Compositions and methods for single molecule placement on substrates

Abstract

Compositions and methods for single molecule placement on substrates are provided. Provided herein are methods and compositions for placing single target molecules on patterned substrates.

Description

[0001] This application is a divisional application of a Chinese invention patent application filed on December 14, 2015, with application number 201580068109.1 and invention name “Compositions and methods for single molecule placement on a substrate”.

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 092,171, filed December 15, 2014, which is hereby incorporated by reference in its entirety for all purposes. Technical Field

[0003] The present disclosure relates to the field of molecular biology, and more particularly to compositions and methods for placing single target molecules on patterned substrates. Background Art

[0004] Single-molecule placement has useful applications in fields such as biomolecular dynamics studies, drug discovery, and nucleic acid sequencing. For example, single-molecule DNA sequencing has been achieved by placing a single polymerase in a waveguide with nanometer dimensions. Single-molecule DNA sequencing has been achieved using nanopore structures that load a single nanopore-forming protein into a lipid bilayer.

[0005] Currently, single molecule placement is mainly achieved by molecular size exclusion effects that limit the anchoring space to the level of single molecule size. This method requires the use of high-resolution lithography (lithography) and results in relatively low loading efficiency due to the small target area. Loading a single nanopore into a lipid bilayer is mainly achieved by monitoring ionic current changes during the loading process, which lacks compatibility with large-scale production.

[0006] Therefore, there exists a need for an efficient method of placing single molecules on a target area. The present invention also satisfies this need and provides related advantages. Summary of the invention

[0007] Substrates and methods that can be used to place single molecules on a target region are particularly provided herein. In a first aspect, a substrate is provided that comprises a plurality of first and second capture primers immobilized to a feature on the substrate. At least one target polynucleotide, one end attached to one of the capture primers and the other end connected to a target molecule, wherein the target polynucleotide comprises a target region flanked by a first and second capture primer binding region complementary to the first and second capture primers, the second capture primer binding region comprising a base pair mismatch with the second capture primer, and a plurality of clonal amplicons complementary to the target polynucleotide immobilized to the feature. In some embodiments, the base pair mismatch is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pair mismatches. In some embodiments, the base pair mismatch is 3 base pair mismatches.

[0008] In some embodiments, the substrate further comprises a plurality of features. In some embodiments, a feature comprises a single target molecule. In some embodiments, the feature is filled to capacity with the plurality of clonalamplicons. The feature is filled to capacity with the plurality of clonalamplicons. In some embodiments, a plurality of features comprises a single target molecule. In some embodiments, two or more of the features comprise different single target molecules. In some embodiments, the feature is filled to capacity with the plurality of clonalamplicons.

[0009] In some embodiments, the target polynucleotide comprises one or more polynucleotides selected from the group consisting of RNA, DNA, and PNA. In some embodiments, the target polynucleotide comprises double-stranded DNA (dsDNA). In some embodiments, the target polynucleotide comprises less than 1,000 nucleotides. In some embodiments, the target polynucleotide comprises 10-25, 26-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 nucleotides.

[0010] In some embodiments, the target molecule comprises a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound or an inorganic compound. In some embodiments, the target molecule comprises a polypeptide. In some embodiments, the polypeptide is selected from a nanopore, a binding polypeptide and an enzyme. In some embodiments, the nanopore is selected from MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, ion channel, protein nanopore and DNA origami nanopore.

[0011] In some embodiments, the binding polypeptide is selected from the group consisting of: antibody, Fab, Fab', F(ab') 2 , scFv, diabody, triabody, minibody and single domain antibody (sdAB), T cell receptor, microcin, neuropeptide, G protein coupled receptor, antibody, epidermal growth factor receptor and HER2. In some embodiments, the enzyme is selected from the group consisting of: recombinase, polymerase, helicase, transposase, ligase, deaminase, oxidase and kinase.

[0012] In some embodiments, the substrate comprises one or more materials selected from glass, silicon, plastics and biopolymers. In some embodiments, the feature is separated by a gap region lacking a target polynucleotide. In some embodiments, the feature comprises a bead, a hole, a channel, a ridge, a projection or a combination thereof. In some embodiments, the hole is a micropore or a nanopore. In some embodiments, the substrate also comprises a hydrogel or a covalently linked gel.

[0013] Also provided herein is a method of placing a single target molecule on a feature of a substrate. In one aspect, the method is a method of placing a single target molecule on a feature of a substrate by hybridizing a plurality of first and second capture primers immobilized to the feature on the substrate with at least one target polynucleotide, wherein the target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, wherein the second capture primer binding region comprises a base pair mismatch with the second capture primer and is connected to the target molecule. The method further comprises amplifying the at least one target polynucleotide at an average amplification rate to produce a plurality of clonal amplicons complementary to the target polynucleotide, the average amplification rate exceeding the average transport rate of the target polynucleotide to the feature. The method also comprises amplifying the at least one target polynucleotide at an average amplification rate to produce a plurality of clonal amplicons complementary to the target polynucleotide, the average amplification rate exceeding the average transport rate of the target polynucleotide to the feature.

[0014] In some embodiments of the methods described herein, the base pair mismatch is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pair mismatches. In some embodiments of the methods described herein, the base pair mismatch is a three base pair mismatch. In some embodiments of the methods described herein, the substrate includes a plurality of features. In some embodiments of the methods described herein, the feature includes a single target molecule. In some embodiments of the methods described herein, the feature is filled with the multiple clonal amplicons. In some embodiments of the methods described herein, the multiple features include a single target molecule. In some embodiments of the methods described herein, two or more of the features include different single target molecules. In some embodiments of the methods described herein, the feature is filled with the multiple clonal amplicons.

[0015] In some embodiments of the methods described herein, amplification includes isothermal amplification. In some embodiments of the methods described herein, isothermal amplification also includes single-stranded binding polypeptides. In some embodiments of the methods described herein, isothermal amplification includes kinetic exclusion amplification. In some embodiments of the methods described herein, isothermal amplification also includes kinetic exclusion amplification.

[0016] In some embodiments of the method described herein, the average amplification rate of the subsequent amplicon produced at the feature exceeds the average amplification rate of the first amplicon. In some embodiments of the method described herein, the target polynucleotide comprises one or more polynucleotides selected from RNA, DNA and PNA. In some embodiments of the method described herein, the target polynucleotide comprises double-stranded DNA (dsDNA). In some embodiments, the target polynucleotide comprises less than 1,000 nucleotides. In some embodiments of the method described herein, the length of the target polynucleotide comprises 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900 or 901 to 1000 base pairs.

[0017] In some embodiments of the methods described herein, the target molecule comprises a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound, or an inorganic compound. In some embodiments of the methods described herein, the target molecule comprises a polypeptide. In some embodiments of the methods described herein, the polypeptide is selected from a nanopore, a binding polypeptide, and an enzyme. In some embodiments of the methods described herein, the nanopore is selected from MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, and DNA origami nanopores. In some embodiments of the methods described herein, the binding polypeptide is selected from: antibodies, Fab, Fab', F(ab'), 2 , scFv, diabodies, triabodies, minibodies and single domain antibodies (sdAB), T cell receptors, microcins, neuropeptides, G protein-coupled receptors, antibodies, epidermal growth factor receptor and HER2.

[0018] In some embodiments of the methods described herein, the enzyme is selected from: recombinase, polymerase, helicase, transposase, ligase, deaminase, oxidase and kinase. In some embodiments of the methods described herein, the substrate comprises one or more materials selected from glass, silicon, plastic and biopolymer. In some embodiments of the methods described herein, the features are separated by gap regions lacking target polynucleotides. In some embodiments of the methods described herein, the features include beads, holes, channels, ridges, protrusions or combinations thereof. In some embodiments of the methods described herein, the holes are micropores or nanowires. In some embodiments of the methods described herein, the substrate also comprises a hydrogel or a covalently linked gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is kinetic exclusion amplification (KEA) using target polynucleotides attached to target molecules, placing a single target molecule in each of multiple features.

[0020] Figure 2 An exemplary substrate is shown.

[0021] Figure 3 Exemplary target polynucleotides are shown in Figures AD.

[0022] Figure 4 Shown is the design of a capture primer binding region on a target polynucleotide that can be used to amplify a strand attached to the target molecule distally from the capture primer binding region while exponentially amplifying the amplicons generated therefrom.

[0023] Figure 5 An exemplary substrate comprising at least two features is shown.

[0024] Figure 6 Shown are first and second rounds of isothermal amplification using mismatch capture primer regions in a double-stranded target polynucleotide.

[0025] Figure 7 An exemplary workflow for generating a substrate is shown.

[0026] Figure 8 An exemplary kit is shown.

[0027] Fig. 9 Shown are the target polynucleotide and capture primer binding region designs used in kinetic exclusion amplification (KEA) reactions using matched and mismatched capture primer / capture primer binding regions.

[0028] Fig.10 Shows Fig. 9 Amplicon products of the target polynucleotides indicated. DETAILED DESCRIPTION

[0029] The present disclosure relates to compositions and methods for placing single molecules on a selected substrate area. Single molecule placement or single molecule placement can be used in a variety of applications where it is desirable to measure single molecule interactions as opposed to average measurements between populations. Single molecule placement can also be useful when it is desirable to use a single molecule to interrogate another molecular entity at the single molecule level.

[0030] exist Figure 1 In an exemplary embodiment shown, the present disclosure relates to placing a single molecule on a target region of a substrate using a kinetic exclusion assay (KEA). In this embodiment, the target region of the substrate is first functionalized with a KEA primer, and the target molecule is connected to a double-stranded nucleic acid rapidly amplified by KEA. When the target molecule falls on the substrate, the connected nucleic acid is rapidly amplified to fill the entire pad and prevent further landing of different molecules. This exemplary method is not very limited by the critical dimension (critical dimension, CD) of the target region, and the loading flux will be higher than the method based on the size exclusion effect. In addition, KEA is a self-limiting process and is therefore compatible with large-scale production.

[0031] As used herein, the term "substrate" is intended to mean a solid support. The term includes any material that can be used as a solid or semisolid basis for producing features, such as pores for depositing biopolymers, including nucleic acids, polypeptides and / or other polymers. The substrates of the present invention can be modified by various methods well known to those skilled in the art, or can be modified to accommodate the attachment of biopolymers. Exemplary types of substrate materials include glass, modified glass, functionalized glass, inorganic glass, microspheres, including inert and / or magnetic particles, plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or optical fiber bundles, various polymers other than those exemplified above, and porous microtiter plates. Exemplary plastics of specific types include acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane and Teflon TM (eg, polytetrafluoroethylene, perfluoroalkoxy, fluorinated ethylene propylene and derivatives thereof.) Specific types of exemplary silica-based materials include silicon and various forms of modified silicon.

[0032] In the methods and compositions presented herein, polynucleotides can be immobilized to substrates as described herein. In some embodiments, polynucleotides are covalently immobilized to substrates. When referring to immobilizing a molecule (e.g., nucleic acid) to a substrate, the terms "immobilization" and "attachment" are used interchangeably herein, and the two terms are intended to include direct or indirect, covalent or non-covalent attachment, unless clearly or otherwise indicated above. In certain embodiments of the invention, covalent attachment may be preferred, but what is generally required is that the molecule (e.g., nucleic acid) remains immobilized or attached to a support under the conditions under which it is intended to use a substrate.

[0033] Certain embodiments of the present invention may use a substrate comprising an inert substrate or matrix (e.g., a glass slide, polymer beads, etc.) that has been functionalized, for example, by applying a layer or coating of an intermediate material that includes reactive groups that allow covalent attachment to biomolecules, such as polynucleotides. Examples of such supports include, but are not limited to, polyacrylamide hydrogels supported on an inert substrate such as glass, particularly polyacrylamide hydrogels as described in WO 2005 / 065814 and US2008 / 0280773, the contents of which are incorporated herein by reference in their entirety. In such embodiments, the biomolecules (e.g., polynucleotides) may be covalently attached directly to the intermediate material (e.g., hydrogel), but the intermediate material itself may be non-covalently attached to the substrate or matrix (e.g., a glass substrate). The terms "covalently attached to a solid support" and "covalently attached to a substrate" are used interchangeably and are interpreted accordingly to include this type of arrangement.

[0034] Exemplary covalent linkages include, for example, those produced using click chemistry techniques. Exemplary non-covalent linkages include, but are not limited to, nonspecific interactions (e.g., hydrogen bonding, ionic bonding, van der Waals interactions, etc.) or specific interactions (e.g., affinity interactions, receptor-ligand interactions, antibody-epitope interactions, avidin-biotin interactions, streptavidin-biotin interactions, lectin-carbohydrate interactions, etc.). Exemplary linkages are described in U.S. Patent Nos. 6,737,236; 7,259,258; 7,375,234 and 7,427,678; and U.S. Patent Publication No. 2011 / 0059865A1, each of which is incorporated herein by reference. Covalent linkages may include those formed using conjugation chemistry as described herein.

[0035] The terms "solid surface", "solid support" and other grammatical equivalents herein refer to any material that is suitable or can be modified to be suitable for attaching polynucleotides. As will be appreciated by those skilled in the art, the number of possible substrates is very large. Possible substrates include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon TM The invention relates to a method for preparing a solid support or solid surface. The method comprises the steps of: preparing a solid support or solid surface by using a flow cell device ...

[0036] Those skilled in the art will know or understand that the composition and geometry of the substrate of the present invention can vary according to the intended use and preferences of the user. Therefore, although planar substrates such as slides, chips or wafers are exemplified herein with reference to the microarrays for illustration, in view of the teaching and guidance provided herein, those skilled in the art will understand that extremely various other substrates exemplified herein or well known in the art can also be used in the method and / or composition of the present invention.

[0037] In some embodiments, the solid support comprises one or more surfaces of a flow cell. As used herein, the term "flow cell" refers to a chamber comprising a solid surface through which one or more fluid reagents can flow. Examples of flow cells and related fluid systems and detection platforms that can be readily used in the methods of the present disclosure are described, for example, in Bentley et al., Nature 456: 53-59 (2008), WO 04 / 018497. US 7,057,026; WO 91 / 06678; WO 07 / 123744; US 7,329,492; US 7,211,414; US 7,315,019; US 7,405,281 and US 2008 / 0108082, each of which is incorporated herein by reference.

[0038] In some embodiments, the solid support or its surface is non-planar, such as the inner or outer surface of a tube or container. In some embodiments, the solid support comprises microspheres or beads. "Microspheres" or "beads" or "particles" or grammatical equivalents herein refer to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methyl styrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex or cross-linked dextran such as agarose gel, cellulose, nylon, cross-linked micelles and Teflon, and any other materials of solid supports outlined herein can be used. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In certain embodiments, the microspheres are magnetic microspheres or beads. The beads need not be spherical; irregular particles can be used. Alternatively or additionally, the beads can be porous. The beads range in size from nanometers, ie, 100 nm, to millimeters, ie, 1 mm, with beads of about 0.2 to about 200 microns being preferred, and about 0.5 to about 5 microns being particularly preferred, although in some embodiments smaller or larger beads may be used.

[0039] In some embodiments, the solid support includes a patterned surface. "Patterned surface" refers to the arrangement of different regions in or on the exposed layer of the solid support. For example, one or more regions can be features in which one or more amplification primers are present. Features can be separated by gaps where there are no amplification primers. In some embodiments, the pattern can be an xy format of features in rows and columns. In some embodiments, the pattern can be a repeated arrangement of features and / or gap regions. In some embodiments, the pattern can be a random arrangement of features and / or gap regions. Exemplary patterned surfaces that can be used in the methods and compositions proposed herein are described in US Ser. No. 13 / 661,524 or U.S. Patent Application Publication No. 2012 / 0316086 A1, each of which is incorporated herein by reference.

[0040] In some embodiments, the solid support comprises an array of holes or depressions in the surface. This can be made using various techniques as generally known in the art, including but not limited to photolithography, stamping techniques, molding techniques, and micro-etching techniques. As will be appreciated by those skilled in the art, the techniques used will depend on the composition and shape of the array substrate.

[0041] The features in the patterned surface can be holes in an array of wells (e.g., microwells or nanowells) on glass, silicon, plastic, or other suitable solid supports with patterned covalently attached gels such as poly(N-(5-azidoacetamidopentyl)acrylamide-co-acrylamide) (PAZAM, see, e.g., U.S. Provisional Patent Application Serial No. 61 / 753,833, which is incorporated herein by reference). This process produces a gel pad for sequencing that can be stable over sequencing runs with a large number of cycles. During various uses, covalent attachment of the polymer to the wells helps maintain the gel in the structured features throughout the life of the structured substrate. However, in many embodiments, the gel need not be covalently attached to the wells. For example, under certain conditions, silane-free acrylamide (SFA, see, e.g., U.S. Patent Application Publication No. 2011 / 0059865A1, which is incorporated herein by reference) (which is not covalently attached to any portion of the structured substrate) can be used as a gel material.

[0042] In a specific embodiment, the structured substrate can be prepared as follows: a solid support material is patterned with holes (e.g., micropores or nanopores), the patterned support is coated with a gel material (e.g., PAZAM, SFA or a chemically modified variant thereof, such as an azidated form of SFA (azido-SFA)), and the gel-coated carrier is ground, for example, by chemical or mechanical polishing, so that the gel is retained in the hole, but substantially all of the gel is removed or inactivated from the interstitial region on the surface of the structured substrate between the holes. Primer nucleic acids can be attached to the gel material. Then, a solution of target polynucleotides (e.g., a fragmented human genome) can be contacted with the polished substrate so that each target polynucleotide is inoculated into a single hole through interaction with the primers attached to the gel material; however, due to the absence or inactivity of the gel material, the target polynucleotide will not occupy the interstitial region. The amplification of the target polynucleotide will be limited to the hole because the absence or inactivity of the gel in the interstitial region prevents the outward migration of the growing nucleic acid colony. The method can be conveniently manufactured, scalable and utilize conventional micro- or nano-manufacturing methods.

[0043] Patterned substrates can include, for example, holes etched into slides or chips. As long as such features are physically or functionally separable from each other, the etching of the holes and the pattern of the geometric shape can be of various shapes and sizes. A particularly useful substrate with such structural features is a patterned substrate that can select the size of solid support particles such as microspheres. An exemplary substrate with these features is an etched substrate used in combination with BeadArray technology (Illumina, Inc., San Diego, Calif). Other examples are described in U.S. Patent No. 6,770,441, which is incorporated herein by reference.

[0044] Array refers to a site population that can be distinguished from each other according to relative position. Different molecules located at different sites of the array can be distinguished from each other according to the position of the site in the array. A single site of an array can include one or more specific types of molecules. For example, a site can include a single target polynucleotide molecule with a specific sequence, or a site can include several nucleic acid molecules with the same sequence (and / or its complementary sequence). The position of the array can be different features located on the same substrate. Exemplary features include but are not limited to holes in the substrate, beads (or other particles) in or on the substrate, projections from the substrate, ridges on the substrate, or channels in the substrate. The sites of the array can be separate substrates, each with different molecules. Different molecules attached to a separation substrate can be identified according to the substrate position on the surface combined with the substrate or according to the substrate position in a liquid or gel. Exemplary arrays of separated substrates located on the surface include but are not limited to those with beads in the holes.

[0045] As used herein, the term "microsphere" or "bead" or "particle" or grammatical equivalents herein refers to small discrete particles. Suitable bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex or cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles and teflon, and any other material for solid supports outlined herein can be used. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0046] As used herein, the term "plurality" is intended to mean a population of two or more different members. The size range of the plurality can be from small, medium, large to very large. The range of smaller sized plurality can be, for example, from a few members to tens of members. The range of medium sized plurality can be, for example, from tens of members to about 100 members or hundreds of members. For example, larger plurality can range from about hundreds of members to about 1000 members, to thousands of members and up to tens of thousands of members. Very large plurality can range from tens of thousands of members to hundreds of thousands, one million, millions, tens of millions, or even greater than hundreds of millions of members. Therefore, the size range of the plurality can be from two to completely exceeding 100 million members and all sizes between and greater than the above exemplary ranges, as measured by the number of members. The exemplary number of features in the microarray includes 1.28 cm 2 A plurality of about 500,000 or more discrete features. Exemplary nucleic acid plurality includes, for example, about 1×10 5 , 5×10 5 and 1×10 6 or more populations of different nucleic acid species. Therefore, the definition of the term is intended to include all integer values ​​greater than 2. The upper limits of the present invention can be set, for example, by the theoretical diversity of the nucleotide sequences in the nucleic acid sample of the present invention.

[0047] As used herein, when used in relation to polynucleotides, the term "immobilization" is intended to mean that the polynucleotides are directly or indirectly attached to a solid support by covalent or non-covalent bonds. In certain embodiments of the present invention, covalent attachment may be used, but all that is required is that the polynucleotides remain stationary or attached to a support under the conditions of the desired use of a carrier, such as in applications requiring nucleic acid amplification and / or sequencing. The oligonucleotides used as capture primers or amplification primers may be immobilized so that the 3'-ends may be used for enzymatic extension, and at least a portion of the sequence may be hybridized with a complementary sequence. Immobilization may occur by hybridization with the oligonucleotides attached to the surface, in which case the immobilized oligonucleotides or polynucleotides may be 3'-5' oriented. Alternatively, immobilization may occur by methods other than base pairing hybridization, such as the above-mentioned covalent attachment.

[0048] As used herein, the term "polynucleotide" is intended to mean ribonucleic acid or deoxyribonucleic acid or analogs thereof, including polynucleotide analytes presented in any context; for example, probes, targets or primers. Specific forms of polynucleotides of the present invention include all types of nucleic acids found in organisms, as well as synthetic nucleic acids, such as polynucleotides produced by chemical synthesis. Specific examples of nucleic acids suitable for analysis by incorporation into a microarray produced by the methods of the present invention include genomic DNA (gDNA), messenger RNA of DNA replication (cDNA), messenger RNA of RNA replication (cRNA), mitochondrial DNA or genome, RNA, messenger RNA (mRNA) and / or other RNA populations. Additional examples of polynucleotides include double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), a gene or gene fragment (e.g., a probe, a primer, an expressed sequence tag (EST) or a serial analysis of gene expression (SAGE) tag), genomic DNA, an exon, an intron, a transfer RNA (tRNA), a ribosomal RNA (rRNA), a ribozyme, a recombinant polynucleotide, a synthetic polynucleotide, a branched polynucleotide, a plasmid, a vector, an isolated DNA of any sequence, an isolated RNA of any sequence, or an amplified copy of any of the foregoing. Fragments and / or portions of the above exemplary nucleic acids are also included within the meaning of the term as used herein.

[0049] The terms "nucleic acid," "polynucleotide," and "oligonucleotide" are used interchangeably herein. Unless otherwise specifically stated, the different terms are not intended to represent any particular differences in size, sequence, or other characteristics. For clarity of description, when describing a particular method or composition involving several nucleic acid species, the terms may be used to distinguish one nucleic acid from another.

[0050] As used herein, the term "double-stranded" when used in relation to a polynucleotide means that some or all of the nucleotides between the complementary strands of the polynucleotide are hydrogen bonded together to form a partial or complete double helix. A partially double-stranded polynucleotide may have at least 10%, 25%, 50%, 60%, 70%, 80%, 90% or 95% of its nucleotides bonded to complementary nucleotides.

[0051] A single-stranded polynucleotide refers to a polynucleotide that has few or no hydrogen bonds with another polynucleotide such that a duplex is not formed or is unstable under a given set of hybridization conditions.

[0052] As used herein, the term "target polynucleotide" is intended to mean a polynucleotide that is an object of analysis, action, inquiry or use. Analysis, action or inquiry includes replication, amplification, sequencing and / or other procedures for nucleic acid inquiry of the target polynucleotide. Analysis or use may also include using the target polynucleotide as a component in a system to analyze, perform actions or inquire other molecular entities. The component may be structural or functional. The target polynucleotide may include additional nucleotide sequences of the target sequence to be analyzed or used. For example, the target polynucleotide may include one or more adapters, including adapters used as primer binding sites, which flank the target polynucleotide sequence to be analyzed or used in the system. The target polynucleotide hybridized with the capture oligonucleotide or capture primer may contain nucleotides that extend beyond the 5' or 3' end of the capture oligonucleotide in the following manner, so that not all target polynucleotides are suitable for extension. In a specific embodiment, as described in more detail below, a plurality of target polynucleotides include different target polynucleotide sequences, but have different types of adapters that are the same for two or more different types. Two adapters that may be located on the flanks of a specific target polynucleotide sequence may have the same sequence, or two adapters may have different sequences. Therefore, a plurality of different target polynucleotides can have the same adapter sequence or two different adapter sequences at each end of the target polynucleotide sequence.Therefore, the kind in a plurality of target polynucleotides can include the known sequence region flanking the region of the unknown sequence to be evaluated by, for example, sequencing.In addition, a plurality of target polynucleotides can be the same kind or different kinds, and can include the known sequence region flanking the region of the known sequence to be used as the component in the system.In the case where the target polynucleotide carries an adapter at a single end, the adapter can be located at the 3' end or the 5' end of the target polynucleotide.The target polynucleotide can be used without any adapter, in which case the primer binding sequence can be directly from the sequence found in the target polynucleotide.

[0053] As used herein, the term "target molecule" is intended to represent any molecule that is the object of analysis, action, inquiry or use. Analysis, action or inquiry include subjecting molecules to, for example, sequencing, binding assays, affinity measurements, catalytic measurements, substrate or product specificity and other molecular assays. Analysis or use may also include using target molecules as components in a system to analyze, perform actions or inquire about other molecular entities. Components may be structural or functional. For example, a target molecule may be a nanopore for nanopore sequencing of nucleic acids. Other target molecules suitable for nanopore sequencing include, for example, helicases and polymerases. Other exemplary analyses or uses of target molecules include, for example, molecular binding affinity screening, enzyme screening, compound screening and all other assays in which it is desired to determine or measure molecular interactions. Therefore, a target molecule may be the subject of analysis, action or inquiry, or may be used to analyze, perform actions or inquire about other molecules. Exemplary target molecules include, for example, biopolymers, including polypeptides, polynucleotides or polysaccharides, amino acids, nucleotides, monosaccharides, disaccharides, other carbohydrates, receptors, haptens, ligands, antigens, analytes, small molecule organic or inorganic compounds, vitamins, metabolites, antioxidants, immunosuppressants, anticancer drugs, or any combination thereof.

[0054] In addition, it should be understood that the target molecule can be attached to the polypeptide (target polypeptide) in any number of ways. For example, the target molecule can be attached using conjugation chemistry techniques known in the art and described herein. Advantageous conjugation chemistry types include chemical reactions performed under relatively mild conditions. These include, but are not limited to, nucleophilic substitutions (e.g., reactions of amines and alcohols with acyl halides, active esters), electrophilic substitutions (e.g., enamine reactions), and additions to carbon-carbon and carbon-heteroatom multiple bonds (e.g., Michael reactions, Diels-Alder additions). These and other useful reactions are discussed in, for example, March, ADVANCED ORGANIC CHEMISTRY, 3rd Ed., John Wiley & Sons, New York, 1985; Hermanson, BIOCONJUGATE TECHNIQUES, Academic Press, San Diego, 1996; and Feeney et al., MODIFICATION OF PROTEINS; Advances in Chemistry Series, Vol. 198, American Chemical Society, Washington, DC, 1982.

[0055] Exemplary reactive functional groups that can be used for conjugation chemistry techniques include carboxyl groups (e.g., N-hydroxysuccinimide esters, N-hydroxybenzotriazole esters, acid halides, acyl imidazoles, thioesters, p-nitrophenyl esters, alkyl, alkenyl, alkynyl, and aromatic esters); hydroxyl groups (i.e., conversion to, e.g., esters, ethers, or aldehydes); alkyl halides; dienophiles (e.g., Diels-Alder reactions); aldehyde groups; ketone groups; sulfonyl groups; amine groups; olefins (i.e., cycloadditions and Michael additions); epoxides; phosphoramidite groups; phosphine groups; azide groups (i.e., for click chemistry); and thiol groups. Such reactive groups can be functionalized on target molecules or polypeptides.

[0056] In an illustrative embodiment, the reduced thiol (-SH) group (i.e., sulfhydryl) of a cysteine ​​residue can react with a tether having a thiol-reactive group. Examples of these groups include maleimide and iodoacetamide. Primary thiol-reactive reagents, including iodoacetamide, maleimide, benzyl halides, and bromomethyl ketones can react by S-alkylation of thiols to produce stable thioether products; arylating agents such as 7-nitrobenz-2,1,3-oxadiazole (NBD) halides can react with thiols or amines, proceeding by analogous substitution of aromatic halides with nucleophiles; and since the thiolate anion is a better nucleophile than a neutral thiol, cysteine ​​is more reactive above its pKa. Additional sulfhydryl-reactive chemical groups include haloacetyl, maleimide, aziridine, acryloyl, arylating agents, vinyl sulfone, pyridyl disulfide, TNB-thiol (2-nitro-5-thiobenzoic acid) and disulfide reducing agents. Such groups can be coupled to sulfhydryl groups by alkylation (e.g., by forming a thioether bond) or disulfide exchange (e.g., forming a disulfide bond). Sulfhydryl exchange reactions can also be appropriately used.

[0057] Alternatively, amine (-NH 2 ) can be targeted. For example, the primary amine of a lysine residue and the N-terminus of a polypeptide are relatively reactive. Amine residues can be targeted with N-hydroxysuccinimide esters (NHS esters) that can form stable amide bonds or imidoester crosslinkers that can react with primary amines to form amidine bonds. There are many other amine-reactive compounds. For example, synthetic chemical groups that can form chemical bonds with primary amines include isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, ethylene oxides, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters; such groups can be combined with amines, for example, by acylation or alkylation.

[0058] In other embodiments, the modified target molecule can be used to introduce novel functionalities such as azides or alkynes for use with click chemistry. For example, thiol or amine reactivity as described above can be used with a linker that allows the addition of an azide or alkyne functional group for further use in a click chemistry reaction.

[0059] The ability to create single-molecule arrays spaced apart on a patterned surface includes many features and applications for chemical and biological sensing. One feature of a single-molecule array includes optimization of packing density subject to detection constraints. Detection can be performed using standard optical detection. For standard optical detection, the intermolecular spacing can be about 500 nm (e.g., on the order of the wavelength of the detection light). Detection can be performed using an electronic readout such as, for example, an electronic readout on a CMOS chip.

[0060] The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 15 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 12 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 10 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 9 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 8 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 7 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 6 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 5 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 4 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 to about 3 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 1 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 2 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 3 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 4 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 5 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 6 um. The spacing of single molecules for electronic readout (e.g., on a CMOS chip) may be such that a single molecule occupies a size of about 7 um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 8um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 9um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 10um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 11um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 12um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 13um. The spacing of single molecules for electronic readout (for example, on a CMOS chip) may allow a single molecule to occupy a size of about 14um.The spacing of single molecules for electronic readout (eg, on a CMOS chip) may be such that a single molecule occupies a size of about 15 um. In an embodiment, a single molecule occupies about a single pixel (eg, 1-10 um in size).

[0061] Single molecule sensors can be used for a variety of applications, including but not limited to DNA sequencing using antibodies, etc., chemical sensing and protein / antigen assays, etc. In embodiments, a single polymerase molecule can be used to sequence DNA in real time in a DNA sequence. In other embodiments, nanopore-based DNA sequencing can be used, where placement of a single nanopore in a bilayer can be used to generate interpretable data.

[0062] As used herein, the term "amplicon" when used to refer to nucleic acid, means the product of nucleic acid replication, wherein the product has a nucleotide sequence identical or complementary to at least a portion of the nucleotide sequence of the replicated nucleic acid. Amplicon can be produced by any of the various amplification methods using nucleic acid or its amplicon as a template, including, for example, recombinase polymerase amplification, kinetic exclusion amplification, polymerase extension, polymerase chain reaction (PCR), rolling circle amplification (RCA), connection extension or connection chain reaction. Amplicon can be a nucleic acid molecule with a single copy of a specific nucleotide sequence (such as a PCR product) or multiple copies of a nucleotide sequence (such as a concatenated product of RCA). The first amplicon of a target nucleic acid can be a complementary copy. The subsequent amplicon is a copy produced after the target polynucleotide or the first amplicon produces the first amplicon. The subsequent amplicon can have a sequence that is substantially complementary to the target polynucleotide or substantially identical to the target polynucleotide.

[0063] The number of template copies or amplicons that can be generated can be adjusted by appropriate modification of the amplification reaction, including, for example, changing the number of amplification cycles, using different sustainable polymerases in the amplification reaction and / or varying the length of time the amplification reaction is run, and other conditions known in the art that affect amplification yield. The number of copies of the nucleic acid template can be at least 1, 10, 100, 200, 500, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 and 10,000 copies, and can vary depending on the specific application.

[0064] When used to refer to an amplicon or multiple amplicons, the term "clone" is intended to mean a homogeneous nucleic acid population with respect to a particular nucleotide sequence. A homogeneous sequence can be at least 10 nucleotides long, or longer, such as at least 50, 100, 250, 500 or 1000 nucleotides long. A clonal population can be derived from a single target polynucleotide or template nucleic acid. Substantially all nucleic acids in a clonal population have the same nucleotide sequence. It should be understood that a small amount of mutation (e.g., due to amplification artifacts) can occur in a clonal population without departing from clonality.

[0065] As used herein, the term "capture primer" is intended to mean an oligonucleotide having a nucleotide sequence that is capable of specifically annealing to a single-stranded polynucleotide sequence that is to be analyzed or subjected to nucleic acid interrogation under conditions encountered, for example, in the primer annealing step of an amplification or sequencing reaction. The term is also intended to mean an oligonucleotide having a nucleotide sequence that is capable of specifically annealing to a single-stranded polynucleotide sequence that is used to analyze, interrogate, or act on another molecular entity.

[0066] As used herein, when referring to capture primers or other oligonucleotides, the term "target specificity" is intended to represent capture primers or other oligonucleotides, which include nucleotide sequences specific to the target polynucleotide sequence, i.e., nucleotide sequences that can selectively anneal to the identification zone of the target polynucleotide. The target-specific capture primer can have a single oligonucleotide, or it can include two or more with different sequences. Therefore, the target-specific capture primer can be two or more sequences, including 3,4,5,6,7,8,9 or 10 or more different sequences. The target-specific capture oligonucleotide can include a target-specific capture primer sequence and a universal capture primer sequence. Other sequences such as sequencing primer sequences etc. can also be included in the target-specific capture primer.

[0067] In contrast, when referring to capture primers or other oligonucleotide sequences, the term "universal" is intended to mean capture primers or other oligonucleotides having a common nucleotide sequence in a plurality of capture primers. The common sequence can be, for example, a sequence complementary to the same adapter sequence. Universal capture primers are suitable for interrogating a plurality of different polynucleotides without having to distinguish between different species, whereas target-specific capture primers are suitable for distinguishing between different species.

[0068] The present disclosure provides a substrate comprising: (a) a plurality of first and second capture primers immobilized to a feature on the substrate; (b) at least one target polynucleotide having one end attached to one of the capture primers and the other end linked to a target molecule, wherein the target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, the second capture primer binding region comprising a base pair mismatch with the second capture primer, and (c) a plurality of clonal amplicons complementary to the target polynucleotide immobilized to the feature.

[0069] In one embodiment, the substrate of the present disclosure may include at least one single molecule positioned at a site on the substrate. For single molecule analysis and / or interrogation embodiments, the substrate of the present disclosure will have a single target molecule positioned at a site on the substrate. A single target molecule can be positioned at one or more sites on the substrate using methods and molecular components such as those described herein.

[0070] Substrates for locating a single target molecule can include those materials and forms previously described, as well as other materials and forms known in the art. For example, in some embodiments, the substrate can be any insoluble solid support, semisolid support or matrix to which biomolecules (including, for example, polynucleotides) can be attached. Exemplary solid supports include glass, modified glass, functionalized glass, inorganic glass, microspheres (e.g., inert and / or magnetic particles), plastics, polysaccharides, nylon, nitrocellulose, ceramics, resins, silica, silica-based materials, carbon, metals, optical fibers or fiber bundles, polymers, and porous (e.g., microtiter plates). Exemplary plastics include acrylic resins, polystyrene, copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, and Teflon TM Exemplary silica-based materials include silicon, including silicon wafers and various forms of modified silicon. Other solid supports that can be used as substrates include, for example, latex beads, dextran beads, polyacrylamide gels, gold surfaces, silicon nitride surfaces, or metal oxide surfaces.

[0071] In some embodiments, the surface can be any desired shape, including, for example, a planar, spherical, or porous material suitable for single molecule placement as described herein. For example, the solid support can be a planar glass surface.

[0072] In a specific embodiment, the substrate can be in a container such as a well, tube, channel, cuvette, culture dish, bottle, or portion of a container to allow for interaction with solutions of various reagents. A particularly useful container is a flow cell, such as described in US 2010 / 011 11768A1 or Bentley et al, Nature 456:53-59 (2008). Exemplary flow cells include those commercially available from Illumina, Inc. (San Diego, CA). Another particularly useful container is a well in a multiwell plate or microtiter plate.

[0073] In some embodiments, the site on the substrate for locating at least one target molecule can be a feature. As described above and below, the feature can exist in any of a variety of desired formats. For example, the feature can be a hole, a pit, a channel, a ridge, a raised area, a peg, a column, etc. As previously described, the feature can be a bead or can contain a bead.

[0074] In specific embodiments, features need not comprise beads or particles. Exemplary features include wells present in a substrate for use in commercial sequencing platforms sold, for example, by 454 Life Sciences (a Roche subsidiary, Basel Switzerland) or Ion Torrent (a Life Technologies subsidiary, Carlsbad California). Other substrates with holes include, for example, etched optical fibers and other substrates described in US 6,266,459; US 6,355,431; US ​​6,770,441; US ​​6,859,570; US 6,210,891; US ​​6,258,568; US 6,274,320; US 2009 / 0026082 A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143 A1; US ​​2011 / 0282617 A1 or PCT Publication No. WO 00 / 63437. In several cases, the substrates are exemplified in these references for applications using beads in the holes. In the compositions of the present disclosure, substrates containing holes can be used with or without beads.

[0075] In some embodiments, the pores of the substrate may include a gel material as set forth in U.S. Provisional Application No. 61 / 769,289, which is incorporated herein by reference.

[0076] In one embodiment, the features may be metallic features on a non-metallic surface, such as glass, plastic or other materials exemplified above. The metal layer may be deposited on the surface using methods known in the art, such as wet plasma etching, dry plasma etching, atomic layer deposition, ion beam etching, chemical vapor deposition, vacuum sputtering, etc. Any of a variety of commercial instruments may be used as appropriate, including, for example, or System (Oxford Instruments, UK). Metal layers can also be deposited by electron beam evaporation or sputtering, as listed in Thornton, Ann. Rev. Mater. Sci. 7:239-60 (1977). Metal layer deposition techniques (such as those exemplified above) can be combined with photolithography techniques, for example, to produce metal areas or patches on the surface. An exemplary method for combining metal layer deposition techniques and photolithography techniques is described in U.S. Serial No. US 13 / 492,661.

[0077] The substrate of the present disclosure can be immobilized to the first and second capture primers on the feature on the substrate. The first and second capture primers can be a plurality of first capture primers and a plurality of second capture primers. In some embodiments, the first and second capture primers can be directed to different sequences of the target polynucleotide, thus showing specificity to different regions of the target polynucleotide. In other embodiments, the first and second capture primers can be directed to different target polynucleotides.

[0078] Figure 2 An exemplary substrate is depicted in FIG. Figure 2 As shown in , the substrate (201) can include a plurality of first capture primers (202, 203, 204) and a plurality of second capture primers (205, 206, 207). The substrate (201) can also include a target polynucleotide (208) attached to the capture primer (202) at one end. The substrate (201) can also include amplicons (210, 211, 212, 213 and 214) of the target polynucleotide (208). In addition, the substrate (201) can include a target molecule (209) attached to the target polynucleotide (208).

[0079] In one embodiment, the present disclosure provides a substrate comprising a first feature, wherein the first feature comprises (a) a plurality of first capture primers; (b) a plurality of second capture primers; and (c) a target polynucleotide, wherein: (i) the target polynucleotide is double-stranded; (ii) the target polynucleotide comprises a second capture primer binding region; (iii) the second capture primer binding region comprises at least one nucleotide mismatch; and (iv) one strand of the second capture primer binding region is the reverse complement of the second capture primer, and the other strand of the second capture primer binding region is less than 100% identical to the second capture primer. The second capture primer binding region may contain more than two nucleotide mismatches. The second capture primer binding region may contain more than three nucleotide mismatches. The second capture primer binding region may contain more than four nucleotide mismatches. The target polynucleotide may further comprise one or more additional capture primer binding regions. The target polynucleotide may also comprise a first capture primer binding region that may hybridize with the first capture primer. The first capture primer binding region may hybridize with the first capture primer with 100% complementarity. For example, the first capture primer binding region may comprise a 100% nucleotide match to the first capture primer. The first feature may further comprise a plurality of amplicons. The plurality of amplicons may comprise amplicons of a target polynucleotide. The first feature may further comprise a target molecule. The target molecule may be attached to the target polynucleotide. The first feature may further comprise one or more additional target molecules. The one or more additional target molecules may be suspended in the capture primer, target polynucleotide or amplicon within the feature. Alternatively or additionally, the one or more additional target molecules are not attached or fixed to the feature. The one or more additional target molecules may be in solution in the feature. The substrate may further comprise one or more additional features. The one or more additional features may comprise a plurality of first capture primers, a plurality of second capture primers, a target polynucleotide, a target molecule, a plurality of amplicons or a combination thereof.

[0080] Two or more capture primers can be present in a feature in any ratio. For example, a plurality of first capture primers and a plurality of second capture primers can exist with equal amounts or with any other ratio, for example molar ratio. A feature can have an excess of greater than 1.1x, greater than 1.2x, greater than 1.3x, greater than 1.4x, greater than 1.5x, greater than 2.0x, greater than 2.5x, greater than 3.0x, greater than 5.0x, greater than 10x, greater than 15x, greater than 20x, greater than 20x, greater than 25x, greater than 30x, greater than 50x, greater than 100x, greater than 300x, greater than 500x, or greater than 1,000x of the first capture primer. In the embodiment using a plurality of features, as for example in a microarray, different features can have the same ratio or different ratios of two or more capture primers.

[0081] Capture primers can include one or more capture regions. Capture primer regions can include, for example, universal capture regions, sequencing primer binding sites (SBS), target-specific capture regions, predetermined cleavage sites, such as restriction sites and joint regions, for example, joint regions separating two or more regions of capture primers. Some capture primers can include, for example, universal capture regions and SBS. Other capture primers can include universal capture regions and target-specific capture regions. Other capture primers can include, for example, universal capture regions, SBS and target-specific regions. Capture primers can be closed at the 3' end (3' closed) or unclosed at the 3' end (3' unclosed). Primers with closed 3'-ends can, for example, be unblocked in enzymes or chemical reactions. Capture primers can also include predetermined (non-random) cleavage sites. Exemplary predetermined cleavage sites are described, for example, in U.S. Patent No. 8,715,966B2. Cutting at a predetermined site can occur, for example, as enzymatic cutting or non-enzymatic cutting, such as chemical cutting using methods well known to those skilled in the art. Given the teachings and guidance provided herein, one of skill in the art will appreciate that a capture primer can contain any number of different regions useful in one or more applications.

[0082] The universal capture region can be any known or predetermined nucleotide sequence included as a common sequence in a plurality of capture primers. Typically, the universal capture region will be long enough to have a unique nucleotide sequence in a population of different sequences, such as the number of different nucleotide sequences included in a genome. It will be appreciated by those skilled in the art that in view of the sequence diversity of the target population, what length of the universal capture region is sufficient to be unique.

[0083] Universal capture region can be designed from scratch, or can be obtained from sources well known in the art. For example, universal capture primers or regions with such universal capture primer specific hybridization can be obtained from commercial sources. Two typical universal capture primers include, for example, nucleotide sequences 5'-AATGATACGGCGACCACCGA-3' and 5'-CAAGCAGAAGACGGCATACGA-3' (respectively P5 and P7, Illumina, San Diego, CA). The region with the above-mentioned capture primer specific hybridization can include, for example, the reverse complementary sequence corresponding to 5'-TCGGTGGTCGCCGTATCATT-3' or 5'-TCGTATGCCGTCTTCTGCTTG-3' (respectively P5' and P7', Illumina, San Diego, CA).

[0084] As with the universal capture zone, the SBS can be any known or designed sequence of sufficient length to specifically anneal with a complementary sequencing primer. Exemplary SBSs include, for example, nucleotide sequences 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' and 5'-CGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT-3' (respectively SBS3 and SBS8, Illumina, San Diego, CA). The region specifically hybridized with the above-mentioned SBS can include, for example, corresponding to 5'-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT-3' and 5'-AGATCGGAAGAGCGGTTCAGCAGGAATGCCGAGACCG-3' (respectively "SBS3" and "SBS81" directional complementary sequences, Illumina, San Diego, CA).

[0085] As previously described, the capture primers can have any combination of regions, such as any combination of the above-exemplified P5, P7, SBS3 or SBS8 primer regions or their complementary sequences (e.g., P5', P7'SBS3' or SBS8'). Exemplary combinations of the above-mentioned specific sequences include, for example, combinations such as P5-SBS3, P5-SBS8, P7-SBS8 and P7-SBS3, their complementary sequences or combinations thereof.

[0086] The first and second capture primers immobilized to a feature can include any capture region or any combination of capture regions. For example, the first capture primer can include a first universal capture region, and the second capture primer can include the same universal capture region or the second universal capture region. The first and second capture primers can further include the same or different SBS. For example, the first capture primer can include a first universal capture primer region and a first SBS, and the second capture primer can include a second universal capture region and a second SBS.

[0087] For example, in some embodiments, the first capture primer comprises a P5 primer nucleotide sequence and the second capture primer comprises a P7 primer nucleotide sequence.

[0088] In other embodiments, the first capture primer includes a P5 primer nucleotide sequence and a SBS3 primer nucleotide sequence, and the second capture primer includes a P7 primer nucleotide sequence and a SBS8 primer nucleotide sequence. In other embodiments, the first capture primer includes a P5 primer nucleotide sequence and a SBS8 primer nucleotide sequence, and the second capture primer includes a P7 primer nucleotide sequence and a SBS3 primer nucleotide sequence.

[0089] The capture primers of the features immobilized on the substrate can be multiple capture primers. In some embodiments, the feature can have a single multiple capture primer, such as when a primer extension reaction is required or when the amplification of polynucleotides uses the same capture primer binding region at both ends. In other embodiments, the feature can have multiple first capture primers and multiple second capture primers. In addition, in addition to multiple first and second capture primers, the feature on the substrate can also have multiple capture primers. For example, in certain embodiments, the feature on the substrate can include multiple third, fourth, fifth and / or sixth or more capture primers. The number of including multiple capture primers will depend on the application. For example, in certain embodiments, it can be expected to perform the first bridge amplification using multiple first and second capture primers, and then perform the second bridge amplification using multiple third and fourth capture primers. In such embodiments, the target polynucleotide will have a capture primer binding region for each of the first, second, third and fourth capture primers. In view of the teaching and guidance provided herein, those skilled in the art will understand the use of a given specific application or the number of capture primers and corresponding capture primer binding regions for the desired structure.

[0090] A plurality of can be a colony of two or more members. In some embodiments, a plurality of are two or more different polynucleotides or other reference molecules, such as capture primers or target molecules. In other embodiments, a plurality of are colonies of two or more identical members (such as polynucleotides or other reference molecules, such as capture primers, target polynucleotides or target molecules). Alternatively or in addition, a plurality of can be colonies of two or more similar members (such as, polynucleotides or other reference molecules, such as capture primers, target polynucleotides or target molecules). Therefore, unless otherwise expressly stated, the term "plurality" is used synonymously with colonies.

[0091] In some embodiments, a plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more different members of a population. In other embodiments, a plurality is 200, 300, 400, 500, 1000, 5000, 10000, 50000, 1 x 10 5 ,2x10 5 ,3x10 5 ,4x10 5 ,5x10 5 ,6x10 5 ,7x10 5 ,8x10 5 ,9x10 5 ,1x10 6 ,2x10 6 ,3x10 6 ,4x10 6 ,5x106 ,6x10 6 ,7x10 6 ,8x10 6 ,9x10 6 or 1x10 7 or more different members.

[0092] In some embodiments, a plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more similar members of a population. In other embodiments, a plurality is 200, 300, 400, 500, 1000, 5000, 10000, 50000, 1 x 10 5 ,2x10 5 ,3x10 5 ,4x10 5 ,5x10 5 ,6x10 5 ,7x10 5 ,8x10 5 ,9x10 5 ,1x10 6 ,2x10 6 ,3x10 6 ,4x10 6 ,5x10 6 ,6x10 6 ,7x10 6 ,8x10 6 ,9x10 6 or 1x10 7 or more similar members.

[0093] In some embodiments, a plurality is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more identical members of a population. In other embodiments, a plurality is 200, 300, 400, 500, 1000, 5000, 10000, 50000, 1 x 10 5 ,2x10 5 ,3x10 5 ,4x10 5 ,5x10 5 ,6x10 5 ,7x10 5 ,8x10 5 ,9x10 5 ,1x10 6 ,2x10 6 ,3x10 6 ,4x10 6 ,5x10 6,6x10 6 ,7x10 6 ,8x10 6 ,9x10 6 or 1x10 7 or more of the same members.

[0094] In some embodiments, a plurality is a mixture of different and similar members of a population. In other embodiments, a plurality is a mixture of different and identical members of a population. In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more of the members of the population in a plurality are identical. In some embodiments, at least 10% of the members of the population in a plurality are identical. In some embodiments, at least 20% of the members of the population in a plurality are identical. In some embodiments, at least 30% of the members of the population in a plurality are identical.

[0095] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more of the members of the plurality of populations are different. In some embodiments, in some embodiments, at least 10% of the members of the plurality of populations are different. In some embodiments, at least 20% of the members of the plurality of populations are different. In some embodiments, at least 30% of the members of the plurality of populations are different.

[0096] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% or more homologous. In some embodiments, at least two or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous. In some embodiments, at least three or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous. In some embodiments, at least four or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous. In some embodiments, at least five or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous. In some embodiments, in some embodiments at least ten or more members of a plurality of populations are at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homologous.

[0097] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more members of a plurality of populations are identical. In some embodiments, at least 2 members of a plurality of populations are identical. In some embodiments, at least 3 members of a plurality of populations are identical. In some embodiments, at least 5 members of a plurality of populations are identical. In some embodiments, at least 10 members of a plurality of populations are identical. In some embodiments, at least 20 members of a plurality of populations are identical.

[0098] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more members of a plurality of populations are similar. In some embodiments, at least 2 members of a plurality of populations are similar. In some embodiments, at least 3 members of a plurality of populations are similar. In some embodiments, at least 5 members of a plurality of populations are similar. In some embodiments, at least 10 members of a plurality of populations are similar. In some embodiments, at least 20 members of a plurality of populations are similar.

[0099] In some embodiments, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more members of a plurality of populations are different. In some embodiments, at least 2 members of a plurality of populations are different. In some embodiments, at least 3 members of a plurality of populations are different. In some embodiments, at least 5 members of a plurality of populations are different. In some embodiments, at least 10 members of a plurality of populations are different. In some embodiments, at least 20 members of a plurality of populations are different.

[0100] Substrate of the present disclosure may include at least one target polynucleotide, one end of which is attached to the capture primer being immobilized to a feature. In some embodiments, substrate of the present disclosure will include a target polynucleotide, one end of which is attached to the capture primer being immobilized to a feature. Although this paper is described with reference to the attachment of at least one target polynucleotide or with reference to the target polynucleotide of the capture primer being attached to a feature at one end, it is understood that in other embodiments, substrate may contain two or more target polynucleotides, which are attached to the capture primer being immobilized to a feature at one end. In these other embodiments, the number of target polynucleotides may be, for example, 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 or more different target polynucleotides. Alternatively, the number of target polynucleotides can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more identical target polynucleotides. In other embodiments, the number of target polynucleotides is, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more similar target polynucleotides.

[0101] With reference to at least one target polynucleotide attached to a capture primer immobilized to a feature at one end, the target polynucleotide can be any polynucleotide capable of amplifying in copy number, including, for example, DNA, RNA or protein nucleic acid (PNA). The target polynucleotide can comprise two or more nucleotides. The two or more nucleotides can comprise ribonucleotides, deoxyribonucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) or any combination thereof. The target polynucleotide can comprise purine bases, pyrimidine bases or both. The target polynucleotide can comprise natural, chemically modified, biochemically modified, non-natural or derivatized nucleotide bases. The target polynucleotide can comprise one or more paired nucleotide bases. The target polynucleotide can be a double-stranded nucleic acid. In some embodiments, the target polynucleotide can be a double-stranded nucleic acid, such as double-stranded DNA (dsDNA). The target polynucleotide can be a double-stranded RNA (dsRNA). The target polynucleotide can be a double-stranded DNA / RNA hybrid. The target polynucleotide can comprise one or more unpaired nucleotide bases. The target polynucleotide can be single-stranded. In other embodiments, the target polynucleotide can be a single-stranded nucleic acid, such as single-stranded DNA (ssDNA). The target polynucleotide may be a single-stranded RNA.The target polynucleotide may comprise a mixture of paired and unpaired nucleotide bases.

[0102] In order to place a single molecule on a feature, the target polynucleotide acts as a tether or anchor of the target molecule to a feature. Therefore, the target polynucleotide can be any sequence of desired length. In some embodiments, when a plurality of target molecules are anchored to a plurality of different features, a plurality of target polynucleotides can have the same sequence or different sequences. In other embodiments, some or all of the plurality of target polynucleotides anchoring a plurality of target molecules to different features can have different sequences. Therefore, the target polynucleotide can be any desired sequence or sequence mixture. In view of the teaching and guidance provided herein, those skilled in the art will understand whether to use the same target polynucleotide sequence or different target polynucleotide sequences when a plurality of target molecules are immobilized to a plurality of different features.

[0103] The target polynucleotide may comprise one or more target polynucleotide regions. The target polynucleotide region may comprise a capture primer binding region, a target region, a primer binding region, a barcode region, a joint region, or an adapter region. For example, the target polynucleotide may contain a target polynucleotide region comprising a capture primer binding region. The target polynucleotide may contain a target polynucleotide region comprising a first capture primer binding region. The target polynucleotide may contain a target polynucleotide region comprising a second capture primer binding region. Alternatively or in addition, the target polynucleotide may contain a target polynucleotide region comprising a target region. The target polynucleotide may contain a target polynucleotide region comprising a primer binding region. The target polynucleotide may contain a target polynucleotide region comprising a barcode region. The target polynucleotide may contain a target polynucleotide region comprising a joint region. Alternatively or in addition, the target polynucleotide may contain a target polynucleotide region comprising an adapter region.

[0104] The target polynucleotide may include multiple target polynucleotide regions. The target polynucleotide may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more target polynucleotide regions. The target polynucleotide may include 2 or more target polynucleotide regions. The target polynucleotide may include 3 or more target polynucleotide regions. The target polynucleotide may include 4 or more target polynucleotide regions. The target polynucleotide may include 5 or more target polynucleotide regions. The target polynucleotide may include 6 or more target polynucleotide regions. The target polynucleotide may include multiple target polynucleotide regions, which include one or more first capture primer binding regions, second capture primer binding regions, target regions, primer binding regions, barcode regions, joint regions, adapter regions, or any combination thereof. The target polynucleotide may include multiple target polynucleotide regions, which include two or more first capture primer binding regions, second capture primer binding regions, target regions, primer binding regions, barcode regions, joint regions, adapter regions, or any combination thereof. The target polynucleotide may comprise a plurality of target polynucleotide regions comprising three or more first capture primer binding regions, second capture primer binding regions, target regions, primer binding regions, barcode regions, linker regions, adapter regions, or any combination thereof.

[0105] Figure 3 Exemplary target polynucleotides comprising two or more target polynucleotide regions are depicted in FIG. Figure 3 As shown in A in FIG. 1 , the target polynucleotide may include two or more target polynucleotide regions (301, 302). The first target polynucleotide region (301) may be a first capture primer binding region. The second target polynucleotide region (302) may be a second capture primer binding region. Figure 3 As shown in B in FIG. 1 , the target polynucleotide may include three or more target polynucleotide regions (301, 302, 303). The first target polynucleotide region (301) may be a first capture primer binding region. The second target polynucleotide region (302) may be a second capture primer binding region. The third target polynucleotide region (303) may be a target region. Figure 3 As shown in C in FIG. 1 , the target polynucleotide may include four or more target polynucleotide regions (301, 302, 303, 304). The first target polynucleotide region (301) may be a first capture primer binding region. The second target polynucleotide region (302) may be a second capture primer binding region. The third target polynucleotide region (303) may be a target region. The fourth target polynucleotide region (304) may be a primer binding region. Figure 3 As shown in D in , the target polynucleotide can include five or more target polynucleotide regions (301, 302, 303, 304, 305). The first target polynucleotide region (301) can be a first capture primer binding region. The second target polynucleotide region (302) can be a second capture primer binding region. The third target polynucleotide region (303) can be a target region. The fourth target polynucleotide region (304) can be a primer binding region. The fifth target polynucleotide region (305) can be a barcode region. Alternatively or additionally, a target polynucleotide region can be a joint region. Alternatively or additionally, a target polynucleotide region can be an adapter region.

[0106] The target polynucleotide or target polynucleotide region (target polynucleotide or region thereof) comprising, for example, a target region, a capture primer binding region and / or other regions described herein or known to those skilled in the art can comprise two or more nucleotides. The target polynucleotide or region thereof can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more nucleotides. The target polynucleotide can comprise 25, 30, 35, 40, 45, 50, 55, 70, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides. The target polynucleotide or its region may contain 150, 200, 250, 300, 350, 400, 450, 500, 550, 700, 650, 700, 750, 800, 850, 900, 950, 1000 or more nucleotides. The target polynucleotide or its region may contain 1 100, 1200, 1300, 1400, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 7000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10000 or more nucleotides. The target polynucleotide or its region may contain 100 or more nucleotides. The target polynucleotide or its region may contain 300 or more nucleotides. The target polynucleotide or region thereof may contain 400 or more nucleotides. The target polynucleotide or region thereof may contain 500 or more nucleotides. The target polynucleotide or region thereof may contain 600 or more nucleotides.

[0107] The target polynucleotide or region thereof may comprise 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900 or 901 to 1000 nucleotides. The target polynucleotide or region thereof may comprise about 5 to 2000, 5 to 1500, 5 to 1000, 5 to 800, 5 to 600, 5 to 400, 5 to 200, 5 to 100 or 5 to 50 nucleotides. The target polynucleotide or region thereof may comprise about 10 to 2000, 10 to 1500, 10 to 1000, 10 to 800, 10 to 600, 10 to 400, 10 to 200, 10 to 100 or 10 to 50 nucleotides. The target polynucleotide or region thereof may comprise about 20 to 2000, 20 to 1500, 20 to 1000, 20 to 800, 20 to 600, 20 to 400, 20 to 200, 20 to 100, or 20 to 50 nucleotides.

[0108] The target polynucleotide or its region may contain less than about 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500 or 1000 or less nucleotides. The target polynucleotide or its region may contain less than about 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 or less nucleotides. The target polynucleotide or its region may contain less than about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 or less nucleotides.

[0109] The target polynucleotide or its region may comprise a double-stranded polynucleotide. The target polynucleotide or its region may comprise one or more base pairs. The target polynucleotide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more base pairs. The target polynucleotide or its region may comprise 25, 30, 35, 40, 45, 50, 55, 70, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs. The target polynucleotide or its region may comprise 150, 200, 250, 300, 350, 400, 450, 500, 550, 700, 650, 700, 750, 800, 850, 900, 950, 1000 or more base pairs. The target polynucleotide or its region can contain 1100,1200,1300,1400,1500,2000,2500,3000,3500,4000,4500,5000,5500,7000,6500,7000,7500,8000,8500,9000,9500,10000 or more base pairs. The target polynucleotide or its region can contain 100 or more base pairs. The target polynucleotide or its region can contain 300 or more base pairs. The target polynucleotide or its region can contain 400 or more base pairs. The target polynucleotide or its region can contain 500 or more base pairs. The target polynucleotide can contain 600 or more base pairs.

[0110] The target polynucleotide or region thereof may comprise 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900, or 901 to 1000 base pairs. The target polynucleotide or region thereof may comprise about 5 to 2000, 5 to 1500, 5 to 1000, 5 to 800, 5 to 600, 5 to 400, 5 to 200, 5 to 100, or 5 to 50 base pairs. The target polynucleotide or region thereof may contain about 10 to 2000, 10 to 1500, 10 to 1000, 10 to 800, 10 to 600, 10 to 400, 10 to 200, 10 to 100, or 10 to 50 base pairs. The target polynucleotide or region thereof may contain about 20 to 2000, 20 to 1500, 20 to 1000, 20 to 800, 20 to 600, 20 to 400, 20 to 200, 20 to 100, or 20 to 50 base pairs.

[0111] The target polynucleotide or region thereof may contain less than about 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500 or 1000 or less base pairs. The target polynucleotide or region thereof may contain less than about 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 150 or 100 or less base pairs. The target polynucleotide or region thereof may contain less than about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15 or 10 or less base pairs.

[0112] A double-stranded target polynucleotide or region thereof may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more mismatches. A double-stranded target polynucleotide or region thereof may contain one or more mismatches. A double-stranded target polynucleotide or region thereof may contain two or more mismatches. A double-stranded target polynucleotide or region thereof may contain three or more mismatches. A double-stranded target polynucleotide or region thereof may contain four or more mismatches. A double-stranded target polynucleotide or region thereof may contain five or more mismatches.

[0113] A double-stranded target polynucleotide or region thereof may contain less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mismatch. A double-stranded target polynucleotide or region thereof may contain five or fewer mismatches. A double-stranded target polynucleotide or region thereof may contain four or fewer mismatches. A double-stranded target polynucleotide or region thereof may contain three or fewer mismatches. A double-stranded target polynucleotide or region thereof may contain two or fewer mismatches. A double-stranded target polynucleotide or region thereof may contain one or fewer mismatches.

[0114] As previously illustrated, the target polynucleotide may include one or more regions that include one or more capture primer binding regions. Typically, the capture primer binding region is complementary to the capture primer. The target polynucleotide region may include a capture primer binding region at the 5' end. The target polynucleotide region may include a capture primer binding region at the 3' end. The target polynucleotide region may include a first capture primer binding region at the 5' end of the target region and a second capture primer binding region at the 3' end of the target region. Alternatively or in addition, the target polynucleotide region including the capture primer binding region may be located in an internal region of the target polynucleotide.

[0115] The target polynucleotide may comprise two or more capture primer binding regions. The target polynucleotide may comprise three or more capture primer binding regions. The target polynucleotide may comprise four or more capture primer binding regions. The two or more capture primer binding regions may be identical. Alternatively or additionally, the two or more capture primer binding regions may be different. The two or more capture primer binding regions may be close to each other on the target polynucleotide. Alternatively or additionally, the two or more capture primer binding regions may not be close to each other on the polynucleotide.

[0116] As with corresponding capture primers, the capture primer binding region can include one or more capture primer binding regions. The capture primer binding region can include, for example, a universal capture primer binding region, a sequencing primer binding region (SBS), a target-specific capture primer binding region, a predetermined cleavage site, such as a restriction site, and a joint region, for example, a joint region that separates two or more regions of the capture primer binding region. Some capture primer binding regions can include, for example, a universal capture primer binding region and an SBS. Other capture primer binding regions can include, for example, a universal capture primer binding region, an SBS, and a target-specific capture primer binding region. The capture primer binding region can be closed or unclosed at 3' or 5' ends. In view of the teaching and guidance provided herein, it will be appreciated by those skilled in the art that the capture primer binding region can be included in any number of different regions useful in one or more applications.

[0117] For example, the previously illustrated universal capture primer can also be used as the universal capture primer binding region, as can other capture primer regions shown in this article or well known in the art. With reference to the aforementioned universal capture primer, the illustrated P5, P7, P5 'and / or P7 'sequence can be used as the universal capture primer binding region to anneal with complementary capture primers (for example, respectively P5 ', P7 'P5 and / or P7). Similarly, the previously listed SBS sequence or other sequences designed or known in the art can be included in the region in the capture primer region. In one embodiment, for example, exemplary SBS sequences SBS3, SBS8, SBS3 'and / or SBS8 'can be included.

[0118] For example, the plurality of target polynucleotide binding regions can have any combination of capture primer binding regions, for example, any combination of the above-exemplified P5, P7, SBS3 or SBS8 primer regions or any combination of their complementary sequences (e.g., P5', P7', SBS3' or SBS8'). Exemplary combinations of the above-mentioned specific sequences include, for example, combinations such as P5-SBS3, P5-SBS8, P7-SBS8 and P7-SBS3, their complementary sequences or combinations thereof.

[0119] like Figure 4 As shown, an exemplary combination of target regions flanked by capture primer binding regions is a P5-SBS3 capture primer binding region at the first end of the target polynucleotide and a P7'-SBS8' capture primer binding region at the second end. The corresponding complementary strand includes a P5'-SBS3' capture primer binding region at the first end and a P7-SBS8 capture primer binding region at the second end.

[0120] The capture primer binding region may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. The capture primer binding region may comprise 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more nucleotides. The capture primer binding region may comprise 5 or more nucleotides. The capture primer binding region may comprise 10 or more nucleotides. The capture primer binding region may comprise 15 or more nucleotides. The capture primer binding region may comprise 20 or more nucleotides. The capture primer binding region may comprise 25 or more nucleotides.

[0121] The capture primer binding region may comprise 400, 375, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100 or less nucleotides. The capture primer binding region may comprise 300 or less nucleotides. The capture primer binding region may comprise 200 or less nucleotides. The capture primer binding region may comprise 100 or less nucleotides. The capture primer binding region may comprise 50 or less nucleotides. The capture primer binding region may comprise 40 or less nucleotides. The capture primer binding region may comprise 30 or less nucleotides.

[0122] The capture primer binding region of the target polynucleotide may be 100% complementary to the capture primer. The capture primer binding region of the target polynucleotide may be at least 60%, 65%, 70%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95% or 97% or more complementary to the capture primer. The capture primer binding region of the target polynucleotide may be 80% complementary to the capture primer. The capture primer binding region of the target polynucleotide may be 85% complementary to the capture primer. The capture primer binding region of the target polynucleotide may be 9% complementary to the capture primer. The capture primer binding region of the target polynucleotide may be 95% complementary to the capture primer.

[0123] The primer binding region of the target polynucleotide may contain 1,2,3,4,5,6,7,8,9 or 10 or more nucleotide mismatches with the capture primer. The capture primer binding region of the target polynucleotide may contain one or more nucleotide mismatches with the capture primer. The capture primer binding region of the target polynucleotide may contain two or more nucleotide mismatches with the capture primer. The capture primer binding region of the target polynucleotide may contain three or more nucleotide mismatches with the capture primer. The capture primer binding region of the target polynucleotide may contain four or more nucleotide mismatches with the capture primer.

[0124] The target polynucleotide can comprise one or more target regions. The target polynucleotide can have a target region that is located in the internal region of the target polynucleotide. For example, the target region can have a capture primer binding region or other region at its 5' and 3' end flanks. Alternatively or in addition, the target region can be located at, for example, the 5' end of the target polynucleotide. The target polynucleotide can comprise the capture primer binding region at the 3' end, 5' end or both ends of the target polynucleotide, as illustrated above. The target polynucleotide can comprise the first target region at the 5' end of the target polynucleotide and the second target region at the 3' end of the target polynucleotide. The target region can have one or more capture primer binding regions at either end or both ends.

[0125] The target polynucleotide may comprise two or more target regions. The target polynucleotide may comprise three or more target regions. The target polynucleotide may comprise four or more target regions. The two or more target regions may be the same. Alternatively or additionally, the two or more target regions may be different. The two or more target regions may be close to each other on the target polynucleotide. Alternatively or additionally, the two or more target regions may not be close to each other on the target polynucleotide.

[0126] The size or length of the target region can be any length exemplified above for a target polynucleotide or a target polynucleotide region. Exemplary lengths include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more nucleotides. The target region can include 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more nucleotides. The target region can include 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 100 or more nucleotides. The target region can include 5 or more nucleotides. The target region can include 10 or more, 20 or more, 50 or more, 100 or more, 200 or more or 400 or more nucleotides. Other exemplary lengths include, for example, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 375, 350, 325, 300, 275, 250, 225, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100 or less nucleotides. Other exemplary lengths include, for example, 700 or less, 500 or less, 300 or less, 200 or less, 100 or less or 50 or less nucleotides.

[0127] The substrate of the present disclosure may include at least one target molecule.At least one target molecule may be attached to a target polynucleotide.At least one target molecule may be attached to a target polynucleotide, which is attached to a capture primer.At least one target molecule may be attached to a target polynucleotide immobilized to a capture primer, which is immobilized to a feature.Although this article is described with reference to at least one target molecule, with reference to at least one target molecule attached to a target polynucleotide, with reference to at least one target molecule attached to a target polynucleotide attached to a capture primer, or with reference to at least one target molecule attached to a target polynucleotide attached to a capture primer immobilized to a feature, it should be understood that in other embodiments, the substrate may include multiple two or more target molecules in each reference case.As further illustrated below, multiple target molecules may be immobilized to different features on a substrate individually.Or, multiple target molecules may be immobilized to the same feature on a substrate.In the embodiment where multiple target molecules are immobilized to different features individually, multiple target molecules may be target molecules of the same type or types or kinds as illustrated above and below. In these embodiments, the number of target molecules in the plurality can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 250, 500, 1,000, 5,000, 10,000 or more different, identical and / or similar target molecules.

[0128] For example, multiple target molecules can include two or more identical target molecules. In this embodiment, the first and second or more target molecules can be, for example, specific polypeptides, such as MspA nanopores. Alternatively or in addition, multiple target molecules can include two or more different target molecules. For example, the first target molecule of multiple target molecules can be an enzyme, and the second target molecule of multiple target molecules can be a polynucleotide. Multiple target molecules can include three or more different target molecules. For example, the first target molecule of multiple target molecules can be an enzyme, the second target molecule of multiple target molecules can be a polynucleotide, and the third target molecule of multiple target molecules can be a small organic compound. Alternatively or in addition, multiple target molecules can include two or more similar target molecules. Two or more similar target molecules can be molecules of the same type. For example, the first target molecule can be an MspA nanopore, and the second target molecule can be a NaIP nanopore. In this case, the first and second target molecules have molecules of the same type (e.g., nanopores). Two or more similar target molecules can be homologues or variants. For example, the first target molecule can be a human form of a polypeptide, and the second target molecule can be a mouse form of a polypeptide. Variants can include polypeptides encoded by different alleles or polypeptides obtained by site-specific mutagenesis, directed evolution, etc. Two or more similar target molecules can be alternative splicing variants of the same protein. For example, the first target molecule can be a full-length form of a polypeptide, and the second target molecule can be a truncated form of a polypeptide. Two or more similar target molecules can be chemical analogs or derivatives. For example, the first target molecule can be compound X, and the second target molecule can be a derivative of compound X. In an exemplary embodiment, a plurality of similar target molecules can be a library of enzyme variants. Exemplary target molecules are further described below.

[0129] In some embodiments, at least one target molecule or multiple target molecules can be attached to a substrate and / or a feature attached to a substrate. At least one target molecule or multiple target molecules can be attached to a capture primer or multiple capture primers, which are respectively immobilized to a feature on a substrate. At least one target molecule or multiple target molecules can be attached to a target polynucleotide, which is attached to a capture primer or multiple thereof, which are respectively immobilized to a feature on a substrate. Attachment can be a covalent or non-covalent attachment of a target molecule to any region or component described above. With reference to at least one target molecule, for example, at least one target molecule can be covalently attached to a substrate and / or a feature on a substrate. At least one target molecule can be covalently attached to a capture primer, which is immobilized to a feature on a substrate. At least one target molecule can be covalently attached to a target polynucleotide, which is covalently attached to a capture primer, which is immobilized to a feature on a substrate. The following further describes an exemplary embodiment of covalently attaching a target polynucleotide to a capture primer via a covalent phosphodiester bond with reference to a method for placing at least one target molecule on a feature of a substrate. In addition, for example, at least one target molecule can be non-covalently attached to a substrate and / or a feature on a substrate. At least one target molecule can be non-covalently attached to a capture primer that is immobilized to a feature on a substrate. At least one target molecule can be non-covalently attached to a target polynucleotide that is non-covalently attached to a capture primer that is immobilized to a feature on a substrate. Methods for covalent and non-covalent attachment of polynucleotides and target molecules to substrates and to each other are well known in the art.

[0130] In addition to at least one target molecule immobilized by the above-mentioned structure, the feature or multiple features on the substrate can also include multiple amplicons of the target polynucleotide. The amplicons can be complementary to the target polynucleotide. In the embodiment where the target polynucleotide or the target polynucleotide region includes one or more nucleotide mismatches, the amplicons can be complementary to the target polynucleotide except for one or more nucleotide mismatches. Similarly, in this embodiment, the double-stranded amplicon of the target polynucleotide can have a chain complementary to the target polynucleotide (except for one or more nucleotide mismatches) and another chain identical in sequence to the target polynucleotide (except for one or more nucleotide mismatches). In other embodiments, the amplicon can be a plurality of different amplicon species exemplified above. A plurality of amplicons can minimize filling features, partially fill features or fill up features on the substrate. Therefore, the amplicon density can be low, medium or high. The target polynucleotide design, the method for generating such an amplicon and the method for controlling the density are described below with further reference to the method for placing at least one target molecule on the feature of the substrate. The density of amplicons may be about 10 to 100 in a feature of about 100 nm in size, or about 100 to 10,000 in a feature of about 100 nm to 1 μm in size.

[0131] The density of the amplicons may be about 10 to 100 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 95 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 90 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 85 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 80 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 75 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 70 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 65 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 60 in features having a size of less than about 100 nm. The density of the amplicons may be about 10 to 55 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 50 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 45 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 40 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 35 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 30 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 25 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 20 in features having a size of less than about 100 nm. The density of amplicons may be about 10 to 15 in features having a size of less than about 100 nm.

[0132] In features having a size less than about 100 nm, the density of amplicons can be greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100.

[0133] The density of amplicons can be about 100 to 10,000 in features having a size of about 100 nm to 1 μm. The density of amplicons can be about 100 to 9,000 in features having a size of about 100 nm to 1 μm. The density of amplicons can be about 100 to 8,000 in features having a size of about 100 nm to 1 μm. The density of the amplicons may be about 100 to 7000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 6000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 5000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 4000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 3000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 2000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 1000, in features having a size of about 100 nm to 1 μm, the density of the amplicons may be about 100 to 90 0, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 800, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 700, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 600, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 500, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 400, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 300, in features having a size of about 100nm to 1μm, the density of amplicons can be about 100 to 200, in features having a size of about 100nm to 1μm.

[0134] The target molecule suitable for single molecule placement on the substrate of the present disclosure and for the method of the present disclosure can be any desired molecule. Exemplary categories of target molecules include, for example, polypeptides, polynucleotides, carbohydrates, amino acids, nucleotides, monosaccharides, haptens, ligands, antigens, analytes, ion channels, small molecule organic compounds or inorganic compounds. Exemplary species of each of the above categories are further described below.

[0135] The target molecule of the present disclosure may comprise a polypeptide. The polypeptide may comprise one or more amino acids. The polypeptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more amino acids. The polypeptide may comprise 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 or more amino acids. The polypeptide may comprise 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 or more amino acids. The amino acid may be a natural amino acid. The amino acid may be a non-natural amino acid. The amino acid may be a D-amino acid. The amino acid may be an L-amino acid. The one or more amino acids may comprise one or more natural amino acids, non-natural amino acids, D-amino acids, L-amino acids or combinations thereof. Examples of amino acids include, but are not limited to, p-acetylphenylalanine, m-acetylphenylalanine, alanine, β-alanine, γ-aminobutyric acid (GABA), aminoisobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), arginine, asparagine, asparagine, p-phenoxy-1-phenylalanine, citrulline, cystathionine, cysteine, cystine, diaminopimelic acid, djenkolic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, lanthionine, leucine, lysine, methionine, ornithine, phenylalanine, phenylselenidylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine and valine. Examples of polypeptides include, but are not limited to, ribosomal peptides, non-ribosomal peptides, peptones, peptide fragments, or any combination thereof. The polypeptides of the present disclosure may be recombinant, synthetic, or naturally occurring.

[0136] The target molecule of the present disclosure may comprise a ribosomal polypeptide. Typically, a ribosomal peptide may be a peptide synthesized by translating mRNA. Ribosomal proteins may undergo one or more post-translational modifications. Examples of post-translational modifications include, but are not limited to, phosphorylation, hydroxylation, sulfonation, palmitoylation, glycosylation, ubiquitination, SUMOylation, and disulfide bond formation. Examples of ribosomal peptides include, but are not limited to, enzymes, receptors, antibodies, transcription factors, hormones, ligands, antigens, and haptens.

[0137] The target molecule of the present disclosure may comprise a non-ribosomal peptide. Generally, a non-ribosomal peptide may be a peptide assembled by an enzyme specific to each peptide rather than by a ribosome. A non-ribosomal peptide may have a cyclic and / or branched structure. A non-ribosomal peptide may contain one or more non-protein amino acids, such as D-amino acids. A non-ribosomal polypeptide may include modifications such as N-methyl and N-formyl (N-formal). A non-ribosomal polypeptide may be glycosylated, acylated, dehydrated or hydroxylated. A non-ribosomal polypeptide may undergo dehydration of one or more serines, resulting in dehydroalanine. A non-ribosomal peptide may be a polymer. A non-ribosomal peptide may be a dimer or a trimer. Examples of non-ribosomal peptides include, but are not limited to, toxins, siderophores, pigments, antibiotics, antibiotic precursors, cytostatics and immunosuppressants.

[0138] The target molecule of the present disclosure may comprise one or more polypeptides assembled into a nanopore. The nanopore may be selected from Mycobacterium smegmatis porin A (MspA), outer membrane phospholipase A (OmpA), OmpC, OmpF, OmpG, Neisseria autotransporter (NalP), WZA, ClyA toxin, α-hemolysin, anthrax toxin, gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40, leukocidin and DNA origami nanopore.

[0139] The target molecule of the present disclosure may comprise an antibiotic. The antibiotic may be a penicillin, a cephalosporin, a polymyxin, a rifamycin, a lipiarmycin, a quinolone, a sulfonamide, a macrolide, an aminoglycoside, a cyclic lipopeptide, a glycylcycline, an oxazolidinone or a microcin. Examples of antibiotics include, but are not limited to, actinomycin, bacitracin, calcium-dependent antibiotics, daptomycin, gramicidin, tyrocidine, vancomycin, amphotericin A, plazomicin, eravacycline, brilacidin, avibactam, azithromycin, clarithromycin, erythromycin, carbomycin, josamycin, kitasamycin, midecamycin, oleandomycin, solitromycin, spiramycin, troleandomycin, tylosin, roxithromycin, cethromycin, ansamycin, carbomycin, tylosin and telithromycin.

[0140] The target molecules of the present disclosure may comprise toxins. A toxin may be a hemotoxin. Hemoglobin may cause destruction of red blood cells. A toxin may be a phototoxin. A phototoxin may cause photosensitivity. A toxin may be an exotoxin. An exotoxin may be excreted by an organism or cell. A toxin may be an endotoxin. An endotoxin may be a toxin released when a cell or organism is lysed. A toxin may be a biotoxin. A biotoxin may be a toxin of biological origin. For example, a toxin produced by a fungus may be referred to as a biotoxin. A toxin may be a necrotoxin. Necrotic proteins may cause necrosis. A toxin may be a neurotoxin. A neurotoxin may affect the nervous system of an animal. A toxin may be a cytotoxin. A cytotoxin may be toxic at the level of individual cells. A toxin may be a mycotoxin. Examples of mycotoxins include aflatoxins, ochratoxins, citrinin, ergot alkaloids, patulin, and Fusarium. Examples of toxins include, but are not limited to, microcystins, nodularins, anatoxin-a, anatoxin-a(S), cylindrospermopsins, sphingomyelin a, saxitoxins, dolastatins, cyanobacterial toxins, HC-toxins, AM toxins, venom toxins, ricin, melittin, fumonisins, trichothecenes, zearalenone, beauvercin, enniatins, butenolide, equisetin, tetanus toxin, botulinum toxin, tetrodotoxin, and fusarins.

[0141] The target molecule of the present disclosure may comprise a hormone. The hormone may be an amine hormone, a peptide hormone, a protein hormone, a steroid hormone, or a combination thereof. An amine hormone may comprise one or more amino acids having one or more modification groups. For example, the carboxyl group of an amino acid may be replaced by a chemical group such as a benzene ring. A peptide hormone may comprise a short chain of linked amino acids. A protein hormone may comprise a long chain of linked amino acids. A steroid hormone may be derived from a lipid cholesterol. Examples of hormones include, but are not limited to, melatonin, triiodothyronine, thyroxine, prostaglandins, leukotrienes, prostacyclin, thrombaxane, amylin, anti-Mullerian hormone, hormone), adinopectin, adrenocorticotropic hormone, corticotrophin, angiotensinogen, angiotensin, antidiuretic hormone, vasopressin, atrial-natriuretic peptide, atrial peptide, brain natriuretic peptide, calcitonin, cholecystokinin, corticotropin-releasing hormone, cortistatin, enkephalin, endothelin, erythropoietin, follicle-stimulating hormone, galanin, gastric inhibitory polypeptide, gastrin, ghrelin, glucagon, glucagon-like peptide-1, gonadotropin-releasing hormone, growth hormone-releasing hormone, h epcidin, human chorionic gonadotropin, human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor, somatomedin, leptin, lipolysis, luteinizing hormone, melanocyte stimulating hormone, motilin, orexin, oxytocin, pancreatic polypeptide, parathyroid hormone, pituitary adenylate cyclase-activating peptide, prolactin, prolactin-releasing hormone, relaxin, renin, secretin, somatostatin, thrombopoietin, thyroid stimulating hormone, thyrotropin, vasoactive intestinal polypeptide, androgens, mineralocorticoids, estrogens, glucocorticoids, progestins, and secosteroids.

[0142] The target molecule of the present disclosure may comprise a hapten. Typically, a hapten may be a small molecule that can elicit an immune response when attached to a large carrier such as a protein. Examples of haptens include, but are not limited to, aniline, anthranilic acid, m-aminobenzoic acid, p-aminobenzoic acid, uroshiol, quinine, hydralazine, fluorescein, biotin, digoxigenin, and dinitrophenol.

[0143] The target molecules of the present disclosure may include ion channels (i.e., porins) or receptors. The receptor may be from an immune cell. The immune cell may be a T cell, a B cell, a K cell or a phagocyte. The phagocyte may be a monocyte, an eosinophil, a macrophage, a neutrophil, a mast cell or a basophil. The receptor may be from a non-immune cell. For example, the non-immune cell may be an organ or tissue. The non-immune cell may be a skin cell, a lung cell, a heart cell, a breast cell, a muscle cell, a kidney cell, a glioma and an ovarian cell. The receptor may be a cell surface receptor. The receptor may be an internal receptor. The receptor may be an ion channel receptor, a G protein receptor or an enzyme-linked protein receptor. The receptor may be a transmembrane receptor. The receptor may include an external ligand binding domain (e.g., an extracellular domain), a domain that spans a hydrophobic transmembrane, an intracellular domain or any combination thereof. Examples of receptors include, but are not limited to, nicotinic acetylcholine receptors, glycine receptors, GABA receptors, glutamate receptors, 5-HT3 receptors, P2X receptors, cyclic nucleotide-gated ion channels, IP3 receptors, intracellular ATP receptors, ryanodine receptors, T cell receptors (TCR), B cell receptors (BCR), pattern recognition receptors (PRR), toll-like receptors (TLR), killer activated receptors (KAR), killer inhibitory factor receptors (KIR), complement receptors, Fc receptors, chemokine receptors, interferon receptors, growth factor receptors, including, for example, epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2). The receptor can be a cytokine receptor, a serine / threonine kinase receptor, a tyrosine kinase receptor, an IFN-α / β receptor (IFNAR), IFNGR, IL10R2, IFNLR1 or a tumor necrosis factor receptor (TNF-R).

[0144] The target molecule of the present disclosure may include cytokines. Cytokines include, but are not limited to, chemokines, interferons, interleukins, lymphokines and tumor necrosis factor (TNF). Chemokines may be from CXC, CC, CX3C or XC subfamily. Chemokines may be CCL2, CCL3, CCL5, CCL7, CCL8, CCL13, CCL17 and CCL22. Chemokines may be CCR1, CCR2, CCR3, CCR4, CCR5, CXCR2 and CXCR4. Chemokines may be CCL1 1, CCL24, CCL26, CCL5, CCL7, CCL13 and CCL3. Chemokines may be CCL2, CCL1, CCL22 and CCL17. Chemokines may be CXC chemokines. Chemokines may be CXC8. Interferons may be type I interferons, type II interferons, or type III interferons. Interferon can be IFN-a, IFN-β, IFN-ε, IFN-κ, IFN-co, IFN-γ. Interleukin (IL) can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, or IL-17. Lymphokine can be granulocyte-macrophage colony stimulating factor or interferon-gamma. Tumor necrosis factor can be TNF, lymphokine-alpha, CD40L, CD27L, CD30L, FASL, 4-1BBL, OX40L, and TNF-related apoptosis-inducing ligand (TRAIL). The target molecule of the present disclosure can also be a neuropeptide.

[0145] The target molecule of the present disclosure may comprise an enzyme. Enzymes may be divided into oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. Oxidoreductases may catalyze oxidation / reduction reactions. Transferases may transfer functional groups (e.g., methyl or phosphate groups). Hydrolases may catalyze the hydrolysis of various bonds. Lyases may cut various bonds by means other than hydrolysis and oxidation. Isomerases may catalyze isomerization changes within a single molecule. Ligases may connect two molecules with a covalent bond. Enzymes may be lactases, reductases, dehydrogenases and polymerases. The enzyme can be an alcohol dehydrogenase, an alcohol dehydrogenase, a homoserine dehydrogenase, an aminopropanol oxidoreductase, a diacetyl reductase, a glycerol dehydrogenase, a propanediol-phosphate dehydrogenase, a glycerol-3-phosphate dehydrogenase, a D-xylulose reductase, a L-xylulose reductase, a lactate dehydrogenase, a malate dehydrogenase, an isocitrate dehydrogenase, an HMG-CoA reductase, a decarboxylase, a dehydratase, an aldehyde lyase, an oxoacid lyase, a carbon-carbon lyase, a carbon-nitrogen lyase, a carbon-sulfur lyase, a carbon-halide lyase, a phosphorus-oxygen lyase, a ferrochelatase, an adenylate cyclase, a deaminase, an oxidase, a kinase or an enzyme of a bacterial autoinducer.

[0146] The target molecule of the present disclosure may comprise a polymerase. The polymerase may be a DNA polymerase, an RNA polymerase or a DNA / RNA polymerase. The polymerase may be a eukaryotic polymerase. The polymerase may be a prokaryotic polymerase. The DNA polymerase may be Pol I, PolII, Pol III, Pol IV or Pol V. The DNA polymerase may be polymerase beta, polymerase lambda, polymerase sigma, polymerase mu, polymerase alpha, polymerase δ, polymerase epsilon, polymerase eta, polymerase iota, polymerase kappa, polymerase Rev1, polymerase zata, telomerase, polymerase gamma and polymerase theta. The RNA polymerase may be RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA polymerase IV or RNA polymerase V. The RNA polymerase may be RNAP. The RNA polymerase may comprise subunits of RNAP. The subunits of RNAP may be β', β, α', a", and ω, and the polymerase may be an RNA-dependent DNA polymerase (RdDp), and RdDp may be a reverse transcriptase. The polymerase may be DNA polymerase, VentR TM (Exo)DNA polymerase, DNA polymerase, Taq DNA polymerase, T4 DNA polymerase, High-fidelity DNA polymerase, Taq DNA polymerase, Hot Start DNA Polymerase, DNA Polymerase I, Large (Klenow) Fragment, High-fidelity DNA polymerase, SP6 RNA polymerase, T7 RNA polymerase, E. coli RNA polymerase, holoenzyme, E. coli RNA polymerase, core enzyme, poly(U) polymerase, and E. coli poly(A) polymerase.

[0147] The target molecule of the present disclosure may include a ligase. The ligase may be a eukaryotic ligase. The ligase may be a prokaryotic ligase. The ligase may be a single-strand ligase. The ligase may be a double-strand ligase. The ligase may be a DNA ligase. The DNA ligase may be T4 DNA ligase, Taq DNA ligase, T7 DNA ligase, T3 DNA ligase, 9°N TM DNA ligase and E. coli DNA ligase. The ligase may be an RNA ligase. The RNA ligase may be T4 RNA ligase 1, T4 RNA ligase 2, T4 RNA ligase truncated, T4 RNA ligase 2 truncated K227Q, T4 RNA ligase 2 truncated KQ and thermostable 5'App DNA / RNA ligase. The ligase may be a thermostable ligase. The ligase may be a DNA / RNA ligase. The ligase may be Ligase, blunt / TA ligase and sticky-end ligase. The ligase can be CircLigaseTM, CircLigaseTM II.

[0148] The target molecule of the present disclosure may include a reverse transcriptase. The reverse transcriptase may be a viral reverse transcriptase. The viral reverse transcriptase may be a retroviral reverse transcriptase. The reverse transcriptase may be a eukaryotic reverse transcriptase. The reverse transcriptase may be an AMV reverse transcriptase, II reverse transcriptase, M-MuLV reverse transcriptase, Reverse transcriptase, II reverse transcriptase, III reverse transcriptase, HIV-1 reverse transcriptase or telomerase reverse transcriptase.

[0149] The target molecule of the present disclosure may comprise a nuclease. The nuclease may be a DNA enzyme. The nuclease may be an RNA enzyme. The nuclease may be an exonuclease. The exonuclease may be a lambda exonuclease, an exonuclease VII, a T5 exonuclease, a T7 exonuclease, an exonuclease T, an exonuclease I (E. coli), an exonuclease V (RecBCD) or an exonuclease III (E. coli). The nuclease may be an endonuclease. The endonuclease may be an endonuclease IV, a T7 endonuclease I, an endonuclease V, an endonuclease VIII, a Tma endonuclease III, an endonuclease III (Nth), a T4 PDG (T4 endonuclease V) or a Tth endonuclease IV. The RNA enzyme may be an RNA enzyme H, an RNA enzyme HII, an RNA enzyme If, RNase III, XRN-1. Examples of nucleases include, but are not limited to, Afu uracil-DNA glycosylase (UDG), Tma endonuclease III, Tth endonuclease IV, Antarctic heat labile UDG, APE 1, Cas9 nuclease, Streptococcus pyogenes (S. pyogenes), DNase I, endonuclease III (Nth), endonuclease IV, endonuclease V, endonuclease VIII, exonuclease I (E. coli), exonuclease III (E. coli), exonuclease T, exonuclease V (RecBCD), exonuclease VII, Fpg, hAAG, hOGG1, hSMUG1, lambda exonuclease, lambda exonuclease reaction buffer, micrococcal nuclease, mung bean nuclease, nuclease BAL-31, RecAf, RecJf, T4 PDG (T4 endonuclease V), T5 exonuclease, T7 endonuclease I, T7 exonuclease, uracil glycosylase inhibitor (UGI) and uracil-DNA glycosylase (UDG).

[0150] The target molecule of the present disclosure may include a helicase. The helicase may be a eukaryotic helicase. The helicase may be a prokaryotic helicase. The helicase may be a DNA helicase. The helicase may be an RNA helicase. The helicase may be a DNA / RNA helicase. The helicase may be an ATP-dependent helicase. The helicase may be a single-stranded helicase. The helicase may be a double-stranded helicase. The helicase may be a chromodomain helicase DNA binding protein. The helicase may be a DEAD box / DEAD / DEAH box helicase. Examples of helicases include, but are not limited to, ATXR, XPD, RecQ, ASCC3, BLM, BRIP1, DNA2, FBX018, FBXO30, HELB, HELLS, HELQ, HELZ, HFM1, HLTF, IFIH1, NAV2, PIF1, RECQL, RTEL1, SHPRH, SMARCA4, SMARCAL1, WRN, WRNIP1, DDX3X, DDX5, DDX6, DDX10, DDX11, DDX12, DDX58, DHX8, DHX9, DHX37, DHX40, DHX58, CHD1, CHD1L, CHD2, CHD3, CHD4, CHD5, CHD6, CHD7, CHD8, and CHD9.

[0151] The target molecule of the present disclosure may comprise a transposase. The transposase may be a DNA transposase. The transposase may be a Tn5 transposase or a Mos1 DNA transposase. The transposase may be an integrase. The integrase may be a viral integrase. The transposase may be HIV-1 IN, ASV IN and MuA transposase.

[0152] The target molecule of the present disclosure may comprise an antibody or an antibody fragment. The antibody may comprise an immunoglobulin (Ig). The immunoglobulin may be IgG, IgM, IgA, IgD, IgE. The antibody may comprise an antibody light chain. The antibody light chain may be a kappa or lambda light chain. The antibody may comprise an antibody heavy chain. The antibody heavy chain may comprise an alpha, gamma, delta, epsilon or mu heavy chain. The antibody may comprise an antibody fragment. The antibody may comprise a fragment antigen binding (Fab), Fab2, a crystallizable fragment (Fc), a variable fragment (Fv), a variable single chain fragment (scFv), a dimeric single chain variable fragment (di-scFv), a single domain antibody sdAb), scFv-Fc, a mini antibody, a double antibody, a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), a heavy chain constant domain (CH domain), a light chain constant domain (CL domain), a complementary determining region (CDR). The antibody may be a monomer. The antibody may be a dimer. The dimer may be a homodimer. The dimer may be a heterodimer. The antibody may be a multimer. The antibody may be a pentamer. The antibody may be a bispecific antibody. The antibody may be a camelid. The antibody may be a trifunctional antibody. The antibody may be a bispecific T cell binder (BiTE). The antibody may be a monoclonal antibody. The antibody may be a polyclonal antibody. The antibody may be a chimeric antibody. A chimeric antibody may refer to an antibody comprising regions derived from two or more sources. The two or more sources may be from different species. Alternatively, the two or more sources may be from different cells. The cells may be of the same cell type. Alternatively, the cells may be of different cell types. The antibody may be a human antibody. The antibody may be a humanized antibody. The antibody may be from mammals, reptiles, birds, and fish. The mammal may be a human, a non-human primate, a dog, a cat, a cow, a sheep, a rabbit, a goat, a rat, a mouse, a bear, a horse, a camel, and a pig. Examples of antibodies include, but are not limited to, abagovomab, abciximab, actoxumab, adalimumab, adecatumumab, aducanumab, afelimomab, afutuzumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anifrolumab,anrukinzumab, apolizumab, arcitumomab, aselizumab, atinumab, torolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bimagrumab, bivatuzumab mertansine, blinatumomab blosozumab, brentuximab vedotin), briakinumab, brodalumab, canakinumab, cantuzumabmertansine, cantuzumab ravtansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cbr96-doxorubicin immunoconjugate, cedelizumab, certolizumab pegol, cetuximab, citatuzumabbogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan,conatumumab,concizumab,crenezumab,cr6261,dacetuzumab,daclizumab,dalotuzumab,daratumumab,demcizumab,denosumab,detumomab,dorlimomab aritox,drozitumab,duligotumab,dupilumab,dusigitumab,ecromeximab,eculizumab,edobacomab,edrecolomab,efalizumab,efungumab,eldelumab,elotuzumab,elsilimomab,enavatuzumab,enlimomab pegol,enokizumab,enoticumab,ensituximab,epitumomab cituxetan, epratuzumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, guselkumab, ibalizumab, ibritumomabtiuxetan, icrucumab, igovomab, imab362, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lambrolizumab, lampalizumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab,libivirumab, ligelizumab, lintuzumab, linlumab, lodelcizumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, margetuximab, maslimomab, mavrilimumab, matuzumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, ontuxizumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, otlertuzumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, panitumumab, pankomab, panobacumab, parsatuzumab, pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab pexelizumab, pidilizumab, pinatuzumab vedotin, pintumomab, placulumab, polatuzumab vedotin, ponezumab, pnliximab, pritoxaximab, pritumumab, pro 140, quilizumab,racotumomab,radretumab,rafivirumab,ramucirumab,ranibizumab,raxibacumab,regavirumab,reslizumab,rilotumumab,rituximab,robatumumab,roledumab,romosozumab,rontalizumab,rovelizumabbruplizumab,samalizumab,sacurumab pendetide,secukinumab,seribantumab,setoxaximab,sevirumab,sibrotuzumab,sgn-cdl9a,sgn-cd33a,cephalimumab,siltuximab,simtuzum ab,siplizumab,sirucumab,solanezumab,solitomab,sonepcizumab,sontuzumab,stamulumab,sulesomab,suvizumab,tabalumab,tacatuzumab tetraxetane,tadocizumab,talizumab,tanezumab,taplitumomab paptox,tefibazumab,telemomab aritox,tenatumomab,teneliximab,teplizumab,teprotumumab,tgnl412,ticilimumab,tremelimumab,tildrakizumab,tigatuzumab,tnx-650,t ocilizumab,atlizumab,toralizumab,tositumomab,tovetumab,tralokinumab,trastuzumab,trbs07,tregalizumab,tremelimumab,tucotuzumab celmoleukin,tuvirumab,ublituximab,urelumab,urtoxazumab,ustekinumab,vantictumab,vapaliximab,vatelizumab,vedolizumab,veltuzumab,vepalimomab,vesencumab,visilizumab,voloximab,mafotuzumabVotumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

[0153] The target molecule of the present disclosure may comprise a polynucleotide. A polynucleotide may comprise one or more nucleotides. A polynucleotide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more nucleic acids. A polynucleotide may comprise 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 or more nucleic acids. The polynucleotide may comprise 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 or more nucleic acids. One or more nucleotides may comprise ribonucleotides, deoxyribonucleotides, locked nucleic acids (LNAs), peptide nucleic acids (PNAs) or any combination thereof. The polynucleotide may comprise purine bases, pyrimidine bases or both. The polynucleotide may comprise or natural, chemically modified, biochemically modified, non-natural or derived nucleotide bases. The polynucleotide may comprise one or more unpaired nucleotide bases. The polynucleotide may be single-stranded. The polynucleotide may comprise one or more paired nucleotide bases. The polynucleotide may be double-stranded. The polynucleotide may comprise double-stranded DNA, double-stranded RNA or double-stranded DNA / RNA hybrids. The polynucleotide may be a synthetic polynucleotide. In some cases, the sequence of a polynucleotide is known. For example, a polynucleotide may include a polyadenylated sequence. In some cases, the sequence of a polynucleotide is unknown. For example, a polynucleotide of random sequence may include a degenerate sequence. In some cases, only a portion of the polynucleotide sequence is known. For example, a polynucleotide may include a polyadenylated sequence, but the rest of the sequence is unknown.

[0154] The target molecule of the present disclosure may include an oligonucleotide primer. An oligonucleotide primer may be an amplification or sequencing primer. An amplification or sequencing primer may be used for the amplification or sequencing of another polynucleotide. For example, an amplification or sequencing primer may include a sequence that is partially complementary to a gene. An amplification or sequencing primer may hybridize with a gene and is capable of amplifying or sequencing a gene. An amplification or sequencing primer may be a specific primer. Typically, a specific primer is complementary to a sequence of a polynucleotide (e.g., a gene, an exon region, etc.). The sequence of a polynucleotide may be unique to a polynucleotide. For example, a specific primer may hybridize with at least a portion of an antibody gene. An amplification or sequencing primer may be a universal primer. Typically, a universal primer is complementary to a sequence common to two or more types of polynucleotides. For example, a universal primer may include an oligodT sequence that may hybridize with a polyadenylation sequence of two or more polynucleotides (e.g., mRNA). An amplification or sequencing primer may hybridize with a natural sequence of a polynucleotide (e.g., a polyA sequence of mRNA, a restriction site, an exon region). Amplification or sequencing primers can hybridize with non-natural sequences of polynucleotides (e.g., adapter sequences or barcode sequences). Amplification or sequencing primers can contain random or degenerate sequences. For example, amplification or sequencing primers can contain random hexamer sequences. Examples of amplification or sequencing primers include, but are not limited to, oligodT, random primers, gene-specific primers, and promoter primers. The oligodT primer can be an oligo(dT)20, oligo(dT)18, or oligo(dT)12-18 primer. The random primer can be a random 9-mer primer or a random 6-mer primer. The random primer can contain oligodeoxyribonucleotides. Alternatively, the random primer includes an oligodeoxynucleotide. Examples of gene-specific primers include, but are not limited to, cytokine gene primers and housekeeping gene primers. The promoter primer can be, for example, an SP6 primer, a T7 promoter primer, a Runx2 promoter primer, a GAPDH promoter primer, a U6 promoter primer, a T3 promoter primer, and a CaMV 35S promoter primer. The target molecule may comprise an oligonucleotide primer selected from the group consisting of: 3'OX1 (for a sequence having an AOX1 terminator, a reverse primer), 5'AOX1 (for a sequence having an AOX1 promoter, a forward primer), 35S promoter (CaMV 35S promoter, a forward primer), AC5 (drosophila actin 5C promoter, a forward primer), Alpha-factor (alpha factor signal sequence, a forward primer), Amp-R (5' end of the ampicillin resistance gene, a reverse primer), AUG1 forward (for a sequence having an AUG1 promoter, a forward primer), AUG1 reverse (for a sequence having an AUG1 promoter, a reverse primer), BGH reverse (bovine growth hormone terminator, a reverse primer), Bglob-intron-F (rabbit beta-globin intron, a forward primer),Bglob-intron-R (rabbit beta-globin intron, reverse primer), Bglob-pA-R (rabbit beta-globin polyA region, reverse primer), CAT-R (5' end of chloramphenicol resistance gene, reverse primer), CMV forward (human CMV immediate early promoter, forward primer), CRE-R (5' end of Cre recombinase, reverse primer), CYC1 (CYC1 transcription termination signal, reverse primer), DsRed1-C (3' end of DsRed1, forward primer), DsRed1-N (5' end of DsRed1, reverse primer), EBV reverse (SV40 polyA terminator, reverse primer), ecdysone forward (Drosophila heat shock promoter, forward primer), EF-1a forward (human elongation factor-1a promoter, forward primer), EGFP-C (3' end of EGFP, forward primer), EGFP-N (5' end of EGFP, reverse primer), EXFP-R (for distinguishing EGFP from ECFP and EYFP, reverse primer), F1ori-F (F1 origin, forward primer), GAL1 (Saccharomyces cerevisiae GAL1 promoter, forward primer), Gal10pro-F (Saccharomyces cerevisiae GAL 10 promoter, forward primer), Gal4 N-term (Gal4 DNA binding end 3', forward primer), Gal4-AD (Gal4 activation domain 3', forward primer), GFP-F (GFP 3', forward primer), GFP-R (GFP 5', reverse primer), GPDpro-F (Saccharomyces cerevisiae GPD promoter, forward primer), GW-3' (Gateway cassette 3', forward primer), GW-5' (Gateway cassette 5', reverse primer), H1 (human H1 promoter, forward primer), HA-F (HA tag, forward primer), HA-R (HA tag, reverse primer), HAT (histidine affinity tag, forward primer), hGH-PA-R (human growth hormone terminator, reverse primer), hrGFP-R (hrGFP (humanized Renilla GFP), forward primer), hUBCpro-F (human ubiquitin C (UbC) promoter, forward primer), IRES-F (3' end of IRES, forward primer), IRES-R (5' end of IRES, reverse primer), L4440 (5' end of MCS in L4440 vector, forward primer), LacI-R (5' end of LacI, reverse primer), LacZ-R (5' end of LacZ, reverse primer), LexA (3' end of LexA DNA binding domain, forward primer), LKO.1 5' (human U6 promoter, forward primer), LNCX (human CMV promoter, forward primer), Luc-F (3' end of luciferase, forward primer), LucNrev (5' end of luciferase,reverse primer), M13(-21) forward (in the lacZ gene), M13(-40) (in the lacZ gene), M13 reverse (in the lacZ gene), M13 / pUC forward (in the lacZ gene), M13 / pUC reverse (in the lacZ gene), MBP-F (3' end of maltose binding protein, forward primer), mCherry-F (3' end of mCherry, forward primer), mCherry-R (5' end of mCherry, reverse primer), MT forward (Drosophila metallothionein promoter, forward primer), MMLV-F (Moloney murine leukemia virus LTR (MoMuLV)), forward primer), mPGK-F (mouse PGK promoter, forward primer), MSCV (murine stem cell virus, forward primer), MSCV-rev (murine stem cell virus, reverse primer), MT1-F (mouse metallothionein 1 promoter, forward primer), mU6-F (mouse U6 promoter, forward primer), Myc (Myc tag, forward primer), Neo-F (3' end of neomycin resistance gene, forward primer), Neo-R (5' end of neomycin resistance gene, reverse primer), NOS-F (Nopaline synthase promoter, forward primer), Nmtl-F (schizosaccharomyces pombe nmt1 promoter, forward primer), OpIE2 forward (OpIE2 promoter, forward primer), pACYC-F (p15A origin, forward primer), pAd-CMV (cloning site after SalI in pAd-CMV vector), pBABE 3' (SV40 enhancer, 3' of MCS in pBABE vector, reverse primer), pBABE 5' (Psi packaging signal, 5' of MCS in pBABE vector, forward primer), pBAD forward (for sequence with E. coli araBAD promoter, forward primer), pBAD reverse (for sequence with E. coli araBAD promoter, reverse primer), pBluescriptKS (for pBluescript vector sequence), pBluescriptSK (for pBluescript vector sequence), pBMN 5' (MMLV sequence, for insert in pBMN retrovirus), pBR322ori-F (pBRS322 origin, forward primer), pBRforBam (in pBR322 tet region, upstream of BamHI, forward primer), pBRforEco (in pBR322, upstream of EcoRI site, forward primer), pBRrevBam (in pBR322 tet region, downstream of BamHI, reverse primer), pCAG-F (rabbit beta-globin intron, for pCAG plasmid, forward primer), pCasper-F (5' end of Drosophila mini-white gene,reverse primer), pCasper-hs (Drosophila Hsp70 promoter, forward primer), pcDL-F (5' of EcoRI site in pcDL vector, forward primer), pENTR-F (5' of attL1 in pENTR vector, forward primer), pENTR-R (3' of attL2 in pENTR vector, reverse primer), pGEX 3' (3' of MCS in pGEX vector, reverse primer), pGEX 5' (3' of glutathione-S-transferase, forward primer), pGP704-R (R6K gamma origin, 3' of MCS in pGP704 vector, reverse primer), pHybLex reverse (ADH terminator, reverse primer), pLTet-F (lambda phage early left (pL) promoter, forward primer), p1XSN 5' (murine stem cell virus, same as MSCV, forward primer), pMRB101-F (HCMV major immediate early protein (IE), forward primer), pMT2-F (3' end of synthetic intron, forward primer), pMX-S1811 (MMLV sequence, 5' of MCS in pMX vector, forward primer), polyhedrin forward (polyhedrin promoter, forward primer), polyhedrin reverse (for baculovirus vector with polyhedrin promoter, reverse primer), pQE promoter (5' of MCS in pQE vector, forward primer), pREP forward (Rous sarcoma virus (RSV) promoter, forward primer), pRS-marker (to sequence yeast selection marker in pRS vector), Pry 1 (PZ P-element, reverse primer), pTrcHis forward (5' of MCS in pTrcHis vector, forward primer), pTrcHis reverse (3' of MCS in pTrcHis vector, same as pBAD-R, reverse primer), Puro-F (3' of puromycin resistance gene, forward primer), pZIP (murine leukemia virus (MuLV), reverse primer), RCAS-F (3' of Rous sarcoma virus (RSV) env gene, forward primer), Rluc-F (3' of Renilla luciferase, forward primer), RVprimer3 (5' of MCS in pGL3 vector, forward primer), SFFV-F (Spleen focus forming virus 5'LTR, forward primer), SP6 (SP6 promoter, forward primer), SV40pA-R (SV40 poly A, reverse primer), SV40pro-F (SV40 promoter / origin, forward primer), SV40-spliceR (SV40 splicing sequence, reverse primer), T3 (T3 promoter, forward primer), T7 (T7 promoter, forward primer),T7 Terminal (T7 terminator, reverse primer), Tac Promoter (Tac promoter, forward primer), tdTomato-Fwd (3' end of tdTomato, forward primer), tdTomato-Rev (5' end of tdTomato, reverse primer), Tet-R (5' end of tetracycline resistance gene, reverse primer), TK-pA-R (thymidine kinase poly A, reverse primer), Tn7-end (bacterial transposon Tn7), TRC-F (human U6 promoter, forward primer), Ubx-F (Drosophila Ultrabithorax gene, forward primer), V5 reverse (V5 epitope, reverse primer), WPRE-R (5' end of WPRE, reverse primer), XBG-R (Xenopus beta-globin 3'UTR, reverse primer), XEF1a (Xenopus EF1 alpha enhancer / promoter, forward primer), and Xpress forward (Xpress epitope, forward primer).

[0155] The target molecule of the present disclosure may comprise an RNA molecule. RNA may be a protein-coding RNA. Protein-coding RNA may be an mRNA. RNA may be a non-coding RNA (ncRNA). ncRNA may be a ribosomal RNA (rRNA), a transfer RNA (tRNA), a small interfering RNA (siRNA), a microRNA (miRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), a long non-coding RNA (IncRNA), and a Piwi-interacting RNA (piRNA). RNA may be a synthetic RNA. Examples of synthetic RNA include, but are not limited to, antisense RNA, short hairpin RNA (shRNA), complementary RNA (cRNA). RNA may be derived from exons. RNA may be derived from introns. RNA may be derived on an untranslated region (UTR). RNA may be polyadenylated. In some cases, RNA is not polyadenylated.

[0156] The target molecule of the present disclosure may comprise a DNA molecule. The DNA may be genomic DNA (gDNA) or complementary DNA (cDNA). The DNA may be B-DNA. The DNA may be A-DNA. The DNA may be Z-DNA. The DNA may comprise at least a portion of a protein coding region. The DNA may comprise at least a portion of a gene. The DNA may comprise exons. The DNA may comprise introns. The DNA may comprise an untranslated region (UTR). The DNA may comprise at least a portion of a non-coding DNA. The non-coding DNA may comprise a sequence transcribed into a non-coding RNA. The non-coding DNA may comprise a regulatory element. The regulatory element may be a cis-regulatory element. The regulatory element may be a trans-regulatory element. The regulatory element may be a promoter. The promoter may promote transcription of a gene. The promoter may be located upstream of the coding region. The regulatory element may be an enhancer. The enhancer may exert a long-range effect on the transcription level of a gene. The non-coding DNA may comprise a pseudogene. The non-coding DNA may comprise a transposon or a retrotransposon. The non-coding DNA may comprise a repetitive sequence, such as long interspersed nuclear elements (LINES) and short interspersed nuclear elements (SINES). The SINE may comprise an Alu sequence. The non-coding DNA may comprise viral elements. The non-coding DNA may comprise telomeres.

[0157] The target molecule of the present disclosure may comprise carbohydrates. Carbohydrates may be monosaccharides, disaccharides, oligosaccharides or polysaccharides. Examples of monosaccharides include, but are not limited to, glyceraldehyde, galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfoquinovose, pyranose, pyranose glucose, furanose, glucose, fructose and galactose. Disaccharides may be sucrose, lactose, trehalose, cellobiose or maltose. Oligosaccharides may be fructo-oligosaccharides, galactooligosaccharides and mannan oligosaccharides. Polysaccharides may be storage polysaccharides. Polysaccharides may be structural polysaccharides. Examples of polysaccharides include, but are not limited to, starch, glycogen, chitin, amylase, pullulan, callose or laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, galactomannan and cellulose.

[0158] The target molecules of the present disclosure may comprise organic compounds. Generally, an organic compound is a compound whose molecule contains carbon. The organic compound may be a natural compound. Generally, a natural organic compound refers to a compound produced by a plant or an animal. Examples of natural organic compounds include most sugars, some alkaloids and terpenoids, and certain nutrients such as vitamin B12. The organic compound may be a synthetic compound. Generally, synthetic organic compounds can be prepared by reactions of other compounds, referred to as "synthetic". Polymers, including plastics and rubbers, may be organic synthetic or semi-synthetic compounds. Some organic compounds may be made using the biochemistry of organisms such as bacteria and yeast. The organic compound may be a small molecule, an alcohol, a fatty acid, a polyketide, a hormone, or a carbohydrate.

[0159] The target molecules of the present disclosure may include inorganic compounds. Generally, compounds that are not called organic compounds are inorganic compounds. Examples of carbon-containing inorganic compounds include, but are not limited to, carbides, carbonates, simple oxides of carbon (e.g., CO and CO2), and cyanides. Inorganic compounds may include quantum dots, metal nanoparticles, metal oxide nanoparticles, and the like. Inorganic compounds may also include minerals, sulfides, organometallic compounds, and bioorganic compounds. Minerals may be talc, gypsum, calcite, fluorite, apatite, orthoclase, quartz, topaz, corundum, or diamond.

[0160] The target molecule of the present disclosure may comprise a small molecule. The small molecule may comprise a peptide. The peptide comprises one or more amino acids. The amino acid may be a natural amino acid. The amino acid may be a non-natural amino acid. The amino acid may be a D-amino acid. The amino acid may be an L-amino acid. Examples of amino acids include, but are not limited to, p-acetylphenylalanine, m-acetylphenylalanine, alanine, β-alanine, γ-aminobutyric acid (GABA), aminoisobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), arginine, asparagine, asparagine, p-phenoxy-1-phenylalanine, citrulline, cystathionine, cysteine, cystine, diaminopimelic acid, djenkolic acid, glutamic acid, glutamine, glycine, histidine, isoleucine, lanthionine, leucine, lysine, methionine, ornithine, phenylalanine, phenylselenidylalanine, proline, selenocysteine, serine, threonine, tryptophan, tyrosine and valine. Small molecules may comprise therapeutic agents.

[0161] The target molecules of the present disclosure may comprise metabolites. Examples of metabolites include, but are not limited to, alkaloids, glycosides, lipids, non-ribosomal peptides such as actinomycin d, phenazines, natural phenols (including flavonoids), polyketides, terpenes, including steroids and tetrapyrroles.

[0162] The target molecule of the present disclosure may comprise a therapeutic agent. The therapeutic agent may comprise an organic compound, an inorganic compound, a peptide, a hormone, a small molecule, an antibody, an antigen, a hapten or any combination thereof. The therapeutic agent may be an antipyretic, an analgesic, an antimalarial, an antibiotic, an antiseptic, a mood stabilizer, a hormone replacement, an oral contraceptive, a stimulant, a tranquilizer, an antiviral, an anticancer drug, an immunosuppressant and a statin. The tranquilizer may be meprobamate, chlorpromazine, reserpine, chlordiazepoxide, diazepam and alprazolam. The statin may be lovastatin, pravastatin or simvastatin.

[0163] In some embodiments, the substrates provided herein comprise a plurality of features. The plurality of features on a substrate is also referred to herein as an array of features or an array.

[0164] Figure 5 An exemplary substrate comprising two or more features is depicted in FIG. Figure 5As shown in , the substrate (501) can include two or more features (502, 503). The first feature (502) can include a plurality of first capture primers (504, 505, 506) and a plurality of second capture primers (507, 508, 509). The first feature (502) can further include a first target polynucleotide (510). The first target polynucleotide (510) can be attached to the first feature (502) by attaching the first target polynucleotide (510) to the first capture primer (504). The first feature (502) can further include a plurality of amplicons (512, 513, 514, 515, 516). The plurality of amplicons (512, 513, 514, 515, 516) can include amplicons of the first target polynucleotide (510). The first feature (502) can further include a first target molecule (511). The first target molecule (511) can be attached to the first target polynucleotide (510). The second feature (503) may include a plurality of first capture primers (520, 521, 522) and a plurality of second capture primers (523, 524, 525). The second feature (503) may further include a second target polynucleotide (526). The second target polynucleotide (526) may be attached to the second feature (503) by attaching the second target polynucleotide (526) to the second capture primer (520). The second feature (503) may further include a plurality of amplicons (528, 529, 530, 531, 532). The plurality of amplicons (528, 529, 530, 531, 532) may include amplicons of the second target polynucleotide (526). The second feature (503) may further include a second target molecule (527). The second target molecule (527) may be attached to the second target polynucleotide (526). The substrate may include two or more identical features. For example, the first feature (502) and the second feature (503) can be the same. The substrate can include two or more different features. For example, the first feature (502) and the second feature (503) can be different. The first feature (502) and the second feature (503) can differ in the first capture primer bound to each feature. For example, the multiple first capture primers (504, 505, 506) of the first feature (502) can be different from the multiple first capture primers (520, 521, 522) of the second feature (503). The first feature (502) and the second feature (503) can differ in the second capture primer bound to each feature. For example, the multiple second capture primers (507, 508, 509) of the first feature (502) can be different from the multiple second capture primers (523, 524, 525) of the second feature (503). The first feature (502) and the second feature (503) can differ in the target polynucleotide bound to each feature.For example, the first target polynucleotide (510) of the first feature (502) can be different from the second target polynucleotide (526) of the second feature (503). Subsequently, the multiple amplicons (512, 513, 514, 515, 516) of the first feature (502) can be different from the multiple amplicons (528, 529, 530, 531, 532) of the second feature (503). The first feature (502) and the second feature (503) can differ in the target molecules bound to each feature. For example, the first target molecule (511) of the first feature (502) can be different from the second target molecule (527) of the second feature (503). The first feature can further include one or more additional target molecules. The second feature can further include one or more additional target molecules. The one or more additional target molecules from the first feature can be the same as the one or more additional target molecules in the second feature. Alternatively or additionally, the one or more additional target molecules from the first feature can be different from the one or more additional target molecules from the second feature.

[0165] A substrate with multiple features can be presented as a grid of dots or patches. Features can be positioned in a repeating pattern. Alternatively or in addition, features can be positioned in an irregular non-repeating pattern. In some embodiments, the pattern is a hexagonal pattern, a rectilinear pattern, a grid pattern, a pattern with reflection symmetry, a pattern with rotational symmetry, etc. Asymmetric patterns can also be useful. The pitch can be the same between different pairs of nearest neighboring features, or the pitch can vary between different pairs of nearest neighboring features.

[0166] Substrates having a plurality of features and methods described herein using such arrays can have features at any of a variety of densities, including, for example, at least about 10 features / cm 2 , 100 features / cm 2 , 500 features / cm 2 , 1,000 features / cm 2 , 5,000 features / cm 2 , 10,000 features / cm 2 , 50,000 features / cm 2 , 100,000 features / cm 2 , 1,000,000 features / cm 2 , 5,000,000 features / cm 2 or higher.

[0167] In specific embodiments, the features of the array may each have a diameter greater than about 100 nm. 2 , 250nm 2 , 500nm 2 , 1μm2 , 2.5μm 2 , 5μm 2 , 10μm 2 , 100μm 2 , or 500μm 2 Alternatively or additionally, the features of the array may each have an area of ​​less than about 1 mm 2 , 500μm 2 , 100μm 2 , 25μm 2 , 10μm 2 , 5μm 2 , 1μm 2 , 500nm 2 or 100nm 2 In practice, the region may have a size within a range between upper and lower limits selected from those exemplified above.

[0168] In some embodiments, the feature on the surface of the array substrate is discontinuous, separated by the gap region on the surface. The gap region with a capture primer or other capture agent having a substantially lower amount or concentration compared to the feature of the array can be useful. The gap region lacking a capture primer or other capture agent is particularly useful. For example, a relatively small amount or absence of a capture agent at the gap region is conducive to the location of at least one target polynucleotide, and subsequently produces an amplicon cluster to the desired feature.

[0169] In a specific embodiment, the features are concave features in a surface (e.g., a well), and the features can contain a gel material. The gel-containing features are separated from each other on the surface by interstitial regions where there is substantially no gel, or if present, the gel is substantially unable to support the localization of a polynucleotide comprising, for example, a capture primer.

[0170] In some embodiments, the pores are micropores or nanopores.Methods and compositions for making and using substrates having gels containing features such as pores are listed, for example, in US 2014 / 0243224, which is incorporated herein by reference.

[0171] In some embodiments, the size of features on the array (e.g., area, diameter, etc.) is selected within a range that is favorable for kinetic exclusion amplification (KEA) and formation of single molecule placement in the features. By way of example with reference to embodiments in which holes are used as features, the size of the holes (e.g., diameter) can vary between about 30 nm and about 1 μm, between about 50 nm and about 800 nm, between about 70 nm and about 600 nm, or between 100 nm and about 400 nm. In some embodiments, the holes have a diameter of about 400 nm. In some embodiments, the holes have a diameter of less than about 1 μm. Exemplary microarrays include HiSeq-X10 microarray on patterned flow cell.

[0172] In some embodiments including an array of features on a surface, the features can be discrete, separated by gap regions. The size of the features and / or the spacing between regions can vary, so that the array can be high density, medium density or low density.

[0173] In some embodiments, the array is a high density, medium density or low density array. High density arrays are characterized by having regions separated by less than about 15 μm. Medium density arrays have regions separated by about 15 to 30 μm, while low density arrays have regions separated by more than 30 μm. Arrays useful in the present invention may have regions separated by less than 100 μm, 50 μm, 10 μm, 5 μm, 1 μm or 0.5 μm.

[0174] In some embodiments, the array contains beads. In some embodiments, the beads are located on the surface, including those in which the beads are located in wells, such as the BeadChip array (Illumina Inc., San Diego CA) or the substrates used in the sequencing platforms from 454 Life Sciences (a subsidiary of Roche, Basel Switzerland) or Ion Torrent (a subsidiary of Life Technologies, Carlsbad California). Other arrays with beads on the surface are described in US 6,266,459; US 6,355,431; US ​​6,770,441; US ​​6,859,570; US 6,210,891; US ​​6,258,568; US 6,274,320; US 2009 / 0026082A1; US ​​2009 / 0127589 A1; US ​​2010 / 0137143A1; US ​​2010 / 0282617 A1 or PCT Publication No. WO 00 / 63437, each of which is incorporated herein by reference. Several of the above references describe methods of attaching target polynucleotides (or capture primers) to beads before loading the beads in or on the array substrate. However, it should be understood that the beads can be made to include capture primers, and the beads can then be used to load the array, thereby forming amplification sites for the methods described herein. As previously listed herein, the substrate may be used without beads.

[0175] In some embodiments, exemplary bead compositions include, but are not limited to, plastics, ceramics, glass, polystyrene, methylstyrene, acrylic polymers, paramagnetic materials, thoria sols, carbon graphite, titanium dioxide, latex or cross-linked dextran such as Sepharose, cellulose, nylon, cross-linked micelles and teflon, and any other material for solid supports outlined herein can be used. The "Microsphere Detection Guide" from Bangs Laboratories, Fishers Ind. is a useful guide. In certain embodiments, the microspheres are magnetic microspheres or beads.

[0176] Thus, the present disclosure provides a substrate having (a) a plurality of first and second capture primers immobilized to a feature; (b) at least one target polynucleotide, one end attached to one of the capture primers and the other end linked to a target molecule, wherein the target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, the second capture primer binding region comprising a base pair mismatch with the second capture primer, and (c) a plurality of clonal amplicons complementary to the target polynucleotide immobilized to the feature. The substrate may have one feature or multiple features. One or more features may be beads, wells, including microwells or nanowells, channels, ridges, protrusions, or a combination thereof. One or more features contain a single target molecule. One or more features may be filled with multiple clonal amplicons of a target polynucleotide. The interstitial regions between multiple features may lack target polynucleotides. A substrate having multiple features, including two or more features, may have a different single target molecule in each different feature within the multiple. The target polynucleotide on a feature comprises one or more polynucleotides selected from the group consisting of RNA, DNA, PNA, or double-stranded DNA (dsDNA). The target polynucleotide on the feature can be less than 1,000 nucleotides in length. The target polynucleotide on the feature can be 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900, or 901 to 1000 nucleotides in length. The target molecule can be a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound, or an inorganic compound. The polypeptide target molecule can be a nanopore, a binding polypeptide, and an enzyme. The nanopore can be MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, and a DNA origami nanopore. The binding polypeptide can be an antibody, Fab, Fab', F(ab') 2, scFv, diabody, triabody, minibody and single domain antibody (sdAB) or T cell receptor. The enzyme can be a polymerase, a helicase, a recombinase, a transposase or a ligase. The substrate can be one or more materials selected from the group consisting of glass, silicon, plastic or a biopolymer. The substrate can further comprise a hydrogel or a covalently linked gel.

[0177] The present disclosure further provides a method of placing a single target molecule on a feature of a substrate. The method comprises (a) hybridizing a plurality of first and second capture primers immobilized to the feature on the substrate with (b) at least one target polynucleotide comprising a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, wherein the second capture primer binding region comprises a base pair mismatch with the second capture primer and is attached to the target molecule, and (c) amplifying the at least one target polynucleotide at an average amplification rate to produce a plurality of clonal amplicons complementary to the target polynucleotide, the average amplification rate exceeding an average transport rate of the target polynucleotide to the feature.

[0178] Kinetic exclusion amplification (KEA) allows a single feature amplification of a single target polynucleotide on a substrate including, for example, a patterned flow cell with a hole, and produces a monoclonal target polynucleotide population in one or more holes. The method for implementing KEA is well known in the art, and is described in, for example, US 2013 / 0338042 A1. In KEA, the amplification rate of the first capture target polynucleotide in a feature is much faster than the much slower rate of target polynucleotide transport and capture. The first target polynucleotide captured in the feature can be rapidly amplified, and fills the entire feature, preventing other target polynucleotides from being captured in the same feature.

[0179] The present disclosure is based in part on the recognition that the effectiveness of KEA for generating a monoclonal target polynucleotide population in a feature (such as a nanopore of a patterned flow cell) decreases as the size of the feature or nanopore increases. Amplification of the first captured target polynucleotide and filling the feature with a monoclonal population of the target polynucleotide is higher in a larger feature than in a smaller feature, while capture of the second target polynucleotide is faster in a larger feature than in a smaller feature. Therefore, the probability of capturing and amplifying more than one target polynucleotide in a feature increases with the size of the feature. The data quality of the molecular interrogation from the feature is optimal for a monoclonal population of the target polynucleotide. The data quality from the feature decreases as the share of target polynucleotides other than the first immobilized target polynucleotide increases.

[0180] exist Figure 1A specific example of using KEA for placing a single target molecule in each of multiple features is shown in FIG. Briefly, features of a substrate are patterned with immobilized capture primers, such as PAZAMs embedded in a nanopore with a P5 and P7 capture primer pair. A target molecule, such as a polymerase or a nanopore polypeptide ( Figure 1 The target polynucleotide can be amplified using KEA (also known as recombinase polymerase amplification (RPA)) using, for example, the commercial TwistDX kit. RPA is a class of amplification methods primarily used in free solutions. However, KEA is a class of localized RPA reactions in which amplification can be performed in a semi-enclosed space such as a solid substrate or bead surface to produce a monoclonal cluster of targeted nucleotides. Amplification rapidly consumes primers in the feature, preventing and / or significantly reducing seeding and amplification from different target polynucleotides, thereby eliminating or reducing the possibility of loading of other target molecules.

[0181] In some embodiments, kinetic exclusion amplification (KEA), also referred to as exclusion amplification (ExAmp), can be used to carry out isothermal amplification.Nucleic acid library of the present disclosure can be prepared using a method, and the method comprises making an amplification reagent react to produce a plurality of amplification sites, and the plurality of amplification sites each comprise a substantially cloned population of the amplicon from the single target nucleic acid inoculated in the site.In some embodiments, amplification reaction is carried out until a sufficient number of amplicon is produced to fill the capacity of the corresponding amplification site.Filling up the site inoculated in this way suppresses the landing and amplification of the target nucleic acid at the site, thereby producing a clonal population of the amplicon at the site.In some embodiments, even if the amplification site is not filled up before the second target nucleic acid arrives at the site, apparent clonality can also be realized.Under some conditions, the amplification of the first target nucleic acid can proceed to the following point, i.e., produce a sufficient number of copies to effectively outperform or overwhelm the second target nucleic acid produced from the site. For example, in an embodiment using a bridge amplification method for circular features less than 500 nm in diameter, it has been determined that after 14 cycles of exponential amplification of a first target nucleic acid, contamination from a second target nucleic acid at the same site will produce a number of contaminating amplicons that is insufficient to adversely affect synthesis sequencing analysis on an Illumina sequencing platform.

[0182] As demonstrated by the above examples, in specific embodiments, the amplification sites in the array can be but not necessarily completely cloned. On the contrary, for some applications, individual amplification sites can be mainly colonized with amplicons from the first target nucleic acid, and can also have low-level contamination amplicons from the second target nucleic acid. The array can have one or more amplification sites with low-level contamination amplicons, so the contamination level does not have an unacceptable impact on the subsequent array use. For example, when the array is used in detection applications, an acceptable contamination level will be a level that will not affect the resolution of the signal and noise or detection technology in an unacceptable manner. Therefore, apparent clonality is generally related to the specific use or application of the array made by the method described herein. The exemplary contamination levels acceptable in the individual amplification sites of specific applications include but are not limited to at most 0.1%, 0.5%, 1%, 5%, 10% or 25% of the contamination amplicons. The array can include one or more amplification sites with these exemplary levels of contamination amplicons. For example, up to 5%, 10%, 25%, 50%, 75% or even 100% of the amplification sites in the array can have some contamination amplicons. It will be appreciated that at least 50%, 75%, 80%, 85%, 90%, 95% or 99% or more of the sites in an array or other collection of sites may be clonal or apparent clonal.

[0183] In some embodiments, when a process occurs at a sufficiently fast rate to effectively exclude another event or process from occurring, kinetic exclusion can occur. For example, a nucleic acid array is prepared, wherein the sites of the array are randomly inoculated with target nucleic acids in solution, and copies of the target nucleic acids are produced in the amplification process to fill up each inoculated site. According to the kinetic exclusion method of the present disclosure, inoculation and amplification processes can be carried out simultaneously under the condition that the amplification rate exceeds the inoculation rate. Therefore, the relatively fast rate of producing copies at the site inoculated by the first target nucleic acid will effectively exclude the second nucleic acid from being inoculated at this site for amplification. The kinetic exclusion amplification method can be implemented as discussed in detail in the disclosure of U.S. Application Publication No. 2013 / 0338042, the entire contents of which are incorporated herein by reference.

[0184] Kinetics excludes the relatively fast rate of the subsequent copy (or the first copy of the target nucleic acid) for preparing the target nucleic acid using a relatively slow rate to start amplification (e.g., the slow rate of the first copy of the target nucleic acid). In the example of the previous paragraph, kinetics excludes the relatively fast rate of the amplification to fill the site with a nucleic acid seed copy due to the relatively slow rate (e.g., relatively slow diffusion or transport) of the target nucleic acid inoculation. In another exemplary embodiment, kinetics excludes the relatively fast rate of the subsequent copy to fill the site due to the delay (e.g., delay or slow activation) of the first copy of the target nucleic acid that has been inoculated in the site. In this example, a single site may have been inoculated with several different target nucleic acids (e.g., several target nucleic acids may be present in each site before amplification). However, the first copy formation for any given target nucleic acid may be randomly activated so that the average rate of the first copy formation is relatively slow compared to the rate of producing subsequent copies. In this case, although a single site may have been inoculated with several different target nucleic acids, kinetics excludes and will allow only one of those target nucleic acids to be amplified. More specifically, once a first target nucleic acid has been activated for amplification, the site will quickly fill up with its copies, thereby preventing the generation of copies of the second target nucleic acid at that site.

[0185] Amplification reagents can include other components that promote amplicon formation, and in some cases increase the rate of amplicon formation. An example is a recombinase. Recombinases can promote amplicon formation by allowing repeated invasion / extension. More specifically, recombinases can promote invasion of target nucleic acids by polymerases and use target nucleic acids as templates for amplicon formation by polymerase extension primers. This process can be repeated as a chain reaction, wherein the amplicon produced from each round of invasion / extension serves as a template in subsequent rounds. Since no denaturation cycle (e.g., by heating or chemical denaturation) is required, this method can occur faster than standard PCR. Therefore, the amplification promoted by the recombinase can be carried out isothermally. It is generally desirable to include ATP or other nucleotides (or its analogs that are not hydrolyzable in some cases) in the amplification reagent promoted by the recombinase to promote amplification. A mixture of recombinase and single-stranded binding (SSB) protein is particularly useful because SSB can further promote amplification. Exemplary preparations for promoting amplification by recombinases include those sold commercially by TwistDx (Cambridge, UK) with TwistAmp test kits. Useful components and reaction conditions for recombinase-facilitated amplification reagents are listed in US 5,223,414 and US 7,399,590, each of which is incorporated herein by reference.

[0186] Another example of a component that can be included in the amplification reagent to promote amplicon formation and increase the amplicon formation rate in some cases is a helicase. Helicases can promote the formation of amplicon by a chain reaction that allows amplicon formation. Because no denaturation cycle (e.g., by heating or chemical denaturation) is required, the method can occur faster than standard PCR. Therefore, the amplification promoted by the helicase can be carried out isothermally. A mixture of helicase and single-stranded binding (SSB) protein is particularly useful because SSB can further promote amplification. Exemplary preparations for promoting amplification by helicases include those sold commercially as IsoAmp test kits by Biohelix (Beverly, MA). In addition, examples of useful preparations including helicase proteins are described in US 7,399,590 and US 7,829,284, each of which is incorporated herein by reference.

[0187] Another example of a component that can be included in an amplification reagent to promote amplicon formation, and in some cases increase the rate of amplicon formation, is an origin binding protein.

[0188] KEA or other isothermal amplification methods can be used in bridge amplification schemes to anchor a single target polynucleotide with a single target molecule to a feature. Although the amplification of target polynucleotide can be carried out from both ends, it is useful to amplify linearly from the end of the target molecule away from attachment to avoid or reduce the loss of target polynucleotide (and the target molecule attached) from some features. The linear amplification of target polynucleotide can be realized by incorporating one or more base pair mismatches in the capture primer binding region of the target polynucleotide attached to the near-end of the target molecule. The capture primer binding region that is not attached to the complementary chain of the target molecule can be complementary to its corresponding capture primer on substrate. According to this exemplary structure, the first round of KEA will linearly amplify two chains of target polynucleotide. However, the subsequent amplification round will linearly amplify the target polynucleotide chain attached to the target molecule, and exponentially amplify the amplicon produced by the complementary chain.

[0189] Mismatches within the capture primer binding region can be placed anywhere as long as it inhibits or delays invasion of the cognate capture primer and / or extension of the primer by a polymerase. Figure 4 and 6 Placing a mismatch at the end of the binding region complementary to the cognate capture primer as exemplified in is particularly useful because it creates a low probability of strand invasion using a recombinase and / or reduces extension by a polymerase since the capture primer does not hybridize to the target polynucleotide at the 3' end.

[0190] exist Figure 4The above exemplary designs for placing at least one target molecule in a feature or placing at least one target molecule in each of multiple features by KEA are shown in FIG. In brief, since the target polynucleotide can diffuse when KEA displaces the polynucleotide chain bound to the target molecule, the target polynucleotide can be constructed with one or more mismatched base pairs in the capture primer region. Figure 4 A target polynucleotide consisting of a PhiX target region flanked by capture primer regions is shown. The 3' end (lower strand) of the ds target polynucleotide shows a capture primer binding region (P5') comprising a sequencing primer binding region (SBS3'). The 5' end (lower strand) represents a capture primer binding region (P7) comprising a sequencing primer binding region (SBS8). The complementary strand structure is shown in the upper strand of the target polynucleotide. Although any base pair matching can be used, in this illustrative embodiment, the P7 / P7' capture primer binding region contains a three base pair mismatch corresponding to the CCC / CTA mismatch sequence introduced at the 3' end of the P7 sequence. At the 5' end of the mutated or mismatched P7 strand, chemical conjugation can be used to connect the target polynucleotide to the target molecule, which is Figure 4 Alternatively, an abasic site or poly-8oxo-G (7,8-dihydro-8-oxo-guanine) can be used to retain the primary template molecule (P7 sequence).

[0191] Figure 6 Depicted are the first and second rounds of KEA designed using the target polynucleotide exemplified above. The features are shown by hatched plane markers at the right and left ends of the target polynucleotide. Attached to the features are capture primers, denoted P5 and P7. The ovals decorating the capture primers indicate the inclusion of SSB proteins. These capture primers are complementary to the P5' and P7' capture primer binding regions flanking the target region of the target polynucleotide. The P7 capture primer binding region adjacent to or in proximity to the attached target molecule is not identical or complementary to P7, thereby preventing or reducing strand invasion during KEA. As shown in FIG. Figure 6 As shown, a regular or standard strand of P5-SBS3-phiX-SBS8'-P7' complementary to the P7 capture primer will be exponentially amplified during KEA to consume features such as the P5 / P7 primers on the nanopore. In contrast, strand invasion will be inhibited from the P7 end of the strand that is mutated or mismatched. Therefore, the target molecule attached to the target polynucleotide will remain hybridized with the substrate-bound amplicon.

[0192] Therefore, using the first pair of capture primers as exemplified above, the methods provided herein can employ patterned substrates, such as initial capture and immobilization of a single target polynucleotide of each feature or hole of a flow cell. Initial target polynucleotide capture can be followed by preliminary amplification of a single target polynucleotide to produce a monoclonal population of target polynucleotide amplicons within a feature, such as produced by KEA. The initial target polynucleotide with an attached target molecule will remain immobilized to a feature by hybridizing, for example, with a capture primer immobilized at the distal end of the target molecule.

[0193] In addition, the methods provided herein can employ any of the components previously described, such as capture primers, capture primer binding regions, target polynucleotides, target molecules, etc., with reference to the substrates provided herein, or any combination thereof. For example, these components can be employed together with the exemplified mismatch capture primer binding region design and an isothermal amplification method that amplifies at least one target polynucleotide at an average amplification rate that exceeds the average transport rate of the target polynucleotide to a feature to produce any configuration of a substrate described herein having at least a target polynucleotide attached to a target molecule immobilized to a feature.

[0194] Thus, the methods provided herein can be performed using any of the exemplary substrates described above, including, for example, any insoluble solid support, semi-solid support or matrix of any shape to which biomolecules (including, for example, polynucleotides) can be attached. Exemplary solid supports include glass, modified glass, functionalized glass, inorganic glass, microspheres (e.g., inert and / or magnetic particles), plastics, nylon, silica-based materials, etc., as described above.

[0195] Additionally, for example, the methods provided herein can be performed using a substrate comprising a flow cell, for example, as described in US 2010 / 0111768A1 or Bentley et al, Nature 456:53-59 (2008) and previously exemplified.

[0196] The methods provided herein can be performed on one or more features on a substrate, including, for example, holes, pits, channels, ridges, raised areas, spikes, columns, beads, metals, etc., as previously described with reference to the substrates provided herein. In some embodiments, the method of placing at least one target molecule on a feature can utilize holes as features and can include a gel material as described in US 2014-0243224 and previously described.

[0197] The method provided herein can utilize the substrate of the first and second capture primers with the feature that is immobilized on the substrate.The first and second capture primers can be a plurality of first capture primers and a plurality of second capture primers.In some embodiments, the first and second capture primers can be directed to the different sequences of the target polynucleotide, therefore showing specificity to the different regions of the target polynucleotide.In other embodiments, the first and second capture primers can be directed to different target polynucleotides. Figure 2 Exemplary substrates that can be used in methods for placing at least one target molecule on a feature are described in , as previously described.

[0198] Two or more capture primers used in the methods provided herein can be present in a feature in any ratio. For example, a plurality of first capture primers and a plurality of second capture primers can be present in about the same amount or in any other ratio, such as a molar ratio, including, for example, a first capture primer greater than 1.1x to greater than 1,000x excess relative to the second capture primer, as previously illustrated. Similarly, in the methods provided herein using multiple features, different features can have the same ratio or different ratios of two or more capture primers.

[0199] Briefly, for example, the capture primer used in the method of placing at least one target molecule on a feature can include one or more capture regions, including, for example, a universal capture region, a sequencing primer binding site (SBS), a target-specific capture region, a predetermined cleavage site, such as a restriction site, and a connector region, such as a connector region separating two or more regions of the capture primer. The capture primer can use any of the designs previously exemplified, such as a universal capture region and an SBS, including any combination of P5, P5', P7, P7', SBS3, SBS3', SBS8 and SBS8' and / or any other sequence that is unique enough to function as a universal primer, a sequencing primer or any other sequence such as an adapter.

[0200] Therefore, the method provided herein can adopt the first and second capture primers, which are immobilized to the feature that can include any capture region or any combination of capture regions. For example, the first capture primer can include the first universal capture region, and the second capture primer can include the same universal capture region or the second universal capture region. The first and second capture primers can further include the same or different SBS. For example, the first capture primer can include the first universal capture primer region and the first SBS, and the second capture primer can include the second universal capture region and the second SBS.

[0201] The methods provided herein can utilize multiple capture primers immobilized to features, as previously described with reference to substrates made by the methods provided herein. Multiple can be single plurality, multiple first capture primers, and multiple second capture primers. Alternatively or additionally, the methods provided herein can use multiple third, fourth, fifth and / or sixth or more capture primers. The number of multiple capture primers included will depend on the application as previously exemplified. Multiple can be a population of two or more members, including, for example, multiple is 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 or more different population members. In other embodiments, multiple is 200, 300, 400, 500, 1000, 5000, 100, 000, 50000, 1x10 5 , 2x10 5 , 5x10 5 , 4x10 5 , 5x10 5 , 6x10 5 , 7x10 5 , 8x10 5 , 9x10 5 , 1x10 6 , 2x10 6 , 3x10 6 , 5x10 6 , 6x10 6 , 7x10 6 , 8x10 6 , 9x10 6 or 1x10 7 , or more different members and any of the other members of the plurality exemplified previously. A plurality may contain different members, similar members and / or identical members, including, for example, a mixture of any combination of different, similar and / or identical members from at least 1% up to and including 99% or more of the members.

[0202] The method provided herein can be used for immobilizing at least one target polynucleotide on a feature, which is attached to a capture primer at one end. The method provided herein can also be used for immobilizing a single target polynucleotide on a feature, which is attached to a capture primer at one end. Although the attachment of at least one target polynucleotide or the single target polynucleotide description attached to the capture primer immobilized to a feature at one end is described herein, it should be understood that in other embodiments, the method provided herein can be used for immobilizing two or more target polynucleotides, one end of which is attached to the capture primer immobilized to a feature. In these other embodiments, the number of target polynucleotides can be, for example, 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95 or 100 or more different target polynucleotides. Alternatively or in addition, the method provided herein can be used for fixing, for example, 2 to 100 or more identical, similar or different target polynucleotides, including mixtures thereof, as previously illustrated.

[0203] Any of the target polynucleotides previously exemplified can be used in a method in which at least one target molecule is placed on a feature or each of the multiple features provided herein. Therefore, the target polynucleotide in the method provided herein can include two or more nucleotides, including, for example, ribonucleotides, deoxyribonucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA) or any combination thereof. The target polynucleotide can include purine bases, pyrimidine bases, both purine and pyrimidine bases, natural, chemically modified, biochemically modified, non-natural or derivatized nucleotide bases, one or more paired nucleotide bases. The target polynucleotides used in the method provided herein can be double-stranded nucleic acids, such as dsDNA, dsRNA, double-stranded DNA / RNA hybrids, and can also include or more unpaired nucleotide bases. Single-stranded target polynucleotides can also be used in the method provided herein, as previously exemplified. Therefore, the target polynucleotide can be a mixture of the type of polynucleotides, sequences or polynucleotides and / or sequences of any desired type.

[0204] Similarly, the target polynucleotides used in the methods provided herein can include one or more target polynucleotide acid regions as listed above, including, for example, capture primer binding regions, target regions, primer binding regions, barcode regions, joint regions, and / or adapter regions. One or more target polynucleotide acid regions can be multiple target polynucleotide acid regions, including, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more target polynucleotide regions. Exemplary target polynucleotides comprising two or more target polynucleotide acid regions are described in Figure 3 Described in AD, as described above.

[0205] The methods provided herein can employ a target polynucleotide or a target polynucleotide region (target polynucleotide or region thereof), including, for example, a target region, a capture primer binding region and / or other regions described herein or known to those skilled in the art having two or more nucleotides. Other lengths include any of 2 to 100 or more nucleotides and up to 10,000 or more nucleotides and the various sizes previously exemplified.

[0206] Methods for placing at least one target polynucleotide on a feature can employ target polynucleotides, or regions thereof, comprising double-stranded polynucleotides of any of the various lengths listed and / or exemplified above, including, for example, 2, 25, 150, 1100, 10000 or more base pairs and all integers therebetween.

[0207] The methods provided herein can employ double-stranded target polynucleotides or regions thereof, including regions of capture primer regions thereof, that can contain any number of mismatches. Particularly useful numbers of mismatches include, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more mismatches with their complementary sequences or, for example, capture primers or other capture agents. Such mismatches are Figure 4 and 6 exemplified in, and described with respect to substrates produced by the methods provided herein.

[0208] As previously illustrated about substrate provided herein, the method of the present disclosure can adopt the target polynucleotide comprising one or more regions with one or more capture primer binding regions. The capture primer binding region can be located at the 5' end, 3' end or 5' and 3' end of the target polynucleotide acid region, or alternatively or in addition it can be located at the internal region of the target polynucleotide. The target polynucleotide can comprise two or more capture primer binding regions, until any number of capture primer regions are suitable for any desired purpose. Similarly, as with corresponding capture primers, the capture primer binding region can comprise one or more capture primer binding regions, including for example universal capture primer binding region, sequencing primer binding site (SBS), target-specific capture primer binding region, predetermined cleavage site, such as restriction site, and joint region, for example separation capture primer binding region of two or more regions of the joint region. Any and all possible combinations and arrangements can be adopted in the method as previously illustrated provided herein. For example, the methods provided herein can employ any capture primer binding region design previously exemplified, such as universal capture regions and SBS, including any combination of P5, P5', P7, P7', SBS3, SBS3', SBS8' and / or any other sequence that is sufficiently unique to function as a universal primer, a sequencing primer, or any other sequence such as an adapter. The length of the capture primer binding region can include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 or more nucleotides, and 400 down to 100 or less nucleotides, as exemplified with respect to substrates produced by the methods provided herein.

[0209] The capture primer binding region of target polynucleotide can be 100% complementary with capture primer. The capture primer binding region of the target polynucleotide adopted in the method provided herein can be for example at least 60%, 65%, 70%, 75%, 77%, 80%, 82%, 85%, 87%, 90%, 92%, 95% or 97% or more complementary with capture primer, including for example comprising 1,2,3,4,5,6,7,8,9 or 10 or more nucleotide mispairings with capture primer. Except one or more capture primer binding regions, target polynucleotide can include one or more target regions, including two or more target regions, as previously illustrated. The size or length of target region can be any length illustrated above about target polynucleotide or target polynucleotide region.

[0210] The method provided herein can place at least one target molecule on the feature of substrate by, for example, being attached to target polynucleotide, and the target polynucleotide can, for example, be attached to capture primer. The method provided herein can also place a plurality of two or more target molecules in each reference situation as exemplified above, including, for example, 2,3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,250,500,1,000,5,000,10,000 or more different, identical and / similar target molecules, as exemplified above. The attachment of target molecule to target polynucleotide, capture primer, feature and / or substrate can be covalent or non-covalent.

[0211] In addition to at least one target molecule immobilized by the above-described configurations, the methods provided herein can also generate multiple amplicons of a target polynucleotide on a feature or features on a substrate, such as Figure 4 and 6 . Multiple amplicons can minimally fill a feature, partially fill a feature, or completely fill a feature on a substrate. Thus, the density of amplicons can be low, medium, or high. Target polynucleotide designs, methods for generating such amplicons, and methods for controlling density are further described below with reference to methods for placing at least one target molecule on a feature of a substrate. The density of the amplicons is as described herein.

[0212] The method for placing a target molecule on a substrate or feature by single molecules provided herein can utilize any desired molecule. Exemplary categories of target molecules include, for example, polypeptides, polynucleotides, carbohydrates, amino acids, nucleotides, monosaccharides, haptens, ligands, antigens, analytes, small molecule organic compounds or inorganic compounds. Exemplary species for each of the above-mentioned categories are further described below. Exemplary species for each of these categories have been previously described, each of which can be similarly employed in the method provided herein to generate a substrate provided herein, as previously described.

[0213] As an example, the target molecules used in the methods provided herein can include, for example, polypeptides of any size or activity and containing any natural or unnatural amino acids; ribosomal polypeptides, including but not limited to enzymes, receptors, antibodies, transcription factors, hormones, ligands, antigens and haptens; non-ribosomal peptides, including but not limited to toxins, siderophores, pigments, antibiotics, antibiotic precursors, cytostatics and immunosuppressants; including MspA, outer membrane phospholipase A (OmpA), OmpC, OmpF, OmpG, Neisseria autotransporter (NalP), W ZA, ClyA toxin, α-hemolysin, anthrax toxin, gramicidin A, maltoporin, PhoE, Tsx, F-pilus, SP1, mitochondrial porin (VDAC), Tom40, leukocidin and DNA origami nanopores; antibiotics as previously exemplified; toxins as previously exemplified; hormones, such as hormones as previously exemplified; haptens, including but not limited to aniline, anthranilic acid, m-aminobenzoic acid, p-aminobenzoic acid, uroshiol, quinine, hydralazine, fluorescein, biotin, digoxigenin and dihydrotestosterone. nitrophenol; receptors, including, for example, receptors from immune cells or non-immune cells, as previously exemplified; cytokines, such as chemokines, interferons, interleukins, lymphokines, tumor necrosis factor (TNF) and neuropeptides, as previously exemplified; enzymes of any type, including, for example, metabolic enzymes, polymerases, ligases, reverse transcriptases, nucleases, helicases and transposases, as previously exemplified; antibodies or antibody fragments, such as previously exemplified; polynucleotides or oligonucleotides, including, for example, DNA or RNA of any length or sequence, as previously exemplified; inorganic compounds, including, but not limited to, carbides, carbonates, carbon Simple oxides (e.g., CO and CO2), cyanide quantum dots, metal nanoparticles, metal oxide nanoparticles, etc., minerals, sulfides, organometallic compounds and bioorganic compounds and minerals, as previously exemplified; small molecules as previously exemplified; metabolites, including but not limited to alkaloids, glycosides, lipids, non-ribosomal peptides, such as actinomycin d, phenazines, natural phenols (including flavonoids), polyketides, terpenes, including steroids and tetrapyrroles; therapeutic agents, including, for example, organic compounds, inorganic compounds, peptides, hormones, small molecules, antibodies, antigens, haptens, or any combination thereof. As previously mentioned, the therapeutic agent can be an antipyretic, analgesic, antimalarial, antibiotic, antiseptic, mood stabilizer, hormone replacement, oral contraceptive, stimulant, tranquilizer, antiviral, anticancer, immunosuppressant, and statin.

[0214] In some embodiments, the methods provided herein can be used to place at least one target molecule in each of a plurality of features or (also referred to herein as an array or feature array) on a substrate, such as Figure 5For example, the methods provided herein can be used with a substrate having a plurality of features arranged in any particular pattern, as previously exemplified, including, for example, a grid of dots or patches, a repeating pattern, an irregular non-repeating pattern, a hexagonal pattern, a straight line pattern, a grid pattern, a pattern with reflection symmetry, a pattern with rotational symmetry, etc. The features can be any of the various densities previously exemplified, for example, from at least about 10 features / cm 2 Up to approximately 5,000,000 features / cm 2 The characteristic areas of the arrays used in the methods provided herein can each have a surface area greater than about 100 nm. 2 to about 500 μm 2 The area and any area therebetween. The features on the array substrate surface can be discontinuous, separated by the gap area of ​​the surface. In a specific embodiment, the method is used together with features that are concave features in a surface (e.g., hole) including, for example, micropores or nanopores, and the features can include gel materials as previously illustrated. The methods and compositions for preparing and using a substrate containing a gel of features (e.g., holes) are listed in, for example, US 2014 / 0243224, which is incorporated herein by reference. The method can be performed with an array having features of high density, medium density, or low density, as described above. The method described herein can be performed with features of beads or beads in holes. Beads can be composed of any material that immobilizes polynucleotides to a surface, including but not limited to plastics, ceramics, glass, polystyrene, carbon graphite, etc., and any other material for solid supports outlined herein can be used.

[0215] In some embodiments, the method provided is used to place at least one target molecule on a feature or in each of a plurality of features, wherein the feature size is selected from a range that is conducive to kinetic exclusion amplification (KEA) and formation of a single molecule placement in a feature. As previously illustrated by reference to nanopores, the pore diameter can be between about 30nm and about 1 μm, between about 50nm and about 800nm, between about 70nm and about 600nm, or between about 100 μm and about 400nm. In some embodiments, the pore has a diameter of about 400nm. In some embodiments, the pore has a diameter less than about 1 μm.

[0216] As an example, in a specific embodiment, the method disclosed herein may include (a) attaching a plurality of first capture primers to a surface; (b) attaching a plurality of second capture primers to a surface; and (c) attaching a double-stranded target polynucleotide to a second capture primer of a second capture primer, wherein (i) the double-stranded target polynucleotide comprises a first parent strand and a second parent strand; (ii) the double-stranded target polynucleotide comprises a second capture primer binding region in the first and second parent strands; (iii) the second capture primer binding region of the first parent strand is 100% complementary to the second capture primer of the plurality of second capture primers; and (iv) the second capture primer binding region of the second parent strand is at least 100% identical to the second capture primer of the plurality of second capture primers. The method may further include attaching a target molecule to the target polynucleotide. The target molecule may be attached to the target polynucleotide before attaching the target polynucleotide to the second capture primer. Attaching the plurality of first capture primers to the surface may occur before attaching the plurality of second capture primers to the surface. Alternatively, attaching the plurality of first capture primers to the surface may occur simultaneously with attaching the plurality of second capture primers to the surface. The target polynucleotide may further comprise a first capture binding region. At least one strand of the first capture binding region may hybridize with a first capture primer of the plurality of first capture primers. The method may further comprise generating a plurality of amplicons. The plurality of amplicons may comprise copies of the target polynucleotide. The plurality of amplicons may be generated by performing an amplification reaction. The amplification reaction may comprise a first recombinase polymerase amplification (RPA). The first RPA may comprise extending a first capture primer and a second capture primer hybridized to the target polynucleotide to generate a plurality of first amplicons of the target polynucleotide. Amplicons may be generated by performing a plurality of one or more additional amplification reactions. The one or more additional amplification reactions may comprise one or more additional recombinase polymerase amplifications (RPA). The one or more additional RPAs may comprise (a) attaching one or more first capture primers of the plurality of capture primers to one or more amplicons of the plurality of first amplicons; and (b) extending the one or more first capture primers to generate a plurality of additional amplicons. The one or more additional RPAs may include (a) extending one or more second capture primers of the plurality of second capture primers; and (b) extending the one or more second capture primers to generate a plurality of additional amplicons. In some instances, the second capture primer does not hybridize to an amplicon containing a complement of the second parental strand. In some instances, the second capture primer does not hybridize to a second capture primer binding region of an amplicon that is a complement of the second capture primer binding region of the second parental strand.

[0217] Figure 7 An exemplary method of generating a substrate is depicted in FIG. Figure 7As shown in step 1 of , a surface (706) comprising a plurality of first capture primers (707, 711, 714) and a plurality of second capture primers (708, 713, 715) is contacted with a target polynucleotide (701). The target polynucleotide (701) comprises a first parent strand (703) and a second parent strand (702). The first parent strand (703) and the second parent strand (702) comprise a first capture primer binding region (717) and a second capture primer binding region (718). Within the second capture primer binding region (718), there is a mismatch region between the first parent strand (703) and the second parent strand (702). The second capture primer binding region (718) of the second parent strand (702) comprises at least one nucleotide difference (704) with a second capture primer of the plurality of second capture primers (708, 713, 715). The second capture primer binding region (718) of the first parent strand (703) is 100% complementary to the second capture primer of the plurality of second capture primers (708, 713, 715). As shown in step 2, the first capture primer (707) of the plurality of first capture primers (707, 711, 714) hybridizes with the first capture primer binding region (717) of the second parent strand (702). The second capture primer (708) of the plurality of second capture primers (708, 713, 715) hybridizes with the second capture binding region (718) of the first parent strand (703). As shown in step 3, the first and second parent strands are amplified to generate a first plurality of amplicons. The first capture primer (707) displaces the first parent strand (703) and is extended to generate an amplicon of the second parent strand (709). The amplicon of the second parent strand (709) contains the complement of the second parent strand. Thus, the amplicon of the second parent strand (709) contains the complement of the second capture primer binding region (718) of the second parent strand (702). As also shown in step 3, the second capture primer (708) displaces the second parent strand (702) and is extended to generate the amplicon of the first parent strand (710). As shown in step 4, one or more additional amplification reactions are performed to generate a second plurality of amplicons. In step 4a, the first capture primer (711) hybridizes with the first capture primer binding region of the second parent strand and displaces the first capture primer (707). In step 4b, the second capture primer (713) attempts to hybridize with the second capture primer binding region of the amplicon of the second parent strand (709).

[0218] However, since the second capture primer binding region of the amplicon of the second parent chain (709) is the complement of the second capture primer binding region of the second parent chain, the second capture primer binding region of the amplicon of the second parent chain (709) is not 100% complementary to the second capture primer (711). The second capture primer (711) cannot displace the amplicon of the second parent chain (709) from the second parent chain. Therefore, the amplicon of the second parent chain containing the second capture primer binding region complementary to the second capture primer binding region of the second parent chain cannot serve as a template to extend the second capture primer. As shown in step 4c, since the first capture primer binding region of the amplicon of the first parent chain (710) is 100% complementary to the first capture primer (714), the first capture primer (714) can displace the first parent chain (703) and hybridize with the first capture primer binding region of the amplicon of the first parent chain (710). The amplicon of the first parent strand (710) can serve as a template to extend the first capture primer (714). As shown in step 4d, a second capture primer, second capture primer (715) can hybridize with the second capture primer binding region of the first parent strand (703), thereby displacing the second capture primer (708). As shown in step 5, the first capture primer (711) can be extended to generate an amplicon of the first parent strand (716). Also shown in step 5, the first capture primer (714) can be extended to generate an amplicon of the first parent strand (717). Also shown in step 5, the second capture primer (715) can be extended to generate an amplicon of the first parent strand (718).

[0219] Thus, the present disclosure provides a method for placing a single target molecule on a feature of a substrate. The method comprises: (a) hybridizing a plurality of first and second capture primers immobilized to a feature on a substrate with at least one target polynucleotide, the target polynucleotide comprising a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, wherein the second capture primer binding region comprises a base pair mismatch with the second capture primer and is attached to the target molecule, (c) amplifying the at least one target polynucleotide at an average amplification rate to produce a plurality of clonal amplicons complementary to the target polynucleotide, the average amplification rate exceeding the average transport rate of the target polynucleotide to the feature. The method may utilize a substrate having one feature or multiple features. One or more features may be beads, holes, including micropores or nanopores, channels, ridges, protrusions, or combinations thereof. The method may place at least one target molecule, including a single target molecule, in each of the feature or multiple features. The method may fill one or more features with multiple clonal amplicons of the target polynucleotide. The interstitial regions between multiple features may lack target polynucleotides. The method can be used with a substrate having multiple features, including two or more features, and different single target molecules can be placed in each of the different features within the multiple. The method can be used with a target polynucleotide selected from the group consisting of RNA, DNA, PNA or double-stranded DNA (dsDNA). The length of the target polynucleotide used by the method provided herein can be less than 1,000 nucleotides and / or it can be 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900, or 901 to 1000 nucleotides. The target molecule placed on one or more features by the method can be a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound or an inorganic compound. The polypeptide target molecule can be a nanopore, a binding polypeptide and an enzyme. The nanopore can be MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin and DNA origami nanopore. The binding polypeptide can be an antibody, Fab, Fab', F(ab') 2 , scFv, diabody, triabody, minibody and single domain antibody (sdAB) or T cell receptor. The enzyme can be a polymerase, a helicase, a recombinase, a transposase or a ligase. The method can be used with a substrate of one or more materials selected from the group consisting of glass, silicon, plastic or biopolymer. The substrate can further comprise a hydrogel or a covalently linked gel.

[0220] The substrate of the present disclosure having a single target molecule in a feature or a single target molecule in each of a plurality of features can be used to interrogate a single molecule entity. One or more molecules to be interrogated can be in a solution, and an immobilized target molecule can be used to determine the features of one or more molecules to be interrogated. For example, the sequence of a single polynucleotide or a plurality of single polynucleotides can be determined by nanopore sequencing, wherein the nanopore corresponds to an immobilized target molecule. Alternatively, one or more molecules to be interrogated can be an immobilized target molecule, and one or more molecules in a solution can be used to detect or determine the features of each fixed target molecule. For example, the immobilized target molecule can be a library of different enzyme variants, and the probe in the solution can be a substrate or a plurality of different substrates to determine the catalytic properties of each different enzyme variant. The opposite direction can also be used, wherein the substrate, for example, corresponds to an immobilized target molecule on a feature, and is contacted with different enzyme variants to measure catalytic properties. In view of the teachings and guidance provided herein, it will be understood by those skilled in the art that a large number of different target molecules can be used in the substrate provided herein to interrogate or be interrogated to determine one or more molecular features. Exemplary features include, for example, determining polynucleotide sequences, polypeptide sequences, binding activity, catalytic activity, etc.

[0221] As further illustration of sequencing, some embodiments can utilize nanopore sequencing (Deamer, DW & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147-151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis". Acc. Chem. Res. 35: 817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and JA Golovchenko, "DNA molecules and configurations in a solid-state nanopore microscope" Nat. Mater. 2: 611-615 (2003), the disclosures of which are incorporated herein by reference in their entirety). In such embodiments, the polynucleotide to be sequenced passes through a nanopore. The nanopore can be a synthetic pore or a biological membrane protein, such as α-hemolysin. When the target nucleic acid passes through the nanopore, each base pair can be identified by measuring the fluctuation of the conductivity of the pore. (US Pat. No. 7,001,792; Soni, GV & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore-based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, SL, Chu, J., Amorin, M. & Ghadiri, MR "A single-molecule nanopore device detects DNA polymerase activity with single-nucleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008), the disclosure of which is incorporated herein by reference in its entirety). The data obtained from nanopore sequencing can be stored, processed and analyzed as described herein. In particular, the data may be processed as images according to the exemplary processing of optical images and other images described herein.

[0222] The method here can also be used for molecular binding affinity screening. In embodiments, the monomolecular antibody can be anchored on a substrate as described herein. Single antigen binding events can be detected by fluorescent staining. Fluorescent staining techniques include techniques well known in the art, including, for example, Illumina Infinium assays or ELISA (enzyme-linked immunosorbent assay) techniques. In certain embodiments, the concentration of the antigen in the solution can be quantitatively analyzed by counting monomolecular fluorescent spots (such techniques known in the art). (T. Blicharz, et al. "Fiber-Optic Microsphere bead antibody array for the analysis of inflammatory cytokines in saliva" Anal. Chem. 2009, 81, 2106.). In embodiments, the method described herein is suitable for in vitro diagnosis.

[0223] The advantage of the method listed herein is that they provide parallel detection of multiple target nucleic acids quickly and effectively. Therefore, the present disclosure provides an integrated system that can use technology known in the art, such as those technologies illustrated above to prepare and detect nucleic acids. Therefore, the integrated system of the present disclosure can include a fluid assembly that can deliver amplification reagents and / or sequencing reagents to one or more immobilized target molecules, and the system includes components such as pumps, valves, reservoirs, fluid pipelines, etc. Can configure and / or use flow cells to inquire in the integrated system. Exemplary flow cells are described in, for example, US 2010 / 0111768 A1 and US Ser.No.13 / 273,666, which are each incorporated herein by reference. As illustrated in the flow cell, one or more fluid assemblies of the integrated system can be used for different steps in the inquiry. Taking the binding reaction embodiment as an example, one or more fluid assemblies of the integrated system can be used for the delivery of the reagent for binding steps and for detection. Or, the integrated system can include a separate fluid system to perform the binding steps and implement the detection method. Examples of integrated sequencing systems capable of producing amplified nucleic acids and also determining nucleic acid sequences include, but are not limited to, the MiSeq™ platform (Illumina, Inc., San Diego, CA) and US Ser. No. 13 / 273,666, which is incorporated herein by reference.

[0224] Further disclosed herein are kits comprising any substrate disclosed herein. The kit can comprise (a) a substrate comprising features, wherein the features comprise (i) a plurality of first capture primers; and (ii) a plurality of second capture primers; and (b) a target polynucleotide comprising a first double-stranded target polynucleotide region, wherein (i) the first double-stranded target polynucleotide region comprises a second capture primer binding region; (ii) the first strand of the second capture primer binding region is 100% complementary to the second capture primer of the second capture primer; and (iii) the second capture primer binding region comprises at least one nucleotide mismatch between the first strand and the second strand.

[0225] The kit can include a substrate comprising features, wherein the features include (a) a plurality of first capture primers; (b) a plurality of second capture primers; and (c) a target polynucleotide comprising a first double-stranded target polynucleotide region, wherein (i) the first double-stranded target polynucleotide region includes a second capture primer binding region; (ii) the first strand of the second capture primer binding region is 100% complementary to the second capture primer of the second capture primer; and (iii) the second capture primer binding region includes at least one nucleotide mismatch between the first strand and the second strand.

[0226] The kit may include (a) a substrate including features, wherein the features include (i) a plurality of first capture primers; and (ii) a plurality of second capture primers; and (b) a target polynucleotide comprising a first double-stranded target polynucleotide region and a second double-stranded target polynucleotide region, wherein (i) the first double-stranded target polynucleotide region comprises a second capture primer binding region; (ii) the first strand of the second capture primer binding region is 100% complementary to the second capture primer of the second capture primer; and (iii) the second capture primer binding region comprises at least one nucleotide mismatch between the first strand and the second strand. The second double-stranded target polynucleotide region may include the first capture primer binding region.

[0227] The kit may include (a) a substrate including features, wherein the features include (i) a plurality of first capture primers; and (ii) a plurality of second capture primers; and (b) a first target polynucleotide comprising a first double-stranded target polynucleotide region, wherein (i) the first double-stranded target polynucleotide region includes a second capture primer binding region; (ii) the first strand of the second capture primer binding region is 100% complementary to the second capture primer of the second capture primer; and (iii) the second capture primer binding region includes at least one nucleotide mismatch between the first strand and the second strand; and (c) a second target polynucleotide region comprising a second double-stranded target polynucleotide region. The second double-stranded target polynucleotide region may include the first capture primer binding region.

[0228] The kits disclosed herein may comprise a substrate, such as Figure 2 As described in Figure 2As shown in , the kit can include a substrate (201) comprising (a) a plurality of first capture primers (202, 203, 204); (b) a plurality of second capture primers (205, 206, 207); (c) a target polynucleotide (208); (d) a target molecule (209); and (e) a plurality of amplicons (210, 211, 212, 213, 214).

[0229] The kits disclosed herein may comprise a substrate, such as Figure 5 As described in Figure 5 As shown in , the kit can include a substrate (501) comprising (a) a first feature (502), the first feature comprising (i) a first plurality of first capture primers (504, 505, 506); (ii) a first plurality of second capture primers (507, 508, 509); (iii) a first target polynucleotide (510); (iv) a first target molecule (511); and (v) a first plurality of amplicons (512, 513, 514, 515, 516); and (b) a second feature (503), the second feature comprising (i) a second plurality of first capture primers (520, 521, 522); (ii) a second plurality of second capture primers (523, 524, 525); (iii) a second target polynucleotide (526); (iv) a second target molecule (527); and (v) a second plurality of amplicons (528, 529, 530, 531, 532).

[0230] Figure 8 Exemplary components of the kits disclosed herein are exemplified in AB. Figure 8 As shown in FIG. 8A , the kit may include (a) a substrate ( 801 ) comprising (i) a plurality of first capture primers ( 802 , 803 , 804 ); and (ii) a plurality of second capture primers ( 805 , 806 , 807 ); and (b) a target polynucleotide ( 808 ). Figure 8 As shown in B, the kit may further comprise one or more additional target polynucleotides (809), amplicons (810), target molecules (811), or a combination thereof.

[0231] The target polynucleotide of any kit disclosed herein can further include one or more target polynucleotide regions disclosed herein. For example, the target polynucleotide can further include a second target polynucleotide region, which includes a first capture primer binding domain. The first capture primer binding region can hybridize with the first capture primer of a plurality of first capture primers. In another example, the target polynucleotide can further include another target polynucleotide region, which includes a target region. The target polynucleotide can include one or more other target polynucleotide regions, which include a barcode, a primer binding region, a joint region, an adapter region, or a combination thereof.

[0232] The target polynucleotide of any kit disclosed herein can be part of the substrate. For example, the target polynucleotide can be attached to the second capture primer of the substrate. Alternatively, the target polynucleotide of any kit disclosed herein can be separated from the substrate. For example, the kit can include a first container containing the substrate and a second container containing the target polynucleotide.

[0233] The kit disclosed herein may further comprise a target molecule. The target molecule may be any target molecule disclosed herein. For example, the target molecule may be a nanopore. The target molecule may be attached to a target polynucleotide. The target molecule may be part of a substrate. For example, the target molecule may be attached to a target polynucleotide, which is attached to a first capture primer or a second capture primer of a substrate. Alternatively or additionally, the target molecule is not attached to the target polynucleotide. For example, the kit may comprise a first container containing a target polynucleotide and a second container containing a target molecule. The target molecule may be provided with a substrate. For example, the kit may comprise a first container containing a substrate and a target molecule and a second container containing a target polynucleotide. Alternatively, the kit may comprise a first container containing a substrate, a target polynucleotide, and a target molecule.

[0234] The kit may further comprise a plurality of amplicons. The plurality of amplicons may be part of a substrate. For example, the plurality of amplicons may be attached to a plurality of first primers, a plurality of second primers, or a combination thereof. The amplicons may be copies of a target polynucleotide.

[0235] The substrate of any of the kits disclosed herein may comprise two or more features.

[0236] It should be understood that modifications that do not substantially affect the activity of the various embodiments of the invention are also included in the definition of the invention provided herein. Therefore, the following examples are intended to illustrate, but not to limit, the present invention.

[0237] Example

[0238] Kinetic exclusion of amplification using mismatched capture primer binding regions

[0239] This example shows that mismatched capture primer binding regions prevent recombinase polymerase amplification (RPA) at the mismatched target polynucleotide termini.

[0240] To verify that mismatched base pairs between the capture primer binding region and the capture primer can prevent the RPA reaction, a commercial kit TwistDX was used in the RPA reaction according to the manufacturer's instructions. Fig. 9Target polynucleotides and capture primers shown. Briefly, the target polynucleotide uses an approximately 500 bp phiX target region flanked by capture primer regions called standard (std) or mismatch (msm). By design, the primer set P5-std / P7-std is complementary to the capture primer binding region in the target polynucleotide phiX-std, and P5-msm / P7-msm is complementary to the capture primer binding region in the target polynucleotide phiX-msm. P5-std / P7-std has a 3 bp mismatch with the template phiX-msm, while P5-msm / P7-msm has a 3 bp mismatch with the template phiX-std.

[0241] The phiX target (13.2uL) containing 5E-16mol of the phiX target molecule was mixed with 2.4μl of 10μM matched or mismatched primers, and then 29.5μl of TwistDx rehydration buffer was added. The above solution was transferred to the TwistDx reaction pellet and mixed by pipetting. The reaction was started by adding 2.5μl of 280mM magnesium acetate and allowed to react for 8 to 10 minutes. The reaction was stopped by the PCR product purification process for gel analysis.

[0242] After KEA reaction, the amplicons were separated by gel electrophoresis (2% E-Gel electrophoresis, Life Technologies), and the results are shown in Fig.10 In brief, RPA reactions with complementary capture primer-capture primer binding region combinations, such as P5-msm / P7-msm plus phiX-msm, resulted in good yields of amplicon formation. However, mismatched capture primer-capture primer binding region combinations in RPA reactions did not produce or resulted in low yields of 500 bp amplicon products.

[0243] These results demonstrate that mismatch design can effectively stop the RPA reaction at the mismatched ends of the target polynucleotide.

[0244] Throughout this application, various publications have been referenced within parentheses. The entire contents of these publications (including GenBank and GI publications) are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0245] Although the present invention has been described with reference to the disclosed embodiments, it will be readily appreciated by those skilled in the art that the specific embodiments and studies detailed above are merely illustrative of the present invention. It should be understood that various modifications may be made without departing from the spirit of the present invention. Therefore, the present invention is limited only by the claims.

[0246] This application involves the following contents.

[0247] 1. A substrate comprising:

[0248] a. a plurality of first and second capture primers immobilized to a feature on the substrate;

[0249] b. at least one target polynucleotide attached at one end to one of said capture primers and linked at the other end to a target molecule, wherein said target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to said first and second capture primers, said second capture primer binding region comprising a base pair mismatch with said second capture primer, and

[0250] c. a plurality of clonal amplicons complementary to the target polynucleotide immobilized to the feature.

[0251] 2. The substrate of item 1, wherein the base pair mismatches are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pair mismatches.

[0252] 3. The substrate of item 2, wherein the base pair mismatch is a three-base pair mismatch.

[0253] 4. The substrate of item 1, further comprising a plurality of features.

[0254] 5. The substrate of claim 1 , wherein the feature comprises a single target molecule.

[0255] 6. The substrate of claim 5, wherein said features are filled with said plurality of clonal amplicons.

[0256] 7. The substrate of claim 4, wherein each of the plurality of features comprises a single target molecule.

[0257] 8. The substrate of claim 7, wherein two or more of said features comprise different single target molecules.

[0258] 9. The substrate of item 7 or 8, wherein the features are filled with the multiple clonal amplicons.

[0259] 10. The substrate of item 1, wherein the target polynucleotide comprises one or more polynucleotides selected from the group consisting of RNA, DNA and PNA.

[0260] 11. The substrate of item 10, wherein the target polynucleotide comprises double-stranded DNA (dsDNA).

[0261] 12. The substrate of claim 1 , wherein the target polynucleotide comprises less than 1,000 nucleotides.

[0262] 13. The substrate of claim 12, wherein the target polynucleotide comprises 10-25, 26-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 nucleotides.

[0263] 14. The substrate of item 1, wherein the target molecule comprises a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound or an inorganic compound.

[0264] 15. The substrate of claim 14, wherein the target molecule comprises a polypeptide.

[0265] 16. The substrate of claim 15, wherein the polypeptide is selected from the group consisting of a nanopore, a binding polypeptide, and an enzyme.

[0266] 17. The substrate of claim 16, wherein the nanopore is selected from the group consisting of MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, ion channels, protein nanopores, and DNA origami nanopores.

[0267] 18. The substrate of item 16, wherein the binding polypeptide is selected from: antibody, Fab, Fab', F(ab') 2 , scFv, diabody, triabody, minibody and single domain antibody (sdAB), T cell receptor, microcin, neuropeptide, G protein-coupled receptor, antibody, epidermal growth factor receptor and HER2.

[0268] 19. The substrate of item 16, wherein the enzyme is selected from: a recombinase, a polymerase, a helicase, a transposase, a ligase, a deaminase, an oxidase and a kinase.

[0269] 20. The substrate of item 4, comprising one or more materials selected from glass, silicon, plastic and biopolymer.

[0270] 21. The substrate of claim 4, wherein the features are separated by gap regions lacking the target polynucleotide.

[0271] 22. The substrate of claim 21, wherein the features comprise beads, holes, channels, ridges, protrusions, or a combination thereof.

[0272] 23. The substrate of claim 22, wherein the pores are micropores or nanopores.

[0273] 24. The substrate of claim 23, further comprising a hydrogel or a covalently linked gel.

[0274] 25. A method of placing a single target molecule on a feature of a substrate, comprising:

[0275] a. hybridizing a plurality of first and second capture primers immobilized to features on a substrate;

[0276] b. at least one target polynucleotide comprising a target region flanked by first and second capture primer binding regions complementary to said first and second capture primers, wherein said second capture primer binding region comprises a base pair mismatch with said second capture primer and is attached to a target molecule, and

[0277] c. amplifying the at least one target polynucleotide at an average amplification rate to produce a plurality of clonal amplicons complementary to the target polynucleotide, the average amplification rate exceeding the average rate of transport of the target polynucleotides to the characteristic.

[0278] 26. The method of claim 25, wherein the base pair mismatches are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pair mismatches.

[0279] 27. The method of claim 26, wherein the base pair mismatch is a three-base pair mismatch.

[0280] 28. The method of claim 25, wherein the substrate comprises a plurality of features.

[0281] 29. The method of claim 25, wherein the feature comprises a single target molecule.

[0282] 30. The method of claim 29, wherein said feature is filled with said plurality of clonal amplicons.

[0283] 31. The method of claim 28, wherein each of the multiple features comprises a single target molecule.

[0284] 32. The method of claim 31 , wherein two or more of the features comprise different single target molecules.

[0285] 33. The method of claim 31 or 32, wherein the feature is filled with the plurality of clonal amplicons.

[0286] 34. The method of claim 25, wherein the amplification comprises isothermal amplification.

[0287] 35. The method of claim 34, wherein the isothermal amplification also comprises a single-chain binding polypeptide.

[0288] 36. The method of claim 34, wherein the isothermal amplification comprises kinetic exclusion amplification.

[0289] 37. The method of claim 36, wherein the isothermal amplification further comprises kinetic exclusion amplification.

[0290] 38. The method of claim 34, wherein the average amplification rate of subsequent amplicons produced at the feature exceeds the average amplification rate of the first amplicons.

[0291] 39. The method of claim 25, wherein the target polynucleotide comprises one or more polynucleotides selected from RNA, DNA and PNA.

[0292] 40. The method of claim 39, wherein the target polynucleotide comprises double-stranded DNA (dsDNA).

[0293] 41. The method of claim 25, wherein the target polynucleotide comprises less than 1,000 nucleotides.

[0294] 42. The method of claim 41, wherein the length of the target polynucleotide comprises 10 to 25, 26 to 50, 51 to 100, 101 to 200, 201 to 300, 301 to 400, 401 to 500, 501 to 600, 601 to 700, 701 to 800, 801 to 900, or 901 to 1000 base pairs.

[0295] 43. The method of claim 25, wherein the target molecule comprises a polypeptide, a polynucleotide, a carbohydrate, an amino acid, a nucleotide, a monosaccharide, a hapten, a ligand, an antigen, an analyte, a small molecule organic compound or an inorganic compound.

[0296] 44. The method of claim 43, wherein the target molecule comprises a polypeptide.

[0297] 45. The method of claim 44, wherein the polypeptide is selected from the group consisting of a nanopore, a binding polypeptide, and an enzyme.

[0298] 46. ​​The method of claim 45, wherein the nanopore is selected from the group consisting of MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, and DNA origami nanopore.

[0299] 47. The method of item 45, wherein the binding polypeptide is selected from: antibody, Fab, Fab', F(ab') 2 , scFv, diabodies, triabodies, minibodies and single domain antibodies (sdAB), T cell receptors, microcins, neuropeptides, G protein-coupled receptors, antibodies, epidermal growth factor receptor and HER2.

[0300] 48. The method of claim 45, wherein the enzyme is selected from the group consisting of: a recombinase, a polymerase, a helicase, a transposase, a ligase, a deaminase, an oxidase, and a kinase.

[0301] 49. The substrate of claim 28, comprising one or more materials selected from the group consisting of glass, silicon, plastic, and biopolymers.

[0302] 50. A substrate of claim 28, wherein the features are separated by gap regions lacking the target polynucleotide.

[0303] 51. The substrate of claim 50, wherein the features comprise beads, holes, channels, ridges, protrusions, or a combination thereof.

[0304] 52. The substrate of claim 51 , wherein the pores are micropores or nanowires.

[0305] 53. The substrate of item 52, further comprising a hydrogel or a covalently linked gel.

Claims

1. A substrate comprising: a. a plurality of first and second capture primers immobilized to a feature on the substrate; b. at least one double-stranded target polynucleotide having one end attached to one of the capture primers and the other end linked to a target molecule, wherein the double-stranded target polynucleotide comprises a target region flanked by first and second capture primer binding regions complementary to the first and second capture primers, wherein the second strand of the double-stranded target polynucleotide is linked to the target molecule, and wherein the first capture primer binding region of the first strand of the double-stranded target polynucleotide is identical to the first capture primer, the second capture primer binding region of the first strand of the double-stranded target polynucleotide is 100% complementary to the second capture primer, and wherein the first capture primer binding region of the second strand of the double-stranded target polynucleotide is 100% complementary to the first capture primer, the second capture primer binding region of the second strand of the double-stranded target polynucleotide contains at least one nucleotide difference from the second capture primer such that the second capture primer binding region in the double-stranded target polynucleotide contains a base pair mismatch, and c. a plurality of clonal amplicons complementary to the target polynucleotide immobilized to the feature; wherein the substrate comprises a plurality of features separated by interstitial regions lacking target polynucleotides; and Wherein the target molecule comprises an analyte.

2. The substrate of claim 1, wherein the base pair mismatches are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 base pair mismatches.

3. The substrate of claim 1 further comprising a plurality of features.

4. The substrate of claim 1, wherein the feature comprises a single target molecule.

5. The substrate of claim 4, wherein the features are filled with the plurality of clonal amplicons.

6. The substrate of claim 3, wherein each of the plurality of features comprises a single target molecule.

7. The substrate of claim 6, wherein the features are filled with the plurality of clonal amplicons.

8. The substrate of claim 1, wherein the target polynucleotide comprises one or more polynucleotides selected from the group consisting of RNA, DNA, and PNA.

9. The substrate of claim 1, wherein the target polynucleotide comprises 10-25, 26-50, 51-100, 101-200, 201-300, 301-400, 401-500, 501-600, 601-700, 701-800, 801-900, or 901-1000 nucleotides.

10. The substrate of claim 1, wherein the target molecule comprises a polypeptide or an antigen.

11. The substrate of claim 10, wherein the target molecule comprises a polypeptide selected from the group consisting of a nanopore, a binding polypeptide, and an enzyme.

12. The substrate of claim 11, wherein the nanopore is selected from the group consisting of MspA, OmpF, OmpG, NalP, WZA, ClyA toxin, α-hemolysin, anthrax toxin, leukocidin, ion channel, protein nanopore and DNA origami nanopore; the binding polypeptide is selected from the group consisting of antibodies, T cell receptors, microcins, neuropeptides, G protein coupled receptors, epidermal growth factor receptor; the enzyme is selected from the group consisting of recombinases, polymerases, helicases, transposases, ligases, deaminases, oxidases and kinases.

13. The substrate of claim 3, wherein the features are separated by gap regions lacking target polynucleotides.

14. The substrate of claim 13, wherein the features comprise beads, holes, channels, ridges, or combinations thereof.

15. The substrate of claim 1, wherein the target molecule comprises a small molecule organic compound or an inorganic compound.

16. The substrate of claim 1, wherein the target molecule comprises a polypeptide, a polynucleotide, a carbohydrate, a ligand, or an antigen.

17. The substrate of claim 1, wherein the target molecule comprises a monosaccharide, an amino acid, a nucleotide, or a hapten.

18. The substrate of claim 6, wherein two or more of the features comprise different single target molecules.

19. The substrate of claim 8, wherein the DNA is double-stranded DNA (dsDNA).

20. The substrate of claim 12, wherein the antibody is a Fab, Fab', F(ab')2, scFv, diabody, triabody, minibody, or single domain antibody (sdAB).

21. The substrate of claim 12, wherein the epidermal growth factor receptor is HER2.

22. The substrate of claim 13, wherein the features comprise protrusions.

23. A method of placing a single target molecule on a feature of a substrate and forming the substrate of any one of claims 1-2 and 4-22, comprising: a. hybridizing a plurality of first and second capture primers immobilized to features on said substrate, b. using at least one target polynucleotide comprising a target region flanked by first and second capture primer binding regions complementary to said first and second capture primers, wherein said second capture primer binding region comprises a base pair mismatch with said second capture primer and is attached to a target molecule, and c. amplifying the at least one target polynucleotide to produce a plurality of clonal amplicons complementary to the target polynucleotide at an average amplification rate that exceeds an average rate of transport of the target polynucleotides to a characteristic, wherein the amplifying comprises isothermal amplification.

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