Compositions and methods for capturing and amplifying target polynucleotides using modified capture primers

Abstract

A composition for capturing a target polynucleotide on a surface of a substrate is provided. The composition may include a plurality of capture primers bound to a surface of the substrate, the plurality of capture primers comprising modified nucleic acids, and a plurality of orthogonal capture primers bound to a surface of the substrate, the plurality of capture primers comprising modified nucleic acids. The modified nucleic acid of the capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T. The modified nucleic acid of the orthogonal capture primers may include locked nucleic acid (LNA), peptide nucleic acid (PNA), or super T.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 128,675, filed December 21, 2020, entitled "Compositions and Methods for Capturing and Amplifying Target Polynucleotides Using Modified Capture Primers", the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application includes a sequence listing submitted electronically in ASCII format, the entire contents of which are incorporated herein by reference. The ASCII copy is named IP - 2054 - PCT_SL.txt and is 4,174 bytes in size.

[0003] This application relates to cluster amplification.

Background Art

[0004] Cluster amplification is an approach for amplifying polynucleotides, for example, intended for use in gene sequencing. A target polynucleotide is captured by primers (e.g., P5 and P7 capture primers) bound to the surface of a substrate within a flow cell, forming "seeds" at random locations on the surface. Amplification cycles are performed to form clusters on the surface around each seed. The clusters contain copies and complementary copies of the seed polynucleotide (collectively referred to as "amplicons"). For example, Figure 1 schematically illustrates the amplification of a polynucleotide on a substrate. Capture and amplification of a single seed 111 on a substrate region 101 can substantially fill the substrate region with amplicons of seed 111, and may result in monoclonal clusters 121 that can be readily used for sequencing-by-synthesis, or SBS (dashed and dotted circles are intended to represent cluster expansion over time). In some scenarios, the substrate is patterned to define regions bound to different clusters, such as wells, which can be filled with each cluster. [Overview of the project]

[0005] Examples provided herein relate to the capture and amplification of target polynucleotides using modified capture primers. Compositions and methods for carrying out such capture and amplification are disclosed.

[0006] In some examples, compositions for capturing target polynucleotides on the surface of a substrate are provided herein. The composition may comprise a plurality of capture primers bound to the surface of the substrate. Each capture primer may comprise a modified nucleic acid. The composition may also comprise a plurality of orthogonal capture primers bound to the surface of the substrate. Each orthogonal capture primer may comprise a modified nucleic acid.

[0007] In some examples, the modified nucleic acid of the capture primer includes lock nucleic acid (LNA), peptide nucleic acid (PNA), or super T. In some examples, the capture primer further includes deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and DNA are distributed between the 5' and 3' ends of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the capture primer, and the DNA is located at the 3' end of the capture primer.

[0008] In some examples, the modified nucleic acid of the orthogonal capture primer includes Loc nucleic acid (LNA), peptide nucleic acid (PNA), or Super T. In some examples, the orthogonal capture primer further includes deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and DNA are distributed between the 5' and 3' ends of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the orthogonal capture primer, and the DNA is located at the 3' end of the orthogonal capture primer.

[0009] In some examples, the composition further comprises a first target polynucleotide, each comprising a first adapter complementary to a capture primer and a second adapter complementary to an orthogonal capture primer. The composition may further comprise a second target polynucleotide complementary to each of the first target polynucleotides.

[0010] In some examples, several first adapters of the first target polynucleotide hybridize to each of the capture primers to form a first double helix, and several second adapters of the first target polynucleotide hybridize to each of the orthogonal capture primers to form a second double helix. In some examples, the first and second double helixes each have a melting temperature (Tm) greater than that of the third double helix formed by the hybridization of the second target polynucleotide to each of the first target polynucleotides complementary to the second target polynucleotide. In some examples, the first and second double helixes each have a Tm of approximately 80°C to approximately 110°C. In some examples, the first and second double helixes each have a Tm of approximately 85°C to approximately 105°C. In some examples, the first and second double helixes each have a Tm of approximately 90°C to approximately 100°C.

[0011] In some cases, virtually none of the second target polynucleotides hybridize in solution with any of the first target polynucleotides.

[0012] In some examples, the composition further comprises about 1% to about 100% formamide (%v / v), or about 5% to about 80% formamide (%v / v).

[0013] In some examples, the composition further contains about 100 to about 800 mM Na+, or about 200 to about 800 mM Na+.

[0014] In some examples, the capture primer is a modified P5 capture primer, and the orthogonal capture primer is a modified P7 capture primer.

[0015] In some examples, each capture primer contains approximately 5 to 20 modified nucleic acids, and each orthogonal capture primer contains approximately 5 to 20 modified nucleic acids.

[0016] In some examples, each capture primer contains at least about nine modified nucleic acids, and each orthogonal capture primer contains at least about nine modified nucleic acids, or each capture primer contains at least about twelve modified nucleic acids, and each orthogonal capture primer contains at least about twelve modified nucleic acids, or each capture primer contains at least about fifteen modified nucleic acids, and each orthogonal capture primer contains at least about fifteen modified nucleic acids.

[0017] In some examples, methods for capturing and amplifying target polynucleotides on the surface of a substrate are provided herein. The method may involve contacting a composition with a fluid. The composition may include a plurality of capture primers bound to the surface of a substrate, each of which contains a modified nucleic acid; and a plurality of orthogonal capture primers bound to the surface of a substrate, each of which contains a modified nucleic acid. The fluid may include a first target polynucleotide comprising a first adapter complementary to the capture primers and a second adapter complementary to the orthogonal capture primers, and a second target polynucleotide complementary to each of the first target polynucleotides. This method may include: hybridizing several first adapters of the first target polynucleotide to each of the capture primers to form a first double helix, while inhibiting hybridization of the second target polynucleotide to the first target polynucleotide in a fluid; hybridizing several second adapters of the first target polynucleotide to each of the orthogonal capture primers to form a second double helix; and then amplifying the first target polynucleotide, wherein the amplification includes generating amplicons of each of the first target polynucleotides.

[0018] In some examples, the modified nucleic acid of the capture primer includes lock nucleic acid (LNA), peptide nucleic acid (PNA), or super T. In some examples, the capture primer further includes deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and DNA are distributed between the 5' and 3' ends of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the capture primer, and the DNA is located at the 3' end of the capture primer.

[0019] In some examples, the modified nucleic acid of the orthogonal capture primer includes Loc nucleic acid (LNA), peptide nucleic acid (PNA), or Super T. In some examples, the orthogonal capture primer further includes deoxyribonucleic acid (DNA). In some examples, the modified nucleic acid and DNA are distributed between the 5' and 3' ends of the capture primer. In some examples, the modified nucleic acid is located at the 5' end of the orthogonal capture primer, and the DNA is located at the 3' end of the orthogonal capture primer.

[0020] In some examples, the first and second double strands each have a greater melting temperature (Tm) than the third double strand formed by the hybridization of the second target polynucleotide to each of the first target polynucleotides complementary to the second target polynucleotide. In some examples, the first and second double strands each have a Tm of approximately 80°C to approximately 110°C. In some examples, the first and second double strands each have a Tm of approximately 85°C to approximately 105°C. In some examples, the first and second double strands each have a Tm of approximately 90°C to approximately 100°C.

[0021] In some cases, virtually none of the second target polynucleotides hybridize in solution with any of the first target polynucleotides.

[0022] In some cases, hybridization is carried out with approximately 1% to 100% formamide (%v / v) or approximately 5% to 80% formamide (%v / v).

[0023] In some examples, hybridization is performed at about 100 to about 800 mM Na+, or at about 200 to about 800 mM Na+.

[0024] In some examples, the capture primer is a modified P5 capture primer and the orthogonal capture primer is a modified P7 capture primer.

[0025] In some examples, each of the capture primers comprises about 5 to about 20 modified nucleic acids, and each of the orthogonal capture primers comprises about 5 to about 20 modified nucleic acids. In some examples, each of the capture primers comprises at least about 9 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 9 modified nucleic acids, or each of the capture primers comprises at least about 12 of the modified nucleic acids, and each of the orthogonal capture primers comprises at least about 12 of the modified nucleic acids, or each of the capture primers comprises at least about 15 modified nucleic acids, and each of the orthogonal capture primers comprises at least about 15 modified nucleic acids.

[0026] It should be understood that any corresponding features / examples of each of the aspects of the disclosure as described herein may be implemented together in any suitable combination. Also, any features / examples from any one or more of these aspects may be implemented in any suitable combination with any of the features of any other aspects as described herein in order to obtain the advantages as described herein.

Brief Description of the Drawings

[0027] [Figure 1] Schematically shows the amplification of polynucleotides on a substrate. [Figure 2A] Schematically shows the compositions and operations in a process flow for capturing polynucleotides on a substrate using a conventionally known capture primer. [Figure 2B] Schematically shows the compositions and operations in a process flow for capturing polynucleotides on a substrate using a conventionally known capture primer. [Figure 2C] The compositions and operations in a process flow for capturing polynucleotides on a substrate using conventionally known capture primers are schematically shown. [Figure 3A] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3B] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3C] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3D] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3E] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3F] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3G] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 3H] The present invention schematically illustrates exemplary compositions and operations in an exemplary process flow for capturing and amplifying polynucleotides on a substrate using the capture primers of the present invention. [Figure 4] A schematic diagram shows an exemplary double-stranded structure between a polynucleotide and one of the capture primers of the present invention, as shown in several examples. [Figure 5A]This plot illustrates the exemplary effect of conditions on the capture of polynucleotides by the capture primers of the present invention. [Figure 5B] This plot illustrates the exemplary effect of conditions on the capture of polynucleotides by the capture primers of the present invention. [Figure 5C] This plot illustrates the exemplary effect of conditions on the capture of polynucleotides by the capture primers of the present invention. [Figure 6] This diagram illustrates an exemplary workflow for a method of capturing and amplifying polynucleotides using the capture primers of the present invention. [Modes for carrying out the invention]

[0028] The examples provided herein relate to the capture and amplification of polynucleotides using modified capture primers. Compositions and methods for carrying out such capture and amplification are disclosed.

[0029] The modified capture primers provided herein can hybridize more cohesively with target polynucleotides than conventionally known capture primers, thereby increasing the efficiency of target polynucleotide capture on the substrate. Exemplarily, due to stronger binding, clusters can be prepared on the substrate even with lower concentrations of target polynucleotides compared to conventionally known capture primers. For example, under conditions in which polynucleotide capture and amplification are carried out using conventionally known primers in the manner further described below with reference to Figures 2A-2C, surface hybridization and solution-based annealing act as competing processes, resulting in some polynucleotides being unavailable for capture or amplification. In contrast, the methods of the present invention, as described with reference to Figures 3A-3H, 4, 5A-5C, and 6, allow the capture primers of the present invention to be carried out under conditions that reduce or inhibit solution-based annealing as a competing process, thereby substantially increasing the effectiveness of polynucleotides for capture and amplification, enabling cluster preparation on the substrate compared to conventionally known capture primers by using higher concentrations of target polynucleotides, and reducing the overall seeding time.

[0030] First, some terms used herein are briefly explained. Next, several exemplary compositions and methods for capturing and amplifying polynucleotides using the capture primers are described.

[0031] term Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. The use of the term “including,” and other forms such as “include,” “includes,” and “included,” is not limited. Furthermore, the use of the term “having,” and other forms such as “have,” “has,” and “had,” is not limited. When used herein, in transitional clauses or in the text of claims, the terms “comprise” and “comprising” should be interpreted as having an open-ended meaning; that is, the above terms should be interpreted as synonymous with the phrases “at least have” or “at least include.” For example, when used in the context of a process, the term “comprising” means that the process includes at least the listed steps, but may include additional steps. When used in the context of compounds, compositions, or devices, the term "comprising" means that the compound, composition, or device includes at least the listed features or components, but may also include additional features or components.

[0032] The terms “substantially,” “approximately,” and “about” as used throughout this specification are used to describe and account for small variations resulting from processing variability, etc. For example, they may refer to ±10%, such as ±5%, ±2%, ±1%, ±0.5%, ±0.2%, ±0.1%, ±0.05%, etc.

[0033] As used herein, “hybridize” means to form a “double helix” by non-covalently bonding a first polynucleotide to a second polynucleotide along the length of those polynucleotides. For example, two DNA polynucleotide strands may associate via complementary base pairing. The strength of the association between the first and second polynucleotides increases with the complementarity between the nucleotide sequences within those polynucleotides. The strength of hybridization between polynucleotides can be characterized by the melting temperature (Tm) at which 50% of the double helix polynucleotide strands separate. Polynucleotides that are “partially” hybridized to one another mean that they have complementary sequences, but such sequences hybridize to each other along only a portion of their length to form a partial double helix. Polynucleotides that are “unable” to hybridize include those that are physically separated from each other with an insufficient number of bases to come into contact with each other in order to hybridize.

[0034] As used herein, the term “nucleotide” is intended to mean a molecule comprising a sugar moiety, a skeletal component containing at least one phosphate group, and, in some examples, a nucleic acid base. A nucleotide lacking a nucleic acid base may be referred to as “debased.” Nucleotides include deoxyribonucleotides, modified deoxyribonucleotides, ribonucleotides, modified ribonucleotides, peptide nucleotides, modified peptide nucleotides, modified phosphate-skeletal nucleotides, and mixtures thereof. Examples of nucleotides include adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), uridine phosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), deoxyadenosine monophosphate (dAMP), and deoxyadenosine Examples include deoxyuridine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxycytidine diphosphate (dCDP), deoxycytidine triphosphate (dCTP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), and deoxyuridine triphosphate (dUTP).

[0035] As used herein, the term “nucleotide” is also intended to encompass any “nucleotide analogue,” i.e., a type of nucleotide that, compared to naturally occurring nucleotides, contains modified nucleic acid bases, sugar moieties, and / or skeletal components (such as phosphates or amides). Nucleotide analogues may also be referred to as “modified nucleic acids.” Exemplary modified nucleic acid bases include inosine, xatianine, hypoxatianine, isocytosine, isoguanine, 2-aminopurine, 5-methylcytosine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyladenine, 6-methylguanine, 2-propylguanine, 2-propyladenine, 2-thiouracil, 2-thiothymine, 2-thiocytosine, 15-halouracil, 15-halocytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6- Examples include azothymine, 5-uracil, 4-thiouracil, 8-halogenated adenine or guanine, 8-aminoadenine or guanine, 8-thioladenine or guanine, 8-thioalkyladenine or guanine, 8-hydroxyladenine or guanine, 5-halogenated uracil or cytosine, 7-methylguanine, 7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, etc. As is known in the art, certain nucleotide analogs, such as adenosine 5'-phosphosulfate, cannot be incorporated into polynucleotides. The nucleotide backbone may contain any suitable number of phosphates, e.g., 3, 4, 5, 6, or more than 6 phosphates. Nucleotide analogs also include locic nucleic acids (LNA), peptide nucleic acids (PNA), and 5-hydroxylbutynyl-2'-deoxyuridine ("Super T"). LNA contains an RNA-like backbone with additional crosslinks linking the ribose portion to the 2' oxygen and 4' carbon. PNA contains a backbone comprising N-(2-aminoethyl)-glycine units linked by peptide bonds and nucleic acid bases attached to the backbone via methylene crosslinks and carbonyl groups.Super T contains the 5-hydroxybutynyl-2'-deoxyuridine nucleic acid base. Exemplary structures of LNA, PNA, and Super T are shown below.

[0036] [ka]

[0037] As used herein, the term “polynucleotide” refers to a molecule containing a sequence of nucleotides linked together. Polynucleotides are a non-exclusive example of polymers. Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and their analogues such as roch nucleic acid (LNA) and peptide nucleic acid (PNA). Polynucleotides may include single-stranded sequences of nucleotides, such as RNA or single-stranded DNA, double-stranded sequences of nucleotides, such as double-stranded DNA, or mixtures of single-stranded and double-stranded sequences of nucleotides. Double-stranded DNA (dsDNA) includes genomic DNA and products of PCR and amplification. Single-stranded DNA (ssDNA) can be converted to dsDNA, and vice versa. Polynucleotides may include enantiomerized DNA, LNA, or non-spontaneously occurring DNA such as PNA. The exact sequence of nucleotides in a polynucleotide may be known or unknown. The following are examples of polynucleotides: genes or gene fragments (e.g., probes, primers, expression sequence tags (ESTs), or gene expression (SAGE) tag serial analysis), genomic DNA, genomic DNA fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, synthetic polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, any of the aforementioned primers, or amplified copies. Polynucleotides may have “chimeric” structures containing adjacent sections of different types of polynucleotides, such as PNA and RNA, DNA and RNA, DNA and PNA, and adjacent sections.

[0038] As used herein, “polymerase” is intended to mean an enzyme having an active site for assembling polynucleotides by polymerizing nucleotides into polynucleotides. A polymerase can bind to a primed single-stranded target polynucleotide and grow it by sequentially adding nucleotides to the primer to form a “complementary copy” polynucleotide having a sequence complementary to the sequence of the target polynucleotide. Another polymerase, or the same polymerase, can then form a copy of the target polynucleotide by forming a complementary copy of its complementary copy polynucleotide. Any such copy may be referred to herein as an “amplicon.” A DNA polymerase can bind to a target polynucleotide and then move the target polynucleotide downstream while growing it by sequentially adding the free hydroxyl group at the 3' end of the polynucleotide chain (amplicon growth). A DNA polymerase can synthesize a complementary DNA molecule from a DNA template, and an RNA polymerase can synthesize an RNA molecule from a DNA template (transcription). Polymerases can initiate chain growth using short RNA or DNA strands (primers). Some polymerases can displace the strand upstream of the site where they add bases to the strand. Such polymerases may also be said to be strand-displacing, meaning they have the activity to remove a complementary strand from the template strand read by the polymerase. Exemplary polymerases with strand-displacing activity include, but are not limited to, large fragments of Bst (Bacillus stearothermophilus) polymerase, exo-Klenow polymerase, or sequencing-grade T7 exo-polymerase. Some polymerases cleave their preceding strand and effectively replace it with the growing strand (5' exonuclease activity). Some polymerases have the activity to degrade their following strand (3' exonuclease activity). Some useful polymerases have been mutated or otherwise modified to reduce or eliminate their 3' and / or 5' exonuclease activity.

[0039] As used herein, the term “primer” is defined as a polynucleotide to which a nucleotide can be added via a free 3'OH group. A primer may include a 3' block that prevents polymerization until the block is removed. A primer may include a 5' end modification to enable a coupling reaction or to allow the primer to be attached to another part. A primer may include one or more parts, such as 8-oxo-G, which can be cleaved under suitable conditions, such as UV light, chemicals, or enzymes. The length of a primer may be the length of any suitable number of bases and may include suitable combinations of natural and / or non-natural nucleotides. A target polynucleotide may include an “adapter” that hybridizes to the primer (having a complementary sequence thereto) and may be amplified to produce a complementary copy polynucleotide by adding a nucleotide to the free 3'OH group of the primer. “Capture primer” is intended to mean a primer that can bind to a substrate and hybridize to a first adapter of the target polynucleotide, and “orthogonal capture primer” is intended to mean a primer that can bind to a substrate and hybridize to a second adapter of its target polynucleotide. The first adapter may have a sequence complementary to the sequence of the capture primer, and the second adapter may have a sequence complementary to that of the orthogonal capture primer. The capture primer and the orthogonal capture primer may have different and independent sequences. In addition, the capture primer and the orthogonal capture primer may differ from each other in at least one other property. For example, the capture primer and the orthogonal capture primer may have different lengths, and either the capture primer or the orthogonal capture primer may contain non-nucleic acid portions (such as blocking groups or removal portions) that the other of the capture primer or the orthogonal capture primer rack does not have, or may contain any preferred combination of such properties. A “modified capture primer” is a capture primer or orthogonal capture primer containing multiple nucleic acid analogs such as LNA, PNA, or Super T. A modified capture primer may further contain multiple naturally occurring nucleic acids, such as but not limited to DNA.

[0040] As used herein, the term “substrate” refers to a material used as a carrier for the compositions described herein. Exemplary substrate materials may include glass, silica, plastics, quartz, metals, metal oxides, organosilicates (e.g., polyhedral organosilsesquioxanes (POSS)), polyacrylates, tantalum oxides, complementary metal oxide semiconductors (CMOS), or combinations thereof. An example of POSS may be found in Kehagias et al., Microelectronic Engineering 86 (2009), pp. 776-778, which is incorporated herein by reference in its entirety. In some examples, substrates used in this application may include silica-based substrates such as glass, condensed silica, or other silica-containing materials. In some examples, the substrate may include silicon, silicon nitride, or hydrogenated silicone. In some examples, the substrates used in this application may include plastic materials or components such as polyethylene, polystyrene, poly(vinyl chloride), polypropylene, nylon, polyester, polycarbonate, and poly(methyl methacrylate). Exemplary plastic materials include poly(methyl methacrylate), polystyrene, and cyclic olefin polymer substrates. In some examples, the substrate is or contains silica-based materials or plastic materials or combinations thereof. In certain examples, the substrate has at least one surface containing glass or silicon-based polymers. In some examples, the substrate may contain metals. In some such examples, the metal is gold. In some examples, the substrate has at least one surface containing metal oxides. In one example, the surface contains tantalum oxide or tin oxide. Acrylamide, enone, or acrylates may also be used as substrate materials or components. Other substrate materials may, but are not limited to, gallium arsenide, indium phosphine, aluminum, ceramics, polyimide, quartz, resins, polymers, quartz, resins, polymers, and copolymers. In some examples, the substrate and / or substrate surface may be or contain quartz. In some other examples, the substrate and / or substrate surface may be or contain semiconductors such as GaAs or ITO.The aforementioned list is intended to be illustrative in this application, but is not limiting. The substrate may comprise a single material or several different materials. The substrate may be a composite or a laminate. In some examples, the substrate comprises an organic silicate material. The substrate may be flat, circular, spherical, rod-shaped, or any other suitable shape. The substrate may be rigid or flexible. In some examples, the substrate is a bead or a flow cell.

[0041] In some examples, the surface is a patterned surface. “Patterned surface” refers to an arrangement of different regions within or on the exposed layer of the substrate. For example, one or more regions may be features where one or more capture primers are present. These features may be separated by gap regions where no capture primers are present. In some examples, the pattern may be an xy format of features in rows and columns. In some examples, the pattern may be a repeating arrangement of features and / or gap regions. In some examples, the pattern may be a random arrangement of features and / or gap regions. In some examples, the substrate includes an array of wells (recesses) on its surface. The wells may be provided by substantially vertical sidewalls. The wells can be manufactured using a variety of techniques, including but not limited to photolithography, stamping techniques, molding techniques, and microetching techniques, as is commonly known in the art. As understood in the art, the techniques used depend on the composition and shape of the array substrate.

[0042] Features within a patterned surface of a substrate may include wells in which patterned, covalently bonded gels, such as poly(N-(5-azidoacetamylpentyl)acrylamide-co-acrylamide) (PAZAM), are arranged in an array on glass, silicon, plastic, or other suitable material. This process creates a gel pad used for sequencing, which can be stable across sequencing operations over numerous cycles. Covalently bonding the polymer to the wells can be useful in maintaining the gel in the structured features throughout the lifespan of the structured substrate during various applications. However, in many examples, the gel does not need to be covalently bonded to the wells. For example, under certain conditions, silane-free acrylamide (SFA) that is not covalently bonded to any part of the structured substrate can be used as the gel material.

[0043] In a specific alternative embodiment, the structured substrate can be prepared by patterning a suitable material using wells (e.g., microwells or nanowells), coating the patterned material with a gel material (e.g., PAZAM, SFA, or a chemically modified variant thereof, e.g., an azidated form of SFA (azido-SFA)), and polishing the gel-coated material, for example, by chemical or mechanical polishing, thereby retaining the gel within the wells, while substantially all of the gel is removed or inactivated from the interstitial regions of the surface of the structured substrate between the wells. Primers can be bound to the gel material. A solution of target polynucleotides (e.g., a fragmented human genome or a portion thereof) can then be brought into contact with the polished substrate so that individual target nucleotides are seeded into individual wells via interaction with primers attached to the gel material, but the target polynucleotides do not occupy interstitial regions because the gel material is absent or inactive. Amplification of the target polynucleotides can be confined to the wells because the absence or inactivity of the gel in the interstitial regions prevents outward movement of clusters. This process is easy to manufacture, scalable, and utilizes conventional micro or nano-fabrication methods.

[0044] Patterned substrates include, for example, wells etched onto slides or tips. The etching and geometric patterns of the wells can be of various different shapes and sizes, and such features may be physically or functionally separable from one another. Particularly useful substrates having such structural features include patterned substrates with selectable solid particle sizes, such as microspheres. An exemplary patterned substrate having these properties is an etched substrate used in connection with BEAD array technology (Illumina, Inc., San Diego, Calif.).

[0045] In some examples, the substrates described herein form at least part of a flow cell, are located within a flow cell, or are coupled to a flow cell. A flow cell may include a fluid chamber divided into multiple lanes or multiple sectors. Examples of flow cells and substrates for the manufacture of flow cells that may be used in the methods and compositions described herein include, but are not limited to, those commercially available from Illumina, Inc. (San Diego, Calif.).

[0046] As used herein, the term “direct” is intended to mean, when used in reference to a layer covering the surface of a substrate, that the layer covers the surface of the substrate without any significant intermediate layer, such as an adhesive layer or a polymer layer. A layer that directly covers a surface can adhere to this surface through any chemical or physical interaction, such as covalent or non-covalent bonds.

[0047] As used herein, and as used in reference to polynucleotides, the term “immobilized” is intended to mean direct or indirect attachment to a substrate via covalent or non-covalent bonds. In certain examples, covalent bonds may be used, or any other suitable bond may be used to allow the polynucleotide to remain stationary or to adhere to the substrate under conditions where the substrate is intended to be used, for example, during amplification or sequencing of the polynucleotide. Polynucleotides used as capture primers or target polynucleotides may be immobilized such that their 3' end is available for enzymatic extension and at least a portion of their sequence can hybridize to a complementary sequence. Immobilization may occur via hybridization to a surface-attached oligonucleotide, in which case the immobilized oligonucleotide or polynucleotide may be oriented 3' to 5'. Alternatively, immobilization may occur by means other than base-pairing hybridization, such as covalent bonds. Exemplarily, a chemical functional group may be incorporated into one of the 5' ends of the capture primer of the present invention via a chemical linker. Chemical functional groups on the capture primer can be bonded to the substrate surface via any preferred combination of one or more non-covalent interactions (e.g., electrostatic, metal-ligand bonding, hybridization, etc.) or covalent interactions (e.g., copper click reaction, copper free click reaction, etc.).

[0048] As used herein, the term “array” refers to a group of substrate regions that can be distinguished from one another according to their relative positions. Different molecules (such as polynucleotides) in different regions of an array can be distinguished from one another according to their positions within the array. Individual regions of an array may contain one or more molecules of a particular type. For example, a substrate region may contain a single target polynucleotide having a particular sequence, or a substrate region may contain several polynucleotides having the same sequence (or complementary sequences). Regions of an array may contain different features on the same substrate. Exemplary features include, but are not limited to, wells in the substrate, beads (or other particles) in or on the substrate, protrusions from the substrate, ridges on the substrate, or channels within the substrate. Regions of an array may each contain different regions on different substrates. Different molecules attached to separate substrates can be identified according to the position of the substrate on the surface where the substrates associate, or according to the position of the substrate in a liquid or gel. Exemplary arrays in which separate substrates are arranged on a surface include, but are not limited to, those having beads in wells.

[0049] As used herein, the term “multiple” is intended to mean a group of two or more distinct members. A multiple can range in size from small, medium, large to very large. A small multiple might range, for example, from a few members to several dozen members. A medium multiple might range, for example, from several dozen members to about 100 or several hundred members. A large multiple might range, for example, from about several hundred members to about 1,000 members, several thousand members, and tens of thousands of members. A very large multiple might range, for example, from tens of thousands of members to about several hundred thousand, several million, tens of millions, or hundreds of millions or more members. Thus, a multiple can also range in size from 200 million to over 100 million, as well as all sizes measured as the number of members between and above the exemplary ranges described above. An exemplary polynucleotide complex might have, for example, about 1 × 10⁶ 5 The above 5 x 10 5 The above, or 1 x 10 6This includes a population of 2 or more different polynucleotides. Therefore, the definition of this term is intended to include all integer values ​​greater than 2. The upper limit of multiple values ​​can be set, for example, by the theoretical diversity of polynucleotide sequences in the sample.

[0050] As used herein, the term “double-stranded,” when used in reference to a polynucleotide, is intended to mean that all or substantially all of the nucleotides in the polynucleotide are hydrogen-bonded to the respective nucleotides in the complementary polynucleotide. A “partially” double-stranded polynucleotide has at least about 10%, at least about 25%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% of its nucleotides, fewer than all of them, hydrogen-bonded to the nucleotides in the complementary polynucleotide.

[0051] As used herein, the term “single-stranded,” when used in reference to a polynucleotide, means that none of the nucleotides in the polynucleotide are hydrogen-bonded to any other nucleotide in the complementary polynucleotide. A polynucleotide that “cannot” hybridize to another polynucleotide may be single-stranded.

[0052] As used herein, the term “target polynucleotide” is intended to mean the polynucleotide that is the object of analysis or action, and may be referred to as “library polynucleotide,” “template polynucleotide,” or “library template.” Analysis or action may include subjecting the polynucleotide to capture, amplification, sequencing, and / or other procedures. The target polynucleotide may include a nucleotide sequence added to the target sequence to be analyzed. For example, the target polynucleotide may include one or more adapters, such as an adapter that functions as a primer binding site, the flank portion of which is the target polynucleotide sequence to be analyzed. The target polynucleotide hybridized to the capture primer may include nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide so that not all of the target polynucleotide is suitable for extension. In certain examples, multiple target polynucleotides may have first and second adapters that are identical to each other but have different sequences. Two adapters that may be adjacent to a particular target polynucleotide sequence may have the same sequence as each other, or complementary sequences as each other, or the two adapters may have different sequences. Therefore, species in multiple target polynucleotides may include known sequence regions adjacent to unknown sequence regions, which can be evaluated, for example, by sequencing (e.g., SBS). In some examples, the target polynucleotide carries an adapter at a single end, and such adapter may be located at either the 3' or 5' end of the target polynucleotide. The target polynucleotide may be used without an adapter, in which case the primer-binding sequence may be the sequence present in the target polynucleotide directly.

[0053] The terms “polynucleotide” and “oligonucleotide” are used interchangeably herein. Unless otherwise specified, the distinction between the terms is not intended to indicate any specific difference in size, sequence, or other properties. For clarity, when describing a particular method or composition involving several polynucleotide species, the terms may be used to distinguish one species of polynucleotide from another.

[0054] As used herein, the term “amplicon,” when used in relation to nucleic acids, means the product of replication of a nucleic acid, which has a nucleotide sequence substantially identical or complementary to at least a portion of the nucleotide sequence of the nucleic acid. “Amplification” and “to amplify” refer to the process of producing an amplicon of a polynucleotide. The first amplicon of a target polynucleotide may typically be a complementary copy. Further amplicons are copies made from the target polynucleotide or the first amplicon after the production of the first amplicon. Subsequent amplicons may have a sequence that is substantially complementary to or substantially identical to the target polynucleotide. It will be understood that a small number of mutations in the polynucleotide (e.g., due to amplification artifacts) may occur when producing an amplicon of that polynucleotide.

[0055] A substrate region containing substantially only the amplicons of a given polynucleotide may be called "monoclonal," while a substrate region containing amplicons of polynucleotides having different sequences may be called "polyclonal." A polyclonal region of a substrate may contain different sub-regions, each of which is monoclonal. Each such monoclonal region may correspond to a "cluster" generated from a "seed," whether within a larger polyclonal region or in itself. A "seed" may refer to a single target polynucleotide, while a "cluster" may refer to an aggregate of amplicons of that target polynucleotide.

[0056] Compositions and methods for capturing and amplifying polynucleotides Examples provided herein relate to compositions and methods for capturing and amplifying target polynucleotides using modified capture primers that can be used under solution-based conditions that reduce or inhibit annealing while enabling the modified capture primers to capture the target polynucleotides from solution. In comparison, conventionally known capture primers can be used under conditions where solution-based annealing competes with the capture of the target polynucleotide from solution, and such competition reduces the availability of the target polynucleotide for capture.

[0057] For example, Figures 2A and 2C schematically illustrate compositions and operations in a process flow for capturing polynucleotides on a substrate using conventionally known capture primers. Composition 2000 shown in Figure 2A comprises a substrate 200 containing a capture primer 231 and an orthogonal capture primer 232 bound thereto, and a fluid (solution) containing a target polynucleotide, e.g., a polynucleotide intended for sequencing using sequencing-by-synthesis (SBS). The target polynucleotide may include or be provided in the form of double helix (exemplary, double-stranded D1, D2, and D3) between target polynucleotides having complementary sequences. For example, double-stranded D1 may include a first target polynucleotide comprising sequence 211, a first adapter 221, and a second adapter 222, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 211', a first complementary adapter 221', and a second complementary adapter 222'. Similarly, double-stranded D2 may include a first target polynucleotide comprising sequence 212, a first adapter 221, and a second adapter 222, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 212', a first complementary adapter 221', and a second complementary adapter 222'. For example, double-stranded D3 may comprise a first target polynucleotide comprising sequence 213, a first adapter 221, and a second adapter 222, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 212', a first complementary adapter 221', and a second complementary adapter 222'. Sequences 211, 212, and 213 may be different from each other, and it may be desirable to determine their sequences using SBS. The first adapter 221 may be complementary to a capture primer 231 so that it can hybridize under suitable conditions, and the second adapter 222 may be complementary to an orthogonal capture primer 232 so that it can hybridize under suitable conditions.Although not specifically shown in the illustration, each capture primer 231 or each orthogonal capture primer 232 may contain cleavable portions such as 8-oxo-G.

[0058] The capture primer 231 may be, for example, a P5 capture primer, and the orthogonal capture primer 232 may be, for example, a P7 capture primer. A P5 capture primer commercially available from Illumina, Inc. (San Diego, CA) has the sequence 5'-AATGATACGGCGACCACCGA-3' (SEQ ID NO: 1). A P7 capture primer commercially available from Illumina, Inc. has the sequence 5'-CAAGCAGAAGACGGCATACGA-3' (SEQ ID NO: 2). The first adapter 221 may be, for example, a complementary P5 adapter (cP5), and the second adapter 222 may be, for example, a complementary P7 adapter (cP7). A complementary P5 adapter commercially available from Illumina, Inc. (San Diego, CA) has the sequence 5'-TCGGTGGTCGCCGTATCATT-3' (SEQ ID NO: 3). A complementary P7 adapter commercially available from Illumina, Inc. (San Diego, CA) may have the sequence 5'-TCGTATGCCGTCTTCTGCTTG-3' (SEQ ID NO: 4).

[0059] Before attempting to capture the target polynucleotides on the substrate 200 for subsequent amplification and sequencing, the double-stranded D1, D2, and D3 are melted in the manner shown in Figure 2B to obtain single-stranded target polynucleotides with a first adapter 221 available for hybridization to the capture primer 231 and a second adapter 222 available for hybridization to the orthogonal capture primer 232. Such melting can be carried out, for example, by changing the temperature and / or composition of the solution in which the double-stranded D1, D2, and D3 are placed. For example, double-stranded D1, D2, and D3 may be exposed to a sufficient amount of formamide solution, e.g., about 1% to 100% formamide (%v / v), or about 5% to 80% formamide (%v / v), or about 10% to 50% formamide (%v / v), or about 1% to 20% formamide (%v / v), in order to dissociate the double-stranded nucleotides at the current solution temperature. Additionally or alternatively, the solution temperature may be raised above the melting temperature (Tm) of the double-stranded nucleotides in the current solution composition. It will be understood that the specific Tm of the double-stranded nucleotides may depend on the composition of the solution (e.g., salt (Na+) concentration and formamide concentration, if present), as well as the length and sequence of the polynucleotides in the double-stranded nucleotides.

[0060] As illustrated with reference to Figure 2B, after the double-stranded D1, D2, and D3 are melted, the first adapter 221 may be available for hybridization to the capture primer 231, and the second adapter 222 may be available for hybridization to the orthogonal capture primer 232. However, the solution and temperature conditions that caused such melting may also inhibit the hybridization of the first adapter 221 to capture primer 231, and may also inhibit the hybridization of the second adapter 222 to the orthogonal capture primer 232. To promote such hybridization and thus promote the capture of the target polynucleotide on the substrate 200, the temperature and / or composition of the solution to which the target polynucleotide is repositioned can be altered. For example, a sufficient amount of formamide may be removed from the solution at the current solution temperature. Additionally or alternatively, the temperature of the solution may be reduced to below the double-strand melting temperature (Tm) between (a) the first adapter 221 and the capture primer 231 and (b) the second adapter 222 and the orthogonal capture primer 232 in the current solution composition.

[0061] In the non-limiting example shown in Figure 2C, further changes in conditions such as solution temperature and / or composition facilitate hybridization between the second adapter 222 bound to sequence 211 and one of the capture primers 232, and between the first adapter 221 bound to sequence 213 and one of the capture primers 231. However, further changes in these conditions also facilitate annealing between target polynucleotides in solution. For example, first and second target polynucleotides, each containing complementary sequences, may reanneal to each other along their lengths, such as sequences 212 and 212', which reanneal along their lengths to reform a double D2. Additionally, the first and second target polynucleotides, which contain sequences that are not complementary to each other, may partially anneal to each other, for example, at adapters 221 and 221' and adapters 222 and 222', such as non-complementary sequences 214 and 211' that anneal substantially only at their adapters to form double-stranded D4, as shown in Figure 2C. Target polynucleotides that anneal in solution to form double-stranded structures, for example, those containing sequences 212 and 214 in the non-limiting example shown in Figure 2C, are unavailable for hybridization with capture primer 231 or orthogonal capture primer 232, and therefore cannot be captured, much less amplified or sequenced.

[0062] In contrast, the modified capture primers of the present invention can hybridize with the adapter of a target polynucleotide under conditions where annealing in solution is substantially ineffective, such as salt concentrations, formamide content, and / or temperatures where annealing in solution is substantially ineffective. Thus, the target polynucleotide may not substantially anneal in such solution, forming a double helix and thus remaining available for hybridization with the present capture primer, which can then be amplified and sequenced, or otherwise used as desired. Additionally, the modified capture primers of the present invention can hybridize with the adapter of a target polynucleotide under conditions that inhibit any nonspecific or unproductive hybridization of the target polynucleotide to the capture primer or adapter in solution, thereby it can be expected that substantially all hybridization events are productive and result in a double helix that can be sequenced.

[0063] Figures 3A to 3H schematically illustrate exemplary compositions and operations in an exemplary process flow for the capture and amplification of polynucleotides on a substrate using the capture primers of the present invention. Composition 3000 shown in Figure 3A comprises a substrate 300 containing a capture primer 331 and an orthogonal capture primer 332 bound thereto, and a fluid (solution) containing a target polynucleotide, e.g., a polynucleotide intended for sequencing using sequencing by capture, amplification, and synthesis (SBS). The target polynucleotide may include or be provided in the form of double hedges (exemplary, double-stranded D5, D6, and D7) between target polynucleotides having complementary sequences. For example, in a manner similar to that described with reference to Figure 2A, double-stranded D5 may include a first target polynucleotide comprising sequence 311, a first adapter 321, and a second adapter 322, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 311', a first complementary adapter 321', and a second complementary adapter 322'. For example, double-stranded D6 may include a first target polynucleotide comprising sequence 312, a first adapter 321, and a second adapter 322, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 312', a first complementary adapter 321', and a second complementary adapter 322'. For example, double-stranded D7 may comprise a first target polynucleotide comprising sequence 313, a first adapter 321, and a second adapter 322, and a second target nucleotide complementary to the first target polynucleotide, for example, comprising complementary sequence 312', a first complementary adapter 321', and a second complementary adapter 322'. Sequences 311, 312, and 313 may be different from each other, and it may be desirable to determine their sequences using SBS. The first adapter 321 may be complementary to a capture primer 331 so that it can hybridize under suitable conditions, and the second adapter 322 may be complementary to a modified orthogonal capture primer 332 so that it can hybridize under suitable conditions.

[0064] Each capture primer 331 may contain multiple nucleic acids that increase the double-stranded Tm between the capture primer 331 and the first adapter 321 compared to the double-stranded Tm between the first adapter 321 and a complementary first adapter 321'. For example, the modified nucleic acids of the capture primer 331 may include lock nucleic acid (LNA), peptide nucleic acid (PNA), or super T, each of which is expected to increase the double-stranded Tm between the capture primer 331 and the first adapter 321 compared to the double-stranded Tm between the first adapter 321 and a complementary first adapter 321'. The capture primer 331 may further contain DNA. The modified nucleic acids and DNA may be distributed between the 5' and 3' ends of the capture primer. For example, the sequence of the capture primer 331 may include one or more DNA molecules, followed by one or more modified nucleic acids, followed by one or more DNA molecules, followed by one or more modified nucleic acids, and so on. Alternatively, modified nucleic acids may be positioned at the 5' end of the capture primer, and DNA may be positioned at the 3' end of the capture primer in a manner as described below, for example, with reference to Figure 4. Although not specifically illustrated, each capture primer 331 may also include cleavable regions such as 8-oxo-G.

[0065] Similarly, each of the orthogonal capture primers 332 may contain multiple modified nucleic acids that increase the double-stranded Tm between the orthogonal capture primer 332 and the second adapter 322 compared to the double-stranded Tm between the second adapter 322 and a complementary second adapter 322'. For example, the modified nucleic acids of the orthogonal capture primer 332 may include lock nucleic acid (LNA), peptide nucleic acid (PNA), or super T, each of which is expected to increase the double-stranded Tm between the orthogonal capture primer 332 and the second adapter 322 compared to the double-stranded Tm between the second adapter 322 and a complementary second adapter 322'. The orthogonal capture primer 332 may further contain deoxyribonucleic acid (DNA). The modified nucleic acids and DNA may be distributed between the 5' and 3' ends of the orthogonal capture primer. For example, the sequence of the orthogonal capture primer 332 may include one or more DNA molecules, followed by one or more modified nucleic acids, followed by one or more DNA molecules, followed by one or more modified nucleic acids, and so on. Alternatively, modified nucleic acids may be positioned at the 5' end of the capture primer, and DNA may be positioned at the 3' end of the capture primer in a manner as described below, for example, with reference to Figure 4. Although not specifically illustrated, each of the orthogonal capture primers 332 may also contain a cleavable moiety such as 8-oxo-G.

[0066] It should be noted that the modified nucleic acid of capture primer 331 may, but is not necessarily, be the same type of modified nucleic acid as that of orthogonal capture primer 332. For example, capture primer 331 and orthogonal capture primer 332 may contain LNA, capture primer 331 and orthogonal capture primer 332 may contain PNA, or capture primer 331 and orthogonal capture primer 332 may contain super T. Alternatively, capture primer 331 may contain LNA while orthogonal capture primer 332 may contain PNA or super T, capture primer 331 may contain PNA while orthogonal capture primer 332 may contain LNA or super T, or capture primer 331 may contain super T while orthogonal capture primer 332 may contain LNA or PNA. In further examples, the orthogonal capture primer 332 may contain LNA while the capture primer 331 may contain PNA or super T; the orthogonal capture primer 332 may contain PNA while the capture primer 331 may contain LNA or super T; or the orthogonal capture primer 332 may contain super T while the capture primer 331 may contain LNA or PNA.

[0067] LNA closely resembles DNA, with the only difference being the linker connecting the 2' and 4' carbons. PNA, like polypeptides, is structured by carboxylic acid and amino groups. SuperT contains modified thymidine bases with butyrine groups. In the case of LNA and SuperT base modifications, the increased rigidity of the molecular structure and stronger base stacking can increase the binding energy of the double interaction (321-331 or 322-332) compared to DNA / DNA double strands (321-321' or 322-322'). On the other hand, the melting point of double strands containing uncharged PNA (321-331 or 322-332) can increase compared to DNA / DNA double strands (321-321' or 322-322') due to reduced electrostatic repulsion. When the capture primer sequence is modified to include the modified nucleic acid, the melting point between the surface / solution double strand (321-331 or 322-332) can be significantly higher than the melting point of the solution / solution DNA double strand (321-321' or 322-322'). This allows the target polynucleotides 311, 312, and 313 to be seeded under a set of conditions (e.g., salt concentration, formamide concentration, and temperature) that do not allow adapter re-annealing in solution. Thus, the target polynucleotides 311, 312, and 313 can be seeded with virtually no loss from adapter re-annealing, as illustrated with reference to Figure 2C.

[0068] Before attempting to capture the target polynucleotides on the substrate 300 for subsequent amplification and sequencing, the double-stranded D5, D6, and D7 are melted in the manner shown in Figure 3B to obtain single-stranded target polynucleotides having a first adapter 321 available for hybridization to the capture primer 331 and a second adapter 322 available for hybridization to the orthogonal capture primer 332. Such melting can be carried out, for example, by changing the temperature and / or composition of the solution in which the double-stranded D5, D6, and D7 are placed, in a manner similar to that described with reference to Figure 2B. For example, double-stranded D5, D6, and D7 may be exposed to a sufficient amount of formamide in the solution, e.g., about 1% to 100% formamide (v / v), or about 5% to 80% formamide (%v / v), or about 10% to 50% formamide (%v / v), or about 1% to 20% formamide (%v / v), in order to dissociate the double-stranded nucleotides at the current solution temperature. Additionally or alternatively, the solution temperature may be raised above the melting temperature (Tm) of the double-stranded nucleotides in the current solution composition. It will be understood that the specific Tm of the double-stranded nucleotides may depend on the composition of the solution (e.g., salt (Na+) concentration and formamide concentration, if present), as well as the length and sequence of the polynucleotides in the double-stranded nucleotides.

[0069] As illustrated with reference to Figure 3B, after the double-stranded D5, D6, and D7 are melted, the first adapter 321 may hybridize to the capture primer 331, and the second adapter 322 may hybridize to the orthogonal capture primer 332 under the same conditions used to melt the double-stranded D5, D6, and D7 in the manner shown in Figure 3C. That is, the solution and temperature conditions that caused such melting also cannot inhibit the hybridization of the first adapter 321 to capture the primer 331, nor can they inhibit the hybridization of the second adapter 322 to the orthogonal capture primer 332. Instead, such conditions may inhibit any annealing between adapter 321 and complementary adapter 321', and any re-annealing between adapter 322 and complementary adapter 322' may inhibit the formation of any double-stranded material as illustrated with reference to Figure 2C. Therefore, each of the target polynucleotides may remain available for capture and can be captured on the substrate without necessarily reducing the concentration of formaldehyde, without necessarily reducing the concentration of formamide, and without necessarily changing the conditions used to melt the double-stranded D5, D6, and D7.

[0070] In the non-limiting example shown in Figure 3C, under conditions that inhibit annealing between target polynucleotides in solution, some first adapters 321 of the target polynucleotides hybridize to each of the capture primers 331 to form a first double helix, and some second adapters 322 of the target polynucleotides hybridize to each of the orthogonal capture primers 332 to form a second double helix. Each of the first and second double helixes may have a higher melting temperature (Tm) than the Tm of the third double helix formed by the hybridization of the second target polynucleotide to each of the complementary first target polynucleotides. For example, a first adapter 321 bound to sequence 312 hybridizes to one of the capture primers 331 to form double-stranded D8, a second adapter 322 bound to sequence 311 hybridizes to one of the orthogonal capture primers 332 to form double-stranded D9, and a second adapter 322 bound to sequence 313 hybridizes to one of the orthogonal capture primers 332 to form double-stranded D10.

[0071] The double-stranded Tm between the first adapter 321 and the capture primer 331 may be greater than the double-stranded Tm between the first adapter 321 and the complementary first adapter 321', for example, it may exceed the double-stranded Tm between the first adapter 321 and the complementary first adapter 321' by at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, or at least about 25°C. Similarly, the double-stranded Tm between the second adapter 322 and the orthogonal capture primer 332 may be greater than the double-stranded Tm between the second adapter 322 and the complementary second adapter 322', for example, it may exceed the double-stranded Tm between the first adapter 321 and the complementary first adapter 321' by at least about 5°C, at least about 10°C, at least about 15°C, at least about 20°C, or at least about 25°C. Therefore, the target polynucleotide can preferably be captured on the substrate 300 at a temperature below the double-strand Tm between the first adapter 321 and the capture primer 331, and below the double-strand Tm between the second adapter 322 and the orthogonal capture primer 332, where it is above the double-strand Tm between the first adapter 321 and the complementary first adapter 321', and above the double-strand Tm between the second adapter 322 and the complementary second adapter 322'.

[0072] Exemplary, the Tm of double-stranded D8, D9, and D10 shown in Figure 3C may exceed the Tm of any of double-stranded D1, D2, D3, and D4 described with reference to Figures 2A and 2C. For example, double-stranded D8, D9, and D10 may each have a melting temperature (Tm) of about 80°C to about 110°C, e.g., about 85°C to about 105°C, or about 90°C to about 100°C. In comparison, the Tm of double-stranded D1, D2, D3, and D4 may be less than about 80°C, e.g., less than about 75°C, less than about 70°C, less than about 65°C, or less than about 60°C. As a result of the significantly different Tm of the double-stranded molecules between the adapter and the surface primer compared to the Tm of the double-stranded molecules between the adapters in solution, substantially none of the second target polynucleotides will hybridize to any of the first target polynucleotides in solution. Therefore, double-stranded D8, D9, and D10 can be readily formed under specific conditions where double-stranded D1, D2, D3, and D4 cannot be formed. Double-stranded nucleotides can be formed on the surface of substrate 300 at different locations, for example, according to a Poisson distribution. Although not specifically illustrated, substrate 300 may be patterned to define different regions where double-stranded nucleotides can each be formed, and it will be understood that clusters can then be formed using amplification within these regions. Additionally, while the examples illustrated with reference to Figures 3A–3H suggest the use of a flat substrate, it will be clear that more complex substrates can be used, such as wells, each seeded with a target polynucleotide, in which substantially monoclonal clusters can be formed.

[0073] As shown in Figure 3D, after the initial hybridization described with reference to Figure 3C, each of the target polynucleotides 311, 312, and 313 can be amplified to form their respective amplicons 311', 312', and 313'. Following such amplification, the target polynucleotides 311, 312, and 313 can be dehydrogenated in the manner shown in Figure 3E, while the amplicons 311', 312', and 313' remain covalently bonded to the substrate 300. It should be noted that such dehydrogenation is not necessarily required. For example, instead of dehydrogenating the target polynucleotides 311, 312, and 313, these polynucleotides may remain hybridized to the substrate and be further amplified using a chain infiltration process known in the art, which may be called ExAmp.

[0074] As shown in Figure 3F, after the initial amplification described with reference to Figures 3D to 3E, the resulting amplicons can be bent to hybridize to other capture primers or orthogonal capture primers on the substrate 300. For example, a complementary first adapter 321' of amplicon 311' may hybridize to one of the capture primers 331, a complementary second adapter 322' of amplicon 312' may hybridize to one of the orthogonal capture primers 332, and a complementary first adapter 321' of amplicon 313' may hybridize to one of the capture primers 331. The double strands between the amplicon adapters and their respective capture primers or orthogonal capture primers may have a Tm similar to, or the same as, the double strands D8, D9, and D10, and thus may remain hybridized to facilitate further amplification of the target polynucleotide.

[0075] Figure 3G shows the composition of Figure 3F after another amplification operation. To make it clear, the composition comprises a further amplicon 311 of amplicon 311', a further amplicon 312 of amplicon 312', and a further amplicon 313 of amplicon 313'. Such further amplicons can hybridize to amplicons derived from them. The solution can be increased to dehydrogenate the adapters of the amplicons from the capture primers or orthogonal capture primers in a manner such as that shown in Figure 3H. The amplification operation can be repeated any preferred number of times to produce further amplicons of amplicons 311, 311', 312, 312', 313, and 313'. The amplification operation can be performed any preferred number of times, for example, using amplicons of the target polynucleotide 311, 312, or 313, so as to substantially occupy each substrate region (not specifically shown) in substantially monoclonal clusters. For example, the amplicons within each substrate region may include at least about 60% of an amplicon of one selected target polynucleotide, or at least about 70% of an amplicon of one selected target polynucleotide, or at least about 80% of an amplicon of one selected target polynucleotide, or at least about 90% of an amplicon of one selected target polynucleotide, or at least about 95% of an amplicon of one selected target polynucleotide, or at least about 98% of an amplicon of one selected target polynucleotide, or at least about 99% of an amplicon of one selected target polynucleotide, or 100% of an amplicon of one selected target polynucleotide.

[0076] As described above, in some examples, certain capture primers and orthogonal capture primers may contain non-nucleotide moieties. Such non-nucleotide moieties include, but are not limited to, removal moieties from which a portion of the capture primer can be selectively removed. The removal moieties may be located at any suitable position along the length of any suitable primer and may, but are not necessarily, be of the same type as each other. Following a desired number of further amplification operations, as described with reference to Figures 3E-3H, a portion of the capture primer 331 or orthogonal capture primer 332 may be removed by reacting the removal moieties with a suitable enzyme or reagent.

[0077] The capture primers and orthogonal capture primers of the present invention may include any preferred number, type, and arrangement of modified nucleic acids that sufficiently increase the double-stranded Tm between the capture primer or orthogonal capture primer and the adapter of the target polynucleotide. Figure 4 schematically shows an exemplary double-stranded structure between a polynucleotide and one of the capture primers of the present invention, in some examples. In the non-limiting example shown in Figure 4, the modified nucleic acid 441 (such as PNA or LNA) is located at the 5' end of the capture primer 331, and DNA 442 is located at the 3' end of the capture primer. An optional T spacer 443 is located between DNA 442 and the surface of the substrate 400 (e.g., a flow cell). The modified nucleic acid 441 and DNA 442 together may be considered to provide a “chimeric” grafting primer structure. The target polynucleotide may include a library template 311 bound to an adapter 321 containing a first subsequence 341 complementary to the sequence of the modified nucleic acid 441, and a second subsequence 342 complementary to the sequence of DNA 442. Alternatively, modified nucleic acids (such as PNA, LNA, or super T) and DNA are distributed between the 5' and 3' ends of the capture primer in the manner further described above. The orthogonal capture primer 332 may be constructed similarly to the capture primer 331 shown in Figure 4, but may include, for example, a chimeric grafted primer structure having a different sequence. Alternatively, modified nucleic acids (such as PNA, LNA, or super T) and DNA may be distributed between the 5' and 3' ends of the orthogonal capture primer in the manner further described above.

[0078] In some examples, each of the capture primers 331 may contain approximately 5 to 20 modified nucleic acids, and each of the orthogonal capture primers 332 may contain approximately 5 to 20 modified nucleic acids. However, it is understood that the number of modified nucleic acids can be optimally adjusted to obtain an appropriate Tm under conditions that allow double-strand formation on the substrate surface while suppressing double-strand formation in solution, and that each of the capture primers and orthogonal capture primers does not need to contain the same number, type, or distribution of modified nucleic acids. Exemplarily, each of the capture primers may contain at least approximately 9 modified nucleic acids, each of the orthogonal capture primers may contain at least approximately 9 modified nucleic acids, or each of the capture primers may contain at least approximately 12 modified nucleic acids, each of the orthogonal capture primers may contain at least approximately 12 modified nucleic acids, or each of the capture primers may contain at least approximately 15 modified nucleic acids, each of the orthogonal capture primers may contain at least approximately 15 modified nucleic acids.

[0079] It should be noted that while capture primer 331 and orthogonal capture primer 332 contain modified nucleic acids, they may have the same or identical sequences as capture primers 231 and 232 unless there is a particular problem. That is, capture primer 331 may be a modified P5 capture primer, and orthogonal capture primer may be a modified P7 capture primer. For example, capture primer 331 may be or include a P5 primer having a sequence provided elsewhere in this specification, in which case at least some of the P5 DNA bases are replaced with their PNA, LNA, or super T analogues. Additionally or alternatively, orthogonal capture primer 332 may be or include a P7 primer having a sequence provided elsewhere in this specification, in which case at least some of the P7 DNA bases are replaced with their PNA, LNA, or super T analogues. Table 1 below shows non-limiting examples of chimeric P5 and P7 sequences containing LNAs of various lengths, and the calculated double-strand Tm between the chimeric P5 or P7 sequence and their respective adapters cP5 or cP7 (750 mM Na+), where bold indicates LNA and non-bold letters indicate DNA. For reference, commercially available P5 and P7 sequences, and the calculated double-strand Tm between the P5 or P7 sequence and their respective adapters cP5 or cP7 are shown.

[0080] [Table 1]

[0081] From the examples provided in Table 1, it can be seen that for P5 sequences, replacing 15 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP5 adapter by approximately 21°C, replacing 12 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP5 adapter by approximately 16°C, and replacing 9 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP5 adapter by approximately 11°C. Additionally, from the examples provided in Table 1, it can be seen that for P7 sequences, replacing 15 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP7 adapter by approximately 21°C, replacing 12 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP7 adapter by approximately 17°C, and replacing 9 DNA bases with LNA can increase the double-stranded Tm between the sequence and the cP7 adapter by approximately 12°C. It will be understood that the number of modified nucleic acids in any sequence is provided herein in order to provide any suitable Tm for use with capture primers or orthogonal capture primers such as those provided herein.

[0082] One consideration for carrying out the modified capture primers of the present invention is that such primers are compatible with suitable enzymes such as recombinases, single-strand binding proteins, and polymerases, so that they can be used for cluster generation and sequencing. Chimeric structures containing PNA or LNA, as illustrated with reference to Figure 4, are expected to be compatible with such enzymes in a manner similar to those described in the following references, whose entire contents are incorporated herein by reference: Levin et al., "Position-dependent effects of locked nucleic acid (LNA) on DNA sequencing and PCR primers," Nucleic Acids Research 34(20):e142,11 pages (2006), and Misra et al., "Polyamide nucleic acid-DNA chimera lacking the phosphate backbone are novel primers for polymerase reaction catalyzed by DNA polymerases," Biochemistry 37:1917-1925 (1998). Further information on LNA can be found in Braasch et al., "Locked nucleic acid (LNA): fine-tuning the recognition of DNA and RNA," Chemistry & Biology 8:1-7 (2001), the full content of which is incorporated herein by reference. Providing DNA to the 3' end of a chimeric structure, as illustrated with reference to Figure 4, may help maintain expected enzymatic activity by placing unmodified DNA bases at positions where the enzyme may interact, while providing modified nucleic acid to the 5' end of the chimeric structure can improve the strength of binding to the adapter.

[0083] Additionally, as described elsewhere in this specification and as will be understood by those skilled in the art, the Tm of any given double-stranded nucleotide can vary depending on the composition of the solution in which the double-stranded nucleotide is placed. For example, adding formaldehyde to the solution can decrease the Tm of a given double-stranded nucleotide, while adding a salt to the solution can increase the Tm of a given double-stranded nucleotide. Thus, the exemplary Tm (and differences between Tm) provided herein are purely illustrative and may vary depending on the specific composition of the solution in which the double-stranded nucleotide is placed. Figures 5A–5C are plots illustrating the exemplary effect of conditions on the capture of polynucleotides by the capture primers of the present invention. In the non-limiting example shown in Figure 5A, the percentage of double-stranded DNA (dsDNA) is shown as a function of temperature for double-stranded DNA formed by hybridization between cP7 and a P7 standard library capture primer (curve 501) and double-stranded DNA formed by hybridization between cP7 and an LNA-modified P7 capture primer (curve 502) in a solution containing 0% v / v formamide and 750 mM Na+. The LNA-modified P7 capture primer has the sequence CAAGCAGAAGACGGCATAC(8-oxoG)AGAT (SEQ ID NO: 13) (bold indicates LNA in the sequence) and was modeled to have a Tm of 20°C when double-stranded with cP7.

[0084] Figure 5A shows that a temperature of approximately 77°C corresponds to the Tm of the double strand formed by hybridization between cP7 and the P7 standard library capture primer (curve 501), and a temperature of approximately 91°C corresponds to the Tm of the double strand formed by hybridization between cP7 and the LNA-modified P7 capture primer (curve 502). Above approximately 85°C, curve 501 shows that the percentage of dsDNA is only slightly (e.g., less than 1%) relative to the double strand formed by hybridization between cP7 and the P7 standard library capture primer. Additionally, below approximately 90°C, curve 501 shows that the percentage of dsDNA is approximately 100% relative to the double strand formed by hybridization between cP7 and the LNA-modified P7 capture primer. Therefore, from Figure 5A, it can be seen that at temperatures of approximately 85°C to 90°C, hybridization between cP7 and the P7 standard library primer can be substantially completely inhibited, while hybridization between cP7 and the LNA-modified P7 capture primer forms a very stable double helix. Thus, within this temperature range, the template polynucleotide can be captured from solution with high efficiency, substantially free from competition from the solution-based annealing process as described with reference to Figure 2C. It will be seen that the specific temperature range suitable for performing such operations may vary depending on the specific composition of the solution. Any other capture primers and adapter sequences can be expected to exhibit similar temperature dependence of double helix formation.

[0085] For example, in the non-limiting example shown in Figure 5B, the Tm of the double strands formed by hybridization between cP7 and the P7 standard library capture primer (curve 503), and the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer (curve 504) are shown as a function of the salt concentration of 750 mM Na+ (%v / v). Similar to Figure 5A, it can be seen that at 0%v / v formamide, a temperature of approximately 77°C corresponds to the Tm of the double strands formed by hybridization between cP7 and the P7 standard library capture primer (curve 503), and a temperature of approximately 91°C corresponds to the Tm of the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer (curve 504). As the concentration of formamide increases, the Tm decreases in both the double strands formed by hybridization between cP7 and the P7 standard library capture primer, and the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer. For example, at a concentration of approximately 5% v / v formamide, the Tm of the double strand formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 69°C, and the Tm of the double strand formed by hybridization between cP7 and the LNA-modified P7 capture primer decreases to approximately 88°C. At a concentration of approximately 10% v / v formamide, the Tm of the double strand formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 66°C, and the Tm of the double strand formed by hybridization between cP7 and the LNA-modified P7 capture primer decreases to approximately 85°C. At a concentration of approximately 15% v / v formamide, the Tm of the double-stranded molecule formed by hybridization between cP7 and a P7 standard library capture primer decreases to approximately 63°C, while the Tm of the double-stranded molecule formed by hybridization between cP7 and an LNA-modified P7 capture primer decreases to approximately 82°C.At a concentration of approximately 20% v / v formamide, the Tm of the double-stranded molecule formed by hybridization between cP7 and a P7 standard library capture primer decreases to approximately 58°C, while the Tm of the double-stranded molecule formed by hybridization between cP7 and an LNA-modified P7 capture primer decreases to approximately 79°C.

[0086] Therefore, from Figure 5B, it can be seen that while hybridization between cP7 and the P7 standard library capture primer can be substantially completely inhibited at any given concentration of formamide (e.g., about 1% to about 20% formamide (%v / v), or about 5% to about 20% formamide (%v / v)), there is a temperature range in which hybridization of cP7 and the LNA-modified P7 capture primer can form a very stable double helix. Thus, within this temperature range, the template polynucleotide can be captured from solution with high efficiency, substantially free from competition from the solution-based annealing process as described with reference to Figure 2C. Any other capture primers and adapter sequences can be expected to exhibit a similar formamide concentration dependence of double helix formation.

[0087] As another example, in the non-limiting example shown in Figure 5C, the Tm of the double strands formed by hybridization between cP7 and the P7 standard library capture primer (curve 505), and the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer (curve 506), are shown as a function of salt concentration (mM Na+). At 750 mM Na+, it can be seen that a temperature of approximately 72°C corresponds to the Tm of the double strands formed by hybridization between cP7 and the P7 standard library capture primer (curve 505), and a temperature of approximately 89°C corresponds to the Tm of the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer (curve 506). As the salt concentration decreases, the Tm decreases in both the double strands formed by hybridization between cP7 and the P7 standard library capture primer, and the double strands formed by hybridization between cP7 and the LNA-modified P7 capture primer. For example, at a Na+ concentration of approximately 500 mM, the Tm of the double-stranded material formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 70°C, and the Tm of the double-stranded material formed by hybridization between cP7 and the LNA-modified P7 capture primer decreases to approximately 87°C. At a Na+ concentration of approximately 100 mM, the Tm of the double-stranded material formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 55°C, and the Tm of the double-stranded material formed by hybridization between cP7 and the LNA-modified P7 capture primer decreases to approximately 77°C. At a Na+ concentration of approximately 0 mM, the Tm of the double-stranded material formed by hybridization between cP7 and the P7 standard library capture primer decreases to approximately 42°C, and the Tm of the double-stranded material formed by hybridization between cP7 and the LNA-modified P7 capture primer decreases to approximately 58°C.

[0088] Therefore, from Figure 5C, it can be seen that at any given salt concentration (e.g., approximately 100–800 mM Na+, or 200–800 mM Na+), hybridization between cP7 and the P7 standard library capture primer can be substantially completely inhibited, while there is a temperature range in which hybridization of cP7 and the LNA-modified P7 capture primer can form a very stable double helix. Thus, within this temperature range, the template polynucleotide can be captured from solution with high efficiency, substantially free from competition from the solution-based annealing process as described with reference to Figure 2C. Any other capture primers and adapter sequences can be expected to exhibit similar salt concentration dependence for double helix formation.

[0089] It will be understood that the exemplary compositions described herein may be used in any preferred method for capturing and amplifying polynucleotides. For example, Figure 6 shows an exemplary flow of operations in Method 600 for capturing and amplifying polynucleotides using the modified primers. Method 600 may be carried out using composition 3000 as described with reference to Figures 3A to 3H, but Method 600 may also be carried out using any other preferred composition.

[0090] Referring here to Figure 6, Method 600 may include providing a composition comprising (a) a plurality of capture primers bound to the surface of a substrate, each containing a modified nucleic acid, and (b) a plurality of orthogonal capture primers bound to the surface of a substrate, each containing a modified nucleic acid (Operation 610). The composition may be similar to composition 3000 described with reference to Figure 3A, and may, for example, include capture primer 331 and orthogonal capture primer 332 bound to substrate 300.

[0091] The method 600 shown in Figure 6 may further include providing a fluid comprising (a) a first target polynucleotide comprising a first adapter complementary to a capture primer and a second adapter complementary to an orthogonal capture primer, respectively, and (b) second target polynucleotides complementary to each of the first target polynucleotides. The fluid may be configured similarly as described with reference to Figure 3A, and may, for example, include target polynucleotides 311, 312, 313 each bound to the first adapter 321 and the second adapter 322, and complementary target polynucleotides 311', 312', 313' each bound to a complementary first adapter 321' and a complementary second adapter 322'.

[0092] Method 600, shown in Figure 6, may further include bringing the composition into contact with a fluid (operation 630). For example, the composition provided in operation 610 may be brought into contact with the fluid provided in operation 620. Exemplarily, the composition may be provided in a flow cell, and the fluid flows in contact with the composition in the flow cell.

[0093] Method 600, shown in Figure 6, may further include (a) hybridizing several first adapters of the first target polynucleotide to each of the capture primers to form a first double helix, and (b) hybridizing several second adapters of the first target polynucleotide to each of the orthogonal capture primers to form a second double helix, while inhibiting the hybridization of the second target polynucleotide to the first target polynucleotide (operation 640). For example, the hybridization of the first adapters for capturing the primers and the hybridization of the second adapters to the orthogonal capture primers may be carried out under conditions that inhibit the annealing of the first and second target polynucleotides to each other, in the manner described with reference to Figures 3B-3C. Exemplary conditions are described elsewhere in this specification. Note that the same set of conditions may be used to induce the dissociation of the first and second polynucleotides to each other and to promote the hybridization of the adapters of the first polynucleotide to the capture primers of the present invention. In contrast, when using conventionally known capture primers, two conditions may be required: a first condition that causes the first and second polynucleotides to dissociate from each other, and a second condition that subsequently promotes the hybridization of the adapter of the first polynucleotide to the conventionally known capture primer.

[0094] Method 600, shown in Figure 6, may further include amplifying a first target polynucleotide, which includes generating each amplicon of the first target polynucleotide (operation 650). Non-limiting examples of how such amplicons may be generated are provided with reference to Figures 3D-3H.

[0095] Therefore, while conventionally known capture primers can be used under conditions where unproductive rehybridization complexes may form in solution, the modified capture primers of the present invention (including orthogonal capture primers) overcome this drawback by using sequences containing modified nucleic acids that confer stronger binding (and higher Tm) to the surface / solution duplex compared to the solution / solution duplex. The difference in Tm between the surface / solution and the solution / solution duplex can be achieved by using modified capture primer sequences that may contain, for example, LNA, PNA, super T, or any combination thereof. By incorporating base modifications into the capture primer, there exists a temperature regime in which the adapter of the target polynucleotide can hybridize to the surface but cannot hybridize to other adapters in solution.

[0096] Therefore, the modified capture primer of the present invention provides one or more of the following advantages: A) Library capture is more efficient. Where appropriate, the input of the target polynucleotide can be used at lower concentrations. B) The modification of surface traps "on the flow cell" can be carried out at high temperatures, thereby reducing or eliminating the need to integrate cooling to reach a temperature that promotes hybridization in a timely manner, and / or C) Flow cells that require the majority of the library can utilize multiple fluid pushes for seeding, which can significantly increase the turnaround time (TAT) from the start of sample preparation to obtaining sequencing data. The higher seeding efficiency provided by these capture primers makes it possible to use higher concentrations of target polynucleotides with fewer fluid pushes. This can save time during seeding.

[0097] It will be understood that the compositions and methods of the present invention are not limited to use in the specific operations described above. For example, Figures 3A-3H may be considered operations corresponding to “crosslinking amplification” or “surface-bound polymerase chain reaction,” but it will be understood that the compositions and methods of the present invention can be readily adapted for use with other amplification modalities. One such amplification modality is “exclusionary amplification” or ExAmp. Exclusionary amplification can enable amplification of a single target polynucleotide per substrate region and the generation of a substantially monoclonal population of amplicons in the substrate region. For example, the amplification rate of a first captured target polynucleotide in a substrate region can be rapid relative to the slower transport rate and capture of target polynucleotides in the substrate region. Thus, a first target polynucleotide captured in a substrate region can be rapidly amplified, occupying the entire substrate region and thus inhibiting the capture of further target polynucleotides in the same substrate region. Alternatively, if a second target polynucleotide binds to the same substrate region after the first polynucleotide, relatively rapid amplification of the first polynucleotide may result in sufficient occupancy of the substrate region to produce a signal strong enough to perform sequencing by synthesis. The use of exclusion amplification may also result in a hyper-Poisson distribution of monoclonal substrate regions; that is, the fraction of substrate regions in an array that is substantially monoclonal may exceed the fraction predicted by the Poisson distribution.

[0098] Increasing the super-Poisson distribution of useful clusters is useful because more substantially monoclonal substrate regions can yield higher quality signals, and therefore improved SBS. However, seeding of target polynucleotides into substrate regions can follow a spatial Poisson distribution, and the trade-off for increasing the number of occupied substrate regions is increasing the number of polyclonal substrate regions. One way to obtain a higher super-Poisson distribution is for seeding to occur rapidly and then delayed among the seeded target polynucleotides. This delay, called "dynamic delay," is thought to occur through biochemical reaction kinetics, thus causing one seeded target polynucleotide to start earlier than other seeded targets. Exclusion amplification works by using a recombinase to facilitate the entry of a primer (e.g., a primer bound to a substrate region) into double-stranded DNA (e.g., a target polynucleotide) when the recombinase mediates sequence matching. The compositions and methods of the present invention can be adapted for use with a recombinase to facilitate the entry of the capture primers and orthogonal capture primers of the present invention into target polynucleotides when the recombinase mediates sequence matching. In fact, the compositions and methods of the present invention can be adapted for use with any surface-based polynucleotide amplification method, such as thermal PCR, chemically denatured PCR, and enzyme-mediated amplification (which may be referred to as recombinase polymerase amplification (RPA) or ExAmp).

[0099] Further comments While various exemplary embodiments have been described above, it will be apparent to those skilled in the art that various changes and modifications can be made therein without departing from the present invention. The appended claims are intended to cover all such changes and modifications that fall within the true spirit and scope of the present invention.

[0100] It should be understood that any corresponding feature / example of each aspect of the present disclosure as described herein may be implemented together in any appropriate combination. Furthermore, any feature / example from any one or more of these aspects may be implemented together with any of the features of any other aspects described herein as described herein in any appropriate combination to obtain the advantages described herein.

Claims

1. A composition for capturing a target polynucleotide on the surface of a substrate, A plurality of capture primers bonded to the surface of the substrate, each of which contains a modified nucleic acid, A plurality of orthogonal capture primers bonded to the surface of the substrate, each of which contains a modified nucleic acid, comprising: A first target polynucleotide comprising a first adapter complementary to the capture primer and a second adapter complementary to the orthogonal capture primer, The present invention further comprises a second target polynucleotide that is complementary to each of the first target polynucleotides, Some of the first target polynucleotides, the first adapters, hybridize to each of the capture primers to form a first double helix, and some of the first target polynucleotides, the second adapters, hybridize to each of the orthogonal capture primers to form a second double helix. A composition wherein each of the first and second double strands has a Tm greater than the melting temperature (Tm) of a third double strand that can be formed by the hybridization of the second target polynucleotide with the first target polynucleotide complementary to the second target polynucleotide.

2. The composition according to claim 1, wherein the modified nucleic acid of the capture primer comprises lock nucleic acid (LNA), peptide nucleic acid (PNA), or Super T®.

3. The aforementioned capture primer further comprises deoxyribonucleic acid (DNA), Optional, (i) The modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer, or (ii) The composition according to claim 1 or 2, wherein the modified nucleic acid is positioned at the 5' end of the capture primer and the DNA is positioned at the 3' end of the capture primer.

4. The composition according to any one of claims 1 to 3, wherein the modified nucleic acid of the orthogonal capture primer comprises Loc nucleic acid (LNA), peptide nucleic acid (PNA), or Super T®.

5. The orthogonal trapping primer further comprises deoxyribonucleic acid (DNA), optionally, (i) The modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer, or (ii) The composition according to claim 4, wherein the modified nucleic acid is positioned at the 5' end of the orthogonal capture primer and the DNA is positioned at the 3' end of the orthogonal capture primer.

6. (i) Each of the first and second double strands has a melting temperature (Tm) of 80°C to 110°C, or (ii) The first and second double strands each have a melting temperature (Tm) of 85°C to 105°C, or (iii) The first and second double strands each have a melting temperature (Tm) of 90°C to 100°C. The composition according to any one of claims 1 to 5.

7. The composition according to any one of claims 1 to 6, wherein none of the second target polynucleotides hybridize in solution with any of the first target polynucleotides.

8. (i) 1% to 100% formamide (%v / v), optionally 5% to 80% formamide (%v / v) and / or (ii) The composition according to any one of claims 1 to 7, further comprising 100 to 800 mM Na+, and optionally 200 to 800 mM Na+.

9. The composition according to any one of claims 1 to 8, wherein the capture primer is a P5 capture primer containing a modified nucleic acid, and the orthogonal capture primer is a P7 capture primer containing a modified nucleic acid.

10. (a) Each of the capture primers contains 5 to 20 of the modified nucleic acids, and each of the orthogonal capture primers contains 5 to 20 of the modified nucleic acids, and / or (b)(i) Each of the capture primers comprises at least nine of the modified nucleic acids, and each of the orthogonal capture primers comprises at least nine of the modified nucleic acids, or (ii) Each of the capture primers contains at least 12 of the modified nucleic acids, and each of the orthogonal capture primers contains at least 12 of the modified nucleic acids, or (iii) The composition according to any one of claims 1 to 9, wherein each of the capture primers comprises at least 15 of the modified nucleic acids, and each of the orthogonal capture primers comprises at least 15 of the modified nucleic acids.

11. A method for capturing and amplifying target polynucleotides on the surface of a substrate, The method involves bringing a composition into contact with a fluid, wherein the composition is A plurality of capture primers bonded to the surface of the substrate, each of which contains a modified nucleic acid, A plurality of orthogonal capture primers bonded to the surface of the substrate, each of which contains a modified nucleic acid, comprising a plurality of orthogonal capture primers. The aforementioned fluid A first target polynucleotide comprising a first adapter complementary to the capture primer and a second adapter complementary to the orthogonal capture primer, Contacting a second target polynucleotide that is complementary to each of the first target polynucleotides, On the other hand, inhibiting the hybridization of the second target polynucleotide to the first target polynucleotide in the fluid, Hybridizing several of the first target polynucleotides to each of the capture primers to form a first double helix, Hybridizing some of the second adapters from the first target polynucleotides to each of the orthogonal capture primers to form a second double helix, and then, The present invention provides a method for amplifying a first target polynucleotide, wherein the amplification includes generating amplicons of each of the first target polynucleotides. A method wherein each of the first and second double strands has a Tm greater than the melting temperature (Tm) of a third double strand that can be formed by the hybridization of the second target polynucleotide with each of the first target polynucleotides which is complementary to the second target polynucleotide.

12. The method according to claim 11, wherein the modified nucleic acid of the capture primer comprises lock nucleic acid (LNA), peptide nucleic acid (PNA), or Super T®.

13. The aforementioned capture primer further comprises deoxyribonucleic acid (DNA), Optional, (i) The modified nucleic acid and the DNA are distributed between the 5' end and the 3' end of the capture primer, or (ii) The method according to claim 11 or claim 12, wherein the modified nucleic acid is positioned at the 5' end of the capture primer and the DNA is positioned at the 3' end of the capture primer.

14. The method according to any one of claims 11 to 13, wherein the modified nucleic acid of the orthogonal capture primer comprises lock nucleic acid (LNA), peptide nucleic acid (PNA), or Super T®.

15. The orthogonal trapping primer further comprises deoxyribonucleic acid (DNA), (i) The modified nucleic acid and the DNA are distributed between the 5' and 3' ends of the capture primer, or (ii) The method according to claim 14, wherein the modified nucleic acid is positioned at the 5' end of the orthogonal capture primer and the DNA is positioned at the 3' end of the orthogonal capture primer.

16. (i) Each of the first and second double strands has a melting temperature (Tm) of 80°C to 110°C, and / or (ii) Each of the first and second double strands has a melting temperature (Tm) of 85°C to 105°C, and / or (iii) Each of the first and second double strands has a melting temperature (Tm) of 90°C to 100°C, and / or (iv) None of the second target polynucleotides hybridize in solution with any of the first target polynucleotides, and / or (v) The hybridization is carried out in 1% to 100% formamide (%v / v), optionally, the hybridization is carried out in 5% to 80% formamide (%v / v), and / or (vi) The hybridization is carried out in 100 to 800 mM Na+, optionally, the hybridization is carried out in 200 to 800 mM Na+, and / or (vii) The method according to any one of claims 11 to 15, wherein the capture primer is a P5 capture primer containing a modified nucleic acid, and the orthogonal capture primer is a P7 capture primer containing a modified nucleic acid.

17. (a) Each of the capture primers contains 5 to 20 of the modified nucleic acids, and each of the orthogonal capture primers contains 5 to 20 of the modified nucleic acids, and / or (b)(i) Each of the capture primers comprises at least nine of the modified nucleic acids, and each of the orthogonal capture primers comprises at least nine of the modified nucleic acids, or (ii) Each of the capture primers contains at least 12 of the modified nucleic acids, and each of the orthogonal capture primers contains at least 12 of the modified nucleic acids, or (iii) The method according to any one of claims 11 to 16, wherein each of the capture primers comprises at least 15 of the modified nucleic acids, and each of the orthogonal capture primers comprises at least 15 of the modified nucleic acids.

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