Low-binding support for improved solid-phase DNA hybridization and amplification

Abstract

To provide a method for determining a nucleic acid sequence, capable of reducing a non-specific background signal, thereby improving a contrast noise ratio (CNR), thereby improving accuracy of base calling and potentially reducing a cycle period, and reducing dependency to an accurate optical system and expensive equipment for a sequence determination process.SOLUTION: A method comprises: a) a step for providing a coupled surface to at least one polymer layer including a polymer coupled to a first nucleic acid molecule; b) a step for bringing the first nucleic acid molecule into contact with a second nucleic acid molecule so that the second nucleic acid molecule is coupled to the first nucleic acid molecule; c) a step for, after step b), magnifying the second nucleic acid molecule or a derivative thereof; and d) a step for acquiring a fluorescent image of the surface.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] cross reference This application claims the interests of U.S. Provisional Patent Application No. 62 / 767,343 filed on 14 November 2018, U.S. Provisional Patent Application No. 62 / 776,898 filed on 7 December 2018, and U.S. Patent Application No. 16 / 363,842 filed on 25 March 2019, all of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Various DNA sequencing methods have been developed and commercialized over the past 20 years (see, for example, E. Mardis (2008), "Next-Generation DNA Sequencing Methods," Annu. Rev. Genomics Hum. Genet. 9:387-402; and J. Heather and B. Chain, (2016), "The Sequence of Sequencers: The History of Sequencing DNA," Genomics 107:1-8). Many "second-generation" and "third-generation" sequencing technologies utilize a massively parallel circular array approach to synthetic sequencing (SBS), in which the accurate decoding of single-stranded template oligonucleotide sequences linked to a solid support depends on the successful polymerase-mediated classification of signals generated from the stepwise addition of A, G, C, and T nucleotides against complementary oligonucleotide chains. These methods typically require modifying oligonucleotide templates with fixed-length known adapter sequences, attaching them to a solid support in random or patterned arrays by hybridization to probes ligated to the surface of known sequences complementary to the adapter sequences, and then probing them using single-molecule (unamplified), synchronous synthesis sequencing (smSBS) approaches (e.g., Helicos technology) or single-molecule, asynchronous synthesis sequencing (smASBS) approaches (e.g., Pacific Biosciences technology). In the smSBS approach, fluorescently tagged terminator nucleotides are used so that the replication enzyme can incorporate only one base per cycle. In the Helicos technology, for example, a single fluorescent tag is used, and sequential induction of A, G, C, T was performed with one base per cycle. During each cycle, an imaging step was performed to classify the exact "base" for each single-molecule template on the array. After the imaging process, the reversibly linked tags are removed so that the replicating enzyme (polymerase) can incorporate the next template base.These cycles are repeated many times to eventually encode template oligonucleotide strands on a random array and determine their respective sequences.

[0003] While the cyclic array approach has been successful in general, it suffers from two inherent drawbacks: (i) long sampling times for adding individual sequences of nucleotides to the complementary strand, and (ii) weak signals resulting from the stepwise addition of single nucleotides (typically detected through the use of fluorescent labeling and fluorescence imaging techniques), exhibiting a low contrast-to-noise ratio (CNR), as discussed in more detail below, and therefore requiring long imaging times and expensive equipment, including high-precision optics, to achieve accurate base-calling.

[0004] Addressing the cycle time problem in cyclic array sequencing approaches has been attempted, for example, through the emergence of the single-molecule asynchronous synthesis sequencing (smASBS) approach, a technique from Pacific Biosciences where, for example, four distinct fluorescent tags are ligated to each of the A, G, C, and T nucleotides, and their addition can then be classified in "real time." In this approach, all four labeled nucleotides are induced simultaneously, and the image is acquired during the entire chain replication process. Each position in the sequence is classified as "A," "G," "C," and "T" based on the spectrum of the detected light. Here, the cycle time can theoretically be as fast as the replication rate of the polymerase catalyst, but the trade-off is a decrease in CNR, thereby introducing classification errors that ultimately lead to reduced accuracy and heavily relying on high-precision optics and expensive equipment.

[0005] Attempts to address the signal limitation problem in several circular array sequencing approaches (i.e., non-single-molecule approaches) have been made by incorporating an amplification step into the process. Solid-phase amplification of template DNA molecules ligated to a solid support in random or patterned arrays increases the number of copies of the target being sequenced, after detectable bases are added stepwise to their respective complementary strands, allowing the signals emanating from the "colonies" of the replicated template molecules to be classified as "A," "G," "C," or "T." The possibility of good classification (and therefore accuracy of base calling) depends on each CNR during each detection event, which is often limited.

[0006] Therefore, there is a need for improved solid supports and solid-phase amplification methods for nucleic acid sequencing that increase the magnitude of the base addition signal, reduce nonspecific background signal, and thus improve the CNR, thereby improving the accuracy of base calling, potentially reducing cycle time, and decreasing the reliance of the sequencing process on high-precision optics and expensive equipment. [Overview of the project]

[0007] Some embodiments relate to a method for performing nucleic acid sequencing, the method comprising the steps of a) providing a surface, wherein the surface is at least one separate region of the surface comprising i) a substrate, ii) at least one hydrophilic polymer coating layer, iii) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer, and iv) a plurality of clone-amplified sample nucleic acid molecules immobilized on the attached oligonucleotide molecules, wherein the plurality of clone-amplified sample nucleic acid molecules are at least 5000 molecules / mm³ 2The method includes a step of a) a) performing a nucleic acid amplification reaction on the sample nucleic acid molecules before or after annealing them to a plurality of oligonucleotide molecules, and c) performing a reaction of at least a single nucleotide binding or incorporation, wherein the nucleotide is labeled with a detectable tag. In some embodiments, the method further includes a step of detecting and characterizing the nucleotide based on the detectable tag.

[0008] Disclosed herein is a surface comprising a substrate, at least one layer of a hydrophilic, nonspecific low-binding (i.e., low-background) coating, and a plurality of oligonucleotide molecules attached to at least one layer of the hydrophilic, low-binding, low-background coating.

[0009] The disclosed nonspecific low-binding solid supports may, but are not limited to, be used in a variety of biological tests, including DNA sequencing and genotyping. These supports comprise a temperature- and chemically stable functionalized substrate, which withstands numerous solvent exchanges and temperature changes, thereby providing nonspecific low-binding over the duration of the assay. The disclosed supports may have some or all of the following properties:

[0010] 1. Surface functionalization performed using any combination of polar protic solvents, polar aprotic solvents, and / or nonpolar solvents results in a greater than 5% increase in the effectiveness of the bioassay function compared to conventional approaches (e.g., improvement in reaction rate and / or formation of the desired product).

[0011] 2. Measurement of the minimum contact angle after functionalization (e.g., less than 35 degrees), which is maintained by continuous solvent and temperature changes.

[0012] 3. Specifically bound molecules versus nonspecific low binding of biomolecules (e.g., more than 1 specifically bound molecules versus less than 0.25 nonspecifically bound molecules / region of interest). When using any variety of detection methods, this can directly lead to an improved contrast-to-noise ratio (CNR).

[0013] Disclosed herein is a surface comprising: a) a substrate; b) at least one hydrophilic polymer coating layer; c) a plurality of oligonucleotide molecules attached to the at least one hydrophilic polymer coating layer; and d) at least one separate region of the surface containing clone-amplified sample nucleic acid molecules annealed to the plurality of attached oligonucleotide molecules, wherein the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 20.

[0014] In some embodiments, a fluorescence image of a surface exhibits a contrast-to-noise ratio (CNR) of at least 20 when the sample nucleic acid molecule or its complementary sequence is labeled with a cyanine dye-3 (Cy3) fluorophore, and the fluorescence image is acquired under non-signal saturation conditions while the surface is immersed in a buffer (e.g., 25 mM ACES, pH 7.4 buffer) by an Olympus IX83 inverted fluorescence microscope equipped with a 20x, 0.75 NA lens, a 532 nm light source, a 532 nm long-pass excitation and a bandpass and dichroic mirror filter set optimized for Cy3 fluorescence emission, and a camera (e.g., Andor sCMOS, Zyla 4.2). In some embodiments, the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 40.

[0015] In some embodiments, the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 60. In some embodiments, the substrate includes glass. In some embodiments, the substrate includes plastic. In some embodiments, at least one hydrophilic polymer coating layer includes PEG. In some embodiments, the surface includes a second hydrophilic polymer coating layer. In some embodiments, at least one hydrophilic polymer layer includes a branched hydrophilic polymer having at least four branches, e.g., PEG. In some embodiments, at least one hydrophilic polymer layer includes a branched hydrophilic polymer having at least eight branches, e.g., PEG. In some embodiments, at least one hydrophilic polymer layer includes a branched hydrophilic polymer having at least sixteen branches, e.g., PEG. In some embodiments, at least one hydrophilic polymer layer includes a branched hydrophilic polymer having at least 32 branches, e.g., PEG. In some embodiments, the plurality of oligonucleotide molecules are at least 50,000 molecules / μm 2 They exist at a surface density of . In some embodiments, multiple oligonucleotide molecules are present at a density of at least 100,000 molecules / μm 2 They exist at a surface density of . In some embodiments, multiple oligonucleotide molecules are present at a density of at least 500,000 molecules / μm 2They exist at a surface density. In some embodiments, the sample nucleic acid molecule was administered at a concentration of 500 nM or less before annealing and clonal amplification. In some embodiments, the sample nucleic acid molecule was administered at a concentration of 20 pM or less before annealing and clonal amplification. In some embodiments, the sample nucleic acid molecule comprises a single-stranded multimeric nucleic acid molecule containing repeats of regularly occurring monomer units. In some embodiments, the length of the single-stranded multimeric nucleic acid molecule is at least 10 kb. In some embodiments, the surface further comprises copies of double-stranded monomers of regularly occurring monomer units. In some embodiments, the surface is located inside a flow channel. In some embodiments, multiple oligonucleotide molecules exist at a uniform surface density across the surface. In some embodiments, multiple oligonucleotide molecules are present at a first location on the surface at a density of at least 100,000 molecules / μm 2It is present at a surface density and at a second local surface density at a second location on the surface. In some embodiments, the background fluorescence intensity measured in a region of the surface laterally displaced from at least one separate region is no more than twice the intensity measured in at least one separate region before clonal amplification. In some embodiments, the surface includes a first layer comprising a monolayer of polymer molecules bonded to the surface of the substrate, a second layer comprising polymer molecules bonded to the polymer molecules of the first layer, and a third layer comprising polymer molecules bonded to the polymer molecules of the second layer, wherein at least one layer comprises branched polymer molecules. In some embodiments, the third layer comprises oligonucleotides bonded to the polymer molecules of the third layer. In some embodiments, the oligonucleotides bonded to the polymer molecules of the third layer are distributed at multiple depths throughout the third layer. In some embodiments, the surface further comprises a fourth layer comprising branched polymer molecules bonded to the polymer molecules of the third layer, and a fifth layer comprising polymer molecules bonded to the branched polymer molecules of the fourth layer. In some embodiments, a single polymer molecule of the fifth layer contains oligonucleotides linked to the polymer molecules of the fifth layer. In some embodiments, the oligonucleotides linked to the polymer molecules of the fifth layer are distributed at multiple depths throughout the fifth layer. In some embodiments, at least one hydrophilic polymer coating layer contains molecules selected from the group consisting of polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, and dextran. In some embodiments, the surface image shows the fluorescence intensity of a specifically amplified Cy3-labeled sample nucleic acid molecule or its complementary sequence, and a nonspecific Cy3 dye adsorption background (Binter ) shows a ratio of at least 3:1 with (...). In some embodiments, the image of the surface is the fluorescence intensity of specifically amplified Cy3-labeled sample nucleic acid molecules, or their complementary sequences, combined with non-specific Cy3-dye adsorption background and non-specific amplification background (B inter + B intra ) shows a ratio of at least 3:1 with (...). In some embodiments, the image of the surface is the fluorescence intensity of specifically amplified Cy3-labeled sample nucleic acid molecules, or their complementary sequences, combined with non-specific Cy3-dye adsorption background (B inter ) shows a ratio of at least 5:1 with (...). In some embodiments, the image of the surface is the fluorescence intensity of specifically amplified Cy3-labeled sample nucleic acid molecules, or their complementary sequences, combined with non-specific Cy3-dye adsorption background and non-specific amplification background (B inter + B intra ) shows a ratio of at least 5:1 with (...).

[0016] Furthermore, disclosed herein is a surface comprising: a) a substrate; b) at least one hydrophilic polymer coating; and c) a plurality of oligonucleotide molecules attached to at least one of the hydrophilic polymer coating layers, wherein the surface exhibits a level of non-specific Cy3-dye adsorption of less than about 0.25 molecules / μm 2 In some embodiments, the surface exhibits a level of non-specific Cy3-dye adsorption of less than about 0.1 molecules / μm

[0017] In some embodiments, the surface exhibits a ratio of specific Cy3-oligonucleotide labeling to non-specific Cy3-dye adsorption greater than about 4:1. In some embodiments, the surface exhibits a ratio of specific Cy3-oligonucleotide labeling to non-specific Cy3-dye adsorption greater than about 10:1. In some embodiments, the plurality of oligonucleotide molecules are at least 10,000 molecules / μm 2 In some embodiments, the surface exhibits a level of non-specific Cy3-dye adsorption of less than about 0.1 molecules / μm. In some embodiments, the surface exhibits a ratio of specific Cy3-oligonucleotide labeling to non-specific Cy3-dye adsorption greater than about 4:1. In some embodiments, the surface exhibits a ratio of specific Cy3-oligonucleotide labeling to non-specific Cy3-dye adsorption greater than about 10:1. In some embodiments, the plurality of oligonucleotide molecules are at least 10,000 molecules / μm 2They are attached at a surface density of . In some embodiments, multiple oligonucleotide molecules are attached at a density of at least 100,000 molecules / μm 2The surface is attached at a surface density. In some embodiments, the surface further comprises multiple clone-amplified clusters of template molecules annealed to multiple oligonucleotide molecules, where the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, at least one of at least two hydrophilic polymer layers comprises branched polyethylene glycol (PEG) molecules. In some embodiments, the surface comprises the surface of a capillary lumen, or at least one inner surface of a flow cell. In some embodiments, the capillary lumen or flow cell is configured for use in performing nucleic acid hybridization, amplification, or sequencing reactions, or any combination thereof. In some embodiments, the surface further comprises a branched polymer barrier layer. In some embodiments, the branched polymer barrier layer is a branched PEG barrier layer. In some embodiments, the branched polymer barrier layer is covalently bonded to the best hydrophilic polymer layer. In some embodiments, the polymer of the first layer contains primary amine functional groups, and the polymer of the second layer contains N-hydroxysuccinimide (NHS) ester functional groups, and after the deposition of the second layer, the second layer is linked to the first layer using covalent amide bonds. In some embodiments, the polymer molecules of the first layer contain N-hydroxysuccinimide (NHS) ester functional groups and the polymer molecules of the second layer contain primary amine functional groups, and after the deposition of the second layer, the second layer is linked to the first layer using covalent amide bonds. In some embodiments, oligonucleotides are linked to the polymer molecules of the second or third layer in a molar ratio of oligonucleotides to polymer molecules of about 1:5. In some embodiments, oligonucleotides are linked to the polymer molecules of the second or third layer in a molar ratio of oligonucleotides to polymer molecules of about 2:5.In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 3:5. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 4:5. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 1:1. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 4:1. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 8:1. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 16:1. In some embodiments, oligonucleotides are linked to polymer molecules in a second or third layer in a molar ratio of approximately 32:1 between oligonucleotides and polymer molecules. In some embodiments, oligonucleotides are present at a surface density of at least 10,000 molecules / square micrometer. In some embodiments, oligonucleotides are present at a surface density of at least 100,000 molecules / square micrometer. In some embodiments, oligonucleotides are uniformly distributed within the third layer.

[0018] Disclosed herein is a method for depositing oligonucleotides on a substrate surface, the method comprising: a) conjugating a first hydrophilic polymer onto the substrate surface in a first layer; b) conjugating a second hydrophilic polymer onto the first layer to form a second layer, wherein the hydrophilic polymer molecules of the second layer are bonded to the first layer by at least two covalent bonds per molecule; and c) conjugating an outermost hydrophilic polymer onto the second layer, wherein the outermost hydrophilic polymer molecules include oligonucleotide molecules that are covalently attached to it before conjugation onto the second layer.

[0019] In some embodiments, the hydrophilic polymer molecules of the second layer are bonded to the first layer by at least four covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the second layer are bonded to the first layer by at least eight covalent bonds per molecule. In some embodiments, the method further includes directly conjugating the outermost hydrophilic polymer to the second layer. In some embodiments, the method further includes conjugating a third hydrophilic polymer to the second layer and conjugating the outermost hydrophilic polymer to the second layer via the third layer to form a third layer. In some embodiments, the method further includes the steps of conjugating a third hydrophilic polymer to the second layer to form a third layer, conjugating a fourth hydrophilic polymer to the third layer to form a fourth layer, and conjugating the outermost hydrophilic polymer to the second layer via the third and fourth layers. In some embodiments, the first hydrophilic polymer includes PEG. In some embodiments, the first hydrophilic polymer comprises PGA. In some embodiments, at least one of the hydrophilic polymer layers comprises a branched polymer. In some embodiments, the branched polymer comprises at least four branches. In some embodiments, the branched polymer comprises at least eight branches. In some embodiments, the branched polymer comprises 16 to 32 branches. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least two covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least four covalent bonds per molecule. In some embodiments, the hydrophilic polymer molecules of the fourth layer are bonded to the third layer by at least eight covalent bonds per molecule. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing ethanol. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing methanol. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing dimethyl sulfoxide (DMSO). In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing acetonitrile.In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing phosphate buffer. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing buffered 3-(N-morpholino)propanesulfonic acid (MOPS). In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing a buffer consisting of 75% acetonitrile and 25% sulfonic acid. In some embodiments, the hydrophilic polymer is delivered to the substrate surface in a solvent containing a buffer consisting of 90% methanol and 10% MOPS.

[0020] Disclosed herein is a surface comprising oligonucleotides having a surface density of at least 10,000 molecules / square micrometer, wherein the oligonucleotides are linked to the surface via a multilayer hydrophilic polymer layer, and wherein the oligonucleotides are evenly distributed throughout the outermost layer of the multilayer hydrophilic polymer layer.

[0021] In some embodiments, the oligonucleotides are distributed at a surface density of at least 50,000 molecules / square micrometer. In some embodiments, the oligonucleotides are distributed at a surface density of at least 100,000 molecules / square micrometer. In some embodiments, the oligonucleotides are distributed at a surface density of at least 500,000 molecules / square micrometer. In some embodiments, at least 10% of the linked oligonucleotides are annealed to the oligonucleotides of the target (or sample). In some embodiments, the multilayer hydrophilic polymer layer is saturated with a hydrophilic solvent. In some embodiments, the surface includes the surface of a glass, fused quartz, silicon, or polymer (e.g., plastic) substrate. In some embodiments, the multilayer hydrophilic polymer layer includes three or more polymer layers. In some embodiments, the multilayer hydrophilic polymer layer includes five or more polymer layers. In some embodiments, one or more layers of the hydrophilic polymer formation contain branched PEG, branched PVA, branched poly(vinylpyridine), branched PVP, branched PAA, branched PNIPAM, branched PMA, branched PHEMA, branched PEGMA, branched PGA, branched polylysine, branched polyglucoside, or dextran. In some embodiments, one or more layers of the hydrophilic polymer formation contain branched PEG molecules. In some embodiments, the branched PEG molecules contain at least four branches. In some embodiments, the branched PEG molecules contain at least eight branches. In some embodiments, the branched PEG molecules contain 16 to 32 branches. In some embodiments, at least the first and second layers of the hydrophilic polymer formation are linked to each other using amide covalent bonds. In some embodiments, at least the first and second layers of the hydrophilic polymer formation are linked to each other by at least two covalent bonds per polymer molecule. In some embodiments, at least a first and a second layer of the hydrophilic polymer formation are linked to each other by at least four covalent bonds per polymer molecule.In some embodiments, at least a first and second layer of the hydrophilic polymer formation are linked to each other by at least eight covalent bonds per polymer molecule. In some embodiments, the surface density of the linked oligonucleotides is at least 50,000 molecules / square micrometer. In some embodiments, the surface density of the linked oligonucleotides is at least 100,000 molecules / square micrometer. In some embodiments, the surface density of the CY3 dye is 0.25 molecules / μm. 2It exhibits nonspecific binding of less than 150. In some embodiments, the surface further comprises clusters of clone-amplified copies of the annealed target oligonucleotide, where substantially all of the clone-amplified copies of the annealed target oligonucleotide comprise a Cy3-labeled nucleotide annealed at a first position, and where the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of at least 20. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, the contrast-to-noise ratio (CNR) is at least 150. In some embodiments, the contrast-to-noise ratio (CNR) is at least 200. In some embodiments, the clone-amplified copies of the annealed target oligonucleotide are prepared using a bridge amplification protocol. In some embodiments, the clone-amplified copies of the annealed target oligonucleotide are prepared using an isothermal bridge amplification protocol. In some embodiments, the clone-amplified copies of the annealed target oligonucleotide are prepared using a rolling circle amplification (RCA) protocol. In some embodiments, cloned amplified copies of the annealed target oligonucleotide are prepared using a helicase-dependent amplification protocol. In some embodiments, cloned amplified copies of the annealed target oligonucleotide are prepared using a recombinase-dependent amplification protocol. In some embodiments, cloned amplified copies of the annealed target oligonucleotide are prepared using a single-strand binding (SSB) protein-dependent amplification protocol. In some embodiments, the surface includes the surface of the capillary lumen or at least one inner surface of the flow cell. In some embodiments, the capillary lumen or flow cell is configured for use in performing nucleic acid hybridization, amplification, or sequencing reactions, or any combination thereof.

[0022] Disclosed herein is a method for performing solid-phase nucleic acid hybridization, the method comprising: a) providing one of the surfaces disclosed herein; and b) performing a solid-phase nucleic acid hybridization reaction in which a template nucleic acid molecule is annealed to a linked oligonucleotide. Further disclosed herein is a method for performing solid-phase nucleic acid amplification, the method comprising: a) providing one of the surfaces disclosed herein; and b) performing a solid-phase nucleic acid amplification reaction using a template nucleic acid molecule hybridized to a linked oligonucleotide.

[0023] In some embodiments, solid-phase nucleic acid amplification includes thermal cycling. In some embodiments, solid-phase nucleic acid amplification includes isothermal amplification. In some embodiments, solid-phase nucleic acid amplification includes rolling circle amplification. In some embodiments, solid-phase nucleic acid amplification includes bridge amplification. In some embodiments, solid-phase nucleic acid amplification includes isothermal bridge amplification. In some embodiments, solid-phase nucleic acid amplification includes polysubstitution amplification. In some embodiments, solid-phase nucleic acid amplification includes helicase treatment. In some embodiments, solid-phase nucleic acid amplification includes recombinase treatment. In some embodiments, there is no change in the surface density of the linked oligonucleotide over at least 30 cycles of the solid-phase nucleic acid amplification reaction. In some embodiments, there is no change in the surface density of the linked oligonucleotide over at least 40 cycles of the solid-phase nucleic acid amplification reaction. In some embodiments, there is no change in the surface density of the linked oligonucleotide over at least 50 cycles of the solid-phase nucleic acid amplification reaction.

[0024] Disclosed herein is a method for performing nucleic acid sequencing, the method comprising: a) providing one of the surfaces disclosed herein; b) performing a solid-phase nucleic acid amplification reaction using a template nucleic acid molecule hybridized to linked oligonucleotides; and c) performing a periodic sequence of single nucleotide linking or incorporation reactions, wherein the nucleotides are labeled with detectable tags.

[0025] In some embodiments, the detectable tag is a fluorophore. In some embodiments, the fluorophore is Cy3, where the fluorescence image of the surface is acquired under non-signal saturation conditions as separately described herein and exhibits a contrast-to-noise ratio (CNR) of at least 20 after binding or incorporation of the first Cy3-labeled nucleotide. In some embodiments, the contrast-to-noise ratio (CNR) is at least 50. In some embodiments, the contrast-to-noise ratio (CNR) is at least 100. In some embodiments, the contrast-to-noise ratio (CNR) is at least 150. In some embodiments, the contrast-to-noise ratio (CNR) is at least 200. In some embodiments, the solid-phase nucleic acid amplification reaction includes a bridge amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes an isothermal bridge amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a rolling circle amplification (RCA) reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a helicase-dependent amplification reaction. In some embodiments, the solid-phase nucleic acid amplification reaction includes a recombinase-dependent amplification reaction.

[0026] Disclosed herein is an apparatus for performing nucleic acid amplification, the apparatus comprising a) any one of the surfaces disclosed herein, wherein the surface comprises the surface of a capillary lumen or at least one inner surface of a flow cell.

[0027] In some embodiments, the device includes at least one fluid inlet within a capillary lumen. In some embodiments, the device further includes at least one fluid outlet. In some embodiments, the device further includes at least one pump. In some embodiments, the device further includes at least one fluid mixing connecting tube. In some embodiments, the device further includes at least one temperature control element. In some embodiments, the device further includes at least one optical window.

[0028] Disclosed herein is a system for performing nucleic acid sequencing, the system comprising: a) at least one of the apparatus disclosed herein; b) a fluid control module; and c) an imaging module.

[0029] Built-in by reference All publications, patents, and patent applications referenced herein are incorporated herein by reference in their entirety to the same extent that each individual publication, patent, or patent application is incorporated herein by reference specifically and individually. In the event of any conflict between the terminology of this specification and that of the incorporated literature, the terminology of this specification shall prevail. [Brief explanation of the drawing]

[0030] The patent or application file must include at least one drawing created in color. A copy of this patent or patent application publication containing the color drawing will be provided by the relevant office after the necessary fees have been requested and paid.

[0031] Novel features of the present invention are described in particular in the appended claims. For a better understanding of the features and advantages of the present invention, please refer to the following detailed description and accompanying drawings illustrating exemplary embodiments in which the principles of the present invention are used. [Figure 1] This provides a schematic diagram of one embodiment of a low-bonding solid support of the present disclosure, in which the support comprises a glass substrate and alternating layers of hydrophilic coatings further comprising chemically reactive functional groups that adhere to the glass covalently or non-covalently and serve as adhesion sites for oligonucleotide primers. [Figure 2] This provides a schematic diagram of the use of a silane reaction to covalently couple a first polymer to the surface of a substrate (e.g., glass) in order to fabricate a first polymer layer. [Figure 3] This provides a schematic diagram of a process in which a branched polymer is covalently coupled to the surface shown in Figure 2 in order to form a second polymer layer on the substrate surface. [Figure 4] This provides a schematic diagram of a coupling reaction used to covalently attach one or more oligonucleotide adapter or primer sequences (e.g., sequence 1 (dotted line) and sequence 2 (dashed line)) to a branched polymer. [Figure 5] This provides a schematic diagram of a process in which a branched polymer, including an oligonucleotide adapter or primer sequence attached by covalent bonds, is covalently coupled to the surface shown in Figure 3 in order to form a third polymer layer on the substrate surface. [Figure 6A] This provides examples of simulated fluorescence intensity data illustrating the difference between using signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) as criteria for evaluating data quality in nucleic acid sequencing and base calling applications. Figure 6A: Examples of simulated data with SNR=2 and CNR=1.25. [Figure 6B] This provides an example of simulated fluorescence intensity data illustrating the difference between using signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) as criteria for evaluating data quality in nucleic acid sequencing and base calling applications. Figure 6B: Example of simulated data with SNR=2 and CNR=12.29. [Figure 7] This provides an example of how improved CNR affects the imaging time required for accurate detection and signal classification (base calling) of cloned nucleic acid colonies on a solid support. [Figure 8] The figures show various images obtained for different cycles of nucleic acid sequencing reactions performed on a solid support with various labeled nucleotides incorporated into complementary strands for each clone-amplified template molecule. The figures also show various background contributions to the overall detection signal for detection platforms requiring repeat spot detection through the distinction of nucleotide-specific signals from noisy intervening and endogenous background images. [Figure 9]This provides an example of image data from a study determining the relative levels of nonspecific binding of a green fluorescent dye to glass substrate surfaces treated according to various surface modification protocols. [Figure 10] This provides an example of image data from a study determining the relative levels of nonspecific binding of a red fluorescent dye to glass substrate surfaces treated according to various surface modification protocols. [Figure 11] This provides examples of data for oligonucleotide primer grafts for substrate surfaces treated according to various surface modification protocols. [Figure 12] This provides examples of replicated images obtained during testing of nonspecific bonding on substrate surfaces treated according to various surface modification protocols. [Figure 13] This provides examples of data for the nonspecific binding of sequencing dye mixtures to substrate surfaces treated according to various surface modification protocols. For comparison, the fluorescence intensity measured under the same set of experimental conditions for the nonspecific binding of the sequencing dye mixture to a single bead grafted with Cy3-labeled oligonucleotides is approximately 1,500 counts. [Figure 14] This provides examples of images and data of nonspecific binding of green and red fluorescent dyes to substrate surfaces treated according to various surface modification protocols. For comparison purposes, the fluorescence intensity of cloned amplified template colonies, measured under the same set of experimental conditions after coupling a single Cy3-labeled nucleotide base, is approximately 1,500 counts. [Figure 15] This provides examples of images and data demonstrating "tunable" nucleic acid amplification on low-binding solid supports by varying the oligonucleotide primer density on the substrate. Blue histogram: low primer density. Red histogram: high primer density. The combination of non-specific low binding and tunable nucleic acid amplification efficiency through adjustment of oligonucleotide primer density results in high CNR and subsequent improvements in nucleic acid sequencing performance. [Figure 16]This provides examples of fluorescence imaging of low-binding solid supports of the present disclosure, where linked oligonucleotides are amplified using various primer densities, isothermal amplification methods, and amplification buffer additives. [Figure 17] This provides an example of a gel image demonstrating the reduction of nonspecific nucleic acid amplification through the use of amplification buffer additives, while maintaining specific amplification of the target sequence. The gel image shows bands corresponding to the specific amplification of the target (arrow) and the amplification product quantified by other gels. [Figure 18] This provides an example of fluorescence imaging demonstrating the effect of formulation changes on improving amplification specificity on low-binding support surfaces. [Figure 19A] This provides non-limiting examples of image data demonstrating improvements in the rigor, speed, and efficacy of hybridization that can be achieved by modifying the composition of the hybridization buffer used for solid-phase nucleic acid amplification, as described herein. Figure 19A provides examples of image data for two different hybridization buffer formulations and protocols. [Figure 19B] This provides non-limiting examples of image data demonstrating improvements in the rigor, speed, and efficacy of hybridization that can be achieved by modifying the composition of the hybridization buffer used for solid-phase nucleic acid amplification, as described herein. Figure 19B provides an example of corresponding image data obtained using standard hybridization buffers and protocols. [Figure 20] This illustrates a nucleic acid sequencing workflow and non-limiting examples of achievable processing times using the disclosed low-binding support and the amplification reaction formulation of the present disclosure. [Figure 21] This provides examples of fluorescence imaging and intensity data of the low-binding supports of this disclosure, in which solid-phase nucleic acid amplification is performed to produce clone-amplified clusters of template oligonucleotide sequences. [Figure 22] This provides a second example of fluorescence imaging and intensity data of the low-binding support of this disclosure, in which solid-phase nucleic acid amplification was performed to produce cloned amplified clusters of a template oligonucleotide sequence. [Figure 23] This provides examples of fluorescence imaging and intensity data of the low-binding supports of this disclosure, in which solid-phase nucleic acid amplification is performed to produce clone-amplified clusters of template oligonucleotide sequences. [Figure 24] This provides an example of a fluorescence calibration curve used to estimate the surface density of primer oligonucleotides linked to a support surface. [Figure 25A] This provides non-limiting examples of modified glass and polymer surfaces of the present disclosure to which amplicons containing fluorescently labeled nucleotides are conjugated. Figure 25A: Modified glass surface. In the inset, it will be understood that the surface produces 226 CNRs. [Figure 25B] This provides non-limiting examples of modified glass and polymer surfaces of the present disclosure to which amplicons containing fluorescently labeled nucleotides are conjugated. Figure 25B: Modified plastic surface. In the inset, it will be understood that the surface produces 10⁹ CNRs. [Figure 26A] This provides an analysis of the images in Figures 25A and 25B. In Figure 26A, the signal intensity for each glass and plastic surface can be seen on the left, and the background intensity on the right. For each, the signal intensity is substantially greater than the background intensity. [Figure 26B] This provides an analysis of the images in Figures 25A and 25B. In Figure 26B, a graphical representation of the CNR values ​​for each glass and plastic surface can be seen. On the left, the glass produces a CNR of 226, while on the right, the plastic produces a CNR of 109, consistent with the insets in Figures 25A and 25B. [Figure 27] This provides surface analysis in relation to the accuracy of the data. The data is collected in two channels and plotted from left to right, for commercially available low-CNR and high-CNR surfaces respectively, as shown from the scattering pod (top) and quantitatively (bottom). [Figure 28]This provides schematic diagrams of oligonucleotide sequences of a multimerized target hybridized to a surface with a high surface density of oligonucleotide adapter or primer molecules (left) and a surface with a lower surface density of oligonucleotide adapter or primer molecules (right). [Figure 29] This provides a comparison of experimental results obtained by performing a conventional hybridization reaction on the low-binding support surface of the present disclosure with experimental results obtained by performing an optimized hybridization reaction on the low-binding support surface of the present disclosure. [Figure 30] This disclosure provides drawings of experimental results in which a conventional hybridization reaction is performed on a low-binding support of this disclosure, followed by RCA or bridge amplification. [Figure 31] This disclosure provides drawings of experimental results in which an optimized hybridization reaction is performed on a low-binding support, followed by RCA or bridge amplification. [Figure 32] The present disclosure provides drawings of experimental results in which an optimized hybridization reaction is performed on a low-binding support of the present disclosure, prepared using improved coupling chemistry to attach oligonucleotide adapter or primer molecules to the surface, followed by RCA or bridge amplification. [Figure 33] This provides non-limiting examples of fluorescence images of conventional support surfaces and fluorescence images of low-binding support surfaces of the present disclosure in which target oligonucleotides are hybridized and amplified. The plots show contrast-to-noise ratios measured from images such as those provided for polyacrylamide surfaces with a bridge amplification protocol, low-binding supports with a standard bridge amplification protocol and a primer density of less than 1000 oligonucleotides / µm², and low-binding support surfaces of the present disclosure in combination with an improved solid-phase nucleic acid amplification method on surfaces with a primer density greater than 1000 oligonucleotides / µm². [Modes for carrying out the invention]

[0032] Disclosed herein are novel solid supports for use in solid-phase nucleic acid amplification and sequencing or other bioassay applications. The solid supports disclosed herein exhibit non-specific low binding to proteins and other amplification reaction components and show improved stability against repeated exposure to various solvents, temperature changes, low pH, or chemical ingress such as long-term storage.

[0033] Either alone or in combination with improved nucleic acid hybridization and amplification protocols, some supports disclosed herein cause one or more of the following: (i) reduced requirements for the amount of starting material needed, (ii) reduced temperature requirements for isothermal or thermal ramping amplification protocols, (iii) increased amplification rate, (iv) increased amplification specificity (i.e., reduced nonspecific amplification of surface primers and primer dimers, while more selective amplification of single-strand template molecules in amplified colonies), and (v) greater distinguishability of sequence-specific signals from background signals (such as signals originating from the background in the interstitium and tissue parenchyma), thereby providing improved contrast-to-noise ratio (CNR) and base-calling accuracy compared to conventional nucleic acid amplification and sequencing methods.

[0034] A starting point for achieving the aforementioned improvements, or any combination thereof, is a disclosed nonspecific low-binding support comprising one or more polymer coatings, e.g., PEG polymer films, that minimize nonspecific binding of proteins and labeled nucleotides to a solid support. Improved nucleic acid hybridization and amplification rates, and subsequent demonstration of specificity, may be achieved through one or more of the following further embodiments of the present disclosure: (i) primer design (sequence and / or modification), (ii) control of the density of linked primers on the solid support, (iii) surface composition of the solid support, (iv) polymer density on the surface of the solid support, (v) use of improved hybridization conditions before and / or during amplification, and / or (vi) use of improved amplification formulations that reduce nonspecific primer amplification or increase template amplification efficiency.

[0035] The advantages of the disclosed nonspecific low-binding support and associated hybridization and amplification methods provide one or more of the following further advantages for any sequencing system: (i) reduced fluid washing time (due to reduced nonspecific binding, and therefore faster sequencing sancle time); (ii) reduced imaging time (and therefore faster time required for assay readout and sequencing cycles); (iii) reduced overall workflow time requirements (due to reduced sancle time); (iv) reduced detection equipment costs (due to improvements in CNR); (v) improved readout (base calling) accuracy (due to improvements in CNR); (vi) improved reagent stability and reduced reagent usage requirements (and therefore reduced reagent costs); and (vii) reduced runtime failures due to nucleic acid amplification failures.

[0036] Low-binding hydrophilic surfaces (multilayer and / or monolayer) for surface bioassays, such as genotyping assays and sequencing assays, are prepared by using any combination of the following:

[0037] Polar protic solvents, polar aprotic solvents, and / or nonpolar solvents for depositing and / or coupling linear or branched hydrophilic polymer subunits on a substrate surface. Some branched hydrophilic polymer subunits may contain functional end groups to facilitate covalent or non-covalent interactions with other polymer subunits. Examples of suitable functional end groups include biotin, methoxyether, carboxylate, amine, ester compound, azide, alkyne, maleimide, thiol, and silane groups.

[0038] Any combination of linear, branched, or polybranched polymer subunits coupled via a modified coupling chemistry / solvent / buffer system may include individual subunits having orthogonal-terminal coupling chemistry, or any combination thereof such that the resulting surface is hydrophilic and exhibits non-specific low binding to assay components of proteins and other molecules. In some examples, the hydrophilic, functionalized substrate surfaces of this disclosure exhibit contact angle measurements of 35 degrees or less.

[0039] In addition to the aforementioned solvents, a suitable buffer system having a desirable pH range of 5–10. Examples include, but are not limited to, phosphate-buffered saline, phosphate buffer, TAPS, MES, MOPS, or any combination thereof.

[0040] Subsequent biomolecular binding (e.g., proteins, peptides, nucleic acids, oligonucleotides, or cells) on a low-binding / hydrophilic substrate via any of the various individual conjugation chemistry methods or any combination thereof described below. The layer deposition and / or conjugation reaction may be carried out using a solvent mixture which may also include any ratio of components such as ethanol, methanol, acetonitrile, acetone, DMSO, DMF, H2O, etc. In addition, a suitable buffer system in a desired pH range of 5–10 may be used to control the rate and efficiency of deposition and coupling, thereby allowing the coupling rate to be more than five times that can be achieved by conventional aqueous buffer-based methods.

[0041] Definitions: Unless otherwise defined, all technical terms used herein have the same meaning as those generally understood by those skilled in the art of the field to which this disclosure belongs.

[0042] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” also include plural references unless explicitly indicated by the context. Any reference to “or” is intended to include “and / or” unless otherwise specified.

[0043] As used herein, a number preceded by the term "approximately" refers to a number that is plus or minus 10% of that number. When used in the context of a range, the term "approximately" refers to a range of minus 10% of the minimum value and plus 10% of the maximum value.

[0044] As used herein, in the context of a series, the phrase “at least one of” includes, on its own or in combination with elements not listed in the list, a single member of the series, two members of the series, all members of the series, or a list containing the following members.

[0045] As used herein, fluorescence is "specific" if it originates from a fluorophore that has a site on the surface that is inversely complementary to the corresponding segment of the oligo and is annealed to the surface, or otherwise linked, such as via a nucleic acid annealed to the corresponding segment. This fluorescence is in contrast to fluorescence originating from fluorophores that are not linked to the surface through such an annealing process, or, in some cases, background fluorescence of the surface.

[0046] Nucleic acids: As used herein, “nucleic acids” (also known as “polynucleotides,” “oligonucleotides,” “ribonucleic acid (RNA),” or “deoxyribonucleic acid (DNA)”) are linear polymers of two or more nucleotides joined by covalent nucleoside bonds, or variants or functional fragments thereof. In spontaneously occurring examples of nucleic acids, the nucleoside bond is typically a phosphate diester bond. However, other examples may optionally include other nucleoside bonds, such as phosphorothiolate bonds, and may or may not contain phosphate groups. Nucleic acids include double-stranded and single-stranded RNA, as well as double-stranded and single-stranded DNA, DNA / RNA hybrids, peptide nucleic acids (PNAs), hybrids between PNAs and DNA or RNA, and may further include other types of nucleic acid modifications.

[0047] As used herein, “nucleotide” refers to a nucleotide, nucleoside, or analogue thereof. In some cases, a nucleotide is an N-glycoside or C-glycoside of a purine or pyrimidine base (e.g., a deoxyribonucleoside containing 2-deoxy-D-ribose, or a ribonucleoside containing D-ribose). Examples of other nucleotide analogues include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, and 2-O-methylribonucleotides.

[0048] Nucleic acids can be optionally bound to one or more non-nucleotide portions, such as labels and other small molecules, large molecules (such as proteins, lipids, and sugars), and solid or semi-solid supports, for example, through covalent or non-covalent bonds to either the 5' or 3' end of the nucleic acid. The labels include any portion that is detectable using any of the various detection methods known to those skilled in the art, and that makes the thus attached oligonucleotide or nucleic acid similarly detectable. Some labels emit electromagnetic radiation that is optically detectable or visible. Alternating or together, some labels include mass tags that make the labeled oligonucleotide or nucleic acid visible in mass spectral data, or redox tags that make the labeled oligonucleotide or nucleic acid detectable by current or voltage measurements. Some labels include magnetic tags that facilitate the separation and / or purification of the labeled oligonucleotide or nucleic acid. Nucleic acids or polynucleotides are often detected directly without attachment to a label.

[0049] The disclosed nonspecific low-binding supports and associated nucleic acid hybridization and amplification methods may be used to analyze nucleic acid molecules derived from any of the various cells, tissues, or samples known to those skilled in the art. For example, nucleic acids may be extracted from cells or tissue samples containing one or more types of cells, derived from eukaryotes (such as animals, plants, fungi, and protists), primordial bacteria, or eubacteria. In some cases, nucleic acids may be extracted from prokaryotes or eukaryotic cells, such as adherent or non-adherent eukaryotic cells. Nucleic acids are extracted in diverse ways from, for example, primitive or immortalized rodents, pigs, cats, dogs, cattle, horses, primates, or human cell lines. Nucleic acids may be extracted from any of the various cells, organs, or tissues (e.g., white blood cells, red blood cells, platelets, epithelial cells, endothelial cells, neurons, glial cells, astrocytes, fibroblasts, skeletal muscle cells, smooth muscle cells, gametes, or cells of the heart, lungs, brain, liver, kidneys, spleen, pancreas, thymus, bladder, stomach, colon, or small intestine). Nucleic acids may be extracted from normal or healthy cells. Alternatingly or together, acids may be extracted from abnormal cells such as cancer cells, or from pathogenic cells that infect the host. Some nucleic acids may be extracted from a distinct subset of cells, such as immune cells (e.g., T cells, cytotoxic (killer) T cells, helper T cells, alpha-beta T cells, gamma-delta T cells, T cell precursors, B cells, B cell precursors, lymphoid stem cells, myeloid progenitor cells, lymphocytes, granulocytes, natural killer cells, plasma cells, memory cells, neutrophils, eosinophils, basophils, mast cells, monocytes, dendritic cells, and / or macrophages, or any combination thereof), undifferentiated human stem cells, human stem cells induced to differentiate, rare cells (e.g., circulating tumor cells (CTCs)), circulating epithelial cells, circulating endothelial cells, circulating endometrial cells, myeloid cells, progenitor cells, foam cells, mesenchymal cells, or trophoblasts), other cells as intended and consistent with the disclosure herein.

[0050] Nucleic acid extraction from cells or other biological samples may be carried out using any of the many techniques known to those skilled in the art. For example, a typical DNA extraction procedure includes (i) taking a cell or tissue sample from which DNA is to be extracted, (ii) disrupting the cell membrane (i.e., cell lysis) to release DNA and other cytoplasmic components, (iii) treating the lysed sample with a concentrated salt solution to precipitate proteins, lipids, and RNA, followed by centrifugation to separate the precipitated proteins, lipids, and RNA, and (iv) purifying the DNA from the supernatant to remove surfactants, proteins, salts, or other reagents used during the cell membrane lysis step.

[0051] Various suitable commercially available nucleic acid extraction and purification kits are consistent with the disclosure herein. Examples include, but are not limited to, the QIAamp kit (for isolation of genomic DNA from human samples) and the DNAeasy kit (for isolation of genomic DNA from animal or plant samples) from Qiagen (Germantown, MD), or the Maxwell® and ReliaPrep® kit series from Promega (Madison, WI).

[0052] Nonspecific Low-Binding Supports for Solid-Phase Nucleic Acid Hybridization and Amplification: Disclosed herein are solid supports comprising a nonspecific low-binding surface composition that enables improved nucleic acid hybridization and amplification performance. Generally, the disclosed supports may comprise a substrate (or support structure), one or more layers of low-binding chemically modified layers, e.g., silane layers, polymer films, attached by covalent or non-covalent bonds, and one or more covalently or non-covalently attached primer sequences that can be used to link single-stranded template oligonucleotides to the support surface (Figure 1). In some examples, the surface formulation, e.g., the chemical composition of one or more layers, the coupling chemistry used to crosslink one or more layers to and / or to each other, and the total number of layers may be varied so that nonspecific binding of proteins, nucleic acid molecules, and other hybridization and amplification reaction components to the support surface is minimized or reduced with respect to a comparable monolayer. Often, the surface formulation may be varied so that nonspecific hybridization on the support surface is minimized or reduced with respect to a comparable monolayer. The surface formulation may be varied so that nonspecific amplification on the support surface is minimized or reduced with respect to a comparable monolayer. The surface formulation may be varied so that the specific amplification rate and / or yield on the support surface is maximized. Amplification levels suitable for detection are achieved in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30 or less, or more than 30 amplification cycles, as disclosed herein.

[0053] Examples of materials from which substrates or support structures can be manufactured include, but are not limited to, glass, fused silica, silicon, polymers (e.g., polystyrene (PS), macroporous polystyrene (MPPS), polymethyl methacrylate (PMMA), polycarbonate (PC), polypropylene (PP), polyethylene (PE), high-density polyethylene (HDPE), cyclic olefin polymer (COP), cyclic olefin copolymer (COC), polyethylene terephthalate (PET), or any combination thereof). Various compositions of both glass and plastic substrates are intended.

[0054] The substrate or support structure may be reproduced in any of the various geometric shapes and dimensions known to those skilled in the art, and may include any of the various materials known to those skilled in the art. For example, in some examples, the substrate or support structure may be locally planar (e.g., including a microscope slide or the surface of a microscope slide). Overall, the substrate or support structure may be cylindrical (e.g., including a capillary or the inner surface of a capillary), spherical (e.g., including the outer surface of a non-porous bead), or irregular (e.g., including the outer surface of a non-porous bead or particle of an irregular shape). In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be a solid, non-porous surface. In some examples, the surface of the substrate or support structure used for nucleic acid hybridization and amplification may be porous, and the coatings described herein may penetrate the porous surface so that the nucleic acid hybridization and amplification reactions performed may occur within the pores.

[0055] A substrate or support structure comprising one or more chemically modified layers, such as a layer of nonspecific low-binding polymer, may be independent or integrated into another structure or assembly. For example, in some examples, the substrate or support structure may comprise one or more surfaces within an integrated or assembled microfluidic flow cell. The substrate or support structure may comprise one or more surfaces within a microplate format (e.g., the bottom surface of a well in a microplate). As described above, in some preferred embodiments, the substrate or support structure comprises the inner surface of a capillary (such as the lumen surface). In alternative preferred embodiments, the substrate or support structure comprises the inner surface of a capillary (such as the lumen surface) etched into a planar chip.

[0056] The chemically modified layer may be applied uniformly across the surface of the substrate or support structure. Alternatively, the surface of the substrate or support structure may be non-uniformly distributed or patterned so that the chemically modified layer is confined to one or more separate areas of the substrate. For example, the substrate surface may be patterned using photolithography techniques to create an ordered array or random pattern of chemically modified areas on the surface. Alternatingly or together, the substrate surface may be patterned using, for example, contact printing and / or inkjet printing techniques. In some examples, an ordered array or random pattern of chemically modified separate regions may include at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10,000, or more separate regions, or any intermediate number extending within the scope of this specification.

[0057] To achieve a non-specific low-bonding surface (also referred to herein as a “low-bonding” or “passivated” surface), the hydrophilic polymer may be non-specifically adsorbed or covalently grafted onto the substrate or support surface. Typically, passivation is carried out using poly(ethylene glycol) (PEG, also known as polyethylene oxide (PEO) or polyoxyethylene), poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM)), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate (PHEMA)), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA)), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, dextran, or other hydrophilic polymers of various molecular weights and having terminal groups linked to the surface using, for example, silane chemistry. The distal terminal groups on the surface are biotin, methoxyether, carboxylate, amine, N The materials may include, but are not limited to, HS esters, maleimides, and bissilanes. In some examples, two or more layers of hydrophilic polymers, e.g., linear polymers, branched polymers, or polybranched polymers, may be deposited on the surface. In some examples, the two or more layers may be covalently coupled to each other or internally crosslinked to improve the stability of the resulting surface. In some examples, oligonucleotide primers (or other biomolecules, e.g., enzymes or antibodies) with various base sequences and base modifications may be linked to the resulting surface layer at varying surface densities. In some examples, for instance, both the surface functional group density and the oligonucleotide concentration may be varied to target a specific primer density range. Furthermore, the primer density can be controlled by diluting the oligonucleotide with other molecules carrying the same functional groups.For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol in reaction with an NHS ester-coated surface to reduce the final primer density. Primers with linkers having different lengths between the hybridization region and the surface-adhering functional groups can also be applied to control surface density. Suitable linker examples include poly T chains and poly A chains (e.g., 0-20 bases), PEG linkers (e.g., 3-20 monomer units), and carbon chains (e.g., C6, C12, C18, etc.) at the 5' end of the primer. To measure primer density, fluorescently labeled primers may be ligated to the surface, and the fluorescence reading may be compared to that of a dye solution of known concentration.

[0058] In some embodiments, the hydrophilic polymer may be a crosslinked polymer. In some embodiments, the crosslinked polymer may include one polymer that is crosslinked with another polymer. Examples of crosslinked polymers include poly(ethylene glycol) crosslinked with polyethylene oxide (PEO) or polyoxyethylene, poly(vinyl alcohol) (PVA), poly(vinylpyridine), poly(vinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), polylysine, polyglucoside, streptavidin, dextran, or other polymers selected from other hydrophilic polymers. In some embodiments, the crosslinked polymer may be poly(ethylene glycol) crosslinked with polyacrylamide.

[0059] As a result of the surface passivation techniques disclosed herein, proteins, nucleic acids, and other biomolecules “do not adhere” to the substrate; i.e., they exhibit nonspecific low binding (NSB). An example is shown below using the preparation of a standard monolayer surface with varying glass preparation conditions. Hydrophilic surfaces passivated to achieve ultra-low NSB for proteins and nucleic acids require novel reaction conditions to improve primer deposition reaction efficiency, hybridization performance, and induce efficient amplification. All of these processes require attaching oligonucleotides to the low-binding surface, followed by protein binding and delivery. As described below, the combination of a novel primer surface conjugation formulation (Cy3 oligonucleotide graft titration) and the resulting ultra-low nonspecific background (NSB function tests were performed using red and green fluorescent dyes) produced results demonstrating the feasibility of the disclosed approach. Several surfaces disclosed herein exhibit both specific binding (e.g., hybridization to a linked primer or probe) and nonspecific binding (e.g., B) of fluorophores such as Cy3. inter ) exhibit a ratio of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value within the specified range. Some surfaces disclosed herein exhibit a ratio of specific fluorescence signals to nonspecific fluorescence signals of fluorophores such as Cy3 (e.g., specific hybridized oligonucleotides to nonspecifically bound, labeled oligonucleotides, or specifically amplified oligonucleotides to nonspecifically bound (B inter ) or nonspecifically amplified (B intra ) Labeled oligonucleotides, or combinations thereof (B inter +B intra(for) at least a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value that falls within the scope of this specification.

[0060] Substrates containing multilayer coatings of PEG and other hydrophilic polymers have been developed to adjust primer surface density and add further dimensionality to hydrophilic or amphoteric surfaces. Significant increases in primer loading density on a surface are possible by using hydrophilic or amphoteric surface layering approaches, including but not limited to the polymer / copolymer materials described below. Conventional PEG coating approaches use monolayer primer deposition, which is commonly reported for single-molecule applications but does not produce high copy numbers for nucleic acid amplification applications. “Layering” as described herein can be achieved using conventional crosslinking approaches with any suitable polymer or monomer subunit, allowing for the sequential construction of surfaces containing two or more highly crosslinked layers. Examples of suitable polymers include, but are not limited to, streptavidin, polyacrylamide, polyester, dextran, polylysine, and copolymers of polylysine and PEG. In some examples, different layers may be bonded to each other through any of the various conjugation reactions, which include, but are not limited to, biotin-streptavidin bonding, azide-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions between positively charged and negatively charged polymers. In some examples, the primer high-density material may be constructed in solution and then layered onto a surface in a number of steps.

[0061] Figure 2 provides a schematic diagram of one non-limiting example of grafting a first hydrophilic polymer layer onto a substrate, for example, a glass substrate. After cleaning the glass surface using any of the various methods known to those skilled in the art (e.g., treatment with piranha solution, plasma cleaning, etc.), the substrate is treated with a silane solution (e.g., silane PEG5K solution), rinsed, dried, and cured at high temperature to form covalent bonds with the surface. The distal ends of the polymer from the surface may contain any of various chemically reactive or protective functional groups. An amine-reactive NHS group is shown in Figure 2.

[0062] Figure 3 provides a schematic diagram of one non-limiting example of coupling a derivatized substrate containing a first polymer layer having NHS functional groups, as shown in Figure 2, with a primary amine-functionalized branched polymer (e.g., a 16-branched or 32-branched PEG polymer, also known as a 16-armed or 32-armed PEG, respectively) to produce a second hydrophilic polymer layer containing excess unreacted functional groups.

[0063] Figure 4 provides a schematic diagram of one non-limiting example of reacting a branched polymer containing a reactive functional group (e.g., 4-branched NHS-PEG) with one or more oligonucleotide adapter or primer sequences in solution (e.g., oligonucleotides containing primary amines, as shown by dotted and dashed lines) before deposition on a substrate surface to create a hydrophilic layer containing covalently attached oligonucleotide molecules. By varying the molar ratio of oligonucleotide molecules (or other biomolecules to be linked, e.g., peptides, proteins, enzymes, antibodies, etc.) to the molar ratio of the branched polymer, the resulting surface density of the attached oligonucleotide sequences can be varied in a controlled manner. In some examples, one or more oligonucleotide molecules (or other biomolecules) may be covalently linked to an existing polymer layer after the layer has been deposited on the surface.

[0064] Figure 5 provides a schematic diagram of one non-limiting example of coupling a branched polymer containing covalently attached oligonucleotide primers to a layered hydrophilic surface, as shown in Figure 3. In this example, a branched polymer containing two different oligonucleotide primers (represented by dotted or dashed lines) and amine-reactive NHS groups is coupled to a primary amine in a preceding layer to create a multilayered, three-dimensional hydrophilic surface with a controlled surface density of the coupled oligonucleotide primers.

[0065] The bonding chemistry used to graft the first chemically modified layer onto the support surface generally depends on both the material from which the support is manufactured and the chemical properties of the layer. In some examples, the first layer may be covalently attached to the support surface. In some examples, the first layer may be non-covalently attached to the surface and may be adsorbed onto the surface, for example, through non-covalent interactions such as electrostatic interactions, hydrogen bonding, or van der Waals interactions between the surface and the molecular elements of the first layer. In either example, the substrate surface may be treated before the attachment or deposition of the first layer. Any of the various surface preparation techniques known to those skilled in the art may be used to clean or treat the support surface. For example, a glass or silicon surface may be acid-cleaned using a piranha solution (a mixture of sulfuric acid (H2SO4) and hydrogen peroxide (H2O2)) and / or cleaned using an oxygen plasma treatment method.

[0066] Silane chemistry constitutes one non-limiting approach to covalently modifying silanol groups on a glass or silicon surface to attach more reactive functional groups (e.g., amines or carboxyl groups), the approach may then be used to couple linker molecules (e.g., linear hydrocarbon molecules of various lengths such as C6, C12, C18 hydrocarbons, or linear polyethylene glycol (PEG) molecules) or layer molecules (e.g., branched PEG molecules, or other polymers) to the surface. Examples of suitable silanes that may be used to produce any surface of the disclosed low-bonding support surface include, but are not limited to, (3-aminopropyl)trimethoxysilane (APTMS), (3-aminopropyl)triethoxysilane (APTES), any of the various PEG silanes (e.g., including molecular weights such as 1K, 2K, 5K, 10K, 20K), amino-PEG silanes (i.e., containing free amino functional groups), maleimide-PEG silanes, biotin-PEG silanes, and the like.

[0067] Any of the various molecules known to those skilled in the art, including but not limited to amino acids, peptides, nucleotides, oligonucleotides, other monomers or polymers, or combinations thereof, may be used to create one or more chemically modified layers on a support surface, and the selection of components used may vary to modify one or more properties of the support surface, such as the surface density of functional groups and / or linked oligonucleotide primers, the hydrophilicity / hydrophobicity of the support surface, or the three-dimensionality (i.e., "thickness") of the support surface. Examples of preferred polymers that may be used to create one or more layers of nonspecific low-binding material on any surface of the disclosed support surface include, but are not limited to, polyethylene glycol (PEG), streptavidin, polyacrylamide, polyester, dextran, polylysine and polylysine copolymers of various molecular weights and branched structures, or any combination thereof. Examples of conjugation chemistry that can be used to graft one or more layers of material (e.g., polymer layers) onto a support surface and / or to crosslink layers to one another include, but are not limited to, biotin-streptoavidin interactions (or their variations), histagu-Ni / NTA conjugation chemistry, methoxyether conjugation chemistry, carboxylate conjugation chemistry, amine conjugation chemistry, NHS esters, maleimides, thiols, epoxy, azides, hydrazides, alkynes, isocyanates, and silanes.

[0068] One or more layers of the multilayer surface may contain branched polymers or may be linear. Suitable examples of branched polymers include, but are not limited to, branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine and branched polyglucosides and dextran.

[0069] In some examples, branched polymers used to produce one or more layers of any of the multilayer surfaces disclosed herein may include at least 4 branches, at least 5 branches, at least 6 branches, at least 7 branches, at least 8 branches, at least 9 branches, at least 10 branches, at least 12 branches, at least 14 branches, at least 16 branches, at least 18 branches, at least 20 branches, at least 22 branches, at least 24 branches, at least 26 branches, at least 28 branches, at least 30 branches, at least 32 branches, at least 34 branches, at least 36 branches, at least 38 branches, or at least 40 branches. Molecules often exhibit a number of branches that is a "power of 2," such as 2, 4, 8, 16, 32, 64, or 128 branches.

[0070] Exemplary PEG multilayers include PEG(8,16,8)(8-arm, 16-arm, 8-arm) on PEG-amine-APTES. Similar concentrations were observed in 3-layer multi-arm PEG(8-arm, 16-arm, 8-arm) and (8-arm, 64-arm, 8-arm) on PEG-amine-APTES exposed to 8uM primers, and 3-layer multi-arm PEG(8-arm, 8-arm, 8-arm) is also intended, using star-shaped PEGamine to replace comparable first, second, and third PEG-layer 16-arm and 64-arm PEG multilayers.

[0071] The linear, branched, or polybranched polymers used to produce one or more layers on any surface of the multilayer surfaces disclosed herein may have a molecular weight of at least 500, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 7,500, at least 10,000, at least 12,500, at least 15,000, at least 17,500, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, or 50,000 Daltons. In some examples, linear, branched, or polybranched polymers used to produce one or more layers on any surface of the multilayer surfaces disclosed herein may have molecular weights of up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 17,500, up to 15,000, up to 12,500, up to 10,000, up to 7,500, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000, or up to 500 Daltons. Any lower and upper limits set forth in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the molecular weight of a linear, branched, or polybranched polymer used to produce one or more layers on any surface of a multilayer surface disclosed herein may range from about 1,500 to about 20,000 daltons. Those skilled in the art will recognize that the molecular weight of a linear, branched, or polybranched polymer used to produce one or more layers on any surface of a multilayer surface disclosed herein may have any value within this range (e.g., about 1,260 daltons).

[0072] In some examples, for instance, if at least one layer of a multilayer surface contains a branched polymer, the number of covalent bonds between the branched polymer molecules of the deposited layer and the molecules of the previous layer may range from about 1 covalent bond per molecule to about 32 covalent bonds per molecule. In some examples, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, at least 22, at least 24, at least 26, at least 28, at least 30, or at least 32, or more than 32, covalent bonds per molecule. In some examples, the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may be up to 32, up to 30, up to 28, up to 26, up to 24, up to 22, up to 20, up to 18, up to 16, up to 14, up to 12, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any values ​​of the lower and upper limits described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may range from about 4 to about 16. Those skilled in the art will recognize that the number of covalent bonds between the branched polymer molecules of the new layer and the molecules of the previous layer may have any value within this range (e.g., about 11 in some examples, or an average of about 4.6 in others).

[0073] Any reactive functional groups remaining after the coupling of a material layer to the support surface may be optionally blocked by coupling with small, inert molecules using high-yield coupling chemistry. For example, when amine coupling chemistry is used to adhere a new material layer to a previous layer, any remaining amine groups may be subsequently acetylated or deactivated by coupling with small amino acids such as glycine.

[0074] The number of layers of nonspecific low-binding material, such as hydrophilic polymer material, deposited on the surface of the disclosed low-binding support may range from 1 to about 10. In some examples, the number of layers is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10. In some examples, the number of layers may be up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1. Any values ​​of the lower and upper limits described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the number of layers may range from 2 to about 4. In some examples, all layers may contain the same material. In some examples, each layer may contain a different material. In some examples, multiple layers may contain multiple materials. In some examples, at least one layer may contain a branched polymer. In some examples, all layers may contain a branched polymer.

[0075] In some cases, one or more layers of nonspecific low-binding material may be conjugated and / or deposited on the substrate surface using polar protic solvents, polar aprotic solvents, nonpolar solvents, or any combination thereof. In some examples, the solvents used for layer deposition and / or coupling may include alcohols (e.g., methanol, ethanol, propanol, etc.), other organic solvents (e.g., acetonitrile, dimethyl sulfoxide (DMSO), dimethylformamide (DMF, etc.), water, aqueous buffer solutions (e.g., phosphate buffer, phosphate-buffered saline, 3-(N-morpholino)propanesulfonic acid (MOPS), etc.), or any combination thereof. In some examples, the organic component of the solvent mixture used may, in balance with water or aqueous buffer solution, constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any proportion extending or approximating the scope of this specification. In some examples, the aqueous component of the solvent mixture used may, in balance with the organic solvent, constitute at least 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the total, or any proportion extending or approximating the scope of this specification. The pH of the solvent mixture used may be any value within or approximately the specified range, such as 5, 5.5, 5.6, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, less than 10, or greater than 10.

[0076] In some examples, one or more layers of nonspecific low-binding material may be deposited and / or conjugated on a substrate surface using an organic solvent mixture, where the dielectric constant of at least one component is less than 40 and the mixture comprises at least 50% by volume of the whole mixture. In some examples, the dielectric constant of at least one component may be less than 10, less than 20, less than 30, or less than 40. In some examples, at least one component comprises at least 20%, at least 30%, at least 40%, at least 50%, at least 50%, at least 60%, at least 70%, or at least 80% by volume of the whole mixture.

[0077] As described above, the nonspecific low-binding supports of this disclosure exhibit reduced nonspecific binding of proteins, nucleic acids, and other components of hybridization and / or amplification formulations used for solid-phase nucleic acid amplification. The degree of nonspecific binding exhibited by a given support surface can be evaluated qualitatively or quantitatively. For example, in some cases, the surface may be exposed to a fluorescent dye (e.g., Cy3, Cy5, etc.), a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and / or a fluorescently labeled protein (e.g., polymerase) under a standardized set of conditions, followed by a specified washing protocol and fluorescence imaging, which may be used as qualitative tools for comparing nonspecific binding on supports containing formulations of various surfaces. In some examples, the surface is exposed to a fluorescent dye, such as Cy3, Cy5, a fluorescently labeled nucleotide, a fluorescently labeled oligonucleotide, and / or a fluorescently labeled protein (e.g., polymerase) under a standardized set of conditions, and the subsequent specified washing protocol and fluorescence imaging are performed under conditions that the fluorescence imaging is linearly related (or related in a predictable way) to the number of fluorophores on the support surface (e.g., under conditions that signal saturation and / or self-quenching of fluorophores is not a problem), and appropriate calibration standards are used, and care has been taken to ensure this. In some examples, other techniques known to those skilled in the art, such as radioisotope labeling and counting methods, may be used to evaluate the quantitative extent to which nonspecific binding is exhibited by the various support surface formulations of this disclosure.

[0078] Some surfaces disclosed herein exhibit a ratio of specific binding to fluorophores such as Cy3 and nonspecific binding of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range disclosed herein. Some surfaces disclosed herein exhibit a ratio of specific fluorescence to nonspecific fluorescence of fluorophores such as Cy3 and nonspecific fluorescence of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range disclosed herein.

[0079] As described above, in some examples, the degree of nonspecific binding exhibited by the disclosed low-binding supports may be evaluated using a standardized protocol for contacting the surface with labeled proteins (e.g., bovine serum albumin (BSA)), streptavidin, DNA polymerase, reverse transcriptase, helicase, single-stranded binding proteins (SSBs), or any combination thereof), labeled nucleotides, labeled oligonucleotide primers, etc., under a standardized set of incubation and rinsing conditions, and then detecting the amount of label remaining on the surface and comparing the resulting signal with a suitable calibration standard. In some examples, the label may include fluorescent labels. In some examples, the label may include radioisotopes. In some examples, the label may include other detectable labels known to those skilled in the art. In some examples, the degree of nonspecific binding exhibited by a given support surface formulation may therefore be evaluated in terms of the number of nonspecifically bound protein molecules (or other molecules) per unit region. In some examples, the low-binding supports of this disclosure have a binding density of 0.001 molecules / μm 2 Less than 0.01 molecules / μm 2 Less than 0.1 molecules / μm 2 Less than 0.25 molecules / μm 2 Less than 0.5 molecules / μm 2Less than 1 molecule / μm 2 Less than 10 molecules / μm 2 Less than 100 molecules / μm 2 Less than 1,000 molecules / μm 2 Nonspecific protein binding (or nonspecific binding of other specified molecules, e.g., Cy3 dyes) of less than 1 / 2 is possible. Those skilled in the art will know that a given support surface of the present disclosure may fall within any value in this range, for example, 86 molecules / μm 2 It will be understood that nonspecific binding of less than 0.5 molecules / µm may be observed. For example, several modified surfaces disclosed herein showed 0.5 molecules / µm after 15 minutes of contact with a 1 µM solution of Cy3-labeled streptavidin (GE Amersham) in phosphate-buffered saline (PBS) buffer, followed by three rinses in deionized water. 2 It exhibits nonspecific binding of proteins less than 0.25 molecules / µm. Some modified surfaces disclosed herein exhibit a similar binding rate of 0.25 molecules / µm. 2This shows nonspecific binding of less than 1 μM of Cy3 dye molecules. In independent nonspecific binding assays, 1 μM labeled Cy3SA (ThermoFisher), 1 μM Cy5SA dye (ThermoFisher), 10 μM aminoallyl-dUTP-ATTO-647N (JenaBiosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (JenaBiosciences), 10 μM aminoallyl-dUTP-ATTO-Rho11 (JenaBiosciences), 10 μM 7-propargylamino-7-deaza-dGTP-Cy5 (JenaBiosciences), and 10 μM 7-propargylamino-7-deaza-dGTP-Cy3 (JenaBiosciences) were incubated at 37°C for 15 minutes on a low-binding substrate in a 384-well plate format. Each well was rinsed 2-3 times with 50 μl of deionized RNase / DNase-free water and 2-3 times with 25 mM ACES buffer pH 7.4. 384-well plates were imaged on a GETyphoon (GE Healthcare Lifesciences, Pittsburgh, PA) instrument using Cy3, AF555, or Cy5 filter sets (depending on the dye tests performed) with 800 PMT sensitivity adjustments and a resolution of 50-100 μm as specified by the manufacturer. For higher resolution imaging, images were acquired on an Olympus IX83 microscope (Olympus Corp., Center Valley, PA) with a total internal reflectivity fluorescence (TIRF) lens (20x, 0.75 NA or 100x, 1.5 NA, Olympus), an sCMOS Andor camera (Zyla 4.2), and an excitation wavelength of 532 nm or 635 nm. Dichroic mirrors were purchased from Semrock (IDEX Health & Science, LLC, Rochester, New York), for example, dichroic reflectors / beam splitters at 405, 488, 532, or 633 nm, and bandpass filters were selected at 532LP or 645LP to match the appropriate excitation wavelength. Several modified surfaces disclosed herein have a molecular weight of 0.25 molecules / µm. 2It shows nonspecific binding of dye molecules to less than a certain number of molecules.

[0080] In some examples, the surfaces disclosed herein exhibit a ratio of specific to nonspecific binding of fluorophores such as Cy3 to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range disclosed herein. In some examples, the surfaces disclosed herein exhibit a ratio of specific to nonspecific fluorescence signals of fluorophores such as Cy3 to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100, or greater than 100, or any intermediate value within the range disclosed herein.

[0081] A low-background surface consistent with the disclosure herein may exhibit a ratio of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or more than 50 specific dye molecules per nonspecifically adsorbed molecule. Similarly, when exposed to excitation energy, a low-background surface consistent with the disclosure herein, to which fluorophores (e.g., Cy3) are attached, may exhibit a ratio of at least 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 30:1, 40:1, 50:1, or greater than 50:1 between a specific fluorescence signal (e.g., generated from an oligonucleotide labeled with Cy3 attached to the surface) and a nonspecific adsorbed dye fluorescence signal.

[0082] In some examples, the degree of hydrophilicity (or "hydration" with aqueous solutions) of the support surface of the Disclosure may be evaluated, for example, by measuring the water contact angle, where a small droplet of water is placed and the contact angle between the droplet and the surface is measured, for example, using an optical tensile meter. In some examples, a static contact angle can be determined. In some examples, an advancing or receding contact angle can be determined. In some examples, the water contact angles of the hydrophilic low-binding support surface disclosed herein may range from about 0 degrees to about 50 degrees. In some examples, the water contact angles of the hydrophilic low-binding support surface disclosed herein may be 50 degrees or less, 45 degrees or less, 40 degrees or less, 35 degrees or less, 30 degrees or less, 25 degrees or less, 20 degrees or less, 18 degrees or less, 16 degrees or less, 14 degrees or less, 12 degrees or less, 10 degrees or less, 8 degrees or less, 6 degrees or less, 4 degrees or less, 2 degrees or less, or 1 degree or less. In many cases, the contact angle is less than or equal to any value within this range, for example, 40 degrees or less. Those skilled in the art will understand that the hydrophilic, low-bonding support surface provided in this disclosure may exhibit a water contact angle of any value within this range (for example, about 27 degrees).

[0083] In some cases, the hydrophilic surfaces disclosed herein often facilitate a reduction in washing time for bioassays by reducing the nonspecific binding of biomolecules to low-binding surfaces. In some cases, a suitable washing step can be performed in less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, less than 15 seconds, less than 10 seconds, or less than 10 seconds. For example, in some cases, a suitable washing step can be performed in less than 30 seconds.

[0084] Some of the low-bonding surfaces of this disclosure exhibit significant improvements in stability or durability against prolonged exposure to solvents and high temperatures, or against repeated cycles of exposure to solvents or temperature changes. For example, in some cases, the stability of the surfaces of this disclosure may be examined by fluorescently labeling functional groups on the surface or biomolecules (e.g., oligonucleotide primers) bonded to the surface, and monitoring the fluorescence signals before, during, and after prolonged exposure to solvents and elevated temperatures, or repeated cycles of exposure to solvents and temperature changes. In some examples, the degree of change over exposure to solvents and / or high temperatures over periods of 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 45 hours, 50 hours, or 100 hours may be less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% (or any combination of these percentages measured over these periods). In some examples, the degree of change over repeated exposure to solvent and / or temperature changes of fluorescence used to assess surface quality over 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 cycles may be less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 10%, less than 15%, less than 20%, or less than 25% (or any combination of these percentages as measured over this range of cycles).

[0085] In some examples, the surfaces disclosed herein may exhibit a high ratio of specificity signals to non-specificity signals or other backgrounds. For example, when used for nucleic acid amplification, some surfaces may exhibit amplification signals that are at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, or 100 times greater than the signals of adjacent sparse surface regions. Similarly, some surfaces may exhibit amplification signals that are at least 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 75 times, 100 times, or 100 times greater than the signals of adjacent amplified nucleic acid population surface regions.

[0086] The fluorescence excitation energy varies among specific fluorophores and protocols, and the excitation wavelength ranges from less than 400 nm to more than 800 nm, consistent with the selection of fluorophores or other parameters for the use of the surfaces disclosed herein.

[0087] Accordingly, the low-background surfaces disclosed herein exhibit lower background fluorescence signals or higher contrast-to-noise ratios (CNRs) compared to surfaces known in the art. For example, in some examples, the background fluorescence of a surface at a given location is spatially distinct or removed from a labeled feature on the surface (e.g., labeled spots, clusters, isolated regions, subdivisions, or subsets of the surface), but includes, for example, hybridized clusters of nucleic acid molecules or cloned-amplified clusters of nucleic acid molecules produced by 20 cycles of nucleic acid amplification via thermal cycling, and may be 20 times or less, 10 times or less, 5 times or less, 2 times or less, 1 time or less, 0.5 times or less, 0.1 times or less, compared to the background fluorescence measured at the same location before performing the hybridization or the 20 cycles of nucleic acid amplification.

[0088] In some examples, the low-background surface fluorescence images of this disclosure exhibit a contrast-to-noise ratio (CNR) of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, or greater than 250 when used in nucleic acid hybridization or amplification applications to create clusters of hybridized or clone-amplified nucleic acid molecules (e.g., directly or indirectly labeled with fluorophores).

[0089] Oligonucleotide primers and adapter sequences: Generally, at least one layer of one or more layers of a low nonspecific binder contains functional groups that covalently or noncovalently attach oligonucleotide molecules, e.g., adapter or primer sequences, to at least one layer, or at least one layer may already contain covalently or noncovalently attached oligonucleotide adapter or primer sequences when it is deposited on the support surface. In some examples, oligonucleotides linked to polymer molecules in at least one-third of the layers may be distributed at multiple depths throughout the layers.

[0090] In some examples, oligonucleotide adapter or primer molecules are covalently coupled to polymer molecules in solution, i.e., before coupling or depositing the polymer molecules onto the surface. In some examples, oligonucleotide adapter or primer molecules are covalently coupled to polymer molecules after coupling or depositing onto the surface. In some examples, at least one hydrophilic polymer layer contains oligonucleotide adapter or primer molecules attached by multiple covalent bonds. In some examples, at least two, at least three, at least four, or at least five hydrophilic polymer layers contain adapter or primer molecules attached by multiple covalent bonds.

[0091] In some cases, oligonucleotide adapter or primer molecules may be coupled to one or more layers of a hydrophilic polymer using one of a variety of suitable conjugation chemistrys known to those skilled in the art. For example, the oligonucleotide adapter or primer sequence may contain moieties that are reactive to amine groups, carboxyl groups, thiol groups, etc. Examples of suitable amine-reactive conjugation chemistrys that may be used include, but are not limited to, reactions involving isothiocyanates, isocyanates, acyladides, NHS esters, sulfonyl chlorides, aldehydes, glyoxal, epoxides, oxiranes, carbonates, aryl halides, imide esters, carbodiimides, anhydrides, and fluorophenyl ester groups. Examples of suitable carboxyl-reactive conjugation chemistrys include, but are not limited to, reactions involving carbodiimide compounds, such as water-soluble EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide HCl). Examples of suitable sulfidyl-reactive conjugation chemistrys include maleimides, haloacetyl groups, and pyridine disulfides.

[0092] One or more types of oligonucleotide molecules may be attached to or ligated to a support surface. In some examples, one or more types of oligonucleotide adapters or primers may include a spacer sequence, an adapter sequence for hybridization to a template library nucleic acid sequence ligated to the adapter, a forward amplification primer, an inversion amplification primer, a sequencing primer, and / or a molecular barcoding sequence, or any combination thereof. In some examples, one primer or adapter sequence may be ligated to at least one layer of the surface. In some examples, at least two, three, four, five, six, seven, eight, nine, ten, or more different primer or adapter sequences may be ligated to at least one layer of the surface.

[0093] In some examples, the length of a linked oligonucleotide adapter and / or primer sequence may range from about 10 nucleotides to about 100 nucleotides. In some examples, a linked oligonucleotide adapter and / or primer sequence may be at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 nucleotides in length. In some examples, the length of a linked oligonucleotide adapter and / or primer sequence may be up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, or up to 10 nucleotides. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, the length of a linked oligonucleotide adapter and / or primer sequence may range from about 20 nucleotides to about 80 nucleotides. Those skilled in the art will recognize that the length of a linked oligonucleotide adapter and / or primer sequence may have any value within this range (e.g., about 24 nucleotides).

[0094] In some cases, the ligated adapter or primer sequences may include modifications designed to enhance the specificity and efficiency of nucleic acid amplification, such as when performed on a low-binding support. For example, in some cases, the primer may include a polymerase termination site such that the extension of the primer sequence between the surface conjugation point and the modification site is always in a single-stranded form and acts as a loading site for the 5' to 3' helicase in certain helicase-dependent isothermal amplification methods. Other examples of primer modifications that may be used to create polymerase termination sites may include, but are not limited to, PEG chain insertions into the primer backbone toward the 5' end between two nucleotides, debasalized nucleotides (i.e., nucleotides without purine or pyrimidine bases), or insertions of obstruction sites that can be avoided by helicases.

[0095] As will be further discussed in the following examples, varying the surface density of linked oligonucleotide adapters or primers on the support surface, and / or spacing the linked adapters or primers away from the support surface (e.g., by varying the length of the linker molecules to the surface used to link the adapters or primers), may be desirable to "tune" the support to optimal performance when using a given amplification method. As described below, adjusting the surface density of linked oligonucleotide adapters or primers may affect the level of specific and / or nonspecific amplification observed on the support, in a manner that varies depending on the selected amplification method. In some examples, the surface density of linked oligonucleotide adapters or primers may be varied by adjusting the ratio of molecular components used to create the support surface. For example, in cases where an oligonucleotide primer-PEG conjugate is used to create a final layer of a low-binding support, the ratio of oligonucleotide primer-PEG conjugate to unconjugated PEG molecules may be varied. The final surface density of linked primer molecules can then be estimated or measured using one of various techniques known to those skilled in the art. Examples include, but are not limited to, using radioisotope labeling and counting methods, covalent coupling of cleavable molecules, which includes an optically detectable tag (e.g., a fluorescent tag) that can be cleaved from the surface of a support in a defined region, collected in a fixed volume of a suitable solvent, and then quantified by comparison of the fluorescence signal against a calibration solution of known optical tag concentrations with that fluorescence signal, or using fluorescence imaging techniques, provided that care is taken with the labeling reaction conditions and image acquisition settings to ensure that the fluorescence signal is linearly related to the number of fluorophores on the surface (e.g., there is no significant self-quenching of fluorophores on the surface).

[0096] In some examples, the final surface density of the oligonucleotide adapter or primer on a low-binding support surface of this disclosure is μm 2 Approximately 100 primer molecules per unit, from μm 2 The surface density of oligonucleotide adapters or primers varies in the range of up to approximately 1,000,000 primer molecules per unit. In some examples, the surface density of oligonucleotide adapters or primers varies in the range of at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, and at least 9,500. 00, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, and at least 400,000. , , may be at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules. In some examples, the oligonucleotide adapter or primer surface density is μm 2Winning combinations include: maximum 1,000,000, maximum 950,000, maximum 900,000, maximum 850,000, maximum 800,000, maximum 750,000, maximum 700,000, maximum 650,000, maximum 600,000, maximum 550,000, maximum 500,000, maximum 450,000, maximum 400,000, maximum 350, 000, up to 300,000, up to 250,000, up to 200,000, 150,000, up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, Large: 45,000, Maximum: 40,000, Maximum: 35,000, Maximum: 30,000, Maximum: 25,000, Maximum: 20,000, Maximum: 15,000, Maximum: 10,000, Maximum: 9,500, Maximum: 9,000, Maximum: 8,500, Maximum: 8,000, Maximum: 7,500, Maximum: 7,000, Maximum: 6,500, Maximum: 6,000, Maximum: 5,5 The number of molecules may be 00, up to 5,000, up to 4,500, up to 4,000, up to 3,500, up to 3,000, up to 2,500, up to 2,000, up to 1,500, up to 1,000, up to 900, up to 800, up to 700, up to 600, up to 500, up to 400, up to 300, up to 200, or up to 100 molecules. Any of the lower and higher values ​​described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the surface density of the adapter or primer is μm 2 Approximately 10,000 molecules per μm 2 This can amount to approximately 100,000 molecules per unit. Those skilled in the art will know, for example, in one example, μm 2 Approximately 3,800 molecules per unit, and in other examples, μm 2You will notice that the surface density of the adapter or primer molecules can take any value within this range, such as approximately 455,000 molecules per unit. In some examples, as further described below, the surface density of the template library nucleic acid sequence (e.g., sample DNA molecule) initially hybridized to the adapter or primer sequence on the support surface may be less than or equal to the surface density indicated for the linked oligonucleotide primer. In some examples, as also further described below, the surface density of the cloned amplified template library nucleic acid sequence hybridized to the adapter or primer sequence on the support surface may be the same as, or different from, the surface density indicated for the linked oligonucleotide adapter or primer.

[0097] The local surface density of adapter or primer molecules as described above does not eliminate variations in density across the surface, and as a result the surface may have, for example, a density of 500,000 / µm. 2 It may include a region with one oligodendrome density, as well as at least a second region with a considerably different local density.

[0098] Hybridization of nucleic acid molecules to low-binding supports: In some aspects of this disclosure, hybridization buffer formulations are described that, when combined with the disclosed low-binding supports, result in improved hybridization rate, hybridization specificity (or stringency), and hybridization efficiency (or yield). As used herein, hybridization specificity is generally an indicator of the ability of a ligated adapter sequence, primer sequence, or oligonucleotide sequence to hybridize precisely only to a perfectly complementary sequence, while hybridization efficiency is generally an indicator of the percentage of all available ligated adapter sequences, primer sequences, or oligonucleotide sequences that hybridize to a complementary sequence.

[0099] Improved hybridization specificity and / or efficiency may be achieved through optimization of the hybridization buffer formulation used with the disclosed low-binding surface, which is described in more detail in the following examples. Examples of hybridization buffer components that may be tuned to achieve improved performance include, but are not limited to, buffer type, organic solvent mixture, buffer pH, buffer viscosity, surfactant and amphoteric components, ionic strength (including adjustment of both monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, and other additives.

[0100] As a non-limiting example, suitable buffers used in the formulation of hybridization buffers may include, but are not limited to, phosphate-buffered saline (PBS), succinic acid, citrate, histidine, acetic acid, Tris, TAPS, MOPS, PIPES, HEPES, MES, etc. The selection of a suitable buffer will generally depend on the target pH of the hybridization buffer. Generally, the desired pH of the buffer solution will vary in the range of approximately pH 4 to approximately pH 8.4. In some embodiments, the pH of the buffer may be at least 4.0, at least 4.5, at least 5.0, at least 5.5, at least 6.0, at least 6.2, at least 6.4, at least 6.6, at least 6.8, at least 7.0, at least 7.2, at least 7.4, at least 7.6, at least 7.8, at least 8.0, at least 8.2, or at least 8.4. In some embodiments, the pH of the buffer may be up to 8.4, up to 8.2, up to 8.0, up to 7.8, up to 7.6, up to 7.4, up to 7.2, up to 7.0, up to 6.8, up to 6.6, up to 6.4, up to 6.2, up to 6.0, up to 5.5, up to 5.0, up to 4.5, or up to 4.0. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the desired pH is μm 2 Approximately 6.4 μm per unit area 2It can be around 7.2. A person skilled in the art will recognize that the pH of the buffer can take any value within this range (e.g., around 7.25).

[0101] Suitable detergents used in hybridization buffer formulations are not limited to, but include, amphoteric surfactants (e.g., 1-dodecanoyl-sn-glycero-3-phosphocholine, (3-(4-tert-butyl-1-pyridinio)-1-propanesulfonate), (3-(N,N-dimethylmyristylammonio)propanesulfonate), (3-(N,N-Dimethylmyristylammonio)propanesulfonate), ASB-C80, C7BzO, CHAPS, CHAPS hydrate, CHAPSO, DDMAB, and Dimethylethylammoniumpropanesulfonate) This includes surfactants such as (sulfonate), N-dodecyl-N,N,N-dimethyl-3-ammonio-1-propanesulfonate, or (N-dodecyl-N,N,N-dimethyl-3-ammonio-1-propanesulfonate), or anionic, cationic, and nonionic surfactants. Examples of nonionic surfactants include poly(oxyethylene) ethers and related polymer molecules (e.g., Brij®, TWEEN®, TRITON®, TRITON X-100, and IGEPAL® CA-630), bile salts, and glycoside surfactants.

[0102] The use of the low-binding supports of this disclosure, either alone or in combination with optimized buffer formulations, can result in relative hybridization rates that are approximately 2 to 20 times faster than those of conventional hybridization protocols. In some examples, the relative hybridization rate may be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 25, 30, or 40 times faster than that of conventional hybridization protocols.

[0103] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized buffer formulation may result in total hybridization reaction times (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the hybridization reaction) of less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, or less than 5 minutes for any of these completion metrics.

[0104] The use of the low-binding support of this disclosure, either alone or in combination with an optimized buffer formulation, may result in improved hybridization specificity compared to that of conventional hybridization protocols. In some cases, this may result in better hybridization specificity than the following: 1 base mismatch in 10 hybridization events, 1 base mismatch in 20 hybridization events, 1 base mismatch in 30 hybridization events, 1 base mismatch in 40 hybridization events, 1 base mismatch in 50 hybridization events, 1 base mismatch in 75 hybridization events, 1 base mismatch in 100 hybridization events, 1 base mismatch in 200 hybridization events, 1 base mismatch in 300 hybridization events, 1 base mismatch in 400 hybridization events, 1 base mismatch in 500 hybridization events, 1 base mismatch in 600 hybridization events, and 700 hybridization events. A single base mismatch in 800 hybridization events, a single base mismatch in 900 hybridization events, a single base mismatch in 1,000 hybridization events, a single base mismatch in 2,000 hybridization events, a single base mismatch in 3,000 hybridization events, a single base mismatch in 4,000 hybridization events, one base mismatch in 5,000 hybridization events, one base mismatch in 6,000 hybridization events, one base mismatch in 7,000 hybridization events, one base mismatch in 8,000 hybridization events, a single base mismatch in 9,000 hybridization events, or a single base mismatch in 10,000 hybridization events may result in better hybridization specificity than a single base mismatch in 10,000 hybridization events.

[0105] In some cases, the use of the low-binding supports of this disclosure alone or in combination with optimized buffer formulations may result in improved hybridization efficiency (e.g., fraction of oligonucleotide primers available on the support surface that successfully hybridize with the target oligonucleotide sequence) compared to the hybridization efficiency of conventional hybridization protocols. In some cases, the achievable hybridization efficiency may be 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% better for any of the target oligonucleotide concentration inputs specified below and any of the hybridization reaction times specified above. For example, in some cases where the hybridization efficiency is less than 100%, the final surface density of the hybridized target nucleic acid sequence on the support surface may be less than the surface density of the oligonucleotide adapter or primer sequence on the surface.

[0106] In some cases, using the low-binding supports of the disclosure for nucleic acid hybridization (or amplification) applications using conventional hybridization (or amplification) protocols, or optimized hybridization (or amplification) protocols, may result in a reduction in the required input concentration of labeled (or sample) nucleic acid molecules in contact with the support surface. For example, in some cases, the target (or sample) nucleic acid molecules may be in contact with the support surface at concentrations ranging from approximately 10 pM to approximately 1 μM (i.e., before annealing or amplification). In some cases, the target (or sample) nucleic acid molecule can be administered at concentrations of at least 10 pM, at least 20 pM, at least 30 pM, at least 40 pM, at least 50 pM, at least 100 pM, at least 200 pM, at least 300 pM, at least 400 pM, at least 500 pM, at least 600 pM, at least 700 pM, at least 800 pM, at least 900 pM, at least 1 nM, at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 200 nM, at least 300 nM, at least 400 nM, at least 500 nM, at least 600 nM, at least 700 nM, at least 800 nM, at least 900 nM, or at least 1 μM.In some examples, the target (or sample) nucleic acid molecule can be up to 1 μM, up to 900 nM, up to 800 nm, up to 700 nM, up to 600 nM, up to 500 nM, up to 400 nM, up to 300 nM, up to 200 nM, up to 100 nM, up to 90 nM, up to 80 nM, up to 70 nM, up to 60 nM, up to 50 nM, up to 40 nM, up to 30 nM, up to 20 nM, and more. It may be administered at concentrations of up to 10 nM, up to 1 nM, up to 900 pM, up to 800 pM, up to 700 pM, up to 600 pM, up to 500 pM, up to 400 pM, up to 300 pM, up to 200 pM, up to 100 pM, up to 90 pM, up to 80 pM, up to 70 pM, up to 60 pM, up to 50 pM, up to 40 pM, up to 30 pM, up to 20 pM, or up to 10 pM. Any of the lower and higher values ​​described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples, the target (or sample) nucleic acid molecule may be administered at concentrations varying in the range from about 90 pM to about 200 nM. Those skilled in the art will recognize that the target (or sample) nucleic acid molecule can be administered at concentrations having any value within this range (e.g., about 855 nM).

[0107] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized hybridization buffer formulation may result in a surface density of hybridized target (or sample) oligonucleotide molecules (i.e., before any subsequent solid-phase reaction or clonal amplification reaction), where the surface density is μm 2 Approximately 0.0001 target oligonucleotide molecules per μm 2 It varies in range from approximately 1,000,000 target oligonucleotide molecules per unit area. In some examples, the surface density of hybridized target oligonucleotide molecules is μm 2Per unit, at least 0.0001, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, at least 0.05, at least 0.1, at least 0.5, at least 1, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1.00 0, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 5,500, at least 6,000, at least 6,500, at least 7,000, at least 7,500, at least 8,000, at least 8,500, at least 9,000, at least 9,500, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, and at least 35,000. At least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 30 It may be 0,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, or at least 1,000,000 molecules. In some examples, the surface density of the hybridized target oligonucleotide molecules is μm 2Winning combinations: Maximum 1,000,000, Maximum 950,000, Maximum 900,000, Maximum 850,000, Maximum 800,000, Maximum 750,000, Maximum 700,000, Maximum 650,000, Maximum 600,000, Maximum 550,000, Maximum 500,000, Maximum 450,000, Maximum 400,000, Maximum 350,000, Maximum 300,000, Maximum 250,000, Maximum 200,000 00, 150,000, maximum 100,000, maximum 95,000, maximum 90,000, maximum 85,000, maximum 80,000, maximum 75,000, maximum 70,000, maximum 65,000, maximum 60,000, maximum 55,000, maximum 50,000, maximum 45,000, maximum 40,000, maximum 35,000, maximum 30,000, maximum 25,000, maximum 20,000, maximum 15,000 0, maximum 10,000, maximum 9,500, maximum 9,000, maximum 8,500, maximum 8,000, maximum 7,500, maximum 7,000, maximum 6,500, maximum 6,000, maximum 5,500, maximum 5,000, maximum 4,500, maximum 4,000, maximum 3,500, maximum 3,000, maximum 2,500, maximum 2,000, maximum 1,500, maximum 1,000, maximum 900, maximum 800, maximum The surface density may be 700, up to 600, up to 500, up to 400, up to 300, up to 200, up to 100, up to 90, up to 80, up to 70, up to 60, up to 50, up to 40, up to 30, up to 20, up to 10, up to 5, up to 1, up to 0.5, up to 0.1, up to 0.05, up to 0.01, up to 0.005, up to 0.001, up to 0.0005, or up to 0.0001 molecules. Any of the lower and higher values ​​described in this paragraph may be combined to form the range included in this disclosure, for example, in some examples the surface density of the hybridized target oligonucleotide molecule is μm 2 Approximately 3,000 molecules per unit, up to μm in size. 2 It can reach up to approximately 20,000 molecules per unit. Those skilled in the art will know that the surface density of the hybridized target oligonucleotide molecules can be any value within this range, for example, μm 2You will realize that each molecule can contain approximately 2,700 molecules.

[0108] In other words, in some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized hybridization buffer formulation results in a certain surface density of hybridized target (or sample) oligonucleotide molecules (i.e., before any subsequent solid-phase reaction or clonal amplification reaction), where the surface density is mm 2 Approximately 100 hybridized target oligonucleotide molecules per mm 2 Approximately 1 x 10 7 Oligonucleotide molecules, or mm 2 Approximately 100 hybridized target oligonucleotide molecules per mm 2 Approximately 1 x 10 12 The surface density of the hybridized target oligonucleotide molecules may vary within a certain range. In some cases, the surface density of the hybridized target oligonucleotide molecules may be 1 mm 2Percentage, at least 100, at least 500, at least 1,000, at least 4,000, at least 5,000, at least 6,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95 ,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 , at least 5 × 10 7 , at least 1 × 10 8 , at least 5 × 10 8 , at least 1x10 9 , at least 5 × 10 9 , at least 1 × 10 10 , at least 5 × 10 10 , at least 1 × 10 11 , at least 5 × 10 11 , or at least 1 × 10 12 It can be a molecule. In some examples, the surface density of the hybridized target oligonucleotide molecule is mm 2 Winning combination: 1 x 10 12 , up to 5 x 10 11 , up to 1 x 10 11 , up to 5 x 10 10 , up to 1 x 10 10 , up to 5 x 10 9 , up to 1 x 109 , up to 5 x 10 8 , up to 1 x 10 8 , up to 5 x 10 7 , up to 1 x 10 7 , up to 5,000,000, 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 The surface density may be up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the surface density of the hybridized target oligonucleotide molecules is mm 2 Approximately 5,000 molecules per mm 2 It can reach up to approximately 50,000 molecules per unit. Those skilled in the art will know that the surface density of the hybridized target oligonucleotide molecules can be any value within this range, for example, mm 2 You will realize that there could be approximately 50,700 molecules per unit.

[0109] In some examples, the length of the target (or sample) oligonucleotide molecule (or nucleic acid molecule) hybridized to an oligonucleotide adapter or primer molecule attached to a low-binding support surface may range from approximately 0.02 kilobases (kb) to approximately 20 kb, or from approximately 0.1 kilobases (kb) to approximately 20 kb. In some examples, the length of the target oligonucleotide molecule may be at least 0.001 kb, at least 0.005 kb, at least 0.01 kb, at least 0.02 kb, at least 0.05 kb, at least 0.1 kb, at least 0.2 kb, at least 0.3 kb, at least 0.4 kb, at least 0.5 kb, at least 0.6 kb, at least 0.7 kb, at least 0.8 kb, at least 0.9 kb, and less Each may be 1kb, with a length of at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, at least 20kb, at least 30kb, or at least 40kb, or any intermediate value across the range described herein, for example, 0.85kb.

[0110] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) includes a single-stranded or double-stranded nucleic acid molecule with multiple bonds, further containing regularly occurring repeating monomer units. In some examples, single-stranded or double-stranded, multiple-bonded nucleic acid molecules may be at least 0.001kb, at least 0.005kb, at least 0.01kb, at least 0.02kb, at least 0.05kb, with a length of at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, with a length of at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, or with a length of at least 20kb, at least 30kb, or at least 40kb, or any intermediate value across the range described herein, for example, 2.45kb.

[0111] In some examples, the target (or sample) oligonucleotide molecule (or nucleic acid molecule) comprises a multimeric nucleic acid molecule containing single-stranded or double-stranded, regularly repeating monomer units ranging from about 2 to about 100 copies. In some examples, the number of copies of regularly repeating monomer units may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100. In some examples, the number of copies of regularly repeating monomer units may be up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, up to 5, up to 4, up to 3, or up to 2. Any of the lower and higher values ​​mentioned in this paragraph may be combined to form a range included in this disclosure, for example, in some examples, the number of copies of regularly repeating monomer units may range from about 4 to about 60. A person skilled in the art will recognize that the number of copies of regularly repeating monomer units may have any value within this range, for example, 17. Therefore, in some cases, the surface density of the hybridized target sequence may exceed the surface density of the oligonucleotide primer in terms of the number of target sequence copies per unit area of ​​the support surface, even if the hybridization efficiency is less than 100%.

[0112] Nucleic Acid Surface Amplification (NASA): As used herein, the phrase “Nucleic Acid Surface Amplification” (NASA) is interchangeable with the phrase “Solid-Phase Nucleic Acid Amplification” (or simply “Solid-Phase Amplification”). In some aspects of this disclosure, nucleic acid amplification formulations resulting in improved amplification rate, amplification specificity, and amplification efficiency are described in combination with the low-binding supports of this disclosure. As used herein, specific amplification refers to the amplification of a template library oligonucleotide chain covalently or non-covalently linked to a solid support. As used herein, non-specific amplification refers to the amplification of a primer dimer or other non-template nucleic acid. As used herein, amplification efficiency is an indicator of the percentage of oligonucleotides linked on a support surface that are successfully amplified during a given amplification cycle or amplification reaction. Nucleic acid amplifications performed on surfaces disclosed herein may achieve amplification efficiencies of at least 50%, 60%, 70%, 80%, 90%, 95%, or greater than 95%, such as 98% or 99%.

[0113] Any of the various thermal cycling or isothermal nucleic acid amplification schemes may be used with the disclosed low-binding supports. Examples of nucleic acid amplification methods that may be used with the low-binding supports of this disclosure include, but are not limited to, polymerase chain reaction (PCR), multiple substitution amplification (MDA), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification (NASBA), strand displacement amplification (SDA), real-time SDA, bridge amplification, isothermal bridge amplification, rolling circle amplification, circle-to-circle amplification, helicase-dependent amplification, recombinase-dependent amplification, or single-strand binding (SSB) protein-dependent amplification.

[0114] Often, improvements in amplification rate, amplification specificity, and amplification efficiency may be achieved by using the low-binding supports of this disclosure alone or in combination with formulations of amplification reaction components. In addition to containing nucleotides, one or more polymerases, helicases, single-chain binding proteins, etc. (or any combination thereof), the amplification reaction mixture can be modified in a variety of ways to achieve improved performance, including, but not limited to, the selection of buffer type, buffer pH, organic solvent mixture, buffer viscosity, surfactant and amphoteric components, ionic strength (including adjustment of both monovalent and divalent ion concentrations), antioxidants and reducing agents, carbohydrates, BSA, polyethylene glycol, dextran sulfate, betaine, etc.

[0115] The use of the low-binding supports of this disclosure alone or in combination with optimized amplification reaction formulations may result in increased amplification rates compared to those achieved using conventional supports and amplification protocols. In some examples, the relative amplification rates that can be achieved may be at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 12 times, at least 14 times, at least 16 times, at least 18 times, or at least 20 times compared to those achieved with the use of conventional supports and amplification protocols in any of the amplification methods described above.

[0116] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized buffer formulation may result in total amplification reaction times (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the amplification reaction) of less than 180 minutes, less than 120 minutes, less than 60 minutes, less than 50 minutes, less than 40 minutes, less than 30 minutes, less than 20 minutes, less than 15 minutes, less than 10 minutes, less than 5 minutes, less than 3 minutes, less than 1 minute, less than 50 s, less than 40 s, less than 30 s, less than 20 s, or less than 10 s for any of these completion metrics.

[0117] Several low-binding support surfaces disclosed herein exhibit a ratio of non-specific binding to specific binding of fluorophores such as Cy3, of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value across these ranges. Some surfaces disclosed herein exhibit a ratio of non-specific fluorophore signals to specific fluorophore signals of fluorophores such as Cy3, of at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 75:1, 100:1, or greater than 100:1, or any intermediate value across these ranges.

[0118] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification buffer formulation may enable faster amplification reaction times of 60 minutes or less, 50 minutes or less, 40 minutes or less, 30 minutes or less, 20 minutes or less, or 10 minutes or less (i.e., the time required to reach 90%, 95%, 98%, or 99% completion of the amplification reaction). Similarly, the use of the low-binding support of this disclosure alone or in combination with an optimized buffer formulation may, in some cases, enable the completion of the amplification reaction in 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 cycles or less, or 30 cycles or less.

[0119] In some cases, the use of the low-binding supports of this disclosure alone or in combination with optimized amplification reaction formulations may result in increased specific amplification and / or decreased nonspecific amplification compared to those obtained using conventional supports and amplification protocols. In some cases, the final ratio of specific amplification to nonspecific amplification that may be achieved is at least 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 20:1, 30:1, 40:1, 50:1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1, 600:1, 700:1, 800:1, 900:1, or 1,000:1.

[0120] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification reaction formulation may result in an increase in amplification efficiency compared to the amplification efficiency achieved using conventional support and amplification protocols. In some cases, the amplification efficiency, which may be achieved, is well above 50%, 60%, 70%, 80%, 85%, 90%, 95%, 98%, or 99% in any of the amplification reaction cycles specified.

[0121] In some examples, the length of a clone-amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) hybridized to an oligonucleotide adapter or primer molecule attached to a low-binding support surface can range from approximately 0.02 kilobases (kb) to approximately 20 kb, or from approximately 0.1 kilobases (kb) to approximately 20 kb. In some examples, the clonally amplified target oligonucleotide molecule may have a length of at least 0.001kb, at least 0.005kb, at least 0.01kb, at least 0.02kb, at least 0.05kb, at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, or at least 20kb, or any intermediate value across the ranges listed herein, for example, at least 0.85kb.

[0122] In some cases, the cloned-amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain a multimeric nucleic acid molecule that further includes repeats of regularly occurring monomer units, either single-stranded or double-stranded. In some cases, the cloned-amplified, single-stranded, double-stranded, multimeric nucleic acid molecule may have a length of at least 0.1kb, at least 0.2kb, at least 0.3kb, at least 0.4kb, at least 0.5kb, at least 1kb, at least 2kb, at least 3kb, at least 4kb, at least 5kb, at least 6kb, at least 7kb, at least 8kb, at least 9kb, at least 10kb, at least 15kb, or at least 20kb, or any intermediate value across the ranges listed herein, for example, at least 2.45kb.

[0123] In some examples, the cloned amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain a multimer nucleic acid molecule with approximately 2 to approximately 100 copies of regularly repeating monomer units, either single-stranded or double-stranded. In some examples, the number of copies of regularly repeating monomer units may be at least 2, at least 3, at least 4, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or at least 100. In some examples, the number of copies of regularly repeating monomer units may be up to 100, up to 95, up to 90, up to 85, up to 80, up to 75, up to 70, up to 65, up to 60, up to 55, up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, up to 5, up to 4, up to 3, or up to 2. Any of the lower and higher values ​​mentioned in this paragraph may be combined to form a range included in this disclosure, for example, in some examples, the number of copies of regularly repeating monomer units may range from about 4 to about 60. A person skilled in the art will recognize that the number of copies of regularly repeating monomer units may be any value within this range, for example, about 12. Therefore, in some cases, the surface density of the cloned target sequence may exceed the surface density of the oligonucleotide primer in terms of the number of copies of the target sequence per unit area of ​​the support surface, even if the hybridization and / or amplification efficiency is less than 100%.

[0124] In some cases, the use of the low-binding supports of this disclosure alone or in combination with optimized amplification reaction formulations may result in an increase in the number of clones compared to the number of clones achieved using conventional supports and amplification protocols. For example, in some cases where the clone-amplified target (or sample) oligonucleotide molecule contains a ligated, multimeric repeat of a monomer target sequence, the number of clones may be considerably smaller compared to the number of clones achieved using conventional supports and amplification protocols. Thus, in some cases, the number of clones may range from about 1 molecule to about 100,000 molecules (e.g., target sequence molecule) per amplified colony.In several examples, the number of clone copies per amplified colony is at least 1, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1,000, at least 2,000, at least 3,000, at least 4,000, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, and fewer. There may be at least 95,000 or at least 100,000 molecules. In some examples, the number of clone copies per amplified colony can be up to 100,000, up to 95,000, up to 90,000, up to 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, and up to 40 The number of copies of a clone may be,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 9,000, up to 8,000, up to 7,000, up to 6,000, up to 5,000, up to 4,000, up to 3,000, up to 2,000, up to 1,000, up to 500, up to 100, up to 50, up to 10, up to 5, or up to 1 molecule. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the number of clone copies may range from about 2,000 molecules to about 9,000 molecules. Those skilled in the art will recognize that the number of clones can be any value within this range, for example, about 2,220 molecules in one example and 2 molecules in another.

[0125] As described above, in some examples, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain a multimeric repeat of the ligated, monomeric target sequence. In some examples, the amplified target (or sample) oligonucleotide molecule (or nucleic acid molecule) may contain multiple molecules each containing a single monomeric target sequence. Thus, the use of the low-binding support of the present disclosure alone or in combination with an optimized amplification reaction formulation results in, per mm 2 from about 100 target sequence copies per mm 2 to about 1×10 12 a surface density of target sequence copies that can vary over a range to target sequence copies per mm 2 where the clonally amplified target sequence molecules per mm are at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 100,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1×10 7 at least 5×10 7 at least 1×10 8 at least 5×10 8 at least 1×109 , at least 5×10 9 , at least 1x10 10 , at least 5×10 10 , at least 1×10 11 , at least 5×10 11 , or at least 1×10 12 and can be. In some examples, the surface density of target sequence copies is per mm 2 at most 1×10 12 , at most 5×10 11 , at most 1×10 11 , at most 5×10 10 , at most 1×10 10 , at most 5×10 9 , at most 1×10 9 , at most 5×10 8 , at most 1×10 8 , at most 5×10 7 , at most 1×10 7 , at most 5,000,000, at most 1,000,000, at most 950,000, at most 900,000, at most 850,000, at most 800,000, at most 750,000, at most 700,000, at most 650,000, at most 600,000, at most 550,000, at most 500,000, at most 450,000, at most 400,000, at most 350,000, at most 300,000, at most 250,000, at most 200,000, at most 150,000, at most 100,000, at most 95,000, at most 90,000, 85,000, at most 80,000, at most 75,000, at most 70,000, at most 65,000, at most 60,000, at most 55,000, at most 50,000, at most 45,000, at most 40,000, at most 35,000, at most 30,000, at most 25,000, at most 20,000, at most 15,000, at most 10,000, at most 5,000, at most 1,000, at most 500, or at most 100 target sequence copy numbers and can be. Any of the lower and higher values described in this paragraph may be combined to form a range included within the present disclosure. For example, in some examples, the surface density of target sequence copies is per mm 2Approximately 1,000 target sequence copies per mm 2 This can amount to approximately 65,000 target sequence copies per unit. Those skilled in the art will know that the surface density of target sequence copies can be any value within this range, for example, mm 2 You should be aware that there could be approximately 49,600 target sequence copies per instance.

[0126] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification buffer formulation results in mm 2 Approximately 100 molecules per mm 2 Approximately 1 x 10 12 This can result in a surface density of clone-amplified target (or sample) oligonucleotide molecules (or clusters) that extends to the colonies. In some examples, the surface density of clone-amplified molecules is mm 2 Per unit, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 , at least 5 × 10 7 , 1 x 10 8(at least) at least 5 × 10 8 , at least 1 × 10 9 , at least 5 × 10 9 , at least 1x10 10 , 1. At least 5 × 10 10 , at least 1 × 10 11 , at least 5 × 10 11 , or at least 1x10 12 It can be a molecule. In some cases, the surface density of cloned amplified molecules is mm 2 The maximum is 1 x 10 12 , up to 5 x 10 11 , up to 1 x 10 11 , up to 5 x 10 10 , up to 1 x 10 10 , up to 5 x 10 9 , up to 1 x 10 9 , up to 5 x 10 8 , up to 1 x 10 8 , up to 5 x 10 7 , up to 1 x 10 7, up to 5,000,000, 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 The number of molecules may be 0, up to 95,000, up to 90,000, 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the surface density of cloned amplified molecules is mm 2 Approximately 5,000 molecules per mm 2 It can reach up to approximately 50,000 molecules per unit. Those skilled in the art will know that the surface density of the cloned amplified colony can be any value within this range, for example, mm 2 You will realize that there could be approximately 48,800 molecules per molecule.

[0127] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification buffer formulation results in mm 2 Approximately 100 molecules per mm 2 Approximately 1 x 10 12 This can result in a surface density of clone-amplified target (or sample) oligonucleotide molecules (or clusters) that extends to the colonies. In some examples, the surface density of clone-amplified molecules is mm 2Per unit, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 , at least 5 × 10 7 , at least 1 × 10 8 , at least 5 × 10 8 , at least 1 × 10 9 , at least 5 × 10 9 , at least 1x10 10 , 1. At least 5 × 10 10 , at least 1 × 10 11 , at least 5 × 10 11 , or at least 1x10 12 It can be a molecule. In some cases, the surface density of cloned amplified molecules is mm 2 The maximum is 1 x 10 12 , up to 5 x 10 11 , up to 1 x 10 11 , up to 5 x 10 10 , up to 1 x 10 10 , up to 5 x 10 9 , up to 1 x 10 9 , up to 5 x 10 8, up to 1 x 10 8 , up to 5 x 10 7 , up to 1 x 10 7 , up to 5,000,000, 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000 The number of molecules may be 0, up to 95,000, up to 90,000, 85,000, up to 80,000, up to 75,000, up to 70,000, up to 65,000, up to 60,000, up to 55,000, up to 50,000, up to 45,000, up to 40,000, up to 35,000, up to 30,000, up to 25,000, up to 20,000, up to 15,000, up to 10,000, up to 5,000, up to 1,000, up to 500, or up to 100 molecules. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the surface density of cloned amplified molecules is mm 2 Approximately 5,000 molecules per mm 2 It can reach up to approximately 50,000 molecules per unit. Those skilled in the art will know that the surface density of the cloned amplified colony can be any value within this range, for example, mm 2 You will realize that there could be approximately 48,800 molecules per molecule.

[0128] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification buffer formulation results in mm 2 Approximately 100 colonies per unit, mm 2 Approximately 1 x 10 12This can result in a surface density of colonies (or clusters) of cloned target (or sample) oligonucleotides, which may extend down to the colonies. In some examples, the surface density of cloned colonies is mm 2 Per unit, at least 100, at least 500, at least 1,000, at least 5,000, at least 10,000, at least 15,000, at least 20,000, at least 25,000, at least 30,000, at least 35,000, at least 40,000, at least 45,000, at least 50,000, at least 55,000, at least 60,000, at least 65,000, at least 70,000, at least 75,000, at least 80,000, at least 85,000, at least 90,000, at least 95,000, at least 1 00,000, at least 150,000, at least 200,000, at least 250,000, at least 300,000, at least 350,000, at least 400,000, at least 450,000, at least 500,000, at least 550,000, at least 600,000, at least 650,000, at least 700,000, at least 750,000, at least 800,000, at least 850,000, at least 900,000, at least 950,000, at least 1,000,000, at least 5,000,000, at least 1 × 10 7 , at least 5 × 10 7 , 1 x 10 8 (at least) at least 5 × 10 8 , at least 1 × 10 9 , at least 5 × 10 9 , at least 1x10 10 , 1. At least 5 × 10 10 , at least 1 × 10 11 , at least 5 × 10 11 , or at least 1x10 12 It can be a colony. In some cases, the surface density of cloned amplified colonies is mm 2 The maximum is 1 x 10 12 , up to 5 x 10 11, up to 1 x 10 11 , up to 5 x 10 10 , up to 1 x 10 10 , up to 5 x 10 9 , up to 1 x 10 9 , up to 5 x 10 8 , up to 1 x 10 8 , up to 5 x 10 7 , up to 1 x 10 7 , up to 5,000,000, 1,000,000, up to 950,000, up to 900,000, up to 850,000, up to 800,000, up to 750,000, up to 700,000, up to 650,000, up to 600,000, up to 550,000, up to 500,000, up to 450,000, up to 400,000, up to 350,000, up to 300,000, up to 250,000, up to 200,000, up to 150,000, up to 100,000, The maximum number of colonies may be 95,000, 90,000, 85,000, 80,000, 75,000, 70,000, 65,000, 60,000, 55,000, 50,000, 45,000, 40,000, 35,000, 30,000, 25,000, 20,000, 15,000, 10,000, 5,000, 1,000, 500, or up to 100 colonies. Any of the lower and higher values ​​described in this paragraph may be combined to form a range included in this disclosure, for example, in some examples the surface density of the cloned amplified colonies may be mm 2 Approximately 5,000 colonies per unit, from mm 2 It can reach up to approximately 50,000 colonies per unit. Those skilled in the art will know that if the surface density of the hybridized target oligonucleotide is any value within this range, for example, mm 2 You will realize that there could be approximately 48,800 colonies in that area.

[0129] In some cases, the use of the low-binding support of this disclosure alone or in combination with an optimized amplification reaction formulation may result in a signal (e.g., a fluorescent signal) from an amplified and labeled nucleic acid population with a coefficient of variation of 50% or less, for example, 50%, 40%, 30%, 20%, 15%, 10%, 5%, or less than 5%.

[0130] In some cases, the support surfaces and methods disclosed herein allow amplification at elevated stretching temperatures such as 15C, 20C, 25C, 30C, 40C, or higher, for example, about 21C or 23C.

[0131] In some cases, the use of the support surfaces and methods disclosed herein enables simplified amplification reactions. For example, in some cases, the amplification reaction may be carried out using only one, two, three, four or fewer, or five or fewer individual reagents.

[0132] In some cases, the use of support surfaces and methods disclosed herein allows for the use of a simplified temperature profile during amplification, and as a result, the reaction is carried out at temperatures ranging from low temperatures of 15C, 20C, 25C, 30C, or 40C to high temperatures of 40C, 45C, 50C, 60C, 65C, 70C, 75C, 80C, or above 80C, for example, in the range of 20C to 65C.

[0133] Amplification reactions also occur at 1 pM, 2 pM, 5 pM, 10 pM, 15 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1,000 pM, 2,000 pM, 3,000 pM, 4,000 pM, 5,000 pM, 6,000 pM, 7,000 pM, and 8,000 pM. The system is improved so that lower amounts of template (e.g., target or sample molecule) are sufficient to produce a recognizable signal on the surface, such as 9,000 pM, 10,000 pM samples, or samples greater than 10,000 pM, such as 500 nM. In a typical embodiment, an input of about 100 pM is sufficient to generate a signal for reliable signal measurement.

[0134] Fluorescence imaging of support surface: The solid-phase nucleic acid amplification reaction formulations and low-binding supports of this disclosure may be used in any application of various nucleic acid analysis, including, for example, nucleic acid base identification, nucleic acid base classification, nucleic acid base calling, nucleic acid detection applications, nucleic acid sequencing applications, and nucleic acid-based (genetic and genetic) diagnostic applications. In many of these applications, fluorescence imaging techniques may be used to monitor hybridization, amplification, and / or sequencing reactions performed on the low-binding supports.

[0135] Fluorescence imaging can be performed using various fluorophores, fluorescence imaging techniques, and fluorescence imaging apparatus known to those skilled in the art. Examples of suitable fluorescence stains that may be used (e.g., by conjugation to nucleotides, oligonucleotides, or proteins) include, but are not limited to, fluorescein, rhodamine, coumarin, cyanine, and cyanine derivatives such as Cyanine dye-3 (Cy3), Cyanine dye-5 (Cy5), and Cyanine dye-7 (Cy7), and their derivatives. Examples of fluorescence imaging techniques that may be used include, but are not limited to, fluorescence microscopy imaging, fluorescence confocal imaging, and two-photon fluorescence. Examples of fluorescence imaging apparatus that may be used include, but are not limited to, fluorescence microscopes, confocal fluorescence microscopes, two-photon fluorescence microscopes equipped with an image sensor or camera, or custom apparatuses that include a light source, lenses, mirrors, prisms, dichroic reflectors, apertures, and an image sensor or camera, etc. A non-limiting example of a fluorescence microscope equipped to obtain images of the low-binding support surface of the present disclosure and images of cloned amplified colonies (or clusters) of target nucleic acid sequences hybridized thereon is the Olympus IX83 inverted fluorescence microscope, equipped with a 20x, 0.75 NA, 532 nm light source, a bandpass and dichroic mirror filter set optimized for 532 nm long-pass excitation and Cy3 fluorescence emission filtering, a Semrock 532 nm dichroic reflector, and a camera (Andor sCMOS (Zyla 4.2)) whose excitation light intensity is adjusted to avoid signal saturation. Often, the support surface may be immersed in a buffer (e.g., 25 mM ACES, pH 7.4 buffer) while the image is being acquired.

[0136] In some examples, the performance of nucleic acid hybridization and / or amplification reactions using the reaction formulations and low-binding supports of this disclosure may be evaluated using fluorescence imaging techniques, where the contrast-to-noise ratio (CNR) of the image provides an important metric for evaluating amplification specificity and nonspecific binding on the support. CNR is generally defined as follows: CNR = (signal - background) = / noise. The background term is well understood as the signal measured for the intervening region surrounding a particular feature (diffraction-limited spot, DLS) in a given region of interest (ROI). While the signal-to-noise ratio (SNR) is often considered a benchmark for overall signal quality, improved CNR can sometimes be shown to offer a significant advantage over SNR as a benchmark for signal quality in applications requiring rapid image acquisition (e.g., sequencing applications where cycle time must be minimized), as shown in the following examples. As shown in Figures 6A and 6B, at high CNR, the imaging time required to achieve accurate identification (and therefore accurate base calling in sequencing applications) can be drastically reduced, even by a moderate improvement in CNR. Figures 6A and 6B provide simulation data (solid lines) and integrated mean values ​​(dashed lines) for signal and background intensity, measured as a function of CNR (CNR=1.25 in Figure 6A and 12.49 in Figure 6B) and integration time (SNR=2 in both figures), illustrating the improved discrimination that can be achieved by using CNR as a metric for signal quality. Figure 7 provides an example of the effect of improving CNR in image data on the imaging integration time required to accurately detect features such as clone-amplified nucleic acid colonies on a support surface.

[0137] In most ensemble-based sequencing approaches, the background term is typically measured as a signal associated with an "intervening" region (see Figure 8). "Intervening" background (B inter) in addition to the "intrinsic" background (B intra The background signals are located within the region occupied by the amplified DNA colonies. The combination of these two background signals defines the achievable CNR and, subsequently, directly influences the optical instrument requirements, structural costs, reagent costs, run time, cost / genome, and ultimately, the accuracy and data quality for circular array-based sequencing applications. inter Background signals arise from various sources; some examples include autofluorescence from commercial flow cells, nonspecific adsorption of detection molecules producing false fluorescent signals that can obscure signals from ROIs, and the presence of nonspecific DNA amplification products (e.g., those arising from primer dimers). In typical next-generation sequencing (NGS) applications, this background signal in the current field of view (FOV) is averaged and subtracted over time. Signals arising from individual DNA colonies (i.e., (S)-B in the FOV) inter ) gives recognizable features that can be classified. In some cases, the intrinsic background (B intra This increases the number of confusing fluorescent signals present within the same ROI but not specific to the target being studied, thereby making averaging and subtracting them far more difficult.

[0138] As will be demonstrated in the following examples, nucleic acid amplification on the low-binding substrate of this disclosure is achieved by reducing nonspecific binding. interBackground signal reduction may occur, resulting in improvements in specific nucleic acid amplification, and may result in a reduction in nonspecific amplification, which may affect background signal originating from both intervening and endogenous regions. In some examples, the low-binding support surface of this disclosure, optionally used in combination with the hybridization and / or amplification reaction formulations of this disclosure, may result in improvements to CNR by multiples of 2x, 5x, 10x, 100x, or 1000x compared to those achieved using conventional supports and hybridization, amplification, and / or sequencing protocols. Although described herein in the context of using fluorescence imaging as the calling or detection mode, the same principles apply to the use of the low-binding support and nucleic acid hybridization and amplification formulations of this disclosure, as well as to other detection modes, including both optical and non-optical detection modes.

[0139] The low-binding supports of this disclosure, optionally used in combination with the hybridization and / or amplification protocols of this disclosure, produce solid-phase reactions exhibiting (i) negligible nonspecific binding of proteins to other reactants (thus minimizing substrate background), (ii) negligible nonspecific nucleic acid amplification products, and (iii) modulable nucleic acid amplification reactions. While described herein primarily in the context of nucleic acid hybridization, amplification, and sequencing assays, this will be understood more in the art. The low-binding supports of this disclosure may be used in any of a variety of other bioassay formats, including but not limited to sandwich immunoassays and enzyme-linked immunosorbent assays (ELISA).

[0140] System: This specification provides a system for performing base identification or base classification reactions using the surface described herein. Furthermore, a system is also provided for performing one or more steps of any sequencing method disclosed herein. Optionally, the system includes components and reagents necessary for coupling oligonucleotide molecules, hybridizing a sample or target nucleic acid to attached oligonucleotide molecules, and detecting or imaging a signal on the surface.

[0141] Furthermore, systems for carrying out one or more steps of any sequencing methods disclosed herein are also provided. Optionally, the system includes components and reagents necessary for analyzing the sequence of nucleic acids in such sequencing techniques based on the detection of fluorescent nucleotides or oligonucleotides. The detection instrument used to read the fluorescent signals on such arrays may be based on either epifluorescence or total internal reflection microscopy. One detector is recommended to be a sequencing-by-synthesis reader. The reader may include a laser that produces fluorescence from a sample in a water channel of a flow cell. The fluorescence is emitted and collected by an imaging optical system including one or more objective lenses and tube lenses. The optical imaging system includes, among other things, a light source for illuminating the sample in a region of interest, one or more detectors, and optical components for guiding light from the region of interest to the detectors. The optical imaging system may also include a focusing mechanism for maintaining the focus of the optical components on the region of interest so that the light received at the detector is focused and received.

[0142] Methods for base pair classification: Provided herein are methods for performing nucleic acid base pair identification or base pair classification, the methods comprising: a) providing a surface, the surface comprising: i) a substrate; ii) at least one hydrophilic polymer coating layer; iii) a plurality of oligonucleotide molecules attached to at least one hydrophilic polymer coating layer; and iv) at least one isolation region of the surface comprising a plurality of clone-amplified sample nucleic acid molecules annealed to the plurality of attached oligonucleotide molecules, wherein the plurality of annealed clone-amplified sample nucleic acid molecules are present at a surface density of at least 10,000 molecules / mm²; b) performing a nucleic acid amplification reaction on the sample nucleic acid molecules before or after annealing them to the plurality of oligonucleotide molecules; and c) performing a periodic sequence of single nucleotide binding or incorporation reactions, wherein the nucleotides are labeled with a detectable tag. Methods for performing nucleic acid sequencing by utilizing the surface or system described herein are also provided herein. In some embodiments, the detectable tag is a fluorophore. In some embodiments, the detectable tag is cyanine dye-3 (Cy3), and here the fluorescence image of the surface is acquired using an Olympus IX83 inverted fluorescence microscope, which comprises a 20x, 0.75 NA, 532 nm light source, a 532 nm long-pass excitation and a set of bandpass and dichroic mirror filters optimized for Cy3 fluorescence emission filtering, a Semrock 532 nm dichroic reflector, and a camera (Andor sCMOS, Zyla4.2) exhibiting a contrast-to-noise ratio (CNR) of at least 20 under non-signal saturation conditions while the surface is immersed in buffer after binding or incorporation of a first Cy3-labeled nucleotide.

[0143] In some embodiments, the nucleic acid amplification reaction includes a bridge amplification reaction. In some embodiments, the nucleic acid amplification reaction includes an isothermal bridge amplification reaction. In some embodiments, the nucleic acid amplification reaction includes a rolling circle amplification (RCA) reaction. In some embodiments, the nucleic acid amplification reaction includes a helicase-dependent amplification reaction. In some embodiments, the nucleic acid amplification reaction includes a recombinase-dependent amplification reaction. In some embodiments, at least one hydrophilic polymer coating layer exhibits a water contact angle of less than 50 degrees. [Examples]

[0144] These embodiments are provided for illustrative purposes only and do not limit the scope of the claims.

[0145] Example 1 - Hydrophilic Substrate We conducted research to prepare and evaluate non-specific low-binding support surfaces using poly(ethylene glycol) (PEG) molecules with different molecular weights and functional end groups. One or more PEG layers were linked to a glass surface via functional group-terminated silane coupling. Examples of functional groups that can be used for coupling include, but are not limited to, biotin, methoxyether, carboxylate, amine, NHS ester, maleimide, and bissilane. Subsequently, oligonucleotide primers with different base sequences and base modifications were linked to the surface layer at various densities. Both the surface functional group density and oligonucleotide concentration were varied to target specific primer concentration ranges. Furthermore, primer density can be controlled by diluting oligonucleotides with other molecules carrying the same functional group. For example, amine-labeled oligonucleotides can be diluted with amine-labeled polyethylene glycol in reaction with an NHS ester coated surface to reduce the final primer density. Primers containing different lengths of linkers positioned between the hybridization region and the surface-attached functional group can also be applied to control density. Exemplary linkers include poly T and poly A chains (0–20 base lengths) at the 5' end of the primer, PEG linkers (3–20 units lengths), and hydrocarbon chain linkers of various lengths (e.g., C6, C12, C18, etc.). To measure primer density, fluorescently labeled primers were immobilized on the surface, and fluorescence readings were compared to readings of dye solutions of known concentrations.

[0146] Non-specific low-NSB (also referred to herein as "passivated") substrate surfaces are desirable because biomolecules such as proteins and nucleic acids do not "adhere" to the surface. Examples of non-specific low-NSB (low NSB) surfaces prepared using standard monolayer surface preparations and various glass surface treatments are provided below. Successfully performing nucleic acid amplification on passivated surfaces presents unique challenges. Because passivated hydrophilic surfaces exhibit extremely low NSBs for proteins and nucleic acids, novel conditions must be utilized to achieve high passivation, improved primer deposition reaction efficiency, hybridization conditions, and effective nucleic acid amplification. Solid-phase nucleic acid hybridization and amplification processes require nucleic acid templates attached to a low-NSB or passivated surface, followed by protein delivery and binding to the surface. The combination of novel primer surface-binding formulations (identified by Cy3 oligonucleotide graft titration) and the resulting extremely low nonspecific background (evaluated using NSB functional test results of red and green dyes) demonstrates the feasibility of these approaches.

[0147] Multilayer coatings were applied and tested with PEG and other hydrophilic polymers to scale primer density and add additional dimensionality to hydrophilic or amphoteric surfaces. By using hydrophilic and amphoteric surface layering approaches, including but not limited to the polymer / copolymer materials described herein, it is possible to significantly increase the primer loading onto the support surface. Conventional PEG-coated supports using monolayer primer deposition processes have been reported for single-molecule sequencing applications, but the copy number for nucleic acid amplification is not high. Herein, “layering” can be achieved using conventional bridge-bonding approaches and chemically compatible monomer or polymer subunits, resulting in the sequential construction of one or more highly crosslinked layers. Non-limiting examples of polymers suitable for use include streptavidin, polyacrylamide (poly The materials include polyacrylamide, poly(N-isopropylacrylamide (PNIPAM), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA), polyester, dextran, polylysine, polyethylene glycol (PEG), polyacrylic acid (PAA), poly(vinylpyridine), poly(vinylimidazole), and polylysine copolymers. Different layers may be bonded to each other using any of the following covalent or non-covalent reactions between positively charged and negatively charged polymers, including but not limited to biotin-streptavidin bonding, azido-alkyne click reactions, amine-NHS ester reactions, thiol-maleimide reactions, and ionic interactions. It is also conceivable that these high-primer-density materials may be constructed in solution and then layered onto a surface in multiple steps.This approach makes it possible to generate low-NSB substrate surfaces / low-background substrate surfaces (Figures 9-13) for performing solid-phase nucleic acid amplification and sequencing chemistry, which provides a significant improvement in nucleic acid amplification, and as a result the signal-to-background ratio can be tuned to meet the needs of specific sequencing applications (Figures 14 and 15). Figure 9 provides an example of image data from a study to determine the relative levels of nonspecific binding of a green fluorescent dye to a glass substrate surface treated according to different surface modification protocols. Figure 10 provides an example of image data from a study to determine the relative levels of nonspecific binding of a red fluorescent dye to a glass substrate surface treated according to different surface modification protocols. Figure 11 provides an example of oligonucleotide primer grafting data of substrate surfaces treated according to different surface modification protocols.

[0148] Method for preparing a two-layer PEG surface using thiol-maleimide chemistry: A glass slide was washed using 2M KOH treatment at room temperature for 30 minutes, rinsed, and then the surface silanol groups were activated using oxygen plasma. Silane PEG5K thiol (Creative PEGWorks, Inc., Durham, NC) was applied at a concentration of 0.1% in ethanol. After a 2-hour coating reaction, the slide was thoroughly washed with ethanol and water, and then reacted with 2.5 mM maleimide-PEG-succinimidyl valerate (MW=20K) in dimethylformamide (DMF) for 30 minutes. The resulting surface was washed and immediately reacted with a 5'-amine labeled oligonucleotide primer at room temperature for 2 hours. After primer immobilization, excess succinimidyl ester on the surface was deactivated by reacting with 100 mM glycine at pH 9.

[0149] Method for preparing multilayer PEG surfaces using NHS ester-amine chemistry: Glass slides were washed and rinsed by 2M KOH treatment at room temperature for 30 minutes, and then the surface silanol groups were activated using oxygen plasma. Silane-PEG2K-amine (Nanocs, Inc., New York, NY) was applied at a concentration of 0.5% in an ethanol solution. After a 2-hour coating reaction, the slides were thoroughly rinsed with ethanol and water. 100 μM 8-arm PEG NHS (MW=10K, Creative PEGWorks, Inc., Durham, NC) was introduced at room temperature for 20 minutes in a solvent composition that may contain 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent organic solvent and 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent low ionic strength buffer. The resulting surface was washed away, and then reacted with 20 μM multi-arm PEG amine (MW=10K, Creative PEGWorks, Inc., Durham, NC) for 2 hours. The resulting amine-PEG surface was then reacted with a mixture of multi-arm PEG-NHS and amine-labeled oligonucleotide primers at various concentrations. This process was repeated to generate additional PEG layers on the surface.

[0150] Solid-Phase Isothermal Amplification: When considering various isothermal amplification methods, each method has a unique optimal primer density range, which can indicate the need for an adjustable surface coating to maximize amplification efficiency. In some cases, a higher primer surface density results in a larger template copy number (Figure 15). In some cases, a higher primer surface density may produce a high foreground signal (i.e., a sequence-specific signal) in some amplification approaches, but it can also cause a high background signal, and is therefore known to be detrimental to other amplification approaches. In Figure 16, the primer deposition density is shown at the top of the figure, and the various helicase isothermal amplification formulations tested are shown in images obtained from the resulting surfaces after the isothermal amplification reaction. If specific amplification occurred, the image of each surface was expected to appear red. As is evident from the series of images of formulation "58" in Figure 16, as the primer density increased, the color faded from red to green, thus indicating a decrease in specific amplification. Furthermore, it has been confirmed that including betaine (a common buffering additive) in the amplification reaction formulation reduces the degree of nonspecific amplification, favoring specific amplification, which in turn results in brighter signals and improved CNR. The combination of a low-binding layered support surface, adjustable primer surface density, improvements in hybridization and / or the amplification reaction formulation (including adjustments to buffer components and additives, e.g., selection of buffers, pH, solvents, ionic strength, purifying agents, formamide, betaine, crowding agents, and other additives), and the resulting improvements in amplification and specificity should lead to unprecedented advancements in next-generation nucleic acid sequencing.

[0151] This disclosure addresses the challenge of achieving high monoclonal amplification of library nucleic acid strands on hydrophilic substrates for various applications requiring signal enhancement, such as nucleic acid detection, sequencing, and diagnostic applications. Conventional isothermal methods for nucleic acid amplification to generate monoclonal clusters of library nucleic acid strands are limited and flawed. Examples of their performance limitations include long clustering times (e.g., more than 2 hours), high temperature requirements (e.g., above 60°C), inability to efficiently amplify / cluster on certain surfaces, high cost, polyclonal issues, and reagent stability problems. While there are many isothermal amplification methods described in the literature, only one or two have been successfully applied to commercial sequencing applications. Here, we propose an isothermal amplification strategy that successfully generates clusters of monoclonal copies of library DNA fragments (or other nucleic acids) for applications such as DNA sequencing, eliminating or mitigating the aforementioned problems.

[0152] Background signal (B inter and B intra The design criteria for developing ensemble DNA amplification / DNA sequencing composition change to reduce interstitial region (B) and promote controlled DNA amplification on low-binding substrates are: (i) compared to conventional technology approaches, inter (ii) reduction of nonspecific DNA amplification (e.g., by primer dimer amplification) in ) and specific DNA colonies (B) compared to conventional approaches. intra (iii) a decrease in the amount of nonspecific DNA amplification products (e.g., primer dimers) within the substrate, and specific DNA amplification on low-binding substrates (e.g., reaction time, cycle time, primer surface density titration, primer surface density, primer sequence, etc.), resulting in a decrease in the signal-to-background (S / B) ratio even without improvements in hybridization and amplification formulations.

[0153] Example 2 - Helicase-dependent amplification on low-binding surfaces with improved specificity It is well known that helicase-dependent amplification is highly susceptible to nonspecific amplification, such as primer dimerization. This nonspecific amplification may be reduced on the surface through any combination of the following methods: (i) designing oligonucleotide primers that produce fewer primer dimers; (ii) adjusting the primer surface density on the multilayer support surface; (iii) performing the reaction at a higher temperature using thermophilic enzymes; (iv) using amplification buffer additives such as those described above in combination with non-self primer sequences; and (v) introducing one or more complete nucleic acid denaturation and primer hybridization steps.

[0154] Specific helicase-dependent amplification on low-NSB surfaces in the absence of SSB proteins: Helicase-dependent amplification of linear template chains can be achieved by reduced nonspecific amplification and high-efficiency clonal amplification on low-binding surfaces. Surface-conjugated forward primers are extended onto a single-stranded template library chain using an amplification reaction mixture containing polymerase and helicase. The template chain is then optionally denatured and washed away. Alternatively, the double strand may be unwound by helicase activity. In either method, a forward strand is left behind, extended from the surface-conjugating primer in a partially or completely single-stranded form. Subsequently, a surface-binding reverse primer hybridizes to and extends this forward strand, thereby creating a double-stranded bridge structure. The helicase present in the reaction mixture unwinds the intermediate double-stranded amplicon chain, which is then available to re-hybridize to other free surface-binding primers for subsequent amplification rounds. For this to occur, the degree of unwinding does not need to be extensive; it is sufficient to simply convert the primer hybridization region into a single-stranded component (terminate the unwinding), after which hybridization of the surface-linked primer becomes possible. The helicase used in this reaction should be able to initiate unwinding from the ends of the bridge structure and may be either a 3'-5' helicase or a 5'-3' helicase. In some cases, it may be a helicase from superfamily 1, 2, 3, 4, 5, or 6. In other cases, it may be a highly processive helicase (i.e., able to unwind many consecutive base pairs without releasing single-stranded or double-stranded structures) or a helicase with limited processivity. Superfamily 1 helicases exhibiting higher processivity, such as the UvrD303 variant of the helicase UvrD, may also be used in this amplification scheme.

[0155] To facilitate unwinding by 5'-3' helicases, all or part of one or both surface-binding primers may include modifications to create a specific loading site for the 5'-3' helicase. On the template nucleic acid extended from such primers, the modification site acts as a polymerase stop site, thereby ensuring that the extension of the primer sequence between the surface conjugate site and the modification site is always in a single-stranded form. This extension would function as a loading site for helicases with 5'-3' orientation, since many helicases have far superior single-stranded nucleic acid binding affinity and induce and enhance 5'-3' helicase unwinding activity when required for helicase-dependent amplification on the support surface. Examples of usable primer modifications include, but are not limited to, insertion of a PEG chain into the primer backbone between two nucleotides toward the 5' end, insertion of a debasalized nucleotide (i.e., a nucleotide without either a purine or pyrimidine base), or lesion sites that can be bypassed by helicases.

[0156] Many helicases possess cofactor proteins and specific configurations that activate or enhance helicase activity. Examples include, but are not limited to, the RepD protein of Bst-derived PcrA helicase, the Phi-X gene protein A of E. coli Rep helicase, and the MutL protein of UvrD helicase. The addition of these accessory proteins may more effectively enable the desired unwinding activity and thus further promote helicase-dependent isothermal amplification. Some of these cofactors have specific binding sequences or moieties that can be added to primers to induce unwinding activity.

[0157] Helicase amplification formulations showed reduced nonspecific amplification when using mesophilic helicases and chain-substituted polymerase formulations lacking single-chain binding (SSB) proteins (e.g., internal reference formulation #58). Unlike most helicase-dependent amplification approaches, it was observed that removing single-chain binding proteins (typically used to interrupt transient nonspecific hybridization) from the formulation reduced nonspecific amplification. Various formulation modifications were shown to mitigate the increase in nonspecific hybridization on low-binding surfaces.

[0158] Modification of formulation composition to improve helicase-dependent amplification: It is generally known that additives such as betaine reduce nonspecific amplification in isothermal amplification reactions carried out in solution. The criteria become more restrictive as higher primer surface density is required to support tunable amplification and high copy number of template nucleic acid colonies. At high primer densities, nonspecific amplification begins to aid the advantage of a high template copy number of the resulting colonies (Figure 16). As a result, additional additive formulations that aid nonspecific amplification within template nucleic acid colonies have been discovered. In the example shown in Figure 16, it can be clearly seen that adding betaine to formulation #58 produces more specific amplification (shown in red) on surfaces with higher primer density. In addition to betaine, it is possible to achieve higher amplification specificity in solution and on low-binding surfaces by combining many different reaction formulation components (Figures 17 and 18).

[0159] Organic solvents such as acetonitrile, DMSO, DMF, ethanol, methanol, and similar compounds alter the structure of single- and double-stranded nucleic acids through the process of oligonucleotide dehydration. Compounds such as 2-pyrrolidone and formamide are known to lower the melting temperature of nucleic acids and reduce the secondary structure from high-GC-content oligonucleotides. Crowding agents are also known to stabilize nucleic acid structures. Low pH buffers favor hybridization and hydrogen bonding in base pairing. Combining the distinct attributes of each of these formulations makes it possible to increase the specific annealing of oligonucleotide sequences by more than two orders of magnitude compared to conventional approaches. By combining these compounds and adding them to the amplification formulations described below, nonspecific amplification, such as that resulting from primer dimers, is dramatically reduced, and good yields of specific amplification products are observed in solution (Figure 17) and on the disclosed low-NSB surface (Figure 18).

[0160] Example 3 - Modified Rolling Circle Multi-Substitution Amplification (Modified RCA-MDA) Nucleic acid library fragments are ligated to adapter sequences (including forward primers, reverse primers, and sequencing primers, as well as any identification / barcode sequences) and then cyclized either in solution or on a low-binding surface.

[0161] Subsequently, the circularized ssDNA is captured by a forward surface primer, either in solution or on the surface, or hybridized to a forward surface primer, and then extended in an RCA reaction by a strand substitution polymerase, producing a single-stranded concatemer copy of the library nucleic acid and adapter sequence. Reverse primers hybridize to this concatemer forward template at multiple positions and are extended by the RCA reaction mixture, thereby producing a concatemer reverse copy. During this process, the reverse strands are substituted by each other. Upstream reverse strand extensions substitute downstream extensions, thus creating a single-stranded concatemer reverse strand. The addition of helicase can restart hybridization and generate a single-stranded region of nucleic acid that lasts long enough to induce a substitution cascade, which can increase the amplified copy number in a relatively controlled manner. Alternatively, the addition of recombinases and accessory proteins can cause the primers to hybridize to homologous regions of double-stranded DNA in a process called strand invasion. This restarts the cascade of substitution and hybridization, increasing the number of colony copies.

[0162] The copy number of RCA-MDA colonies is determined by the primer surface density, which in turn determines how frequently and successfully the initial concatemer or substituted concatemer hybridizes with the forward and reverse primers. Increasing the primer density on low-binding surfaces has been shown to generate a greater number of amplified copies of these clusters (Figure 15). In summary, it is possible to increase the copy number or specific amplification and decrease non-specific amplification on low-binding surfaces using one or a combination of the following methods. (i) The specific copy number can be increased by improving the efficiency of primer template hybridization through formulation modifications (Figure 16), (ii) The specific copy number can be increased by increasing the primer density on a low-binding substrate (Figures 14 and 15), (iii) Nonspecific amplification of primer dimers or chimeric DNA generation can be reduced by using the additives described above, (iv) The amplification incubation temperature can be increased using a thermostable enzyme in combination with the formulation modifications described above to reduce nonspecific amplification, (v) A primer composition containing a non-self-contained hybridizing primer sequence can be used in combination with an additive and / or an increase in the amplification incubation temperature to reduce nonspecific primer dimer amplification.

[0163] Example 4 - Single-stranded DNA-binding (SSB) protein-mediated isothermal amplification SSB proteins alter the secondary structure of nucleic acid strands, stabilizing single-stranded DNA after unwinding, interfering with or disrupting transient, non-broad hybridization of two nucleic acid strands (precursors to primer dimer amplification), promoting specific hybridization of short oligonucleotides to the corrective complementary region of target oligonucleotides, and acting on the branching junctions of single- and double-stranded nucleic acids to induce enzymes. C-terminal truncation mutations in SSBs have been reported to remove cooperative binding to ssDNA, while their monomers exhibit stronger binding to ssDNA and lower the melting temperature of dsDNA. For example, the C-terminal truncation mutation T4gp32 in the phage SSB protein produces a protein (gp32ΔC) that lowers the melting temperature of dsDNA by tens of degrees compared to the performance of the wild-type protein.

[0164] This amplification scheme utilizes both truncated and wild-type SSB proteins (and their chain-displacement polymerases) in a formulation to simultaneously dissolve the ends of double-stranded nucleic acid bridge intermediates, enabling hybridization of surface primers and primer extension. Even if SSBs slow nucleic acid hybridization, they are generally known to promote specific hybridization. Therefore, the use of truncated SSB proteins, such as T4gp32ΔC, allows for more efficient hybridization of the 3' end of the bridged nucleic acid structure to other free-surface ligating primers. While this further disrupts the newly formed primer-template complex, an optimized formulation should enable extension of such complexes by the chain-displacement polymerase. SSB proteins are found in various phages (e.g., T4gp32), bacteria, archaea, fungi, and eukaryotes. Some of these are thermally stable SSB proteins, and their C-terminal truncation morphology allows for more efficient nucleic acid end fusion at optimized higher temperatures, thus enabling SSB-dependent thermophilic amplification while utilizing lower nonspecific amplification at high temperatures.

[0165] Through the use of additives such as those mentioned above, primer sequence design, specific hybridization and / or amplification formulations, and optimized temperatures suitable for use with appropriate enzymes and proteins that have temperature and chemical tolerance, this amplification method can be adjusted to promote highly specific growth while eliminating nonspecific amplification.

[0166] This scheme can be used to amplify circular and linear DNA, where each initiation of extension by strand substitution polymerase may subsequently trigger multiple substitution amplification events, which generate concatemer copies of the circular DNA template (see the modified RCA-MDA section above).

[0167] SSB-mediated amplification of circular library DNA (e.g., using phi-29 SSB) has been shown to be much faster than conventional RCA (e.g., requiring 30 minutes–1 hour amplification time compared to 2–3 hours for conventional RCA). Products of SSB-mediated amplification performed in solution were obtained as separate concatemer ladders on a gel (ran).

[0168] Example 5 - Low-temperature thermal cycling bridge amplification on low-coupling surfaces By using a combination of additives for hybridization and / or amplification formulations, thermally stable SSB proteins, and / or truncation-type SSB proteins, PCR formulations have been developed that can perform thermal cycling at lower temperatures than conventional methods outlined in Mullis' original PCR disclosure. In this scheme, additives can be used to lower the temperatures of nucleic acid hybridization and dehybridization below conventional values. For example, formamide lowers the melting temperature of DNA (T mThis is often used to lower the bond temperature, and subsequent DNA dehybridization can be performed at a temperature of approximately 60 degrees. On the other hand, the re-annealing temperature usually also requires a temperature gradient from high temperatures (95°C) to near room temperature. It is possible to create formulations that can dramatically reduce the strictness of the re-annealing temperature. Using such formulations results in a significant improvement over conventional bridge amplification methods, and the temperature gradient can be performed between 20 and 60 degrees. The reduced temperature gradient requirement and improved strictness of hybridization on low-binding substrates can produce the following advantages over conventional bridge amplification: (i) reduced amplification time, (ii) simplified instrumentation design, (iii) reduced reagent usage through faster and more specific hybridization resulting in more efficient amplification rates, and (iv) reduced reagent costs. It is also possible to demonstrate that the improved strictness of hybridization will reduce the number of amplification cycles required for amplification on the support surface.

[0169] Figures 19A and 19B provide examples of data illustrating the improvement in hybridization efficiency that can be obtained using the nonspecific low-binding support and improved hybridization formulation (Figure 19A) of this disclosure compared to a conventional hybridization formulation (Figure 19B).

[0170] Figure 20 shows a workflow for nucleic acid sequencing using the low-binding support and hybridization / amplification reaction formulation of this disclosure, and an example of the non-limiting processing time that can be achieved thereby.

[0171] Example 6: Preparation of a two-layer PEG surface by thiol-maleimide chemical reaction The glass slide was chemically treated to remove organic matter and activate the hydroxyl groups for silane coupling (various methods include plasma treatment, piranha etching, base wash, base bath, high-temperature glass annealing, and any combination thereof). Silane-PEG5K-thiol (Creative PEGWorks, Inc.) was applied to an ethanol solution at a concentration of 0.1%. After a 2-hour coating reaction, the slide was thoroughly washed with ethanol and water, and then reacted with 2.5 mM maleimide-PEG-succinimidyl valerate (MW=20K) in DMF for 30 minutes. The resulting surface was washed off and immediately reacted with a 5'-amine labeled oligonucleotide primer at room temperature for 2 hours. Excess succinimidyl ester on the surface was deactivated with 100 mM glycine at pH 9 after primer fixation. This approach results in a very small, low-binding solid support surface and efficient, repeatable primer-polymer coupling that surpasses conventional methods by nearly two orders of magnitude.

[0172] Example 7: Preparation of multilayer PEG surfaces by chemical reaction of NHS ester amines Organic matter is removed from the glass slide by chemical treatment, and then hydroxyl groups are activated for silane coupling (various methods include plasma treatment, piranha etching, base wash, base bath, high-temperature glass annealing, and any combination thereof). Silane-PEG-amine (Nanocs, Inc.) is applied to a clean ethanol solution at a concentration of 0.1%–2%. After a 2-hour coating reaction, the slide is thoroughly washed with ethanol and water. Multi-arm PEG NHS is introduced at room temperature for 5–30 minutes in a solvent composition that may include 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent organic solvent and 5, 10, 20, 30, 40, 50, 60, 70, 80, or 90 percent low ionic strength buffer. The resulting surface was washed away and reacted with multi-arm PEG amine (MW 10k, Creative PEGWorks, Inc.). The resulting amine-PEG surface was then reacted with a mixture of multi-arm PEG-NHS and amine-labeled oligonucleotide primers at various concentrations. This process was repeated to generate further PEG layers on the surface. This approach results in a very low-binding solid support surface and efficient, repeatable primer-polymer coupling that surpasses conventional methods by nearly two orders of magnitude.

[0173] Example 8 - Calculation of CNR on cluster data Typically, when fluorescence detection is used in solid-phase assays, the signal is generated by linking and / or amplifying molecules, which then bind to or adhere to a reporter dye molecule. This process produces both specific and nonspecific signals. Nonspecific components are often referred to as nonspecific noise or background (resulting from interstitial or intrastitial contributions) and interfere with the measurement of specific signals, reducing the contrast-to-noise ratio (CNR).

[0174] Nonspecific background can be generated from either dye molecules nonspecifically adsorbed onto the support surface, or from nonspecific amplification, such as primer dimer pairs on the surface. Both mechanisms generate substantial fluorescence background when a fluorescent reporter is used to label a specific molecule of symmetry.

[0175] The disclosed low-binding support surfaces and associated methods for use demonstrate a significant improvement in minimizing nonspecific background. As illustrated in the examples below, using specific amplification methods and support surfaces results in estimated nonspecific background far below 10% of the total signal. In contrast, conventional amplification methods and support surfaces often generate background signals of 30%–50% of the total signal.

[0176] As used herein, nonspecific background or noise is only one component of the overall system noise, which may also include other contributions from the detection system, such as photon shot noise, autofluorescence background, imaging sensor noise, and illumination noise (e.g., resulting from fluctuations in illumination intensity). In this regard, it is possible to achieve greater improvements over NGS and other bioassay techniques by extending the disclosed approaches for CNR improvement with novel support surfaces and associated hybridization and amplification methods to correct signal impurities that may arise from conventional synthetic sequencing, for example, through the use of pre-phasing and phasing, and further by correcting errors caused by DNA strand loss and / or DNA damage resulting from strict washing conditions or unblocking (reversible terminator removal) across multiple assay reaction cycles.

[0177] Generally, an assay for measuring the CNR of a solid-phase bioassay includes the following steps:

[0178] (1) A step of preparing a disclosed low-binding substrate which is functionalized with a symmetric receptor, target, and / or capture oligonucleotide.

[0179] (2) A step of capturing the receptor, target, and / or capture oligonucleotide. The receptor, target, and / or capture oligonucleotide may be directly labeled or may be a precursor to a subsequent labeling reaction. If amplification or additional probe labeling steps are not required, proceed to step 5. If probe labeling steps are required to produce a reporter, proceed to step 4. Otherwise, proceed to step 3.

[0180] (3) A step of performing amplification of receptors, targets, and / or captured oligonucleotides via conventional immunoassay signal amplification, oligonucleotide replication amplification (e.g., using bridging, isothermal, RCA, HDA, or RCA-MDA amplification strategies).

[0181] (4) A step of probing the amplified target with reporter labeling (for example, through the use of a fluorescent species or other type of reporter). This step is applicable to any surface-based bioassay, including but not limited to genotyping, nucleic acid sequencing, or surface-based target / receptor identification.

[0182] (5) The step of performing an appropriate detection method. In the case of fluorescence imaging, the detection method can consist of various methods. For example, a conventional optical microscope method includes all or some of the following components: an illumination or excitation light source, an objective lens, a sample, other optical components (such as a tube lens, optical filter, dichroic reflector), and a detection modality (e.g., an EMCCD camera, CCD camera, sCMOS, CMOS, PMT, APD, or other conventional method for measuring light levels). In the case of non-light-based detection, electrical signals can be measured using various means, including but not limited to field-effect transistor (FET) detection, electrode-based measurement of electrical signals (DC or AC), tunnel current, and measurement of magnetic signals.

[0183] The use of imaging and signal processing from a specified field of view (FOV) to calculate the CNR is shown in Figure 8, where CNR = (signal - background) / (noise), and background = (B intrastitial(組織間) +B interstitial(組織内) )

[0184] In the following example of calculating the CNR for clone-amplified clusters of nucleic acid sequences on a low-binding support of this disclosure, an image analysis program was used to identify representative foreground bright spots ("clusters"). Typically, a spot is defined as a small connected region of image pixels that exhibits a light intensity exceeding a certain intensity threshold. Only connected regions constituting a total number of pixels within a specified range are counted as spots or clusters. Regions that are too large or too small in terms of the number of pixels are ignored.

[0185] Once a sufficient number of spots or clusters have been identified, the average spot or foreground intensity and other signal statistics are calculated, such as maximum, average, and / or interpolated maximum intensity. The median or mean of all spot intensities is used to represent the foreground intensity of the spot.

[0186] Representative estimates of background region intensity can be determined using one of several different methods. One method is to divide the image into a plurality of small "tiles", each containing, for example, 25x25 pixels. Within each tiled region, a certain percentage (e.g., 25%) of the brightest pixels are discarded, and intensity statistics are calculated for the remaining pixels. Another method for determining background intensity is to select a region of at least 500 pixels, or more, that contains no foreground "spots" (as defined in a previous step), and then calculate intensity statistics. Subsequently, for any of these methods, a representative background intensity (median or mean) and a standard deviation are calculated. The standard deviation of the intensity of the selected region is used as the representative background variation.

[0187] Next, the contrast-to-noise ratio (CNR) is calculated as (foreground intensity - background intensity) / (background standard deviation).

[0188] Figures 21-23 provide examples of histograms of raw image data and intensity data used to calculate the CNR for different combinations of the nucleic acid amplification methodologies and low-binding supports described herein. In each of these examples, the upper histogram is the background pixel intensity histogram, the lower histogram is the foreground spot intensity histogram, and a portion of the original image is also included. Note that the visual perception of brightness does not indicate the actual intensity since the images are not on the same intensity scale.

[0189] In these examples, low-binding solid supports were created using the methods discussed previously. Oligonucleotide primers (one or two primer sequences depending on the amplification scheme used) were grafted at various densities using the disclosed methods. The surface density for each of these experiments was approximately 100K primers / μm 2was estimated. The primer surface density was estimated using the following methodology. (i) A fluorescence titration curve was generated using a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) and a capillary flow cell with a known area (40 mm 2 ), height (0.5 mm), and volume (200 μl), (ii) primers grafted to a low-binding support were hybridized to Cy3-labeled complementary oligonucleotides using a conventional hybridization protocol (3X sodium citrate (SSC) at 37 degrees or room temperature (RT); the hybridization conditions should be characterized for completeness), and the fluorescence intensity of the signal on the resulting surface was measured using the same GE Typhoon instrument used to generate the calibration curve, (iii) the number of primer molecules linked per unit area of the surface was calculated based on comparison with the calibration curve of the measured surface signal.

[0190] Subsequently, the DNA library sequence was hybridized to the linked primers. The hybridization protocol used in the library hybridization step varies depending on the surface characteristics, but a controlled library input is required to create resolvable DNA amplification colonies.

[0191] In this example, DNA amplification was performed using the following protocol. (i) Bridge amplification for 28 cycles at a primer density of approximately 1K primers / um 2 , (ii) bridge amplification for 28 cycles at a higher primer density >5K primers / um 2 , (iii) rolling circle amplification (RCA) for 90 minutes at a primer density of approximately 2 - 4K primers / um 2 .

[0192] After amplification, the amplified DNA was hybridized with complementary “sequencing” primers, and a sequencing reaction mixture containing Cy3-labeled dNTPs was added (“first base” assay) to determine the first base CNR for each methodology. The sequencing reaction mixture used in the “first base assay” may include any combination of labeled nucleotides, enzymes for incorporating modified nucleotide triphosphates (dNTPs), and associated incorporation buffers, metal cations, and cofactors to enable the identification of four bases.

[0193] After the initial base incorporation, the sequencing reaction mixture was replaced with buffer, imaging was performed using the same GE Typhoon instrument, and the CNR was calculated for the resulting images.

[0194] Figure 21 provides examples of fluorescence images and intensity data for the low-binding supports of this disclosure, for which approximately 2K primer / µm 2 Solid-phase nucleic acid amplification was performed using 28 cycles of bridge amplification at a primer density to create clonal amplified clusters of the template oligonucleotide sequence. In this example, the background intensity was 592 counts (standard deviation 66.5 counts), the foreground intensity was 1047.3 counts, and the calculated CNR was (1047.3-592) / 66.5=455.3 / 66.5=6.8. Estimated nonspecific noise = (592-100) / (1047-100)=52%.

[0195] Figure 22 provides a second example of fluorescence images and intensity data for the low-binding supports of the present disclosure, for which 5K primer / µm 2 Solid-phase nucleic acid amplification was performed using 28 cycles of bridge amplification at a higher primer density to produce clonely amplified clusters of the template oligonucleotide sequence. In this example, the background intensity was 680 counts (standard deviation 118.2 counts), the foreground intensity was 1773 counts, and the calculated CNR was (1773-680) / 118.2=1093 / 118.2=9.2. Estimated nonspecific noise = (680-100) / (1773-100)=35%.

[0196] Figure 23 provides examples of fluorescence images and intensity data for the low-binding supports of this disclosure, for approximately 100K primer / µm. 2 Solid-phase nucleic acid amplification was performed using rolling circle amplification (RCA) for 90 minutes at a primer density to produce clonely amplified clusters of template oligonucleotide sequences. In this example, the background intensity was 254 counts (standard deviation 22.7 counts), the foreground intensity was 6161 counts, and the calculated CNR was (6161-254) / 22.7=5907 / 22.7=260. Note the dramatic improvement in CNR achieved using this combination of low binding surface and amplification protocol. Estimated nonspecific noise = (254-100) / (6161-100)=3%.

[0197] Example 9 - Modification of the polymer support surface Surface modification for the purposes disclosed herein involves making the surface reactive to a number of chemical groups (-R), including amines. When prepared on a suitable substrate, these reactive surfaces can be stored for extended periods, for example, at least three months, at room temperature. Such surfaces can be further grafted with R-PEG and R-primer oligomers for surface amplification of nucleic acids, as described throughout this specification. Plastic surfaces, such as cyclic olefin polymers (COPs), can be modified using any of a number of methods known in the art. For example, treatment with Ti:sapphire laser ablation, UV-mediated ethylene glycol methacrylate photografting, plasma treatment, or mechanical agitation (sandblasting, polishing, etc.) can create hydrophilic surfaces that can maintain reactivity to a number of chemical groups, such as amines, for several months. These groups can then be conjugated with passivation polymers such as PEG, or biomolecules such as DNA and proteins, without losing biochemical activity. For example, the attachment of DNA primer oligomers enables DNA amplification on a passivated plastic surface, while simultaneously minimizing the nonspecific adsorption of proteins, fluorophore molecules, or other hydrophobic molecules.

[0198] Furthermore, surface modification can also be combined, for example, with laser printing or UV masking to create patterned surfaces. This allows for patterned adhesion of DNA oligomers, proteins, or other parts, enabling surface-based enzymatic activity, binding, detection, or processing. For example, DNA oligomers can be used to capture amplified DNA, or amplified long DNA concatemers, in a patterned manner, only within patterned features. In some embodiments, islands of enzymes may be generated in patterned regions that can react with a solution-based substrate. Plastic surfaces are particularly well-suited to these processing methods and may therefore be considered particularly advantageous in some embodiments as contemplated herein.

[0199] Furthermore, plastics can be formed into any shape, including microfluidic devices, much more easily than glass substrates, by injection molding, embossing, or 3D printing. Therefore, they can be used to create surfaces for binding and analyzing biological samples in multiple configurations, such as sample-to-result microfluidic chips for biomarker detection or DNA sequencing.

[0200] Specific localized DNA amplification was achieved on the modified plastic surface, and when probed with fluorescent labeling, it produced spots with extremely high noise-to-contrast and very low background.

[0201] We discovered that grafting amine-primers and amine-PEG onto the surface of a representative hydrophilic and amine-reactive cyclic olefin polymer facilitated rolling circle amplification. Subsequently, when probed with fluorophore-labeled primers, or when labeled dNTPs were added to primers hybridized by polymerase, bright spots on DNA amplicons exhibiting a signal-to-noise ratio exceeding 100 were observed. These bright spots exhibited extremely low background, highly specific amplification, and extremely low levels of protein and hydrophobic fluorophore binding, characteristic of high-precision detection systems such as fluorescence-based DNA sequencers.

[0202] Here, we test plastic flow cells into which linked DNA clusters are incorporated and the first base of the library sequence is probed. For DNA integration onto the surface, 25-mer amine-primer 1, amine-primer 2, and amine-5K PEG were grafted onto a hydrophilic cyclic olefin polymer (COP) plastic surface, as described in Examples 1 and 7 and elsewhere herein, as well as on PEG-NHS coated glass surfaces. In addition to the library insert, a 5 pM cyclic DNA library containing the primer 2 sequence, the sequencing primer sequence, and a sequence complementary to primer 1 was hybridized onto the surface for 15 minutes. Rolling circle amplification (RCA) was then performed as described in Examples 2–5 and elsewhere herein to produce concatemer sequence DNA coils up to 0.5–1 Mb in length. The sequencing primers were hybridized, and the first base was incorporated using polymerase-set fluorophore-labeled dNTPs to label the clusters in three different colors as shown in Figure 25A.

[0203] To provide a comparison between passivated glass and passivated COP surfaces, parallel experiments were conducted, starting with glass rather than the COP surface (by surface preparation as described in Example 7), and using the same parameters. As shown in Figures 25A and 25B, the signal generated by incorporating the first fluorescently labeled base onto the COP surface corresponds to the signal obtained on a similarly treated glass surface, both in terms of the intensity and resolution of the observed spots. This suggests that the methods disclosed herein provide a general method for surface preparation for nucleic acid immobilization, amplification, and detection.

[0204] The intensity and CNR are determined for both glass and plastic. Figure 26A shows that both glass and plastic exhibit signal intensity under detection conditions that substantially exceed the background. For both glass and plastic, the signal intensity is depicted on the left, and the background on the right. Figure 26B shows that the CNR for both glass and plastic is above 50 under the assayed conditions.

[0205] Example 10 - Surface product A surface exhibiting slight nonspecific binding to organic dyes and organic proteins, stability up to at least 95°C, chemical stability to organic solvents (methanol, ethanol, acetonitrile, formamide, oxidizing agents, phosphine) at high pH (0.1M NaOH) and low pH (>5.0), long-term storage stability, low input library requirements, and scalable primer loading is generated as follows. This process includes surface washing and silanization or passivation.

[0206] The surface was cleaned using a 2M KOH solution combined with an alconox / hellmanex purifying agent, and then rinsed with ethanol. The surface was then heated to 560°C to expose the OH groups. The surfaces were then subjected to plasma treatment, either alternately or together.

[0207] The surfaces were tested using surfaces that had been silanized with 5 mg / mL silane-5kPEG-NHS (99.9% ethanol / 0.01% acetic acid), heated to 65°C, and then cleaned with KOH / purifying agent / heat. Alternatingly or together, the surfaces were tested using surfaces that had been silanized with 10 mg / mL silane-5kPEG-NHS (90% DMF / 10% 100 mM MES, pH 5.5), heated to 25°C, and then cleaned with KOH / purifying agent / heat, or using plasma-treated surfaces.

[0208] Many dyes such as Cy3-C, R11-U, Cy3.5C, 647N-A, Cy5-G, 660-U, Cy5.5-C can be used with these surfaces (Note: only 200 nM of the dye). Exemplary dye mixtures include Cy3-A, Cy3.5-C, Cy5-U, AHO690-G.

[0209] Such surfaces can be loaded with primers at low concentrations (5.0x10 4 primers / μm 2 ), high concentrations (1.0x10 7 primers / μm 2 ), and at concentrations of values within or outside the range defined by these endpoints in an adjustable manner.

[0210] Concentration is optionally measured as follows. Prepare Cy3-dCTP solutions of different concentrations and measure the FL intensity with a GE Typhoon (GE Healthcare Lifesciences, Pittsburgh, PA) or appropriate equipment in a capillary with a fixed dimension (0.5 mm x 5 mm or other area). This gives primer loading when the area is known and the number of molecules is known.

[0211] Concentrations of 80,000, 160,000, 320,000, 640,000, 1,300,000, 2,600,000, and 5,100,000 primers / μm 2 have been measured using this approach, and other concentrations of values within or outside the range defined by these endpoints can be readily achieved. These densities are facilitated by the presence of multilayer PEG or other surfaces as disclosed herein.

[0212] It has been confirmed that the surface shows no significant decrease in stability for storage over one week.

[0213] Densities were measured for numerous surface mutants, and the results were confirmed as follows: Three-layer polybranched PEG(8,16,8) on PEGamine-APTES, when exposed to two layers of 7uM primer preloading, showed concentrations of 2,000,000 to 10,000,000 on the surface. Similar concentrations were observed for three-layer polybranched PEG(8,16,8) and (8,64,8) on PEGamine-APTES exposed to 8uM primers, and for three-layer polybranched PEG(8,8,8) in which the dumbbell-shaped 16-mer and 64-mer were replaced using star-shaped PEG-amine.

[0214] Using these approaches, it was observed that increased primer density resulted in higher foreground intensity, higher colony density, and higher CNR. For example, with an input of 10 pM, a primer density <1.0 x 10⁻¹⁶ was obtained. 4 primer / um 2 On a surface having , a CNR of 10 is obtained, while on the other hand, with the same input, primer density > 1.0 x 10 6 primer / um 2 Then a CNR of 40-60 was obtained.

[0215] Example 11 - Obtaining high-quality data using a high CNR surface. Current state-of-the-art surfaces, including low-CNR and high-CNR surfaces such as those disclosed herein, were tested for their fluorescence when detected in first and second channels corresponding to a first and second dye, respectively.

[0216] As CNR increased, it was observed that individual detection events were seen with sharper resolution. These detection events aligned along separate axes corresponding to the dye emission spectrum, rather than in a "cloud" of higher errors, as seen in the top three files of Figure 27. Looking at the bottom three files of Figure 27, this more accurate data acquisition appears as narrower, higher peaks at specific expected wavelengths and fewer data points at intermediate positions. This more clearly resolved dataset translates to a more accurate fluorescence base call resulting from assays performed on high-CNR surfaces.

[0217] Example 12 - Clonally amplified multimerized target oligonucleotide molecule Figure 28 provides schematic diagrams of clone-amplified, multimeric target oligonucleotide sequences hybridized to surfaces containing high surface density oligonucleotide adapter or primer molecules, such as more than 4,000 molecules per μm² (left), and surfaces containing lower surface density oligonucleotide adapter or primer molecules, such as less than 500 molecules per μm² (right), and shows the resulting and achievable improvement in CNR. Some surfaces have a density of 2000 molecules / μm². 2 It is prepared to have a higher oligonucleotide density, and the surface fluorescence image has a contrast-to-noise ratio of over 20.

[0218] Example 13 - Reduction of Input Nucleic Acid Requirements Figure 29 provides a comparison of results for performing a conventional hybridization reaction on a low-binding support surface of the present disclosure and for performing an optimized hybridization reaction on a low-binding support surface of the present disclosure. The conventional hybridization approach uses SSC buffer and is heated to 95°C, followed by slow cooling for 2 hours. After attaching oligonucleotide primers to the low-binding support surface, efficient hybridization of target nucleic acids to the oligonucleotide primers may suffer from reduced collision frequency on the low-binding surface. At least partially due to the reduced capture oligonucleotide coupling on the conventional surface, conventional hybridization methods for adding target DNA to surface-bound primers require input DNA concentrations of up to 10 nM (see left in Figure 29, showing labeled target oligonucleotides bound to the binding support surface). Even at these high concentrations, coupling of target oligonucleotides is limited. For comparison, non-complementary oligonucleotides were used as negative controls at the same concentrations (bottom of Figure 29). For comparison, using novel hybridization reaction conditions containing a developed PEG-containing mixture, target nucleic acid sequences in solvents less polar than water, such as ethanol, methanol, isopropanol, acetonitrile, and butanol, formamide, and a low pH buffer (<7), were hybridized to oligonucleotides attached to the surface at a low target nucleic acid input concentration of 50 pM. The decrease in target nucleic acid input concentration resulted in an approximately 200-fold increase in hybridization efficiency (see the right in Figure 29, showing labeled target oligonucleotides binding to the disclosed low-binding surface), which provides a significant advantage for use in sequencing techniques where input library DNA may be insufficient. The efficient primer coupling process and hybridization conditions may enable the preparation of surfaces with non-specific low binding and high surface density of oligonucleotide primers, which would not be achieved using conventional primer coupling chemistry or hybridization conditions described in the Art.For comparison, non-complementary oligonucleotides were used as negative controls at the same concentrations (Figure 29, bottom row, right). The detectable tag for each image is cyanine dye-3 (Cy3), where the acquired surface fluorescence images were obtained using a 20x, 0.75 NA, 532 nm light source, a bandpass and dichroic mirror filter set, a 532 nm long-pass excitation and Cy3 fluorescence emission filter Semrock, a 532 nm dichroic reflector, and a camera (Andor, sCMOS, Zyla 4.2, etc.).

[0219] Conventional or standard conditions were tested using a hybridization reporter probe (a complementary oligonucleotide sequence labeled with a Cy(trademark)3 fluorophore at its 5' end) in 2X-5X saline-sodium citrate (SSC) buffer (std) at a reported concentration of 90°C, followed by a slow cooling process (2 hours) to 37°C. The surface used for both test conditions had a particle size of approximately 0.25 molecules / μm. 2 The surface exhibited non-specific ultra-low binding with levels of non-specific Cy3 dye absorption less than 1.5°C. The wells were washed with 50 mM Tris (pH 8.0) and 50 mM NaCl. Images were taken using an inverted microscope (Olympus IX83) with a 100 X TIRF objective lens, NA=1.4 (Olympus), a dichroic mirror optimized for 532 nm light (Semrock, Di03-R532-t1-25x36), a bandpass filter optimized for Cy3 emission (Semrock, FF01-562 / 40-25), and a camera (sCMOS, Andor Zyla) under non-signal saturating conditions for 1 second (Laser Quantum, Gem 532, <1 W / cm² in sample). 2Samples were acquired using ) and immersed in buffer (25 mM ACES, pH 7.4 buffer). Two conditions were selected: 50% ACN + MES with a 1 μM oligonucleotide graft concentration and 25% ACN + MES + 20% PEG + 10% formaldehyde with a 5.1 μM oligonucleotide graft concentration. The feasibility of these conditions was tested to improve existing standard surface hybridization protocols on low-binding substrates. Oligonucleotide probes were added at the specified concentrations, and hybridization was performed at 50°C for 2 minutes. Images were collected as described above, and the results are shown in the figure.

[0220] Example 14 - Comparison of the chemical action of conventional oligo-primer linkage, hybridization reaction conditions, and amplification techniques on low-binding support surfaces. Figures 30-32 provide a comparison of experimental results for the low-binding supports of this disclosure, specifically for the implementation of conventional hybridization reactions or improved hybridization reactions using the methodology outlined in Figure 29, where oligo-primers were attached using the chemistry for conventional coupling, typically performed in sodium biocarbonate buffer at pH=8.3 for NHS-NH2 coupling reactions, or using the chemistry for improved coupling, with the polarity of the coupling buffer (organic-based solvent) altered by the addition of a buffer component with pH>8.0, followed by either RCA or bridge amplification after the hybridization reaction. Amplification of target DNA on low-binding supports with oligonucleotide primer surface densities below a clear surface density threshold, either by PCR-based ("bridge") amplification or rolling circle amplification, results in elongated or diffused target molecules, creating two challenges: 1) reduced packing density, and 2) reduced signal. Consequently, the scaling of systems for such surface-based high-throughput sequencing applications is significantly impaired. Figure 30 shows oligonucleotide density <1000 oligonucleotides / µm 2The images show the results of both PCR amplification ("bridge"; right) and rolling circle amplification (left) on low-binding surfaces using the chemical action of conventional oligonucleotide coupling. In these images, the labeled and amplified target DNA may exhibit extended conformation, which poses serious problems for imaging in sequencing applications. For comparison, Figure 31 shows the results of PCR / "bridge" amplification, and Figure 32 shows the results of rolling circle amplification (RCA), and they are μm 2 The work was performed on surfaces having a surface density of at least 1,000 oligonucleotide primer molecules per unit area. These surfaces compress the amplified target DNA into highly localized regions, which generate high fluorescence intensity from the attached labels and a small pixel area during imaging. In particular, the high fluorescence intensity leads to an increase in signal when calculating spot intensity for sequencing applications. Importantly, the enhanced contrast-to-noise ratio resulting from the use of low-binding surfaces disclosed herein is a function of both the very high signal resulting from this compression of the amplified target DNA and the very low background provided by the hydrophilic coated surface. Each of these images has labeled nucleotides with individual labels, and each nucleotide has a different luminescence label. As an example, the labels consisted of Cy3, Cy3.5, Cy5, and Cy5.5, respectively, and images were captured with an exposure time of at least 0.5 seconds using an Olympus IX83 microscope with a 20x, 0.75NA, 532nm light source, a bandpass and dichroic mirror filter set optimized for 532nm long-pass excitation and Cy3 fluorescence emission filters, a Semrock, a 532nm dichroic reflector, and a camera (Andor sCMOS, Zyla 4.2).

[0221] Example 15 - Comparison of amplification reactions on the surface of a low-binding support Figure 33 provides non-limiting examples of fluorescence images of a conventional support surface and a low-binding support surface of this disclosure, with target oligonucleotides hybridized and amplified. To improve sequencing accuracy, each amplified target nucleic acid must be clearly separable from other target nucleic acids in the image of the support surface. Each target nucleic acid must also present a signal (such as a fluorescence signal) related to the identification of each nucleotide in the sequence during the sequencing cycle (a process called "base calling"). Often simply the fluorescence intensity provided by a label attached to the target nucleic acid molecule, this base calling signal must be clearly and accurately resolvable over both noise (variations in the signal within a spot or target) and background (spurious signals generated nonspecifically by the properties of the materials forming the experimental environment). The contrast-to-noise ratio ("CNR") defines a sequencing system's ability to accurately determine the base present at each position in the target nucleic acid sequence, as well as its read length, reproducibility, and throughput. The currently disclosed low-binding support surface offers reduced non-specific protein and dye binding, resulting in a lower background signal and a more compact and brighter foreground signal, which in turn leads to an enhanced CNR and superior base calling results in sequencing applications. Figure 32 shows a comparison of a PEG-coated surface (top, "Conventional"), prepared according to conventional methods and adapted as needed for cloned amplified target DNA, with the low-binding support surface of this disclosure (top, "Element, Current"). The images clearly show that the currently disclosed surface exhibits sharper and stronger spots than the conventional surface, particularly for the measurement of cloned amplified DNA. Quantitative measurements of both spot intensity (fluorescence attributable to cloned amplified target DNA, equivalent to the sequencing signal) and background intensity show that the CNR of the disclosed low-binding support surface (bottom, "Element, Current Best") far exceeds what is achievable using the conventional surface (bottom, "Improved Conventional"), even under ideal imaging conditions such as when using improved hybridization conditions for target DNA binding.This increase in CNR is greater than what could be reasonably expected using conventional support surfaces and represents both qualitative and quantitative improvements over conventional surfaces. These improvements are achieved by generating support surfaces that meet several criteria, such as reduced nonspecific protein and dye binding, appropriate density of oligonucleotide adhesion to the surface, and hybridization / binding of clonal amplification target nucleic acids to the surface, resulting in images with very high CNR, enabling enhanced base calling in sequencing applications.

[0222] Example 16 - A descriptive example of preparing a low-binding support using other polymers. To remove organic contaminants and activate the hydroxyl groups on the surface for silane coupling, the glass slide is treated physically or chemically (e.g., using plasma treatment, piranha cleaning, acid cleaning agents, base cleaning agents, high-temperature glass annealing, or any combination thereof). The prepared glass surface is then reacted with a silane to covalently bond a functional group (e.g., a primary amine) and / or a first layer of hydrophilic polymer. In some examples, silanes such as (3-aminopropyl)trimethoxysilane (APTMS) or (3-aminopropyl)triethoxysilane (APTES)3(3-acrylopropyl)trimethoxysilane are reacted with the surface using standard protocols to covalently bond a primary amine functional group to the surface. In other examples, silane-modified polymers, such as hydrophilic heterobifunctional polymers containing a silyl group at one end and a second functional group (e.g., a primary amine, carboxyl group, etc.) at the other end, may be reacted directly with the surface. (For example, by contacting a clean glass surface with a silane-modified polymer at a concentration of 0.1% to 2% in ethanol for about 1 to 2 hours, and then rinsing with ethanol and water.)Examples of suitable silane-modified polymers include, but are not limited to, the following: silane-PEG-NH2 (e.g., polyethylene glycol (PEG) with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-COOH (e.g., PEG with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-maleimide (e.g., PEG with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-biotin (e.g., PEG with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane- PEG-acrylates (e.g., PEGs with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane-PEG-silanes (e.g., PEGs with molecular weights of 1000, 2000, 3400, 5000, or 10K Daltons), silane-modified polypropylene glycols (PPGs) of various molecular weights containing additional functional groups, silane-modified poly(vinyl alcohols) (PVAs) of various molecular weights containing additional reactive functional groups, silane-modified polyethyleneimines (PEIs) of various molecular weights containing additional reactive functional groups, silane-modified poly(lysines) of various molecular weights containing additional reactive functional groups, etc., or any combination thereof.

[0223] In some examples, following an initial reaction between the surface and a silane or silane-modified polymer, at least one additional layer of hydrophilic polymer is coupled to or deposited on the glass surface. The polymer coating may use any of the many hydrophilic polymers known to those skilled in the art, including, but not limited to, polyethylene glycol (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyridine, polyvinylpyrrolidone) (PVP), poly(acrylic acid) (PAA), polyacrylamide, poly(N-isopropylacrylamide) (PNIPAM), poly(methyl methacrylate) (PMA), poly(2-hydroxyethyl methacrylate) (PHEMA), poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA), polyglutamic acid (PGA), poly-lysine, poly-glucoside, streptavidin, dextran, or any combination thereof. Here, in the case of covalent coupling, polymers containing appropriate monofunctional, homodifunctional, and / or heterodifunctional reactive groups are selected in relation to their compatibility with the chosen conjugation chemistry. In some cases, derivatized polymers such as PEGamine, PEG-NHS, or PEG acrylate are used. In some cases, difunctional PEG derivatives such as acrylate-PEG-NHS are used. In some cases, these additional hydrophilic polymer layers may be coupled or deposited to the previous layer by contacting the surface with 0.1% to 2% of the polymer in ethanol or ethanol / aqueous buffer at room temperature for about 5 minutes to about 1 hour, and then rinsing with ethanol or ethanol / aqueous buffer.

[0224] In some examples, a second, third, fourth, fifth, or more hydrophilic polymer addition layer may be coupled to or deposited on the initial layer on the support surface. In some examples, polymer molecules within the layer may be crosslinked to each other using appropriate homofunctional or heterofunctional crosslinking reagents. In some examples, polymer molecules in different layers may be crosslinked to each other. In some examples, one or more hydrophilic polymer layers may contain branched polymers, such as branched PEG, branched poly(vinyl alcohol) (branched PVA), branched poly(vinylpyridine), branched poly(vinylpyrrolidone) (branched PVP), branched poly(acrylic acid) (branched PAA), branched polyacrylamide, branched poly(N-isopropylacrylamide) (branched PNIPAM), branched poly(methyl methacrylate) (branched PMA), branched poly(2-hydroxyethyl methacrylate) (branched PHEMA), branched poly(oligo(ethylene glycol) methyl ether methacrylate) (branched POEGMA), branched polyglutamic acid (branched PGA), branched polylysine, branched poly-glucoside, branched dextran, or any combination thereof.

[0225] One of the more hydrophilic polymer layers may contain multiple covalently bonded oligonucleotide adapter or primer molecules, where the oligonucleotide molecules are covalently coupled to the polymer using any of the various suitable conjugation chemistry known to those skilled in the art. In some examples, the oligonucleotide adapter or primer molecules are covalently bonded to the polymer molecules in solution, i.e., before the polymer molecules are coupled to or deposited on the surface. In some examples, the oligonucleotide adapter or primer molecules are covalently bonded to the polymer after they have been bonded or deposited on the surface. In some examples, at least one hydrophilic polymer layer contains multiple covalently bonded oligonucleotide adapter or primer molecules. In some examples, at least two, at least three, at least four, or at least five layers of hydrophilic polymer contain multiple covalently attached adapter or primer molecules.

[0226] The selection of polymers used, the number of layers, the degree of crosslinking within and between layers, the number of layers containing covalently bonded oligonucleotide adapter or primer molecules, and the local or surface density of oligonucleotide adapter or primer molecules may be individually or relatively adjusted to "tune" the surface properties to achieve: desired surface wettability (e.g., indicated by a water contact angle of less than 50 degrees), desired surface stability under prolonged exposure to thermal cycling, requiring sequencing / genotyping reagents and a temperature gradient often of at least 95°C peak temperature, maintained for at least 5 minutes, and cycled multiple times, at least 30 times, and desired surface density of oligonucleotide adapter or primer molecules (e.g., μm 2Each layer contains at least 1,000 adapter or primer molecules, which results in an extreme reduction in nonspecific binding of dye molecules or other labeled sequencing / genotyping reagents, improved hybridization efficiency, improved amplification efficiency and specificity, optimal density of cloned amplified target sequences (in terms of the number of cloned colonies per unit area, the number of target sequence copies per unit area, or the number of amplified target molecules per unit area), a high contrast-to-noise ratio (CNR) in images of the support surface (e.g., fluorescence images) (e.g., a CNR greater than 20), and ultimately, improved detection accuracy or base calling accuracy in genotyping and sequencing applications.

[0227] Example 17 - Acrylate-coupled surface Plasma-treated, KOH-treated, or plasma / KOH-treated glass surfaces, silicon wafers, or plasma-treated COP surfaces were treated with (3-acrylopropyl)trimethoxysilane and then incubated with bifunctional acrylate-PEG-NHS with an average PEG molecular weight ranging from 1K to 6K, particularly including PEG-3.4K. A molecular weight of 500-10K was assumed, with the constraint that the PEG must be soluble in water at 42°C, 37°C, room temperature, in liquid state at 42°C, 37°C, or room temperature. PEG incorporation was optionally assisted by the addition of up to 0.5% (w / w) of 2-hydroxy-2-methylpropiophenone, followed by UV treatment (3.0 mW / cm2 for 10 minutes). Acrylate-PEG-NHS was used at concentrations of 3 mM and 6 mM, with superior results achieved with 6 mM acrylate-PEG-NHS. After washing to remove unbound polymers, the surfaces were cultured at room temperature for a sufficient time to allow autolysis of the NHS groups, leaving terminal carboxylate groups on the bound PEG molecules. These surfaces were then activated by treatment with EDAC-HCl, and 5'-aminooligonucleotides were added to generate oligonucleotide-conjugated surfaces. Combinations of SP6 oligonucleotide (25 NT) and SP5P oligonucleotide (25 NT with a 3' phosphate cap) were added in 1:1, 1:2, 1:5, or 1:10 ratios (SP6 oligonucleotide is a primer for surface-grown rolling circle amplification, while SP5P assists in preventing random priming or nonspecific condensation / binding of RCA amplification products). After washing with 90% EtOH in MES at pH 9 to remove unbound oligonucleotides, a storage buffer containing ACES / KCl / EDTA / Tween20 was added. The surface can be stored in this buffer state for at least 7 days.Next, to amplify the target nucleic acid in a rolling circle on the surface, a surface containing attached primers was used to generate condensed nucleic acid molecules as shown elsewhere in this specification. The prepared RCA product was also bound to the surface to similarly generate a dense nucleic acid structure.

[0228] While preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided only as examples. Many modifications, changes, and substitutions will be conceivable without departing from the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be used in any combination in carrying out the invention. The following claims define the scope of the present invention, and it is intended that methods and structures within the scope of these claims and their equivalents are encompassed thereby.

Claims

1. A method for determining nucleic acid sequences, wherein the method is a) A step of providing a linked surface to at least one hydrophilic polymer coating layer containing a hydrophilic polymer linked to a first oligonucleotide molecule. b) A step of bringing the first oligonucleotide molecule into contact with the sample nucleic acid molecule so that the sample nucleic acid molecule is linked to the first oligonucleotide molecule. c) After b) the step of amplifying the sample nucleic acid molecule, d) A step of acquiring a fluorescence image of the surface, Here, the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of 20 or more when the fluorescence image of the surface is acquired using an inverted fluorescence microscope and camera under non-signal saturation conditions while the surface is immersed in the buffer after b), Here, the sample nucleic acid molecule includes a detectable tag which is a fluorophore. A method wherein the background fluorescence intensity measured in a region of the surface having the at least one hydrophilic polymer coating layer, which is laterally displaced from separate regions of the surface, is no more than twice the intensity measured in the separate regions before amplification.

2. The method according to claim 1, wherein the fluorescence image of the surface exhibits a contrast-to-noise ratio (CNR) of 40 or more when the fluorescence image is acquired after b).

3. The method according to claim 1, wherein the at least one hydrophilic polymer coating layer includes at least a first polymer layer connected to the surface and a second polymer layer connected to the first polymer layer.

4. The method according to claim 1, wherein the at least one hydrophilic polymer coating layer comprises a branched polymer having at least eight branches.

5. The fluorescence image of the surface is obtained using the inverted fluorescence microscope and the camera under non-signal saturation conditions while the sample nucleic acid molecules are amplified and labeled with cyanine dye-3 and the surface is immersed in the buffer, and the fluorescence image is obtained with respect to the fluorescence intensity of the foreground and the nonspecific dye adsorption background (B ｉｎｔｅｒ The method according to claim 1, which exhibits a ratio of at least 5:1 with respect to the fluorescence intensity of ).

6. The method according to claim 1, wherein the nucleic acid sequence is determined by sequencing the sample nucleic acid molecule.

7. The at least one hydrophilic polymer coating layer is (i) A first layer comprising a first monolayer of polymer molecules linked to the surface, (ii) A second layer comprising a second monolayer of polymer molecules linked to the first monolayer of polymer molecules, (iii) A third layer comprising a third monolayer of polymer molecules linked to a second monolayer of polymer molecules, The method according to claim 1, wherein the polymer molecules of the first layer, the second layer, or the third layer include branched polymer molecules.

8. The first oligonucleotide molecule is present at a density of 10,000 molecules / mm³. ２ The method according to claim 1, wherein the surface density is as described above.

9. The method according to claim 1, wherein (b) comprises bringing the surface into contact with a solution containing the sample nucleic acid molecules, wherein the sample nucleic acid molecules are present in the solution at a concentration of less than 1 nM.

10. The method according to claim 1, wherein (b) above comprises bringing the surface into contact with a solution containing the sample nucleic acid molecules, wherein the sample nucleic acid molecules are present in the solution at a concentration of 50 pM or less.

11. The method according to claim 1, wherein the sample nucleic acid molecule is a single-stranded multimeric nucleic acid molecule containing regularly occurring repeating monomer units.

12. The method according to claim 11, wherein the length of the single-stranded multimeric nucleic acid molecule is at least 10 kb.

13. The method according to claim 1, wherein the sample nucleic acid molecule is a double-stranded polymeric nucleic acid molecule containing regularly occurring repeating monomer units.

14. The method according to claim 1, wherein the first oligonucleotide molecule is present at a uniform surface density across the surface.

15. The method according to claim 1, wherein the first oligonucleotide molecule is present at a first position on the surface at a first local surface density and at a second position on the surface at a second local surface density, wherein the first local surface density is different from the second local surface density.

16. The method according to claim 1, wherein the at least one hydrophilic polymer coating layer comprises polyethylene glycol (PEG).

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