about 100-700 nucleotides, about 100-600 nucleotides, or about 100-500 nucleotides.

[0210] "Amplification oligonucleotide" or "amplification oligomer" refers to an oligonucleotide that hybridizes to a target nucleic acid or its complement and participates in a nucleic acid extension or amplification reaction, serving, for example, as a primer and / or promoter primer. Amplification oligomers also encompass promoter providers that include promoters that can initiate transcription, but are not necessarily extendable by DNA polymerase, and may include a 3' blocking portion. Certain amplification oligomers include a target hybridizing sequence of at least about 10 contiguous bases, and optionally at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 contiguous bases, that are complementary to a region of the target nucleic acid sequence or its complementary strand. Other exemplary lengths or length ranges of target hybridizing sequences are described elsewhere herein and can be applied to amplification oligomers. The contiguous bases can be at least about 70%, at least about 80%, at least about 90%, or fully complementary to the target sequence to which the amplification oligomer binds. In some embodiments, the amplification oligomer includes an intervening linker or non-complementary sequence between two segments of complementary sequence, e.g., the two complementary segments of the oligomer collectively include at least about 10 complementary bases, and optionally 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 in length, and can optionally include modified nucleotides. The amplification oligomer can optionally be modified, e.g., by including a 5' region that is non-complementary to the target sequence. Such modifications can include functional additions, such as tags, promoters, or other sequences that are used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0211] "Primer" refers to an oligomer having a 3' end that hybridizes to a template nucleic acid and is extended by polymerization. A primer can optionally be modified, for example, by including a 5' region that is non-complementary to the target sequence. Such modifications can include functional additions such as tags, promoters, or other sequences that are used or useful for manipulating or amplifying the primer or target oligonucleotide.

[0212] The first sequence is the "complement" of (or, equivalently, is "complementary" to) the second sequence, and the first sequence has sufficient length and content to anneal to the second sequence under reasonable binding conditions (which may be, but are not necessarily, the stringent hybridization conditions described herein, and include, for example, annealing conditions used in standard PCR and other techniques involving primer or probe binding and extension).

[0213] "Tag" refers to any additional sequence other than the target hybridizing sequence that may be included in an oligomer. Any sequence present in addition to the target hybridizing sequence may function as a tag. Tags include, but are not limited to, adapters (see below). Additional examples of tags are promoters, mixed nucleotide elements as described elsewhere herein, and stabilizing sequences including clamps.

[0214] An "adapter" is a sequence that adapts the molecule to which it is attached to provide a binding site for another molecule (e.g., a sequencing primer, or the target hybridizing sequence (THS) of a capture oligomer). The binding site can be a universal binding site (e.g., for multiple capture oligomers, all with the same THS, in a multiplex format, or for a universal primer). Additional examples of binding sites are binding sites for displacer oligomers, probes, capture oligomers, or nucleic acid modifying enzymes (e.g., RNA polymerases, primases, ligases, RNAses (such as RNAse H), or restriction enzymes), or for attachment to a solid phase (including by a solid phase primer or capture oligomer), including for use in clonal amplification, or other functional element or elements useful for downstream applications, such as enrichment, library preparation, clonal amplification, or sequencing. Thus, sample barcodes or index sequences, key or calibrator sequences, molecular barcodes (containing unique molecular identifiers), sites for downstream cloning and sites for circularization of target molecules are additional examples of elements that may be included in adapters.

[0215] A "linker" is a sequence or non-sequence element, or combination thereof, that links one portion of an oligomer to another portion. In some embodiments, the sequence linker comprises a sequence that does not hybridize to the target polynucleotide and / or other oligomers in the combination or composition. In some embodiments, the non-sequence linker is an alkyl, alkenyl, amide, or polyethylene glycol group [(-CH2CH2O-) n ] is included.

[0216] A "stabilizing sequence" is a clamp, mixed nucleotide region, or other sequence that functions to increase the stability of a duplex region and / or control the register of hybridization (e.g., when located adjacent to a sequence prone to slippage, e.g., containing repetitive nucleotides, e.g., poly-dA or poly-dT sequences). An "alignment sequence" is a stabilizing sequence that controls the register of hybridization. In addition to the clamps and mixed nucleotide regions described elsewhere herein, stabilizing sequences include GC-rich sequences and sequences containing affinity-enhancing modifications.

[0217] An "internal extension blocker" is an element located within the sequence of a nucleic acid or attached to a nucleic acid that prevents the extension of a complementary strand along the nucleic acid. Examples include non-nucleotidic linkers or one or more abasic sites, non-natural nucleotides, or chemically modified natural nucleotides, as well as reversible extension blockers, as described below.

[0218] A "reversible extension blocker" is an internal extension blocker whose blocking function can be reversed, i.e., capable of allowing the extension of a complementary strand. An exemplary reversible extension blocker is a non-natural nucleotide that has a complementary nucleotide that is accepted by a polymerase and exhibits specificity for a natural nucleotide (i.e., the polymerase does not add a natural base beyond the reversible extension blocker). Providing a complementary nucleotide reverses the blocking function. Examples of non-natural base pairs in which either member of the pair can function as a reversible extension blocker are Iso-dC or Iso-dG, xanthine or 5-(2,4 diaminopyrimidine); 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one; 4-Methylbenzimidizole or 2,4-difluorotoluene; 7-azaindole or isocarbostyril: dMMO2 or d5SICS; or dF or dQ. Other examples of reversible extension blockers are one or more chemically modified nucleotides, where the modification is attached via a reversible linkage, which can be reversed by providing one or more of: a chemical, an enzyme, a change in temperature, a change in reagent composition, etc.; a reversible structural feature of a nucleic acid; or a molecule reversibly attached to the capture oligomer, where optionally the reversibly attached molecule is a protein, an enzyme, a lipid, a carbohydrate, or a chemical moiety.

[0219] "Nucleic acid amplification" refers to any in vitro procedure that creates multiple copies of a target nucleic acid sequence, or its complementary sequence, or a fragment thereof (i.e., an amplified sequence that includes less than the entire target nucleic acid). Examples of nucleic acid amplification procedures include transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA) and others (e.g., U.S. Pat. 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. Pat. No. 4,786,600), polymerase chain reaction (PCR) (e.g., U.S. Pat. Nos. 4,683,195, 4,683,200, ... These include transcription-related methods such as rolling circle amplification (RCA) (e.g., U.S. Pat. Nos. 5,854,033 and 6,143,495), recombinase polymerase amplification (RPA) (e.g., U.S. Pat. No. 7,666,598), ligase chain reaction (LCR) (e.g., European Patent Application Publication No. 0320308) and strand displacement amplification (SDA) (e.g., U.S. Pat. No. 5,422,252). Replicase-mediated amplification uses self-replicating RNA molecules and a replicase such as QB replicase. PCR amplification uses DNA polymerase, primers, and thermal cycling steps to synthesize multiple copies of two complementary strands of DNA or cDNA. LCR amplification uses at least four separate oligonucleotides to amplify a target and its complementary strand by using multiple cycles of hybridization, ligation, and denaturation. SDA uses primers containing recognition sites for restriction endonucleases that nick one strand of a semi-modified DNA duplex containing the target sequence, followed by amplification in a series of primer extension and strand displacement steps. Although certain embodiments use PCR, it will be apparent to one of skill in the art that the oligomers disclosed herein can readily be used as primers in other amplification methods.

[0220] "Hybridization" or "hybridize" refers to the ability of two fully or partially complementary nucleic acid strands to come together in a parallel or antiparallel orientation under certain hybridization assay conditions to form a stable structure with a double-stranded region. "Hybridization" and "hybridize" are synonymous with "annealing" and "annealing", respectively. The two constituent strands of this double-stranded structure, sometimes called a hybrid, are held together by hydrogen bonds. 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 strand, although base pairing can also be formed between bases that are not members of these "canonical" pairs. Non-canonical base pairing is well known in the art. (See, for example, RLP Adams et al., The Biochemistry of the Nucleic Acids (11th ed. 1992))

[0221] As used herein, the term "specifically hybridize" means that under given hybridization conditions, a probe, primer, or other oligomer (e.g., capture oligomer) detectably hybridizes substantially only to the target sequence(s) in a sample containing the target sequence(s) (i.e., there is little or no detectable hybridization to non-target sequences). In particular, an oligomer can be configured to specifically hybridize to any one of a set of targets (e.g., sequences from organisms of a particular taxonomic group, e.g., genus). In some embodiments, a probe, primer, or other oligomer (e.g., capture oligomer) can hybridize to its target nucleic acid to form a stable oligomer:target hybrid, but in some cases fails to form a sufficient number of stable oligomer:non-target hybrids for amplification or capture. Amplification and capture oligomers that specifically hybridize to a target nucleic acid are useful for amplifying and capturing target nucleic acids, but are not useful for amplifying and capturing non-target nucleic acids, especially non-target nucleic acids of phylogenetically closely related organisms. Thus, the oligomer hybridizes to a target nucleic acid to a sufficiently greater extent than non-target nucleic acid, allowing the skilled artisan to accurately capture, amplify, and / or detect the presence (or absence) of nucleic acid from a specific target (e.g., a specific pathogen) as required. In general, reducing the degree of complementarity between an oligonucleotide sequence and its target sequence reduces the degree or rate of hybridization of the oligonucleotide to its target region. However, including one or more non-complementary nucleosides or nucleic acid bases can facilitate the ability of the oligonucleotide to distinguish non-target nucleic acid sequences.

[0222] "Stringent hybridization conditions" or "stringent conditions" refers to conditions that (1) allow an oligomer to hybridize preferentially to a target nucleic acid as opposed to a different nucleic acid (e.g., a nucleic acid that has only one nucleotide difference in identity with the target nucleic acid), or (2) allow only an oligomer having a higher affinity target hybridizing sequence to hybridize to a target (compared to an oligomer having a lower affinity target hybridizing sequence), e.g., the higher affinity target hybridizing sequence is longer than the lower affinity target hybridizing sequence and / or contains affinity-enhancing modifications that the lower affinity target hybridizing sequence does not contain. While the definition of stringent hybridization conditions does not change, the actual reaction environment that may be used for stringent hybridization may vary depending on factors including the GC content and length of the oligomer, the degree of similarity between the oligomer sequence and the sequences of target and non-target nucleic acids that may be present in the test sample. Hybridization conditions include temperature and composition of the hybridization reagent or solution. Exemplary stringent hybridization conditions using oligomers of the present disclosure correspond to temperatures of about 40° C. to 75° C., e.g., 40° C. to 50° C., 50° C. to 60° C., or 60° C. to 75° C., with monovalent cation concentrations in the range of about 0.4 to 1 M, divalent cation concentrations in the range of about 0 to 10 mM, and pH in the range of about 5 to 9. Additional details of hybridization conditions are provided in the Examples section. Other acceptable stringent hybridization conditions will be readily ascertained by one of skill in the art.

[0223] "Label" or "detectable label" refers to a moiety or compound directly or indirectly linked to a probe that is detected or provides a detectable signal. Any detectable moiety can be used, such as radionuclides, ligands such as biotin or avidin, enzymes, enzyme substrates, reactive groups, chromophores such as dyes or particles (e.g., latex or metal beads) that impart a detectable color, luminescent compounds (e.g., bioluminescent, phosphorescent, or chemiluminescent compounds), and fluorescent compounds (i.e., fluorophores). Embodiments of fluorophores include those that absorb light (e.g., have peak absorption wavelengths) in the range of about 495 nm to 690 nm and emit light (e.g., have peak emission wavelengths) in the range of about 520 nm to 710 nm, including those known as FAM™, TET™, HEX, CAL FLUOR™ (orange or red), CY, and QUASAR™ compounds. Fluorophores can be used in combination with a quencher molecule that absorbs light when in close proximity to the fluorophore, reducing background fluorescence. Such quenchers are well known in the art and include, for example, BLACK HOLE QUENCHER™ (i.e., BHQ™), Blackberry Quencher® (i.e., BBQ-650®), Eclipse®, or TAMRA™ compounds.

[0224] An "unextendable" oligomer or an oligomer that includes a "blocking moiety at its 3' end" includes a blocking moiety close enough to its 3' end (also referred to as the 3' terminus) to prevent extension. Any blocking moiety close enough to the 3' end to block extension is considered "at" the 3' end for purposes of this disclosure, even if it is not attached to or instead present at the 3' hydroxyl or oxygen. A blocking moiety near the 3' end is, in some embodiments, within 5 residues of the 3' end and is large enough to restrict binding of a polymerase to the oligomer, while other embodiments include a blocking moiety covalently attached to the 3' end. Many different chemical groups can be used to block the 3' end, including alkyl groups, non-nucleotidic linkers, alkane-diol dideoxynucleotide residues (e.g., 3'-hexanediol residues), and cordycepin. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that prevent extension; inverted nucleotides (e.g., linked via a 3' to 3' phosphodiester to the preceding nucleotide, optionally with an exposed 5'-OH or phosphate); or proteins or peptides linked to the oligonucleotide to prevent further extension of the nascent nucleic acid chain by a polymerase. The non-extendable oligonucleotides of the present disclosure can be at least 10 bases long and can be up to 15, 20, 25, 30, 35, 40, 50 or more nucleotides long. Non-extendable oligonucleotides containing detectable labels can be used as probes.

[0225] A "binding partner" is a member of a pair of moieties that can be used to form a non-covalent association. An exemplary set of binding partners is biotin and a biotin-binding agent. Further examples of binding partners include, but are not limited to, digoxigenin / anti-digoxigenin, more generally, antibodies and their targets.

[0226] A "biotin-binding agent" is an agent (e.g., a polypeptide) that can specifically bind to biotin. Streptavidin, avidin, and NeutrAvidin are examples of biotin-binding agents. Anti-biotin antibodies are also considered biotin-binding agents.

[0227] The term "antibody" encompasses any polypeptide comprising a functional antigen-binding region having complementarity determining regions and framework regions (e.g., VH and VL domains), and includes, but is not limited to, scFv, Fab, and full-length antibodies (e.g., IgA, IgG, IgD, IgE, or IgM antibodies).

[0228] As used herein, a "kit" is a packaged combination of reagents, including, for example, one or more oligonucleotides disclosed herein. For example, the kit may include a packaged combination of one or more vials, tubes, or cartridges with multiple chambers containing reagents for isolating target polynucleotides. The reagents may include capture, primer and probe oligonucleotides, such as those described herein, and nucleotide polymerizing enzymes (e.g., DNA polymerase, reverse transcriptase, RNA polymerase, etc.). In certain embodiments, the reagents may be in liquid form, solid form (e.g., lyophilized), or semi-solid form (e.g., glass). In some embodiments, the oligonucleotide reagent and the enzyme reagent are present in the kit as components of a single lyophilized composition (e.g., pellet). In such an example, the primer, probe, and one or more enzymes (e.g., DNA polymerase) may be placed in the same reaction chamber or vessel in lyophilized form that can be reconstituted with aqueous reagents, and separate vials or tubes containing aqueous reagents are included in the same kit. The kit may further include some optional components, such as, for example, other oligomers. Other reagents that may be present in the kit include reagents suitable for performing in vitro amplification, such as buffers, salt solutions, and / or appropriate nucleotide 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, e.g., magnetic beads) for directly or indirectly immobilizing the oligomers in a sample preparation procedure. In certain embodiments, the kit further includes a set of instructions for practicing the method according to the present disclosure, and the instructions may be associated with the insert and / or packaging of the kit or its components.

[0229] As used herein, a "combination" of oligomers refers to any multiple oligomers in proximity to one another, e.g., in different containers or the same container in a kit, or in a composition or set of compositions juxtaposed to one another, e.g., a plate, rack, or other container.

[0230] 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 DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd edition (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).

[0231] B. Exemplary Compositions, Kits, Methods, and Uses The present disclosure provides oligomers, compositions, and kits useful for isolating target polynucleotides and / or attaching tags such as adapters. Isolation includes limited amount isolation (limited capture) and specific amount isolation (copy control). For example, in certain workflows, it is desirable to capture (or amplify and capture) a target polynucleotide (e.g., which may be natural DNA or RNA or amplicon) in as much amount as possible, but not more than a predetermined amount (e.g., a maximum desired value for downstream applications such as sequencing library preparation). Similarly, in certain workflows, it is desirable to capture (or amplify and capture) a predetermined specific amount (e.g., a specific number of molecules or copies of a molecule) of target polynucleotide (e.g., which may be natural DNA or RNA, amplicon, or sequencing library) for use in downstream applications (e.g., clonal amplification, including next-generation sequencing workflows). Additionally, in certain workflows, it is desirable to incorporate additional sequences into target polynucleotides, such as incorporating adapters into sequencing libraries. Oligomers containing various elements are described herein. Unless otherwise indicated, additional elements may be present before, between, or after the recited elements of the oligomer, so long as they do not interfere with its functionality.

[0232] In some embodiments, the oligomer is provided, for example, in a kit or composition. The oligomer generally comprises a target hybridizing region configured, for example, to specifically hybridize to a target polynucleotide. Although oligomers of different lengths and base compositions can be used, in some embodiments, the oligomers of the present disclosure have a target hybridizing region of a length as described in the section discussing target hybridizing sequences. In some embodiments, the oligomer comprises an additional region of sequence, as described in detail elsewhere herein, which may be located 5' of the target hybridizing region. In some embodiments, the oligomer does not comprise a second region of sequence. In some embodiments, the additional region of sequence comprises a capture sequence.

[0233] 1. Capture oligomers and combinations for copy control and other applications a. a capture oligomer comprising a capture sequence and its complement In some embodiments, a capture oligomer is provided that includes a capture sequence, an internal extension blocker, a complement of the capture sequence, and a target hybridizing sequence, where the complement of the capture sequence is configured to anneal to the capture sequence in the absence of the extended target sequence that anneals to the target hybridizing sequence and the complement of the capture sequence. As shown in Figures 1A-1C for an exemplary capture oligomer, such a capture oligomer can be captured in a target-dependent manner in that the capture sequence is initially annealed to the complement of the capture sequence (C') (Figure 1A), but becomes available for binding once the target polynucleotide is extended through C' (Figure 1B). The internal extension blocker prevents extension of the target along the capture sequence itself. Thus, the extension product of the target capture oligomer is accessible to binding to a secondary capture reagent that includes the complement of the capture sequence and a binding partner or solid substrate (Figure 1C). On the other hand, since the unbound capture oligomer does not function as an extension template, C' remains annealed to the capture sequence, and the unbound capture oligomer does not substantially interact with the secondary capture reagent. Thus, the unbound capture oligomer remains substantially in the original solution after isolation of the target capture oligomer extension product complex, which can be achieved using appropriate standard techniques based on the identity of the binding partner (e.g., biotin) or solid substrate (e.g., magnetic beads). The complex of the target and capture oligomer associated with the bead is shown diagrammatically in FIG. 2A, and the elution of the target from the bead is shown in FIG. 2B.

[0234] Such capture oligomers and their combination with appropriate secondary capture reagents are useful in any application where capture, limited capture, or copy control is desired, and where incorporation of additional sequences is desired. The capture oligomers described herein having an extendable 3' end can be used as amplification oligomers (e.g., primers) with, for example, an amplification primer, target strand, or amplicon strand, having the opposite orientation of the capture oligomer, to generate an extension product or amplicon, which can then be isolated by contacting the complex formed with a secondary capture reagent and performing an appropriate isolation step. In other embodiments, the capture oligomer is not used as an amplification oligomer or is not used for multiple rounds of extension, and the amplicon or natural DNA or RNA can be simply captured by annealing to the capture oligomer (and optionally or where appropriate, a complementary oligomer or additional oligomers as discussed herein, such as a displacer oligomer + reverse amplification oligomer, which can facilitate target-dependent displacement of the complement of the capture sequence, as discussed in detail elsewhere herein), performing an extension step with a polymerase, and then contacting the extended complex with a secondary capture reagent and performing an appropriate isolation step. In either case, the amount of the secondary capture reagent can be selected to set a specific amount or maximum amount desired for capture. Furthermore, the amounts of both the capture oligomer and the secondary capture reagent can be selected to set a specific amount or maximum amount desired 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 secondary capture reagent can be less than the amount of the capture oligomer.

[0235] An exemplary formula for a capture oligomer according to the present disclosure is: 5'-A1-CLB-A2-C'-A3-RB-A4-THS-X-3' (wherein A1 is a first additional sequence that is optionally present; C is a capture sequence, L is an optional linker, B is an internal extension blocker, A2 is an optional second additional sequence, C' is the complement of the capture sequence, A3 is an optional third additional sequence, RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence, THS is the target hybridizing sequence; X is an optional blocking moiety. It is.

[0236] In the embodiments described herein, including but not limited to those according to the above formula, the ordinal numbers (first, second, third, and fourth) preceding each additional sequence are used merely to allow specific reference to each possible individual additional sequence, and do not necessarily mean that any other additional sequences are present. Thus, a capture oligomer may include any one or any combination of two or more of the additional sequences, such as only the fourth additional sequence; the third and fourth additional sequences; the first, third, and fourth additional sequences; etc. Furthermore, any additional sequence may include multiple elements (e.g., a barcode and a primer binding site, or either or both of these + additional sequences) or may consist of a single element.

[0237] If present, the first additional sequence can include any sequence or one or more bases useful, for example, to protect the 5' end of the capture sequence or to function as a cleavage site, an enzyme recognition site (e.g., for a restriction endonuclease), or a stabilizing sequence to stabilize a duplex and / or align registers of duplex regions (e.g., a clamp). A capture oligomer including a clamp sequence is shown in FIG.

[0238] The capture sequence can include any sequence suitable for use as a capture sequence, for example, any of the capture sequence embodiments described herein, including those embodiments above and in the oligomeric element section below.

[0239] When present, the linker can be a sequence or a non-sequence element or a combination thereof. The linker can provide flexibility to facilitate self-hybridization of the complement of the capture sequence to the capture sequence, and can adopt a loop-like conformation (e.g., substantially single-stranded when the linker is a sequence element) when the complement of the capture sequence hybridizes to the capture sequence. Exemplary non-sequence linkers include alkyl, alkenyl, amide and polyethylene glycol groups [(-CH2CHO-) ] with exemplary lengths of 3, 6, 12 or 18 or more atoms. n Additional linker embodiments are provided elsewhere herein.

[0240] An internal extension blocker is an element that cannot be crossed by DNA polymerase, thus preventing the extension of the strand that is complementary to the strand that the internal extension blocker is a part of. In some embodiments, the internal extension blocker is an abasic site, a chemically modified nucleotide, a non-natural nucleotide (e.g., isoC or isoG or other non-natural nucleotides described elsewhere herein; to be an effective blocker, the complementary non-natural nucleotide must not be present in the reaction mixture, e.g., if isoC is a blocker, isoG is not present, and vice versa), or a non-sequence element (e.g., any of the non-sequence linkers discussed above). A non-sequence linker can function as an internal extension blocker, or there can be separate linkers and internal extension blockers. When a non-natural base such as isoC or isoG is used as an internal extension blocker, the DNA polymerase cannot cross the position of the modified base, and thus no complementary base is provided.

[0241] If present, the second additional sequence may comprise a tag, such as a mixed nucleotide element, as discussed below. In some embodiments, the second additional sequence comprises a sequence used to align a cleavage site or a register of a stabilizing and / or duplex region (e.g., a clamp sequence), for example, when used in combination with a corresponding complementary sequence in another additional sequence.

[0242] The complement of the capture sequence may include the complement of any sequence suitable for use as a capture sequence, for example, the complement of any of the capture sequence embodiments described herein, including the embodiments above and the oligomeric element section below. In some embodiments, the complement of the capture sequence has a complementarity level of at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% to the capture sequence. Furthermore, the complement of the capture sequence may be complementary to only a portion of the capture sequence, for example, 50%, 60%, 70%, 80%, or 90% of the capture sequence.

[0243] If present, the third additional sequence may comprise a sequence used to align (e.g., clamp sequence) a tag or cleavage site or a register of stabilized and / or duplexed regions, for example, when used in combination with a corresponding complementary sequence in the first additional sequence. The complementary clamp sequence can make self-hybridization more energetically favorable and / or help keep the complementary regions C and C' in a desired register (alignment), improving the performance of the capture oligomer as shown in the examples. A capture oligomer comprising a clamp sequence is shown in FIG. 3. In some embodiments, the third additional sequence comprises a sequence that can be used as an adapter or tag, e.g., a binding site for a primer (including a sequencing primer), an anchor oligomer or probe binding site in a subsequent reaction or step, or a cleavage or enzyme binding site (e.g., for a restriction endonuclease), or as a barcode or other tag, e.g., to identify the source of a sample or molecule and facilitate approaches involving pooling, e.g., highly parallel sequencing, etc. Such a sequence may be part of a third additional sequence along with a stabilizing (eg, clamp) sequence.

[0244] If present, a reversible extension blocker can be used to limit the extension of the complementary strand along the capture oligomer during the first period, e.g., to maintain the specificity of the binding of the target hybridizing sequence (THS) to the target polynucleotide when the capture oligomer is functioning as an amplification oligomer. Exemplary reversible extension blockers include IsoG and IsoC, as well as others described elsewhere herein, which can be unblocked by adding a complementary nucleotide (e.g., IsoC to IsoG, or vice versa). If a reversible extension blocker is used, an element different from the reversible extension blocker should be used as the internal extension blocker 5' of the complement of the capture sequence. A reversible extension blocker can also be used for the amplification oligomer (e.g., the reverse amplification primer in addition to the capture oligomer in the extension / amplification reaction) located 5' of the THS and 3' of any additional sequence present. As mentioned above, this can maintain the specificity of the binding of the THS of the amplification oligomer to the target polynucleotide during the amplification process, among other advantages. The reversible extension blocker can be reversed after amplification, and the complement to the additional sequence can be made in a single extension step, thus incorporating the additional sequence into the amplicon. If the capture oligomer also contains a reversible extension blocker, it can also be optionally reversed, and the complement of the capture oligomer is made (as described above) to the internal extension blocker simultaneously with a similar process of the amplification oligomer. This single extension step can optionally be performed in the same reaction mixture as the amplification reaction. This represents a quick and simple way to maintain specificity while still incorporating additional sequences into the product (which can be used, for example, for the addition of adapters in sequencing libraries).

[0245] If present, the fourth additional sequence may comprise an adaptor or tag, for example, as a binding site sequence for primers, probes, anchor oligos, etc., or to function as a barcode or other tag, for example, to identify the source of a sample or molecule and facilitate pooling approaches, such as highly parallel sequencing. Such sequences may be part of the fourth additional sequence, along with a stabilizing sequence, an alignment sequence, a cleavage site, or an enzyme binding site. In some embodiments, the fourth additional sequence comprises a stabilizing or alignment sequence.

[0246] In any of the foregoing embodiments in which any of the described additional sequences (or their complements formed by extension along the additional sequences) contain tags useful for downstream processes (e.g., addition of adapters useful for library preparation, clonal amplification, sequencing or data analysis), tagging can be combined with target capture to streamline the overall workflow.

[0247] The target hybridizing sequence can be any sequence of sufficient length and complementarity to hybridize to a given target, for example, any of the target hybridizing sequence embodiments described herein, including the embodiments above and the oligomeric element section below.

[0248] If present, the blocking moiety may be any moiety that prevents the extension of the 3' end by polymerase. Exemplary blocking moieties are described in detail elsewhere herein. The blocking moiety may prevent the extension and subsequent capture of the capture oligomer dimer that would otherwise occur, for example, when the capture oligomer concentration is high and / or when the target hybridization sequence may dimerize. See Figure 6.

[0249] i. Combination In some embodiments, a combination is provided that includes a capture oligomer according to the present disclosure and one or more additional oligomers. The additional oligomers may include any of the following, or any combination of two or more of the following: a complementary oligomer for use in capturing a target molecule (which may be circular) without the capture oligomer binding to a site that includes the 3' end of the target (see Figures 11A-11B for an illustration of this combination and its use to capture a circular target molecule); an amplification oligomer that, for example, binds to a target polynucleotide in a reverse orientation relative to the capture oligomer; a pair of amplification oligomers that, for example, are configured to amplify a target; a secondary capture reagent that, for example, includes a complement of the capture sequence and a binding partner or solid support; a splint oligomer; a blocker oligomer; or a displacer oligomer. Such additional oligomers are described in detail elsewhere herein. The combination may also include one or more additional capture oligomers, for example, for performing multiplex capture of multiple target polynucleotides. The additional capture oligomer may be accompanied by additional oligomers such as suitable reverse amplification oligomers, displacer oligomers, blocker oligomers, etc. When multiple capture oligomers are used, they may have the same or sufficiently similar capture sequences so that a single secondary capture reagent can be used. Alternatively, they may have different capture sequences, e.g., when one target is present in high abundance, the resulting complex with the extended capture oligomer does not saturate the secondary capture reagent to eliminate the low abundance target complex.

[0250] In some embodiments, the combination includes a capture oligomer as described herein, which includes a complement of the capture sequence, at least a second, third, or fourth additional sequence, and other elements, and a complementary oligomer, which includes, in a 5' to 3' direction, a target hybridizing sequence, and a complement of at least a portion of the second, third, or fourth additional sequence. See FIG. 11A. The target hybridizing sequence of the complementary oligomer should bind to a portion of the target that is close to (3', according to the orientation of the target) the target hybridizing sequence of the capture oligomer. The complement of at least a portion of the second or third additional sequence should be configured to anneal to the capture oligomer in a target-dependent manner (i.e., should not anneal in the absence of the target). The ternary complex of target, capture oligomer, and complementary oligomer is a substrate for extension of the 3' end of the complementary oligomer by a polymerase, and displacement of the capture sequence from its complement by such extension makes it available for capture using a secondary capture reagent.

[0251] b. Combinations comprising a capture oligomer and a complementary oligomer In some embodiments, a combination is provided that includes a capture oligomer and a complementary oligomer, where (a) the capture oligomer includes, in a 5' to 3' direction: a capture sequence that includes a first and a second portion, an internal extension blocker, a spacer sequence that includes a first and a second portion, and a target hybridization sequence; (b) the complementary oligomer includes, in a 3' to 5' direction: a complement of the second portion of the capture sequence, and a complement of at least a first portion of the spacer sequence, and the complement of the second portion of the capture sequence and the complement of the first portion of the spacer sequence are configured to anneal to the capture oligomer simultaneously in the absence of the complement of the spacer sequence.See FIG. 10A for a description of such a combination and its use.

[0252] Such capture oligomers have the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3' where A1 is a first additional optional sequence, C1 is a first portion of the capture sequence, C2 is a second portion of the capture sequence, B is an internal extension blocker, A2 is an optional second additional sequence, S1 is a first portion of the spacer sequence, S2 is a second portion of the spacer sequence, A3 is an optional third additional sequence, RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence, THS is the target hybridizing sequence; X is an optional blocking moiety. may have:

[0253] The complementary oligomer has the formula: 5'-S1'-A2'-L-C2'-X-3' where S1' is the complement of the first portion of the spacer sequence, A2' is the optional complement of a second additional sequence optionally present in the capture oligomer; L is an optional linker, C2' is the complement of the second portion of the capture sequence; X is an optional blocking moiety. may have:

[0254] Figure 10A shows an exemplary combination of capture oligomers according to the above description. In the absence of target, complementary oligomers are annealed to the capture oligomers. Following hybridization to the target, the extension of the 3' end displaces the complementary oligomers. The capture sequence remains substantially single-stranded due to the extension of the complementary oligomers, displacement and generation of duplexes in the "S" region (which blocks reannealing of complementary oligomers), and can be contacted by a secondary capture reagent that includes the complement of the capture sequence and a binding partner or bead, facilitating the subsequent isolation step.

[0255] Figure 13 shows a different exemplary workflow for using a combination of capture oligomers according to the above description, in which the complementary oligomer is provided after the extension of the target along the capture oligomer, rather than before the capture oligomer is annealed to the target. Additionally, in the exemplary workflow shown in Figure 13, a reverse amplification oligomer is provided that is utilized in conjunction with the capture oligomer in an amplification reaction (e.g., PCR) to generate a product in which a spacer region is incorporated into the amplicon. This spacer region is an arbitrary sequence. In some embodiments of this workflow, the 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 sequence during the annealing stage of the reaction, and still annealing to the capture oligomer in the absence of the complement of the spacer sequence after the amplification reaction is completed.

[0256] If present, the first additional sequence may comprise a tag. In some embodiments, the first additional sequence comprises one or more bases useful, for example, to protect the 5' end of the capture sequence or to function as a cleavage site or enzyme recognition site (e.g., a restriction endonuclease).

[0257] The first and second portions of the capture sequence form a capture sequence, e.g., any sequence suitable for use as a capture sequence, e.g., any of the capture sequence embodiments described herein, including the embodiments above and the oligomeric element section below.

[0258] An internal extension blocker is an element that cannot be crossed by DNA polymerase. In some embodiments, the internal extension blocker is an abasic site, a chemically modified nucleotide, a non-natural nucleotide (e.g., isoC or isoG, or any other non-natural nucleotide described herein that does not template the addition of a natural nucleotide), or a non-sequence element (e.g., any of the non-sequence linkers described above). A non-sequence linker can function as an internal extension blocker. When a modified base such as isoC or isoG is used as an internal extension blocker, the DNA polymerase cannot cross the position of the modified base, so no complementary base is provided.

[0259] If present, the second additional sequence comprises an adaptor or tag, for example, as a binding site sequence for primers, probes, anchor oligos, etc., or as a barcode or other tag, for example, to identify the source of a sample or molecule and facilitate pooling approaches, such as highly parallel sequencing. Such sequences may be part of the second additional sequence, along with a stabilizing sequence, an alignment sequence, a cleavage site, or an enzyme binding site. In some embodiments, the second additional sequence comprises a stabilizing or alignment sequence.

[0260] The first and second parts of the spacer sequence form a spacer sequence. At least the first part of the spacer sequence is responsible for binding of a complementary oligomer. The spacer sequence is configured to serve as a template for extension when a target polynucleotide including its 3' end is annealed to a capture oligomer, such extension resulting in the displacement of the complementary oligomer, thereby making the capture sequence accessible to a secondary capture reagent. The spacer sequence should be different from the target hybridization sequence, the capture sequence, and their complements, i.e., it should not substantially hybridize to any of them. The spacer sequence or the first or second part thereof may also include any of the elements described above with respect to the second additional sequence.

[0261] If present, the third additional sequence may include an adapter or tag, e.g., as a target binding site sequence that can be used as a primer, or a probe, anchor oligo, etc. in a subsequent reaction or step, or any sequence or function as a barcode or other tag, e.g., to identify the source of a sample or molecule and facilitate approaches involving pooling, e.g., highly parallel sequencing. Such target sequences may be part of the third additional sequence along with a clamp stabilizing sequence, an alignment sequence, a cleavage site, or an enzyme binding site sequence. In some embodiments, the third additional sequence includes a stabilizing or alignment clamp sequence.

[0262] If present, the reversible extension blocker can be used to limit the extension of the complementary strand along the capture oligomer during a first period of time, e.g., to maintain the specificity of the binding of the target hybridizing sequence to the target polynucleotide when the capture oligomer is functioning as an amplification oligomer. Exemplary reversible extension blockers include IsoG and IsoC, as well as other members of the pairs of non-natural nucleotides described elsewhere herein, which can be unblocked by adding a complementary nucleotide (e.g., IsoC for IsoG, or vice versa). If a reversible extension blocker is used, a different element than the reversible extension blocker should be used as the internal extension blocker 5' of the complement of the capture sequence.

[0263] If present, the fourth additional sequence may comprise an adaptor or tag, for example, as a binding site sequence for primers, probes, anchor oligos, etc., or to function as a barcode or other tag, for example, to identify the source of a sample or molecule and facilitate pooling approaches, such as highly parallel sequencing. Such sequences may be part of the fourth additional sequence, along with a stabilizing sequence, an alignment sequence, a cleavage site, or an enzyme binding site. In some embodiments, the fourth additional sequence comprises a stabilizing or alignment sequence.

[0264] In any of the foregoing embodiments in which any of the described additional sequences (or their complements formed by extension along the additional sequences) contain tags useful for downstream processes (e.g., addition of adapters useful for library preparation, clonal amplification, sequencing or data analysis), tagging can be combined with target capture to streamline the overall workflow.

[0265] The target hybridizing sequence can be any sequence of sufficient length and complementarity to hybridize to a given target, for example, any of the target hybridizing sequence embodiments described herein, including the embodiments above and the oligomeric element section below.

[0266] If present, the blocking moiety may be any moiety that prevents the extension of the 3' end by polymerase. Exemplary blocking moieties are described in detail elsewhere herein. The blocking moiety may prevent the extension and subsequent capture of the capture oligomer dimer that would otherwise occur, for example, when the capture oligomer concentration is high and / or when the target hybridization sequence may dimerize. See Figure 6.

[0267] Turning to the complementary oligomer, the complement of the first portion of the spacer sequence serves to stabilize a complex between the capture oligomer and the complementary oligomer that did not serve as a template for extension of the target polynucleotide. Although not required, the complementary oligomer may include the complement of the entire spacer sequence.

[0268] When a complementary oligomer is used in conjunction with a capture oligomer that includes a second additional sequence, it may be useful to include the complement of the second additional sequence in the complementary oligomer to facilitate binding to the capture oligomer.

[0269] If present, the linker, which may be a sequence or a non-sequence element as discussed above for linkers, can provide appropriate spacing depending on the size and nature of the internal extension blocker in the capture oligomer to promote simultaneous annealing of both the complement of the first portion of the spacer and the complement of the second portion of the capture sequence to the corresponding portions of the capture oligomer.

[0270] The complement of the second portion of the capture oligomer should be of sufficient length and content to anneal to the capture oligomer together with the complement of the first portion of the spacer sequence (and, if applicable, other portions of the complementary oligomer that are complementary to sequences in the capture oligomer), but insufficient to anneal independently of the complement of at least the first portion of the spacer sequence. In other words, when the complement of at least the first portion of the spacer sequence is displaced, such as by extension of the target polynucleotide along the spacer sequence, the complement of the second portion of the capture sequence should dissociate, since it lacks sufficient affinity to bind to the second portion of the capture sequence without the energetic contribution of additional hybridization by other portions of the oligomer.

[0271] If present, the blocking moiety may be any moiety that prevents the extension of the 3' end by a polymerase. Exemplary blocking moieties are described in detail elsewhere herein. The blocking moiety may prevent the extension of a complementary oligomer along any sequence that, upon binding thereto, may create undesirable by-products, primer-dimers, etc.

[0272] i. Further Combinations; Kits and Reaction Mixtures In some embodiments, further combinations are provided that include one or more additional oligomers. The additional oligomers may include any of the following, or any combination of two or more of the following: a complementary oligomer for use in capturing a target molecule (which may be circular) without the capture oligomer binding to a site that includes the 3' end of the target; an amplification oligomer that, for example, binds to a target polynucleotide in a reverse orientation relative to the capture oligomer; a pair of amplification oligomers that are configured to amplify the target; a secondary capture reagent that, for example, includes a complement of the capture sequence and a binding partner or solid support; a splint oligomer; a blocker oligomer; or a displacer oligomer. Such additional oligomers are described in detail elsewhere herein. The combinations may also include one or more additional capture oligomers, for example, for performing multiplex capture of multiple target polynucleotides, which may involve one or more additional suitable complementary oligomers (and / or complementary oligomers that bind to two or more or all of the capture oligomers may be provided). The additional capture oligomer may be accompanied by additional oligomers such as suitable reverse amplification oligomers, displacer oligomers, blocker oligomers, etc. When multiple capture oligomers are used, they may have the same or sufficiently similar capture sequences so that a single secondary capture reagent can be used. Alternatively, they may have different capture sequences, e.g., when one target is present in high abundance, the resulting complex with the extended capture oligomer does not saturate the secondary capture reagent to eliminate the low abundance target complex.

[0273] 2. Detailed Description of Oligomeric Elements and Other Reagents and Components A oligomeric element i. Capture sequence Any capture sequence that can be bound by a suitable complement can be used. In some embodiments, the capture sequence comprises a polyA or polyT 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 polyA sequence comprises 25 or 30 consecutive A residues. In some embodiments, the polyT sequence comprises 25 or 30 consecutive T residues. The T residues include U residues for the purpose of qualifying as polyT sequences, unless otherwise indicated. In some embodiments, the capture sequence is any sequence that does not exist in the target polynucleotide. Any sequence can be selected or designed to provide desired properties for a given process, for example, to obtain desired thermostability, affinity, and kinetic properties. In some embodiments, when the target polynucleotide is or comprises a sequence derived from a gene, the capture sequence is any sequence that does not exist in the genome of the gene. In some embodiments, when the target polynucleotide is or comprises a sequence derived from an organism, the capture sequence is any sequence that does not exist 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.

[0274] ii. The complement of the capture sequence Any sequence sufficiently complementary to the capture sequence to bind with an appropriate level of specificity and stability can be used as the complement of the capture sequence. In some embodiments, the complement of the capture sequence comprises at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% complementary residues of the capture sequence. In some embodiments, the complement of the capture sequence is configured to form a complex with the capture sequence that has a melting temperature in the range of 40° C. to 75° C., e.g., 40° C. to 50° C., 50° C. to 60° C., or 60° C. to 75° C., under hybridization conditions such as 0.4 to 1 M monovalent cation, 0 to 10 mM divalent cation (e.g., magnesium), 10 to 100 mM buffer (e.g., citrate, phosphate, Tris, borate) pH 5 to 9, and 0 to 1 mg / mL BSA.

[0275] iii. Additional sequences As described herein, a capture oligomer can include one or more additional sequences that, for example, provide additional functionality to the capture oligomer and / or the associated extension product.

[0276] The capture oligomer can include a stabilizing (e.g., clamp) sequence, such as 5' of the capture sequence and 3' of the complement of the capture sequence, which can make self-hybridization more energetically favorable and / or help align the capture oligomer and the complement sequences as desired to the capture oligomer. In some embodiments, the clamp sequence includes G and / or C residues, such as alternating G and C residues. Exemplary clamp sequences are 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the clamp sequence includes one or more affinity-enhancing modifications, as described elsewhere herein.

[0277] In some embodiments, the additional sequence is a tag, which may be provided to spatially separate other elements or to confer another property.

[0278] In some embodiments, the additional sequence provides a binding site for an adapter, such as a probe or primer. For example, the additional sequence (or its complement formed by extension along the additional sequence) can serve as a binding site for a universal primer or a sequencing primer. Thus, the inclusion of the additional sequence can streamline processes such as sequencing library preparation, which require the addition of adapter sequences, by combining adapter addition with target capture.

[0279] In some embodiments, the additional sequence provides a tag sequence, e.g., a barcode sequence, that facilitates identification of molecules that originate from a particular source or have been processed in a particular way.

[0280] In some embodiments, the additional sequence provides one or more enzyme recognition sites, such as restriction sites. Thus, the additional sequence can facilitate cleavage and subsequent ligation or molecular cloning procedures. Another example of an enzyme recognition site is the site recognized by a site-specific recombinase.

[0281] In some embodiments, the additional sequence comprises an alignment sequence, such as a mixed nucleotide segment. A mixed nucleotide segment is not a contiguous series of the same nucleotides. In some embodiments, the alignment sequence or mixed nucleotide segment is immediately 3' to the internal extension blocker, such that an extension product formed using an amplification oligomer that binds in a reverse orientation to a capture oligomer that includes the alignment sequence has the complement of 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 (e.g., has different base pairing specificity) from each of its directly adjacent nucleotides. The alignment sequence or mixed nucleotide segment may be useful for preparing substrates for circularization reactions, as described elsewhere herein.

[0282] iv. Linker In some embodiments, the linker comprises a sequence. The linker sequence may be any or may have any of the features discussed with respect to the additional sequence. In some embodiments, the linker comprises a non-sequence element. Exemplary non-sequence linker elements include alkyl, alkenyl, amide and polyethylene glycol groups having a chain length of 3-20 atoms or more, i.e., 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 repeat units, e.g., -CH2CHO- units.

[0283] v. Internal elongation blockers Various capture oligomers described herein include internal extension blockers. Any moiety that, when present in the template, prevents polymerase from continuing the extension reaction along the template can be used as an internal extension blocker. Depending on the polymerase, exemplary internal extension blockers can include abasic sites, chemically modified nucleotides, non-natural nucleotides such as IsoG or IsoC (to be an effective blocker, complementary non-natural nucleotides must not be present in the reaction mixture, e.g., if isoC is a blocker, isoG is not present, and vice versa), non-sequence linker elements as discussed above, or combinations thereof.

[0284] In some embodiments, the internal extension blocker comprises an abasic site. An abasic site is a position in a nucleic acid that is missing a nucleobase from the position where it would normally occur.

[0285] In some embodiments, the internal extension blocker comprises chemically modified nucleotide.Various chemically modified nucleotides are known that block DNA polymerase from elongating, such as alkylated nucleotides and modified nucleotides formed by reacting nucleotides with certain DNA damaging agents, such as chemotherapeutic agents.Other modified nucleotides that are useful as internal extension blockers include those that have backbone modification, sugar modification, base modification, and combinations thereof.

[0286] In some embodiments, the internal extension blocker comprises a non-natural nucleotide, such as IsoG (6-amino-2-ketopurine) or IsoC (2-amino-4-keto pyrimidine). IsoG and IsoC are nucleotides that do not form Watson-Crick base pairs with any of the four natural DNA nucleotides, but do pair with each other. See, e.g., Hirao et al., Proc Jpn Acad Ser B Phys Biol Sci. 2012;88(7):345-367. Thus, if (i) the template contains IsoG and (ii) no IsoC nucleotide is available, or vice versa, the polymerase has nothing to incorporate opposite IsoG or IsoC, and extension is blocked.

[0287] In some embodiments, internal extension blockers include non-sequence linker elements, such as alkyl, alkenyl, amide, and polyethylene glycol groups having chain lengths of 3-20 or more atoms, i.e., 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 repeat units, such as -CH2CHO- units.

[0288] vi. Reversible elongation blockers In some embodiments, the capture oligomer comprises a reversible extension blocker. A reversible extension blocker is an element that, when present in the template, prevents the polymerase from continuing the extension reaction along the template until the block is reversed. Examples of reversible extension blockers include members of the unnatural base pairs discussed above, such as IsoG and IsoC. See, e.g., Hirao et al., supra. An IsoG-based block can be reversed by providing an IsoC nucleotide triphosphate so that extension can continue, and vice versa. A general description of the use of reversible extension blockers is provided in FIG. 7A. In some embodiments, the amplification oligomer comprises a reversible extension blocker. Such an amplification oligomer can be used separately in an extension or amplification reaction, or simultaneously with a capture oligomer that does not contain a reversible extension blocker, or simultaneously with a capture oligomer that contains a reversible extension blocker. When an amplification oligomer and a capture oligomer, both of which contain a reversible extension blocker, are used in the same process, the respective reversal steps can be performed simultaneously or separately.

[0289] vii. Target Hybridizing Sequence Various capture oligomers according to the present disclosure include a target hybridizing sequence. The target hybridizing sequence may hybridize to a naturally occurring sequence, for example in the nucleic acid (such as DNA or RNA) of an organism, or may hybridize to a binding site or adapter sequence (e.g., added to an amplicon using an amplification oligomer containing an adapter sequence or using ligation). In some embodiments, the target hybridizing sequence has a length ranging from about 10-60 bases, about 12-50 nucleotides, about 12-40 nucleotides, about 12-35 nucleotides, about 12-30 nucleotides, about 15-30 nucleotides, or about 17-25 nucleotides. A variety of algorithms for selecting appropriate sequences for use as target hybridizing sequences are available to one of skill in the art.

[0290] In some embodiments, the target hybridizing sequence is configured to bind to a site that includes the 3' end of the target. Such binding creates a substrate for extension of the 3' end that results in strand displacement that makes the capture sequence available for binding to a secondary capture reagent, as discussed in more detail elsewhere herein, for the various oligomers and combinations described herein.

[0291] In some embodiments, the target hybridizing sequence is configured to bind to an internal site within the target. Such binding can be used in combination with displacers or other additional oligomers, or more generally in the capture methods described elsewhere herein that do not involve extending the 3' end of the target.

[0292] viii. Blocking part In some embodiments, the capture oligomer or other oligomer described herein includes a blocking moiety at its 3' end. The blocking moiety prevents the extension of the oligomer along the template. Exemplary blocking moieties include alkyl groups, non-nucleotide linkers, alkane-diols (e.g., 3'-hexanediol residues), and cordycepin. Further examples of blocking moieties include 3'-deoxynucleotides (e.g., 2',3'-dideoxynucleotides); 3'-phosphorylated nucleotides; fluorophores, quenchers, or other labels that prevent extension; inverted nucleotides (e.g., linked to the preceding nucleotide via a 3' to 3' phosphodiester, optionally with an exposed 5'-OH or phosphate); or proteins or peptides linked to the oligonucleotide to prevent extension. The use of an exemplary capture oligomer with a blocking moiety at its 3' end is shown diagrammatically in FIG. 5. Such capture oligomers can be useful, for example, in combination with an appropriate reverse amplification oligomer (which may also provide additional sequence to be incorporated into the target) to isolate single-stranded targets. A capture oligomer having a blocking moiety at its 3' end can also provide the advantage of preventing extension and subsequent capture of the dimerized capture oligomer. See Figure 6 for an exemplary illustration.

[0293] ix. Affinity-Enhancing Modifications In some embodiments, the capture oligomer or other oligomers described herein contain one or more affinity-enhancing modifications in the portion of the sequence responsible for binding to another molecule, such as a target polynucleotide or a secondary capture reagent. Affinity-enhancing modifications are useful to ensure that the oligomer effectively competes with its target even in the presence of competing oligomers that contain sequences that bind to the same target, such as primers that are not incorporated by the amplification reaction. Competing oligomers may be present in excess relative to the capture oligomer and / or target, meaning that effectively competing for a relatively high affinity for the target has a significant effect, rather than being dominant, on whether some of the capture oligomers bind to the target.

[0294] Exemplary affinity enhancing modifications include 5-methylation of cytosine; use of 2-aminopurine; 2'-fluoro modification; 2'-methoxy modification; C-5-propyne; and constrained ethyl (cEt) substitutions. Embodiments of oligomers that can affect the stability of a hybridization complex include PNA oligomers, LNAs (locked nucleic acids that stabilize nucleotides in certain conformations and reduce the extent to which entropy impairs the free energy of hybridization), or ZNAs (Zip Nucleic Acids), oligomers that contain charged linkages (e.g., phosphorothioates) or neutral groups (e.g., methylphosphonates) that affect the overall charge, charge density, or steric association of the hybridization complex.

[0295] b. Secondary capture reagent In some embodiments, a secondary capture reagent is present in combination with or used with a capture oligomer as described herein. The secondary capture reagent may comprise a complement of the capture sequence in the capture oligomer and (i) a binding partner or (ii) a solid support, such as a bead (such as a magnetic bead), well, flat surface, packed column, fiber, resin or gel, and may hybridize to a target polynucleotide and / or may have undergone extension to include a copy of the target polynucleotide sequence, facilitating isolation of a complex comprising the secondary capture reagent and the capture oligomer.

[0296] Any suitable binding partner can be used herein, including, for example, any of the binding partners described in the definitions section, hi some embodiments, the binding partner is biotin.

[0297] C. Blocker oligomer In some embodiments, a blocker oligomer is present in combination with or used with a capture oligomer as described herein. A blocker oligomer generally contains a complement of an additional sequence or at least a portion thereof, and can be used to promote specific hybridization of the target hybridizing sequence of the capture oligomer (or an amplification oligomer used to prepare an amplicon for capture by the capture oligomer) to its target by competing for hybridization with the complement of the additional sequence (i.e., blocking, or at least reducing the ability of, the additional sequence in the capture oligomer to hybridize to its complement). See FIG. 9 for an explanation of the general principles of using blocker oligomers in amplification reactions. A blocker oligomer may contain a blocking moiety at its 3' end, for example, when extension of the blocker oligomer is undesirable. The use of blocker oligomers can reduce the extent to which products misprime and undergo further amplification during the amplification reaction, in that only targets that contain substantially the complement of the target hybridizing sequence are good substrates for hybridization to capture or amplification oligomers, regardless of the presence of additional sequence complements.

[0298] D. Splint Oligomer In some embodiments, the splint oligomer is present in combination with the capture oligomer described herein or is used with the capture oligomer described herein. The splint oligomer can be used to facilitate the ligation of the extension product and circularize it. See Figure 12. The splint oligomer can include the complement of the sequence that is not present in the capture oligomer; the second additional sequence that is present in the capture oligomer; and the complement of the capture sequence of the capture oligomer. Optionally, the splint oligomer can further include a fourth additional sequence that is present in the capture oligomer.

[0299] The splint oligomer is configured to anneal to a target polynucleotide extended from its 3' end along a capture oligomer that includes an optional fourth additional sequence, the complement of the capture sequence, and a second additional sequence, followed by an internal extension blocker, in a 3' to 5' direction, such that the complement of the second additional sequence is at the 3' end of the target polynucleotide.

[0300] The complement of the splint oligomer of a sequence not present in the capture oligomer is complementary to a sequence at the 5' end of the target polynucleotide (i.e., distal to the site bound by the capture oligomer), which may be an adapter sequence added during amplification or may correspond to part or all of the amplification oligomer used to amplify the target polynucleotide.

[0301] The second additional sequence is configured to anneal to its complement located at the 3' end of the target polynucleotide. The complement of the capture sequence, and if present, the fourth additional sequence, also anneal to the corresponding sequence near the 3' end of the target polynucleotide. When the target polynucleotide is annealed to the splint oligomer, it becomes a substrate for circularization by ligation. For a description of an exemplary splint oligonucleotide according to the present specification and its use for circularization, see Figure 12. Circularization of a target molecule can be useful, for example, in preparing a target for use in a rolling circle amplification procedure.

[0302] e. Displacer Oligomer In some embodiments, a displacer oligomer (e.g., a displacer primer) is present in combination with or used with a capture oligomer as described herein. The displacer oligomer can be used to displace the extended capture oligomer from the template strand of the target polynucleotide. See, for example, Figures 8A and 8B. In some embodiments, the displacer oligomer is provided in combination with a capture oligomer having a target hybridizing sequence that binds to an internal site within the target (e.g., a site within the target polynucleotide, amplicon, or additional sequence incorporated during a previous extension or amplification step) positioned relative to the binding site of the displacer oligomer to facilitate displacement by extension of the displacer oligomer.

[0303] In some embodiments, the combination comprising a displacer oligomer (e.g., displacer primer) and a capture oligomer further comprises an amplification oligomer (e.g., primer) configured to bind to the extended capture oligomer (and thus have a reverse binding orientation relative to the capture and displacer oligomers). Extension of the amplification oligomer may result in displacement of the complementary oligomer or capture sequence, depending on the configuration of the capture oligomer and / or combination, and thus make the capture sequence of the extended capture oligomer available to interact with a secondary capture reagent. As described elsewhere, the amplification oligomer may also be used to incorporate additional sequences (e.g., tags such as adapter sequences) at the end of the extended capture oligomer distal to the original capture oligomer sequence (e.g., a complement to the additional sequence of the amplification oligomer is created via further extension of the extended and displaced capture oligomer, see FIG. 8A). Thus, in a simple and rapid single-step one-pot method, products can be generated that contain additional sequences as well as accessible capture sequences at both ends.

[0304] f. Amplification oligomers In some embodiments, an amplification oligomer (e.g., a primer) is present in combination with or used with a capture oligomer as described herein. For example, the amplification oligomer can be configured to anneal in an opposite orientation to the target hybridizing sequence of the capture oligomer. See FIG. 4A for an example. The amplification oligomer can include a target hybridizing sequence and, optionally, an additional sequence 5' of the target hybridizing sequence. The additional sequence can be incorporated upon further extension of the strand containing the extended capture oligomer using the additional sequence of the amplification oligomer as a template to provide one or more tags, which can include one or more adapter sequences. In some embodiments, the amplification oligomer includes a reversible internal extension blocker, e.g., 3' of one or more elements other than the THS, such as between the target hybridizing sequence and the additional sequence 5' of the target hybridizing sequence. The combination including the amplification oligomer can further include one or more additional oligomers, such as displacer oligomers, splint oligomers, for example, as described elsewhere herein.

[0305] G. Kit In some embodiments, kits are provided that include a capture oligomer or combination thereof, such as any of the capture oligomers or combinations described herein, and further include one or more additional elements, such as reagents, buffers, or other substances for use with the capture oligomers or combinations thereof, and / or instructions for using the capture oligomers or combinations thereof. Exemplary reagents include dNTPs, DNA polymerase, sodium salts, magnesium salts, and beads, including, for example, a second binding partner that binds to a first binding partner that is present with the complement of the capture sequence or the complement of the capture sequence in a secondary capture reagent.

[0306] In some embodiments, the capture oligomer is 7 ~10 13 Molecules / Reactions, 10 9 ~10 12 molecules / reaction, or 10 10~10 12 Present in the kit in a range of amounts of molecule / reaction.

[0307] In some embodiments, the secondary capture reagent, which comprises the complement of the capture oligomer, is 10 3 ~10 14 molecules / reaction, or 10 3 ~10 9 molecules / reaction, or 10 5 ~10 13 molecules / reaction, or 10 5 ~10 8 molecules / reaction, or 10 6 ~10 13 molecules / reaction, or 10 6 ~10 8 A range of molecules / reactions are present in the kit.

[0308] In some embodiments, the kit comprises a capture oligomer in a liquid, frozen, or lyophilized state.

[0309] In some embodiments, the kits each include at least a first and a second container that includes a capture oligomer as described herein and one or more additional oligomers (e.g., a complementary oligomer, a secondary capture reagent, or an amplification, splint, blocker, second or displacer oligomer). Alternatively, such combinations of oligomers can be supplied in a single container.

[0310] In some embodiments, the kit comprises a solid support / bead that comprises a second binding partner (eg, streptavidin) configured to bind to the binding partner of the secondary capture reagent.

[0311] h. Composition Compositions are also provided that include the capture oligomers or combinations described herein. Any of the oligomers or combinations described herein can be present in the composition. In some embodiments, the composition is in the form of a lyophilizate or solution.

[0312] In some embodiments, a reaction mixture is provided that includes a capture oligomer or combination thereof, such as any capture oligomer or combination described herein. The reaction mixture may further include one or more target polynucleotides and / or one or more reagents (e.g., any of the reagents discussed herein in connection with the kits or methods).

[0313] The reaction mixture may further comprise one or more target polynucleotides (e.g., any of the targets discussed herein in connection with the methods). In some embodiments, the target polynucleotide comprises a sequence derived from the DNA or RNA (e.g., genomic DNA or mRNA) of the target organism, and an additional sequence, and the target hybridizing sequence of the capture oligomer is configured to anneal to the additional sequence of the target polynucleotide. The additional sequence may have been added in a previous reaction, such as an extension, ligation, or amplification reaction. For example, the additional sequence may be a sequence that is not present in the DNA and RNA of the target organism. Such an additional sequence may have been added in a previous extension reaction using a primer that includes the additional sequence. This approach facilitates multiplex capture or reuse of the capture oligomer design with different targets to which the same additional sequence is attached. In some embodiments, the composition comprises a plurality of target polynucleotides that include (i) an additional sequence and (ii) a sequence that is different from the DNA or RNA of the target organism, and the method comprises capturing the plurality of target polynucleotides.

[0314] 3. Detailed Description of the Method for Capturing Polynucleotides A method of capturing a target polynucleotide from a composition is provided, the method comprising: contacting the target polynucleotide with a capture oligomer described herein comprising a capture sequence and a complement of the capture sequence, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site comprising the 3' end of the target polynucleotide; The method further includes extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence that is annealed to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with the complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and isolating the complex from the composition, thereby capturing the target polynucleotide. An exemplary scheme illustrating the contacting and extension steps is shown in Figures 4A-4B. In Figure 4A, the THS of the capture oligomer binds to a sequence at the 3' end of the target. In Figure 4B, the THS of the capture oligomer binds to an additional sequence at the 3' end of the target, which may be added, for example, in a previous reaction.

[0315] Also provided herein is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the composition with a combination described herein comprising a capture oligomer and a complementary oligomer, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide or an additional sequence at a site comprising the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the spacer sequence that is annealed to the capture oligomer such that the complementary oligomer is displaced to a sufficient extent that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. The complementary oligomer of the combination may be provided to the composition at any time prior to contacting the capture sequence of the capture oligomer with a secondary capture reagent, for example, together with the capture oligomer or after extension of the target along the capture oligomer. For a description of an exemplary method according to the present specification, see Figures 10 and 13.

[0316] Also provided herein is a method of capturing a target polynucleotide from a composition, the method comprising: contacting the target polynucleotide with a capture oligomer or combination described herein, wherein the capture oligomer comprises a third or fourth additional sequence 5' of the target hybridizing sequence and a second portion of the 3' of the complement of the capture sequence or spacer sequence; contacting the target polynucleotide with a second oligomer comprising a second target hybridizing sequence 5' that is the complement of at least a portion of a third or fourth additional sequence, wherein the capture oligomer and the second oligomer form a triple-stranded junction with the target polynucleotide; extending the 3' end of the second oligomer with a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence or a complement of the spacer sequence that is annealed to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. In some embodiments of the method, the target polynucleotide is circular. See Figures 11A-11B for an illustration of an exemplary method according to the present specification. In some embodiments, the target polynucleotide is not circular. For example, the target polynucleotide may include a translocation junction (e.g., BCR-ABL, or any other translocation) that brings the binding sites of the capture oligomer and the second oligomer into close proximity. In some embodiments, the second oligomer is provided to the reaction mixture at a higher concentration than the capture oligomer. In some embodiments, the second oligomer is provided to the reaction mixture at a lower concentration than the capture oligomer.

[0317] The following further embodiments apply to any of the aforementioned methods. In some embodiments, the secondary capture reagent comprises a binding partner (e.g., biotin), and isolating comprises contacting the complex with a solid support (e.g., a bead) comprising a second binding partner (e.g., streptavidin) configured to bind to the binding partner of the secondary capture reagent. The capture oligomer may be provided in excess relative to the secondary capture reagent.

[0318] Exemplary concentration and amount ranges for the capture oligomer and, if applicable, the complementary and / or second oligomer are: 10 for the capture oligomer; 7 ~10 13 Molecules / Reactions, 10 9 ~10 12 molecules / reaction, or 10 10 ~10 12 Molecules / reaction; approximately 1.5 x 10 for complementary oligos 7 ~10 14 molecules / reaction or approximately 1.5 x 10 9 ~10 13 molecule / reaction; and 10 for the second oligomer. 6 ~10 14 molecules / reaction or 10 8 ~10 13 Exemplary ranges of secondary capture reagents are provided above.

[0319] The method is useful for capturing target polynucleotide from any source. In some embodiments, the source of target polynucleotide is a clinical specimen, a pathogen, or an environmental sample. In some embodiments, the clinical sample is a sample from a subject having or 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 having or suspected of having cancer. In some embodiments, the target polynucleotide is an extension or amplification product.

[0320] In some embodiments, the target polynucleotide is a member of a sequencing library.

[0321] In some embodiments, the extension product (extended capture oligomer) is formed by extending the capture oligomer along the target polynucleotide, where the capture oligomer comprises a third or fourth additional sequence. In some embodiments, the method further comprises contacting the extension product with a blocker oligomer comprising a non-extendable third or fourth additional sequence, optionally configured to bind to the complement 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 complement of the third or fourth additional sequence that has a higher melting temperature than a complex between the third or fourth additional sequence of the capture oligomer and the complement of the third or fourth additional sequence. This approach is useful for reducing or avoiding non-specific extension, for example, the products of a mispriming event by the capture oligomer may give rise to additional rounds of amplification by hybridization of a third or fourth additional sequence to the reverse extension products that would occur in the absence of the blocker oligomer.

[0322] In some embodiments, the method comprises: Contacting the target polynucleotide with amplification oligomers The method further comprises: an amplification oligomer comprising (i) a reverse target hybridizing sequence that binds to the target polynucleotide in a reverse orientation relative to the capture oligomer; and (ii) an additional sequence located 5' of the second target hybridizing sequence. The method further comprises extending the amplification oligomer along the target polynucleotide to form a reverse extension product, with a portion of the capture oligomer annealing to the reverse extension product. Such an embodiment is useful for incorporating additional sequences (e.g., tags such as adapters) at the end of the target distal to the end bound to the capture oligomer or the end incorporating the capture oligomer. As discussed elsewhere, extension along the capture oligomer (e.g., the extended capture oligomer) can displace the complement of the capture sequence from the capture sequence. An amplification oligomer can also be used to prime such extension.

[0323] For example, in some embodiments where the capture oligomer comprises a blocking moiety at its 3' end, the method further comprises isolating a complex comprising a reverse extension product (e.g., made as described above or using an amplification oligomer that does not necessarily include additional sequence) annealed to the capture oligomer, where the extension product is substantially single-stranded 5' of the target hybridizing sequence of the capture oligomer, and the capture element of the capture oligomer is replaced by the extension of the reverse extension product, as shown, for example, in FIG. 5. An extension product is considered to be substantially single-stranded if at least half of the molecule is not hybridized to another strand. Thus, for example, a short stretch of self-hybridized nucleic acid does not prevent the molecule or a portion thereof from being substantially single-stranded.

[0324] In some embodiments in which reverse extension products are created, the capture oligomer is extendable, and the method further comprises extending the capture oligomer along the reverse extension product, thereby forming a second extension product that includes the complement of the amplification oligomer. The second extension product will include any additional sequence present in the capture oligomer and the complement of any additional sequence present in the reverse amplification oligomer. In this manner, products can be generated that include additional sequences (e.g., tags such as one or more adaptors) near or at one or both ends (e.g., both ends).

[0325] In some embodiments, the target polynucleotide comprises a sequence derived from the DNA or RNA of the target organism and an additional sequence, and the target hybridizing sequence of the capture oligomer is configured to anneal to the additional sequence of the target polynucleotide. The additional sequence may have been added in a previous reaction, such as an extension, ligation, or amplification reaction. For example, the additional sequence may be a sequence that is not present in the DNA and RNA of the target organism. Such an additional sequence may have been added in a previous extension reaction using a primer that includes the additional sequence. This approach facilitates multiplex capture or reuse of the capture oligomer design with different targets to which the same additional sequence is attached. In some embodiments, the composition comprises a plurality of target polynucleotides that include (i) an additional sequence and (ii) a sequence that is different from the DNA or RNA of the target organism, and the method comprises capturing the plurality of target polynucleotides.

[0326] In some embodiments, the capture oligomer comprises a reversible extension blocker located 5' of the target hybridizing sequence, and the method comprises: copying or amplifying the target polynucleotide before unblocking the reversible extension blocker; unblocking the reversible extension blocker; and copying or amplifying the target polynucleotide one or more additional times. In some embodiments, only a single cycle of amplification / extension is performed after unblocking the reversible extension blocker and before capturing the target polynucleotide. In some embodiments, the amplification oligomer comprises a reversible extension blocker and functions similarly to the above. In some embodiments, an amplification oligomer and a capture oligomer, both of which have a reversible extension blocker, are used together in the same process. In such cases, the reversible extension blocker of the amplification capture oligomer can be reversed simultaneously or in a separate step. 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 comprises providing a complementary member of the non-natural nucleotide pair.

[0327] In some embodiments, the method includes circularizing the extension product of the target polynucleotide. For example, when the capture oligomer includes a mixed nucleotide segment between the extension blocker and the first portion of the spacer sequence or the complement of the capture sequence (e.g., immediately 3' of the internal extension blocker). The method includes extending the 3' end of the target polynucleotide along the capture oligomer to the internal extension blocker, thereby forming an extension product that includes the complement of the mixed nucleotide segment at its 3' end. The method further includes contacting the extension product with a splint oligonucleotide in the 5' to 3' direction that includes: the complement of the 5' end segment of the extension product; the mixed nucleotide segment; the complement of the capture sequence; and, optionally, a segment complementary to the segment in the extension product immediately 5' of the capture sequence. The method further includes ligating the 5' end of the extension product to the 3' end of the extension product. In some embodiments, the 5' end segment of the extension product comprises at least a portion of the sequence of the amplification oligomer used to generate the target polynucleotide, e.g., at least a portion of the target hybridizing sequence of the amplification oligomer, or (if applicable) at least a portion of an additional sequence of the amplification oligomer, where the additional sequence is 5' of the target hybridizing sequence of the amplification oligomer. See Figure 12 for a description of an exemplary method according to the present specification.

[0328] In some embodiments, any of the foregoing methods include adding additional sequences to one or both ends of the target polynucleotide. For example, such methods include contacting the target polynucleotide with a capture oligomer described herein, where the target hybridizing sequence of the capture oligomer anneals to a site upstream of the 3' end of the target polynucleotide; extending the 3' end of the capture oligomer along the target polynucleotide, thereby forming a first extended strand; contacting the target polynucleotide with a displacer oligomer that includes a displacer target hybridizing sequence that anneals to the target polynucleotide downstream of the target hybridizing sequence of the capture oligomer; extending the displacer oligomer along the target polynucleotide, thereby displacing the first extended strand. It may include, In some cases, the capture oligomer and the displacer are added to the composition simultaneously or sequentially (e.g., to initiate extension of the capture oligomer prior to displacement by extension of the displacer oligomer). The capture oligomer may further comprise an additional sequence (e.g., a third or fourth additional sequence) located 5' of the target hybridizing sequence. In some embodiments, the THS of the capture oligomer has a higher Tm or higher affinity for binding to its complement than the THS of the displacer oligomer does to its complement, or the capture oligomer is provided at a higher concentration than the displacer oligomer. This can facilitate binding by the capture oligomer prior to extension of the displacer oligomer and extension (or at least initiation of extension) of the capture oligomer. If the Tm and / or affinity of the capture oligomer is higher, this can be further facilitated by first incubating the reaction mixture at a higher temperature that preferentially allows binding and extension of the capture oligomer relative to binding and extension of the displacer oligomer, and then at a lower temperature that allows binding and extension of the displacer oligomer.

[0329] In some embodiments, such methods further include contacting a first extension of the target polynucleotide with a reverse amplification oligomer that includes a reverse target hybridizing sequence configured to bind to the first extension, and extending the reverse amplification oligomer, thereby forming a second extension. The reverse amplification oligomer may include additional sequence 5' of its target hybridizing sequence.

[0330] In some embodiments, such methods further include contacting the first extension strand with a reverse amplification oligomer that includes a reverse target hybridizing sequence configured to bind to the 3' end of the first extension strand, and extending the reverse amplification oligomer, thereby forming a second extension strand. The reverse amplification oligomer may include an additional sequence 5' of the target hybridizing sequence, in which case the first extension strand is further extended using the additional sequence as a template to create the complement of the additional sequence. See Figure 8A.

[0331] In some embodiments, the target polynucleotide comprises a sequence derived from the DNA or RNA of the target organism and an additional sequence not present in the DNA and RNA of the target organism, and the target hybridizing sequence of the capture oligomer is configured to anneal to the additional sequence of the precursor of the target polynucleotide. The displacer oligomer is also configured to anneal to the additional sequence of the precursor of the target polynucleotide, and the binding site of the displacer oligomer in the additional sequence is downstream of the binding site of the target hybridizing sequence of the capture oligomer (see, e.g., FIG. 8B).

[0332] In some embodiments, the first strand of the target polynucleotide comprises a second adapter sequence located proximal to the sequence derived from the DNA or RNA of the target organism and distal to the target hybridizing sequence of the capture oligomer; The method further includes contacting the first strand of the target polynucleotide with a reverse amplification oligomer comprising a reverse target hybridizing sequence configured to bind to a second adapter sequence and extending the reverse amplification oligomer, thereby forming a second strand of the target polynucleotide.

[0333] In some embodiments, any of the aforementioned methods further comprise performing clonal amplification of the captured target polynucleotides. In some embodiments, such methods further comprise sequencing the clonally amplified target polynucleotides. In some embodiments, any of the aforementioned methods further comprise performing sequencing of the captured target polynucleotides. In some embodiments, the sequencing is Sanger sequencing or next-generation sequencing, optionally where next-generation sequencing includes sequencing-by-synthesis, sequencing-by-ligation, sequencing-by-hybridization, or single-molecule sequencing. Next-generation sequencing includes any form of sequencing that generates reads in a largely parallel manner compared to Sanger sequencing.

[0334] 4. Capture Oligomers and Combinations for Limited Capture and Other Applications A capture oligomer containing a self-complementary sequence In some embodiments, a capture oligomer is provided that includes a first self-complementary sequence, a target hybridizing sequence, and a second self-complementary sequence; The first and second self-complementary sequences are configured to anneal to each other when the target hybridization sequence is single-stranded, and are configured not to anneal to each other when the target hybridization sequence is annealed to its target. Such capture oligomers may be combined with a secondary capture reagent that includes the complement and binding partner of the first or second self-complementary sequence. The capture oligomer may be present in combination in an amount greater than the secondary capture reagent. Such oligomers and combinations are useful for capturing a certain amount (e.g., a limited amount or an amount equal to or less than a predetermined amount) of target polynucleotide from a composition.

[0335] In some embodiments, such a capture oligomer has the formula: 5'-SC1-THS2-THS1-L-THS2'-SC2-3'. In some embodiments, such a capture oligomer has the formula: 5'-SC2-THS2'-L-THS1-THS2-SC1-3'. In each of the above formulas, SC1 is the first self-complementary sequence; THS2 and THS1 are the second and first portions of the target hybridizing sequence, respectively; L is an optional linker; THS2' is the optional complement of the second portion of the target hybridizing sequence; and SC2 is the second self-complementary sequence.

[0336] Either the first or second self-complementary sequence can function as a capture sequence. If the first self-complementary sequence is a capture sequence, the second self-complementary sequence is the complement of the capture sequence. If the second self-complementary sequence is a capture sequence, the first self-complementary sequence is the complement of the capture sequence. Capture sequences and the complements of capture sequences are discussed in detail elsewhere herein. Any suitable capture sequence and its complement can be used.

[0337] If present, the linker may be any suitable linker, for example, any linker described elsewhere herein, including sequence and non-sequence linkers. The linker may function as a flexible element that promotes intramolecular hybridization of the first and second self-complementary sequences. If the linker comprises a sequence, the sequence in some embodiments is a sequence that does not exist in the source (e.g., organism, genome, gene, or other nucleic acid, or combination thereof) from which the target hybridizing sequence is derived or to which the target hybridizing sequence is complementary. The linker should generally not be so long as to allow intramolecular hybridization of the first and second self-complementary sequences, even when the target hybridizing sequence is annealed to its target. (See discussion below).

[0338] Target hybridizing sequences are discussed in detail elsewhere herein. Any suitable target hybridizing sequence (and the complement of a portion thereof, if present) can be used.

[0339] Secondary capture reagents are discussed in detail elsewhere herein and include the complement of a capture sequence.Any suitable secondary capture reagent can be used.

[0340] When the capture oligomer is present in the combination in an amount greater than the secondary capture reagent, the ratio can be about 10,000 to 1, 5000 to 1, 2000 to 1, 1000 to 1, 500 to 1, 200 to 1, 100 to 1, 50 to 1, 20 to 1, 10 to 1, 5 to 1 or 2 to 1.

[0341] Such capture oligomers are useful in methods for capturing target polynucleotides. The capture oligomers can assume a self-hybridized conformation in the absence of target polynucleotides. The self-hybridized conformation involves annealing the first and second self-complementary sequences to each other, thus rendering the capture oligomer substantially unable to interact with the complement of the capture sequence of the secondary capture reagent. However, the target hybridization sequence is at least partially single-stranded. If the target polynucleotide is present, the target hybridization sequence can hybridize to it. When the target hybridization sequence is thus substantially double-stranded, intramolecular hybridization of the first and second self-complementary sequences cannot occur because they are too far apart, although there are other potential factors. Thus, the capture sequence (i.e., one of the first and second self-complementary sequences) is substantially single-stranded and can hybridize to the secondary capture reagent. It may be beneficial for hybridization of the target hybridizing sequence to be more energetically favorable than intramolecular hybridization of the first and second self-complementary sequences, so that the capture oligomers preferentially hybridize to available target polynucleotides. Excess capture oligomers may also be used to drive the formation of complexes with target polynucleotides.

[0342] The complex of secondary capture reagent, capture oligomer, and target can then be isolated, for example, using standard techniques appropriate for the binding partner of the secondary capture reagent or the solid support. The amount of captured polynucleotide can be limited or determined by the amount of secondary capture reagent, or by the amount of capture oligomer in combination with the amount of secondary capture reagent.

[0343] b. A combination comprising a capture oligomer and a complementary oligomer having a complement of a portion of the capture sequence. In some embodiments, a combination is provided that includes a capture oligomer and a complementary oligomer, (a) The capture oligomer comprises, in the 5' to 3' direction: a capture sequence comprising a first and a second portion; and A target hybridizing sequence comprising a second and a first portion Including, (b) The complementary oligomer comprises, in the 3' to 5' direction: the complement of a second portion of the capture sequence, and The complement of the second portion of the target hybridizing sequence wherein the complement of the second portion of the capture sequence and the complement of the second portion of the target hybridizing sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the target hybridizing sequence. Figure 10B provides an illustration of an exemplary oligomer according to these embodiments. Optional additional elements may be present as described in the further embodiments listed above and / or as shown in Figure 10B (e.g., any individual element of Figure 10B or any combination thereof).

[0344] Combinations can be used to perform limited capture in that complementary oligomers can be configured to bind to free capture oligomers, rather than to capture oligomers bound to target polynucleotides. For example, the binding of the target hybridization sequence of the capture oligomer to the target polynucleotide can be more energetically favorable than the binding of the complement of the second part of the capture sequence to the second part of the target hybridization sequence. In the absence of target polynucleotide, complementary oligomers bind to the capture oligomer and block access to the capture sequence (C1+C2 in FIG. 10B) to an extent sufficient to block the binding of the capture sequence by the complement of the capture sequence in the secondary capture reagent, which can be any of the secondary capture reagents described elsewhere herein.

[0345] Accordingly, there is also provided a method of capturing a target polynucleotide from a composition, the method comprising: contacting the composition with a combination as described herein above or any further embodiment thereof, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide; contacting the capture oligomer with a complementary oligomer before or after the capture oligomer anneals to the target polynucleotide, where if the complementary oligomer anneals to the free capture oligomer and partially occupies its capture sequence, the complementary oligomer does not anneal to a complex that includes the capture oligomer annealed to the target polynucleotide, and where contacting the capture oligomer with the complementary oligomer occurs before the capture oligomer anneals to the target polynucleotide, annealing of the target hybridizing sequence to the target polynucleotide results in dissociation of the complementary oligomer from the capture oligomer; contacting the capture sequence of the capture oligomer complexed with the target polynucleotide with a secondary capture reagent comprising a complement of the capture sequence and (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and Isolating the complex from the composition, thereby capturing the target polynucleotide. Optional additional elements may be present as described in the further embodiments listed above and / or as shown in FIG. 10B (e.g., any individual element of FIG. 10B or any combination thereof).

[0346] 5. Differential Capture Methods and Oligomer Combinations Also provided herein are methods and oligomer combinations for differentially capturing (e.g., with different affinities) capture oligomers based on whether they are complexed with target polynucleotides. Such methods allow for the elution of target polynucleotides without substantially eluting capture oligomers that are not associated with target polynucleotides. The capture sequences, target hybridization sequences, and other elements of oligomers in such combinations and methods can be, for example, any of the capture sequences, target hybridization sequences, etc. described herein, consistent with the characteristics of oligomers, combinations, and methods described below.

[0347] In some embodiments, such methods include contacting a target polynucleotide with a capture oligomer that includes, in a 5' to 3' direction: a capture sequence; an optional internal extension blocker; an optional spacer sequence; and a target hybridizing sequence configured to anneal to the target polynucleotide. Such contacting can thereby cause at least some of the capture oligomers (i.e., a fraction of the population of capture oligomers) to anneal to the target polynucleotide. The method further includes contacting the capture oligomer (before, during, or after contacting the target polynucleotide with the capture oligomer) with a first capture reagent that includes a complement of the capture sequence; and providing a second capture reagent that includes a complement of a sequence in the capture oligomer other than the capture sequence, and if some or all of the capture oligomers are not annealed to the target polynucleotide, the second capture reagent contacts the capture oligomers that are not annealed to the target polynucleotide. The sequence in the capture oligomer other than the capture sequence can be part or all of the target hybridizing sequence, or a sequence that overlaps with the target hybridizing sequence, or a sequence that is otherwise inaccessible to a second capture reagent when the capture oligomer is annealed to the target polynucleotide. The method further includes isolating the first and second complexes from the composition, where the first complex comprises the target polynucleotide and the second complex comprises the capture oligomer that is not annealed to the target polynucleotide; and selectively eluting the target polynucleotide or a subcomplex that comprises the target polynucleotide from the first complex.

[0348] In some embodiments, the target polynucleotide is contacted with an excess amount of capture oligomer. In some embodiments, the first capture reagent 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, the fraction of the capture oligomer contacted with the second capture reagent comprises unbound capture oligomer.

[0349] In some embodiments, the first capture reagent comprises a first solid support that comprises the complement of the capture sequence. Additional aspects of such embodiments are described elsewhere herein, including as summarized above.

[0350] In some embodiments, the first capture reagent further comprises a second capture sequence that is not complementary to the capture oligomer or the target polynucleotide, and the method comprises contacting the annealed capture oligomer with the first capture reagent and then annealing the second capture sequence to a solid support that comprises the complement of the second capture sequence. Additional aspects of such embodiments are described elsewhere herein, including as summarized above.

[0351] Also provided herein is a combination comprising a capture oligomer, a first solid support, and a second solid support. The capture oligomer has, in the 5' to 3' direction: A first capture sequence, an internal extension blocker, a second capture sequence, and a target hybridizing sequence. Includes. The first solid support comprises a complement of a first capture sequence. The second solid support comprises a complement of a second capture sequence. A first complex formed by annealing the first capture sequence with the complement of the first capture sequence has a lower melting temperature than a second complex formed by annealing the second capture sequence with the complement of the second capture sequence, and / or the complement of the second capture sequence has an affinity for the second capture sequence that is greater than the affinity of the complement of the first capture sequence for the first capture sequence.

[0352] Also provided herein is a combination comprising a capture oligomer, a first capture reagent, a second capture reagent, a first solid support, and a second solid support. The capture oligomer is, in the 5' to 3' direction: First capture sequence, internal extension blocker, and target hybridizing sequence Includes. The first capture reagent comprises a second capture sequence and a complement of the first capture sequence, where the second capture sequence is not complementary to the capture oligomer. The second capture reagent comprises a third capture sequence and a complement of a sequence of the capture oligomer other than the first capture sequence, where the third capture sequence is not complementary to the capture oligomer. The first solid support comprises a complement of the second capture sequence. The second solid support comprises a complement of the third capture sequence. The first complex formed by annealing the second capture sequence with the complement of the second capture sequence has a lower melting temperature than the second complex formed by annealing the third capture sequence with the complement of the third capture sequence, and / or the complement of the third capture sequence has an affinity for the third capture sequence that is greater than the affinity of the complement of the second capture sequence for the second capture sequence.

[0353] Also provided herein is a combination comprising a capture oligomer and a secondary capture reagent. The capture oligomer comprises a target hybridizing sequence and a capture sequence comprising one or more affinity enhancing nucleotides. The secondary capture reagent comprises a complement and a binding partner of the capture sequence. In some embodiments, the capture sequence is located 5' of the target hybridizing sequence. In some embodiments, the target hybridizing sequence is configured to anneal to an adapter sequence. In some embodiments, the secondary capture reagent is present in a smaller amount than the capture oligomer in the combination.

[0354] Also provided herein is a method for capturing target polynucleotide from a composition, comprising contacting target polynucleotide with the above-mentioned combination.Capture oligomer and secondary capture reagent are added simultaneously or sequentially.The target hybridization sequence of capture oligomer anneals to target polynucleotide, and secondary capture reagent anneals to capture sequence of capture oligomer, thereby forming a complex. The method further includes contacting the complex with a second binding partner configured to bind to the binding partner of the secondary capture reagent, where the second binding partner is associated with a solid support and the second binding partner binds to the binding partner of the secondary capture reagent; and isolating the complex from the composition, thereby capturing the target polynucleotide.

[0355] In some embodiments, the secondary capture reagent is present in the combination in a smaller amount than the capture oligomer and / or the target polynucleotide. In some embodiments, the capture oligomer is present in the combination in a smaller amount than the target polynucleotide, and the secondary capture reagent is present in the combination in a smaller amount than the capture oligomer. In some embodiments, the target polynucleotide comprises an adapter sequence, and the target hybridizing sequence anneals to the adapter sequence. In some embodiments, the target polynucleotide comprises a sequence derived from the DNA or RNA of the target organism, and the adapter sequence is not present in the DNA and RNA of the target organism.

[0356] IV. Examples The following examples are provided to illustrate certain disclosed embodiments and should not be construed in any way as limiting the scope of the disclosure.

[0357] A. Capture of a given amount of amplicon with a capture oligomer containing a capture sequence and its complement Oligomer.

[0358] PCR to amplify a segment from the E. coli uidA gene was performed using the primers: Ec_uidA_F: GTATCAGCGCGAAGTCTTTATACC (SEQ ID NO: 1) Ec_uidA_R: GGCAATAACATACGGAGTGACATC (SEQ ID NO: 2)

[0359] The primers were designed to generate an amplicon with the following sequence: GTATCAGCGCGAAGTCTTTATACCGAAAGGTTGGGCGGGCCAGCGTATTGTACTGCGTTTCGATGCGGTCACTCATTACGGCAAAGTGTGGGTAAATAATCAGGAAGTGATGGAGCATCAGGGCGGCTATACGCCATTTGAAGCCGATGTCACTCCGTATGTTATTGCC (SEQ ID NO: 3)

[0360] A capture oligomer, designated uidA_PA_1.2, was prepared with the following sequence: AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAACCTCTA / iSp18 / TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTAGACGCAAGCTACTGGTGATTTGGCAATAACATACGGAGTGACATCGGCTTC (SEQ ID NO: 4) (iSp18 = hexaethylene glycol (HEG) internal spacer (IDT))

[0361] In this oligomer, the 5' polyA sequence is the capture sequence. CCTCTA is the linker sequence. iSp18 is an internal extension blocker. The polyT sequence following iSp18 is the complement of the capture sequence. AGACGCAAGCTACTGGTGATTT (SEQ ID NO:5) is a fourth additional sequence. The target hybridizing sequence (THS) is GGCAATAACATACGGAGTGACATCGGCTTC (SEQ ID NO:6), which specifically hybridizes to a segment of the uidA gene sequence in the target amplicon. In this example, the THS is longer than the reverse PCR primer (see sequence above) which it overlaps, increasing the Tm of the THS and giving it a competitive advantage over the reverse primer in hybridizing to the target.

[0362] A secondary capture reagent was used with the following sequence: dT 30 - Biotin: TTTTTTTTTTTTTTTTTTTT / 3' Biotin (SEQ ID NO: 7)

[0363] The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products: Ec_uidA_F GTATCAGCGCGAAGTCTTTATACC (SEQ ID NO: 1) uidA_Probe 5'FAM / TAGCCGCCCTGATGCTCCATCACTTCCTG / 3'IowaBlack (SEQ ID NO: 8) TQ_R AGACGCAAGCTACTGGTGAT (SEQ ID NO: 9)

[0364] Protocol / reaction conditions.

[0365] (1) The above primers were used to generate a PCR amplicon of the target uidA. The amplicon was purified using AMPure XP (Beckman Coulter) using the manufacturer's recommended protocol and quantified by qPCR using the uidA forward and reverse primers together with the uidA_Probe.

[0366] (2) Capture oligomer annealing and amplicon strand extension - Purified uidA amplicons were diluted 2, 10 or 100-fold. A 20 μL aliquot of each dilution of the amplicon was added to 0.07 U / μl SDPol (Bioron), 1× SDPol reaction buffer, 0.17 mM dNTPs, 3 mM MgCl2, 1 mg / ml BSA and 5×10 10 A capture oligomer was added to the annealing / extension reaction consisting of 10 copies of the capture oligomer (final volume of 30 μL). The capture oligomer was annealed to the 3' end of the complementary strand of the uidA amplicon and the amplicon strand was extended using a thermal cycler following the following thermal profile: 92°C for 2 min, 54°C for 2 min, 68°C for 10 min, 54°C for 2 min, followed by a controlled ramp down to 20°C (0.3°C / sec). In this example, the 3' end of the capture oligomer was also extended.

[0367] (3) Hybridization of the complement of the capture sequence of the capture oligomer - The entire capture oligomer / amplicon extension reaction mixture was added to 10 μL of secondary capture reagent (4x concentration) to a final concentration of 125 mM NaCl, 0.25 mg / ml BSA, and 10 7 , 10 8 or 10 9 The complement of the captured sequence of the copy was obtained (i.e., three different amounts were tested). Hybridization was performed by incubating the reaction mixture at room temperature (20-24 °C) for 15 min.

[0368] (4) Capture of amplicon and capture oligomer extension product / capture oligomer complexes - A 5 μL aliquot (50 μg) of MyOneC1 streptavidin beads (ThermoFisher Scientific) in 250 mM NaCl and 1 mg / ml BSA was added to the hybridization mixture (step 3 above) to capture the capture oligomer / amplicon extension product / complement of the capture sequence complex onto the beads, and the beads were washed according to the manufacturer's recommendations.

[0369] (5) Elution - After the final wash was completed and the wash buffer removed, 10 μL of water was added to the bead pellet, the beads resuspended and incubated for 2 minutes at 70° C. The beads were pelleted with a magnet and the eluate removed.

[0370] (6) Quantification - The amount of elution product and capture oligomer extension product (see step 2 above) was quantified by qPCR using primers targeting the uidA_F primer site and the TQ primer-adapter site with the uidA_Probe.

[0371] Results and conclusions:

[0372] 2-fold, 10-fold and 100-fold dilutions of the amplicons generated in the targeted enrichment step (see step 1 above) were 7 , 10 8 or 10 9Each PCR dilution was subjected to a copy control process (see steps 2-5 above) using a capture oligomer of 100 copies. As shown in Table 1, the amount of amplicon recovered was proportional to the amount of capture oligomer added for each PCR dilution (approximately in the expected ratio; see the "Ratio" column in the table). The variability between replicates at each data point was low (see the "Standard Deviation (Std Dev)" column).

[0373] [Table 1]

[0374] Furthermore, despite a 50-fold difference in amplicon input, the variation in output was 10 7 , 10 8 and 10 9 The copy number for the capture oligomer was 2.36-fold, 3.11-fold, and 3.44-fold, respectively (Table 1). As shown above, the variation between replicates was small.

[0375] These data demonstrate that the capture oligomers described herein can be used to obtain a given amount of target output from a range of different input target amounts.

[0376] Additional experiments were performed essentially as described above, but in a multiplex format (i.e., four capture oligomers were used per reaction) using capture oligomers whose THS regions were 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. The uidA, nuc, and vanA genes were each amplified separately using PCR as described above, using the primers shown above for uidA and additional primers designed for the nuc and vanA genes. The resulting amplicons were diluted 10- or 100-fold, and 20 μL aliquots of each dilution of each individual target were mixed with 5 × 10 of each of the four capture oligomers described above. 10 A separate capture oligomer containing 10 copies of the capture sequence was added to the annealing and extension reaction of the amplicon strand (i.e., 4-plex capture oligos, but with only one target present). The reaction conditions were approximately the same as described, except for the following thermal profile: 92°C for 2 min, 64°C for 2 min, 68°C for 10 min, 98°C for 2 min, 57°C for 2 min, followed by a controlled ramp down to 20°C (0.3°C / sec). After this reaction was completed, 5x10 copies of the complement of the capture sequence of the capture oligomer were added to the amplicon strand extension reaction. 8 A copy was added to each reaction, which was followed by the capture, wash, elution and quantification steps described above.

[0377] The output amounts after capturing amplicons using input amounts that differed by 10-fold are shown in Table 2. The results show that for each of the three individual target amplicons tested in multiplex format, a 10-fold difference in input amplicon levels was reduced to a difference in average output levels after copy control of 1.4-fold or less. Furthermore, the range of average output levels after copy control was spread approximately 1.6-fold across all three target amplicons, even though their input levels were spread over 270-fold.

[0378] [Table 2]

[0379] Additional experiments were performed essentially as above in a singleplex format, but using a capture oligomer in which THS anneals to a universal binding site in a tag sequence that was incorporated into the target of interest during the PCR amplification step. Primers were designed to target the bacterial 23S rRNA gene, and PCR amplicons were generated from bacterial genomic DNA. The reverse primer contained a universal sequence tag that was incorporated into the amplicon during PCR. Undiluted (i.e., no dilution; approximately 9 × 10 12 copy) or 50-fold dilution (approximately 2 × 10 11 A 40 μL aliquot of 10 copies of each 1000-fold diluted 100% PBS containing 0.02 U / μL SD polymerase (Bioron), 0.4× SD polymerase reaction buffer (Bioron), 0.012 mM 4dNTPs, 1.8 mM MgCl2, 0.6 mg / mL BSA, and 5×10 10 Capture oligomer annealing and amplicon strand extension were performed as described above, except that 10 copies of capture oligomer were added to the reaction mixture containing the capture oligomer, resulting in a final reaction volume of 100 μL. The thermal profile used was 92°C for 2 min, 54°C for 2 min, 68°C for 10 min, 54°C for 2 min, followed by a controlled ramp down to 20°C (0.3°C / sec). The total volume of the reaction was adjusted to 1 mg / ml BSA, 125 mM NaCl, and 5×10 8A 50 μL volume of 3× annealing mix containing 10 copies of secondary capture reagent was added. Annealing was performed by incubating the reaction mixture in a thermal block at 25° C. for 10 min. A 50 μL volume (200 μg in this experiment) of MyOneC1 streptavidin beads (ThermoFisher Scientific) was added to the 150 μL reaction above in 4× wash buffer consisting of 4 M NaCl, 20 mM Tris-HCl pH 7.5, 2 mM EDTA, 0.20% Tween 20, 2 mg / mL BSA. The resulting complex containing the capture oligomer, amplicon extension product, and the complement of the capture sequence was captured on the beads, and the beads were washed according to the manufacturer's recommendations (in this case, 1× wash buffer was used; see 4X formulation immediately above). A 20 μL volume of water was used for elution (protocol and others the same as above). qPCR was performed using a specific forward primer and a reverse primer targeting the universal tag.

[0380] Despite a 50-fold difference in amplicon input levels, the output varied by only 2.5-fold after capture oligomer annealing and amplicon strand extension steps, and by 1 (i.e., perfectly normalized) after contact with secondary capture reagent and isolation of the resulting complex (see Table 3). Furthermore, these results demonstrate an embodiment of the present disclosure in which THS binds to a universal tag sequence.

[0381] [Table 3]

[0382] Additional experiments were performed essentially as immediately above (singleplex, universal THS), but in a multiplex format, with the THS using a capture oligomer that anneals to a universal tag sequence incorporated into each of the targets of interest during the PCR amplification step. Eight different amplicon targeting regions in the bacterial 16S rRNA gene, 23S rRNA gene and antibiotic resistance marker KPC were generated from K. pneumoniae genomic DNA in eight separate singleplex reactions. One amplicon targeting a synthetic internal control (IC) DNA was also generated, for a total of nine individual amplicons. Equal amounts of all nine amplicons were pooled and diluted undiluted (i.e., no dilution; approximately 1 × 10 13 copy) or a 10-fold dilution (approximately 1 × 10 12 54 μL aliquots of 5 × 10 copies were added to separate capture oligomer annealing and amplicon strand extension reaction mixtures (final volume of 100 μL each). 11 Capture oligomer and 5 x 10 copies 9 The remainder of the workflow was essentially the same as above, except that a second copy of the capture reagent was used. qPCR using a specific forward primer and a reverse primer targeting the universal tag was performed to quantify each target (9 separate PCRs). The amount recovered for each individual target was summed to determine the overall recovery of the capture method.

[0383] Despite a 10-fold difference in amplicon input levels, the overall output varied by only 2.5 after capture oligomer annealing and amplicon strand extension steps, and 1.6 after contact with secondary capture reagent and isolation of the resulting complex (see table). Furthermore, these results demonstrate an embodiment of the invention in which THS binds to a universal tag sequence in a multiplexed format, thereby capturing all target amplicons present in the mixture.

[0384] [Table 4]

[0385] B. Capture of a given amount of amplicon with a capture oligomer containing a capture sequence, its complement, and a clamp sequence. Target amplicons were prepared essentially as described above for uidA and used in experiments with capture oligomers with and without a clamp sequence (GCGCGC) inserted as the first and third additional sequences (see FIG. 3). Capture was performed essentially as described above using undiluted, 10-fold diluted and 100-fold diluted amplicons. The amount of captured product was quantified essentially as described above. The results are shown in FIG. 14. The use of capture oligomers containing a clamp sequence improved the ability to normalize the amount of output across different dilutions of the amplicon.

[0386] C. Capture of a given amount of amplicon with a capture oligomer and a complementary oligomer Oligomer. PCR to amplify a segment from the E. faecium vanA gene was performed using the following primers: Efm_vanA_F: GGCTGCGATATTCAAAGCTCAG (SEQ ID NO: 10) Efm_vanA_R: CTGAACGCGCCGGCTTAAC (SEQ ID NO: 11) The primers were designed to generate an amplicon with the following sequence: GGCTGCGATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATACATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAG (SEQ ID NO: 12) A capture oligomer, designated CC_Blo_vanA_001, was prepared with the following sequence: AAAAAAAAAAAAAAAAAAAAA / iSp18 / CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTCCTGAACGCGCCGGCTTAAC (SEQ ID NO: 13) (iSp18 = hexaethylene glycol (HEG) internal spacer (IDT))

[0387] This oligomer contains the elements shown for the exemplary capture oligomer in Figure 10A. In this oligomer, the 5' polyA sequence is a capture sequence having a first and second portion. iSp18 is an internal extension blocker. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC (SEQ ID NO: 14) is a spacer sequence having a first and second portion. The target hybridizing sequence (THS) is CTGAACGCGCCGGCTTAAC (SEQ ID NO: 15), which specifically hybridizes to a segment of the vanA gene sequence in the target amplicon.

[0388] A complementary oligo called Blocker_vanA_001, containing the elements shown for the exemplary complementary oligomer in Figure 10A, was prepared with the following sequence: CGGTGCCAGAGGAGTTTTTTTTTT / invdt / (SEQ ID NO: 16), where invdt is an inverted T nucleotide that serves as a blocking moiety. In this oligomer, CGGTGCCAGAGGAG (SEQ ID NO: 17) is the complement of a first portion of the spacer sequence of the capture oligomer, and TTTTTTTTTT (SEQ ID NO: 18) is the complement of a second portion of the capture sequence.

[0389] A secondary capture reagent was used with the following sequence: dT 20 - Biotin: TTTTTTTTTTTTTTTTTTTT / 3' Biotin (SEQ ID NO: 19)

[0390] The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products: vanA_PCR2_Fwd TTGTATGGACAAATCGTTGACATACA (SEQ ID NO: 20) Efm_Probe_FAM 5'FAM / TGCTGGGATAGCTACTCCCGCCTTTTGG / 3'IowaBlack (SEQ ID NO:21) CC_Univ_Inner_Rev ACCGTGCTGCCTTGGCTTC (SEQ ID NO: 22)

[0391] Protocol / reaction conditions.

[0392] (1) PCR amplicons of target vanA were generated using the Efm_vanA_F and Efm_vanA_R primers described above. The amplicons were quantified by chip-based capillary electrophoresis using an Agilent BioAnalyzer.

[0393] (2) Annealing of capture oligomers and extension of amplicon strands - vanA amplicons were either diluted (undiluted) or diluted 10-fold (approximately 2 × 10 13 and 2 × 10 12 An 80 μL aliquot of each amplicon (undiluted and 10-fold diluted) was combined with 20 μL of capture oligomer annealing / extension reaction mixture containing 0.02 U / μL Deep Vent (exo-)Pol (NEB), 0.4× Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, 1×10 11 Capture oligomer (CC_Blo_vanA_001) and 1 × 10 copies 12 A final mixture consisting of a copy of the complementary oligomer (Blocker_vanA_001) was obtained in a final volume of 100 μL. The capture 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 following the following thermal profile: 92°C for 2 min, 64°C for 2 min and 68°C for 10 min. In this example, the 3' end of the capture oligomer was also extended.

[0394] (3) Hybridization of the complement of the capture sequence of the capture oligomer - 50 μL of secondary capture reagent (3x concentration) was added to the entire capture oligomer / amplicon extension reaction mixture to a final concentration of 42 mM NaCl, 0.33 mg / ml BSA, and 10 9 Copy the complement of the capture sequence (dT 20 -biotin). Hybridization was carried out by incubating the reaction mixture at 30° C. for 10 minutes.

[0395] (4) Capture of amplicons and capture oligomer extension products / capture oligomer complexes - A 50 μL aliquot (200 μg) of streptavidin-coated magnetic beads was added to the total hybridization mixture (150 μL) to give 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 25° C. and the beads were washed using a washing reagent with the same composition as detailed immediately above.

[0396] (5) Elution - After the final wash was completed and the wash buffer removed, 30 μL of water was added to the bead pellet, the beads resuspended and incubated for 2 minutes at 70° C. The beads were pelleted with a magnet and the eluate removed.

[0397] (6) Quantification - The amount of elution product and capture oligomer extension product (see step 2 above) was quantified by qPCR using primers targeting the vanA_PCR2_Fwd primer site and the universal primer-adapter site (CC_Univ_Inner_Rev) with Efm_Probe_FAM.

[0398] Results and conclusions:

[0399] 0-fold (undiluted) and 10-fold dilutions of the amplicon generated in step 1 (PCR) above were 9Each replicate was subjected to a copy control process (see steps 2-5 above) using a secondary capture oligomer. As shown in Table 5, despite a 10-fold difference in the amount of input target, the output levels were essentially identical. [Table 5]

[0400] These data demonstrate that the capture and complementary oligomers described herein can be used to obtain a given normalized amount of target output across a 10-fold difference in input target amount.

[0401] Additional experiments were performed essentially as above, with the following differences:

[0402] (1) PCR amplicons were purified using a QIAGEN QIAquick PCR Purification kit according to the manufacturer's instructions and then quantified by chip-based capillary electrophoresis using an Agilent BioAnalyzer.

[0403] (2) Annealing of capture oligomers and extension of amplicon strands - vanA amplicons were diluted undiluted, 10-fold, and 100-fold (approximately 8 × 10 11 , 8×10 10 and 6×10 9 The capture oligomer (CC_Blo_vanA_001) was used at 1 × 10 12 Use the capture oligomer for the copy / reaction and add the complementary oligo (Blocker_vanA_001) at 0 or 1 × 10 13 The capture 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 following the following thermal profile: 95°C for 2 min and 64°C for 15 min. All other conditions in this step were the same as in step 2 above.

[0404] (3-6) Steps 3-6 were carried out as described above for steps 3-6, except that in step 4, the complexes were captured on the beads at 30°C instead of 25°C.

[0405] Results and conclusions:

[0406] 0x (undiluted), 10x and 100x dilutions of the amplicon generated in step 1 (PCR) above were subjected to the copy control process (see steps 2-5 above). In this experiment, the amounts of the various nucleic acid components used were approximately 8 x 10 11 , 8×10 10 and 6×10 9 Copy Target, 1x10 12 Copies of capture oligomer, 0 or 1 x 10 13 Complementary oligomer and 1 x 10 copies 9 The results with and without the complementary oligomer are shown in Table 6. [Table 6]

[0407] In the absence of a complementary oligomer, the secondary capture oligomer (dT 20 -biotin) can be attached to any of the capture oligomer molecules, whether or not they are bound to the target. In this experiment, 1 × 10 12 Capture oligomer and 1 x 10 copies 9 10 copies of secondary capture oligomer were used, i.e., these amounts differ by a factor of 1000. At the highest target level (undiluted), the majority of the capture oligomers bind to the target, and therefore the majority of the secondary capture oligomers bind to the capture oligomers associated with the target, resulting in a relatively high copy output after capture and elution. This is consistent with the relatively high output (9.3×10 7This is supported by the data for undiluted target input (no complementary oligos), where 10 copies (10 copies) are observed. However, at a 10-fold dilution of the target, there is an excess of capture oligomers, and therefore not all of them bind to the target. Some of the secondary capture oligomers bind to the capture oligomers associated with the target, but some to the capture oligomers that are not bound to the target. Thus, as is indeed observed, the output is reduced (output = 2.6 x 10 7 At a 100-fold dilution of the target, the majority of the capture oligomers do not bind to the target, and similarly the majority of the secondary oligomers bind to capture oligomers that are not associated with the target. Thus, the expectation under these conditions would be a significant drop in output, and this is indeed observed (output = 5.0 x 10 5 copy).

[0408] In the presence of complementary oligomers, capture oligomers that are not bound to the target will have complementary oligomers bound, which in turn will block binding of the secondary capture reagent. Conversely, capture oligomers that are bound to the target will not have complementary oligomers bound (which have been displaced), which in turn will allow binding of the secondary capture reagent. Thus, at all levels of target input tested in this experiment, the output would be expected to be higher in the presence of complementary oligomers than in the absence of complementary oligomers (the results of which are discussed above). This is exactly what is observed (see Table 6). Furthermore, the data demonstrates that normalization is taking place, with only about a 2-fold difference between undiluted and 10-fold diluted output target levels, and only a little over 14-fold difference between undiluted and 100-fold output target levels. The output at 100-fold target dilution is slightly lower than theoretical due to slower binding kinetics resulting from this low level of target. Given a longer incubation time, the output would increase and the normalization factor would improve.

[0409] These data demonstrate that the capture and complementary oligomers described herein can be used to obtain a given normalized amount of target output across a 100-fold difference in input target amount.

[0410] D. Generation of a capturable product containing additional sequences (e.g., adapters) on both ends of the target sequence using a capture oligomer, a complementary oligomer, a displacer oligomer, and a forward primer Oligomer. PCR to amplify a segment from the E. faecium vanA gene was performed using the following primers: Efm_vanA_F: GGCTGCGATATTCAAAGCTCAG (SEQ ID NO: 23) Efm_vanA_R: CTGAACGCGCCGGCTTAAC (SEQ ID NO: 24) The primers were designed to generate an amplicon with the following sequence: GGCTGCGATTCAAAGCTCAGCAATTTGTATGGACAAATCGTTGACATACATCGTTGCGAAAAATGCTGGGATAGCTACTCCCGCCTTTTGGGTTATTAATAAAGATGATAGGCCGGTGGCAGCTACGTTTACCTATCCTGTTTTTGTTAAGCCGGCGCGTTCAG (SEQ ID NO: 25)

[0411] A capture oligomer was prepared, called PCR2R_adapter_CC, with the following sequence: AAAAAAAAAAAAAAAAAAAAA / iSp18 / CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTCGTAGCTGCCACCGGCCTAT (SEQ ID NO: 26) (iSp18 = Hexaethylene glycol (HEG) internal spacer (IDT)) This oligomer contains the elements shown for the exemplary capture oligomer in Figure 10A. In this oligomer, the 5' polyA sequence is a capture sequence having a first and second portion. iSp18 is an internal extension blocker. CTCCTCTGGCACCGTGCTGCCTTGGCTTCATTGTGGTC (SEQ ID NO: 27) is a spacer sequence having a first and second portion. The target hybridizing sequence (THS) is GTAGCTGCCACCGGCCTAT (SEQ ID NO: 28), which specifically hybridizes to a segment of the vanA gene sequence in the target amplicon.

[0412] A complementary oligo called Blocker_vanA_001, containing the elements shown for the exemplary complementary oligomer in Figure 10A, was prepared with the following sequence: CGGTGCCAGAGGAGTTTTTTTTTT / invdt / (SEQ ID NO:29), where invdt is an inverted T nucleotide that serves as a blocking moiety. In this oligomer, CGGTGCCAGAGGAG (SEQ ID NO:30) is the complement of a first portion of the spacer sequence of the capture oligomer, and TTTTTTTTTT (SEQ ID NO:31) is the complement of a second portion of the capture sequence.

[0413] An oligomer called PCR1F_adapter was also prepared containing the elements shown for the exemplary forward primer with adapter in Figure 8A, and had the following sequence: AAAACGAGACATGCCGAGCATCCGCGGCTGCGATATTCAAAGCTCAG (sequence number 32).

[0414] A secondary capture reagent was used with the following sequence: dT 20 - Biotin: TTTTTTTTTTTTTTTTTTTT / 3' Biotin (SEQ ID NO: 33) The following primers and probes were used for quantitative PCR (qPCR) analysis of copy control products: CCRPA_uni_F AAAACGAGACATGCCGAGCATC (SEQ ID NO: 34) Efm_Probe_FAM 5'FAM / TGCTGGGATAGCTACTCCCGCCTTTTGG / 3'IowaBlack (SEQ ID NO: 35) CC_Univ_Inner_Rev ACCGTGCTGCCTTGGCTTC (SEQ ID NO: 36)

[0415] Protocol / Reaction Conditions(1). (1) PCR amplicons of target vanA were generated utilizing the Efm_vanA_F and Efm_vanA_R primers described above. The amplicons were purified using a QIAGEN QIAquick PCR Purification kit according to the manufacturer's instructions prior to quantification by chip-based capillary electrophoresis using an Agilent BioAnalyzer. (2) Annealing and extension of the forward primer with the capture oligomer, displacer oligomer and adapter - approximately 1 × 10 12 Aliquots of vanA amplicons containing copies were combined with the annealing / extension reaction mixture containing 0.02 U / μl Deep Vent (exo-)Pol (NEB), 0.4× Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, 5×10 13 Copy capture oligomer (PCR2R_adapter_CC), ±1 × 10 13 Displacer oligomer (Efm_vanA_R), and 5 × 10 copies 13 The final mixture consisting of a forward primer with adapter (PCR1F_adapter) for the copy was obtained in a final volume of 100 μL. Annealing and extension of the capture and displacer oligomers with the input amplicon, as well as annealing and extension of the forward primer with adapter with the extension product of the capture oligomer, were all performed in the same annealing / extension reaction using a thermal cycler following the following thermal profile: 95°C for 5 min, then 64°C for 20 min. (3) Quantification - An aliquot of each of the annealing-extension reactions was diluted 100-fold and the amount of product contained in each was quantified by qPCR using primers CCRPA_uni_F and CC_Univ_Inner_Rev (targeting the universal adapter region in the forward and reverse orientations, respectively) together with Efm_Probe_FAM.

[0416] Results and conclusions: A single cycle annealing and extension reaction (a single cycle is defined as only one denaturation step, e.g., incubation at 95° C.; another cycle would begin with another heat denaturation step) was performed using the above input target and oligomers. As shown in Table 7, products were formed that contained universal adapters on both ends of the molecule, as evidenced by amplification using universal primers. [Table 7]

[0417] These data demonstrate that using the embodiment of the invention shown in FIG. 8A, a single annealing / extension cycle can be used to generate products with adapters (or other desired sequences) on both ends of the molecule. Furthermore, these data demonstrate that at least one of the primer-adapter oligomers (in this case, PCR2R_adapter_CC) can bind to an internal site on the target, not just to the termini. These data also demonstrate that the desired product can be generated in the absence of a displacer oligomer. While not wishing to be bound by any particular theory, it is possible that different mechanisms can function within the disclosed embodiments to obtain the desired product. It is possible that multiple mechanisms can function in the presence of a displacer oligomer to generate the observed results.

[0418] Additional experiments were performed essentially as above, with the following differences: (2) Annealing and extension of the forward primer with the capture oligomer, displacer oligomer and adapter - approximately 1 × 10 13 Aliquots of vanA amplicons containing copies were combined with the annealing / extension reaction mixture containing 0.02 U / μl Deep Vent (exo-)Pol (NEB), 0.4× Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, 5×10 14 Capture oligomer (PCR2R_adapter_CC) and 5 × 10 copies 14 A final mixture consisting of the forward primer with the adapter (PCR1F_adapter) was obtained in a final volume of 100 μL. Annealing and extension of this mixture was performed using a thermal cycler according to the following thermal profile: 95 °C for 5 min, then 64 °C for 15 min. At this point, 5 × 10 14 A copy of the displacer oligomer (Efm_vanA_R) was added to some replicates of the reaction mixture and to some just buffer, and annealing and extension were continued using a thermal profile of 64°C for 5 min, 75°C for 5 min, and 72°C for 15 min.

[0419] Results and conclusions: A single cycle annealing and extension reaction (a single cycle is defined as only one denaturation step, e.g., incubation at 95° C.; another cycle would begin with another heat denaturation step) was performed using the input target and oligomers described above. To further optimize performance, a displacer oligomer was added to the annealing and extension reaction midway through the process. As shown in Table 8, products were formed that contained universal adapters on both ends of the molecule, as evidenced by amplification using universal primers. [Table 8]

[0420] As noted above, these data demonstrate that using the embodiment of the invention shown in FIG. 8A, a single annealing / extension cycle can be used to generate products with adapters (or other desired sequences) at both ends of the molecule. Also, as noted above, these data demonstrate that at least one of the primer-adapter oligomers (in this case, PCR2R_adapter_CC) can bind to an internal site of the target, not just to the termini. Furthermore, these data demonstrate that by adjusting the annealing and extension temperature profile (and, in this case, by adding a displacer oligomer midway through the process), the overall performance can be improved. Of particular note is that under these conditions, greater amounts of the desired product were generated when the displacer oligomer was present than when it was not, demonstrating that the displacement scheme is working as shown in FIG. 8A. Also, without wishing to be bound by any particular theory, different mechanisms may also function within the disclosed embodiments to obtain the desired product.

[0421] Protocol / Reaction Conditions (2). (1) PCR amplicons of target vanA were generated utilizing the Efm_vanA_F and Efm_vanA_R primers described above. The amplicons were purified using a QIAGEN QIAquick PCR Purification kit according to the manufacturer's instructions prior to quantification by chip-based capillary electrophoresis using an Agilent BioAnalyzer. (2) Annealing and extension of the forward primer with the capture oligomer, displacer oligomer and adapter - approximately 1 × 10 12 Aliquots of vanA amplicons containing copies were combined with the annealing / extension reaction mixture containing 0.02 U / μl Deep Vent (exo-)Pol (NEB), 0.4× Deep Vent Pol reaction buffer, 0.012 mM dNTPs, 1.8 mM MgCl2, 0.6 mg / ml BSA, 5×10 13Copy capture oligomer (PCR2R_adapter_CC), ±5×10 12 Displacer oligomer (Efm_vanA_R), and 5 × 10 copies 13 The final mixture consisting of a forward primer with adapter (PCR1F_adapter) for the copy was obtained in a final volume of 100 μL. Annealing and extension of the capture and displacer oligomers with the input amplicon, as well as annealing and extension of the forward primer with adapter with the extension product of the capture oligomer, were all performed in the same annealing / extension reaction using a thermal cycler following the following thermal profile: 95°C for 5 min, then 64°C for 20 min. (3) Hybridization of the complement of the capture sequence of the capture oligomer. Add 50 μL of secondary capture reagent (3x concentration) to the entire reaction mixture to a final concentration of 42 mM NaCl, 0.33 mg / ml BSA, and 5x10 14 Copy the complement of the capture sequence (dT 20 -biotin). Hybridization was carried out by incubating the reaction mixture at 30° C. for 10 minutes. (4) Capture of amplicons and capture oligomer extension products / capture oligomer complexes - A 50 μL aliquot (200 μg) of streptavidin-coated magnetic beads was added to the total hybridization mixture (150 μL) to give 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 using a washing reagent with the same composition as detailed immediately above. (5) Elution - After the final wash was completed and the wash buffer removed, 30 μL of water was added to the bead pellet, the beads resuspended and incubated for 2 minutes at 70° C. The beads were pelleted with a magnet and the eluate removed. (6) Quantification - Elution products were quantified by qPCR using primers CCRPA_uni_F and CC_Univ_Inner_Rev (targeting the universal adapter region in the forward and reverse orientations, respectively) together with Efm_Probe_FAM.

[0422] Results and conclusions: Using the above input target and oligomers, a single cycle annealing and extension reaction was performed, and the products were captured, washed, eluted, and then quantified using qPCR. As shown in Table 8, products were formed that contained universal adapters on both ends of the captured and eluted molecules, as evidenced by amplification using universal primers. [Table 9]

[0423] These data demonstrate that using the embodiment of the invention shown in FIG. 8A, a single annealing / extension cycle can be used to generate products with adapters (or other desired sequences) on both ends of the molecule, which can be isolated by capture onto beads, washing, and elution. Furthermore, these data demonstrate that at least one of the primer-adapter oligomers (in this case, PCR2R_adapter_CC) can bind to an internal site on the target, not just to the termini. These data also demonstrate that the desired product can be generated in the absence of a displacer oligomer. While not wishing to be bound by any particular theory, it is possible that different mechanisms can function within the disclosed embodiments to obtain the desired product. It is possible that multiple mechanisms can function in the presence of a displacer oligomer to generate the observed results.

[0424] Additional experiments were performed essentially as above, with the following differences: (2) Annealing and extension of the capture oligomer, displacer oligomer, and forward primer with adapter - the annealing / extension reaction mixture was 5 × 10 14 The procedure was the same as above, except that a new sample was added that also contained a copy of the complementary oligomer (Blocker_vanA_001). Annealing and extension of the capture and displacer oligomers with the input amplicon, annealing of the complementary oligomer with the capture oligomer, and annealing and extension of the forward primer with adapter with the extension product of the capture oligomer were all performed in the same annealing / extension reaction using a thermal cycler following the following thermal profile shown in Table 9. [Table 10] (3) Hybridization of the complement of the capture sequence of the capture oligomer—the complement of the capture sequence (dT 20 -biotin) 1×10 9 The conditions were the same as above, except that a copy was used.

[0425] Results and conclusions: Using the above input target and oligomers, a single cycle of annealing and extension reaction is performed and the product is fused to a defined amount of the complement of the capture sequence (dT 20 -biotin) and then quantified using qPCR. The results are shown in Table 10. [Table 11]

[0426] These data demonstrate that using the embodiment of the invention shown in Figure 8A, a single annealing / extension cycle can be used to generate products with adapters (or other desired sequences) on both ends of the molecule, which can be isolated by capture onto beads, washing and elution. Furthermore, a 10-fold difference in input target levels is normalized to a 4.7-fold difference, which is greater than a 2-fold normalization factor. Furthermore, these data demonstrate that at least one of the primer-adapter oligomers (in this case, PCR2R_adapter_CC) can bind to an internal site on the target, not just to the termini.

[0427] Additional experiments were performed essentially as above, with the following differences:

[0428] (2) Annealing and extension of the capture oligomer, displacer oligomer, and forward primer with adapter - the annealing / extension reaction mixture used 1E+13 and 1E+12 (10-fold dilution) copies / reaction of input target; 5 × 10 14 The same as above, except that a new sample was added that also contained a copy of the complementary oligomer (Blocker_vanA_001). Annealing and extension of the capture oligomer with the input amplicon, annealing of the complementary oligomer with the capture oligomer, and annealing and extension of the forward primer with the adapter with the extension product of the capture oligomer were all performed in the same annealing / extension reaction using a thermal cycler following the following thermal profile: 95°C for 5 min, then 64°C for 15 min.

[0429] (3) Hybridization of the complement of the capture sequence of the capture oligomer—the complement of the capture sequence (dT 20 -biotin) 1×10 9 The conditions were the same as above, except that a copy was used. Hybridization was carried out at 30° C. for 30 minutes.

[0430] Results and conclusions:

[0431] Using the above input target and oligomers, a single cycle of annealing and extension reaction is performed and the product is fused to a defined amount of the complement of the capture sequence (dT 20 -biotin) and then quantified using qPCR. The results are shown in Table 10.

[0432] [Table 12]

[0433] These data demonstrate that using the embodiment of the invention shown in FIG. 8A, a single annealing / extension cycle can be used to generate products with adapters (or other desired sequences) at both ends of the molecule, which can be isolated by capture onto beads, washing, and elution. Furthermore, the 10-fold difference in input target levels was normalized to a 0.67-fold difference (approximately 1) when complementary oligos were present. Without complementary oligos, as expected, product recovery was reduced by more than 10-fold, but normalization was similar. Furthermore, these data demonstrate that at least one of the primer-adapter oligomers (in this case, PCR2R_adapter_CC) can bind to an internal site of the target, not just to the termini. [Sequence List Free Text]

[0434] Sequence Listing 1 <223> synthesis Sequence Listing 2 <223> synthesis Sequence Listing 3 <223> synthesis Sequence Listing 4 <223> synthesis Sequence Listing 4 <223> iSp18 site (hexaethylene glycol (HEG) internal spacer) Sequence Listing 5 <223> synthesis Sequence Listing 6 <223> synthesis Sequence Listing 7 <223> synthesis Sequence Listing 7 <223> 3' Biotin Sequence Listing 8 <223> synthesis Sequence Listing 8 <223> 5'FAM, 3'IowaBlack Sequence Listing 9 <223> synthesis Sequence Listing 10 <223> synthesis Sequence Listing 11 <223> synthesis Sequence Listing 12 <223> synthesis Sequence Listing 13 <223> synthesis Sequence Listing 13 <223> iSp18 site (hexaethylene glycol (HEG) internal spacer) Sequence Listing 14 <223> synthesis Sequence Listing 15 <223> synthesis Sequence Listing 16 <223> synthesis Sequence Listing 16 <223> 3'invdt Sequence Listing 17 <223> synthesis Sequence Listing 18 <223> synthesis Sequence Listing 19 <223> synthesis Sequence Listing 19 <223> 3' Biotin Sequence Listing 20 <223> synthesis Sequence Listing 21 <223> synthesis Sequence Listing 21 <223> 5'FAM, 3'IowaBlack Sequence Listing 22 <223> synthesis Sequence Listing 23 <223> synthesis Sequence Listing 24 <223> synthesis Sequence Listing 25 <223> synthesis Sequence Listing 26 <223> synthesis Sequence Listing 26 <223> iSp18 site (Hexaethylene glycol (HEG) internal spacer) Sequence Listing 27 <223> synthesis Sequence Listing 28 <223> synthesis Sequence Listing 29 <223> synthesis Sequence Listing 29 <223> 3'invdt Sequence Listing 30 <223> synthesis Sequence Listing 31 <223> synthesis Sequence Listing 32 <223> synthesis Sequence Listing 33 <223> synthesis Sequence Listing 33 <223> 3' Biotin Sequence Listing 34 <223> synthesis Sequence Listing 35 <223> synthesis Sequence Listing 35 <223> 5'FAM, 3'IowaBlack Sequence Listing 36 <223> synthesis

Claims

1. In the 5' to 3' direction: Capture sequence, Internal extension blockers, the complement of the capture sequence, and Target Hybridizing Sequence A capture oligomer comprising: A capture oligomer, wherein the complement of the capture sequence is configured to anneal to the capture sequence in the absence of the target hybridizing sequence and the extended target sequence that anneals to the complement of the capture sequence.

2. The capture oligomer has the formula: 5'-A1-CL-B-A2-C'-A3-RB-A4-THS-X-3' where A1 is a first additional sequence that is optionally present; C is the capture sequence, L is an optional linker, B is the internal extension blocker; A2 is an optional second additional sequence, C' is the complement of the capture sequence; A3 is an optional third additional sequence; RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence; THS is the target hybridizing sequence; X is an optional blocking moiety.

2. The capture oligomer of claim 1 having the following structure:

3. 3. The capture oligomer of claim 1 or 2, comprising a linker between the capture sequence and the internal extension blocker, which linker is optionally a nucleotide sequence or a non-nucleotide linker or a combination thereof.

4. 4. The capture oligomer of any one of claims 1 to 3, wherein the internal extension blocker comprises a non-nucleotidic linker, or one or more abasic sites, non-naturally occurring nucleotides, or chemically modified naturally occurring nucleotides.

5. 5. The capture oligomer of claim 1, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridizing sequence.

6. 6. The capture oligomer of claim 5, wherein the capture oligomer comprises a third additional sequence located 3' of the complement of the capture sequence and 5' of the target hybridizing sequence, and the reversible extension blocker is located 3' of an adapter sequence, and optionally the third additional sequence comprises an adapter sequence.

7. 7. The capture oligomer of claim 1, wherein the capture oligomer comprises a fourth additional sequence located 3' to the reversible blocker and 5' to the target hybridizing sequence, optionally the fourth additional sequence comprising an adapter sequence.

8. 8. The capture oligomer of claim 1, further comprising a second additional sequence between the internal extension blocker and the complement of the capture sequence, wherein the second additional sequence optionally comprises a mixed nucleotide segment.

9. The reversible extension blocker is selected from the group consisting of Iso-dC or Iso-dG, xanthine or 5-(2,4 diaminopyrimidine), 2-amino-6-(N,N-dimethylamino)purine or pyridin-2-one, 4-Methylbenzimidizole or 2,4-difluorotoluene, 7-azaindole or isocarbostyril, dMMO2 or d5SICS, dF or dQ; one or more chemically modified nucleotides, 9. The capture oligomer of claim 8, comprising one or more chemically modified nucleotides, wherein the modification is attached via a reversible linkage that can be reversed by providing one or more of a chemical, an enzyme, a temperature change, or a change in reagent composition; a reversible nucleic acid structural feature; or a molecule reversibly bound to the capture oligomer, optionally wherein the reversibly bound molecule is a protein, an enzyme, a lipid, a carbohydrate, or a chemical moiety.

10. A combination comprising a capture oligomer or combination according to any one of the preceding claims, wherein the capture oligomer comprises an adapter sequence 5' of the target hybridizing sequence, and the combination further comprises a blocker oligomer comprising the adapter sequence which is non-extendable, optionally configured to bind to the complement of the adapter sequence with a higher affinity than the adapter sequence of the capture oligomer or to form a complex with the complement of the adapter sequence which has a higher melting temperature than a complex between the adapter sequence of the capture oligomer and the complement of the adapter sequence.

11. 1. A method for capturing a target polynucleotide from a composition, the method comprising: contacting the target polynucleotide with a capture oligomer of any one of claims 1 to 9, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site that includes the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming a complement of the complement of the capture sequence that is annealed to the capture oligomer such that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and isolating said complex from said composition, thereby capturing said target polynucleotide. A method comprising:

12. A combination comprising a capture oligomer and a complementary oligomer, (a) the capture oligomer comprises, in a 5' to 3' direction: a capture sequence comprising a first and a second portion; Internal extension blockers, a spacer sequence comprising a first and a second portion; and Target Hybridizing Sequence Including, (b) the complementary oligomer comprises, in the 3' to 5' direction: the complement of the second portion of the capture sequence, and the complement of at least the first portion of the spacer sequence wherein the complement of the second portion of the capture sequence and the complement of at least the first portion of the spacer sequence are configured to simultaneously anneal to the capture oligomer in the absence of the complement of the spacer sequence.

13. The capture oligomer has the formula: 5'-A1-C1-C2-B-A2-S1-S2-A3-RB-A4-THS-X-3' where A1 is a first additional sequence that is optionally present; C1 is the first portion of the capture sequence; C2 is the second portion of the capture sequence; B is the internal extension blocker; A2 is an optional second additional sequence, S1 is the first portion of the spacer sequence, S2 is the second portion of the spacer sequence; A3 is an optional third additional sequence; RB is an optionally present reversible elongation blocker; A4 is an optional fourth additional sequence; THS is the target hybridizing sequence; X is an optional blocking moiety.

13. The combination of claim 12, having the formula:

14. The complementary oligomer has the formula: 5'-S1'-A2'-L-C2'-X-3' wherein S1' is the complement of at least the first portion of the spacer sequence; A2' is the optional complement of a second additional sequence optionally present in said capture oligomer; L is an optional linker, C2' is the complement of the second portion of the capture sequence; X is an optional blocking moiety.

14. The combination according to claim 12 or 13, having the formula:

15. 15. The combination of any one of claims 12 to 14, wherein the capture oligomer and / or the complementary oligomer comprises a blocking moiety at its 3' end.

16. 16. The combination of any one of claims 12 to 15, wherein when the spacer sequence of the capture oligomer is occupied by a distinct complement, the complement of the second portion of the capture sequence is insufficient to stably anneal to the capture sequence of the capture oligomer at a temperature of 65°C or greater.

17. 17. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 16, wherein the capture sequence comprises polyA or polyT, and the complement of the capture sequence or the complement of the second portion of the capture sequence comprises polyT or polyA.

18. 18. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 17, wherein the capture oligomer comprises an adapter sequence as part or all of the spacer sequence, or as part or all of the third additional sequence 3' of the spacer sequence or the fourth additional sequence 5' of the target hybridizing sequence.

19. 19. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 18, wherein the capture oligomer comprises, for example, an affinity enhancing modification in the target hybridizing sequence, such as any one or more of 5-Me-C, 2-aminopurine, 2'-fluoro, C-5-propyne, LNA, PNA, ZNA, phosphorothioate, 2'-OMe, or constrained ethyl (cEt) substitutions.

20. 20. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 19, wherein the capture oligomer comprises a reversible extension blocker located 5' to the target hybridizing sequence.

21. 21. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 20, comprising a second additional sequence between the internal extension blocker and the first portion of the spacer sequence, the second additional sequence comprising a mixed nucleotide segment.

22. 22. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 21, further comprising a secondary capture reagent comprising a complement of the capture sequence and (a) a binding partner (e.g., biotin) or (b) a solid support (e.g., a bead or surface).

23. 23. The capture oligomer, reaction mixture, or combination of any one of claims 12 to 22, further comprising a displacer oligomer comprising a displacer target hybridizing sequence configured to bind to a target polynucleotide, the 3' end of the displacer target hybridizing sequence being oriented toward the site bound by the target hybridizing sequence of the capture oligomer.

24. 1. A method for capturing a target polynucleotide from a composition, the method comprising: contacting the composition with the combination of any one of claims 12 to 22, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site that includes the 3' end of the target polynucleotide; extending the 3' end of the target polynucleotide with a DNA polymerase having strand displacement activity, thereby forming the complement of the spacer sequence annealed to the capture oligomer such that the complementary oligomer is displaced to an extent sufficient that the capture sequence of the capture oligomer is available for binding; contacting the capture sequence of the capture oligomer with a complement of the capture sequence and a secondary capture reagent comprising (i) a binding partner or (ii) a solid support, thereby forming a complex comprising the target polynucleotide, the capture oligomer, and the secondary capture reagent; and isolating said complex from said composition, thereby capturing said target polynucleotide. A method comprising:

25. 25. The method of claim 24, wherein the secondary capture reagent comprises a binding partner (e.g., biotin) and isolating comprises contacting the complex with a solid support (e.g., a bead) that comprises a second binding partner (e.g., streptavidin) configured to bind to the binding partner of the secondary capture reagent.

26. 1. A method for capturing a target polynucleotide from a composition, the method comprising: contacting the target polynucleotide with a capture oligomer or combination of any one of claims 1-10 or 12-23, wherein the target hybridizing sequence of the capture oligomer anneals to the target polynucleotide at a site upstream of the 3' end of the target polynucleotide; extending the 3' end of the capture oligomer along the target polynucleotide, thereby forming a first extended strand; contacting the target polynucleotide with a displacer oligomer comprising a displacer target hybridizing sequence that anneals to the target polynucleotide downstream of the target hybridizing sequence of the capture oligomer; extending said displacer oligomer along said target polynucleotide, thereby displacing said first extended strand of said target polynucleotide. Including, Optionally, the method wherein said capture oligomer and said displacer are added to said composition simultaneously or sequentially.

27. 27. The method of claim 26, further comprising contacting the first extended strand with a reverse amplification oligomer comprising a reverse target hybridizing sequence configured to bind to the first extended strand, and extending the reverse amplification oligomer, thereby forming a second extended strand.

28. 28. The method of claim 27, wherein the reverse amplification oligomer comprises an additional sequence 5' of its target hybridizing sequence, and optionally the 3' end of the first extended strand is further extended, thereby forming the complement of the additional sequence of the reverse amplification oligomer.

29. 29. The method of any one of claims 26 to 28, wherein the capture oligomer further comprises an additional sequence located 5' to the target hybridizing sequence, and optionally the extension of the second extension strand, if present, forms the complement of the additional sequence of the capture oligomer.

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