DNA bridging method for capturing DNA molecules

Abstract

This invention relates to methods for suspending stretched nucleic acids on surface features. These methods can be used to prepare stretched nucleic acids that are more active in enzymatic and other reactions than nucleic acids deposited on flat surfaces. These methods can be achieved by applying a photoresist layer to a substrate, stretching the nucleic acid on the substrate surface, and then removing a portion of the photoresist to form a surface feature, thereby suspending the stretched nucleic acid. Furthermore, forming a hydrogel layer on the stretched nucleic acid and the surface feature allows the stretched nucleic acid to be transferred to the hydrogel for further reactions, including enzymatic reactions.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 657,732, filed April 14, 2018, the contents of which are incorporated herein by reference in their entirety. Background Technology

[0003] Genetic information within an organism is contained in polymers called nucleic acids, such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The genome is the genetic material within the chromosomes of a cell. The DNA sequence information of the genome can be used to determine individual characteristics and the presence of susceptibility to many common diseases, such as cancer and some metabolic disorders. Furthermore, because some drugs are more effective in individuals with certain genetic markers, knowledge of an individual's genome offers opportunities for personalized medicine. Tools have been developed to allow for faster and more reliable sequence determination. In addition, miniaturized and mass-producible molecular detection platforms, such as DNA microarrays, have made disease detection accessible to a wider range of people.

[0004] DNA microarrays have been widely used in a range of genomic sequence analyses, including mutation and polymorphism (SNP genotyping), cytogenetics (copy number), nuclear proteomics, gene expression profiling and analysis, and transcriptome analysis. While many of these applications can be read using direct hybridization-based methods, the use of enzyme-catalyzed readouts may offer certain distinct advantages. For example, polymerase extension or ligation of array sequences may provide a higher level of discriminative power compared to detection by hybridization alone. Summary of the Invention

[0005] Stretching nucleic acid molecules onto a surface and imbuing them with enzymatic activity is desirable. This paper presents a solution to a problem in current methods for stretching nucleic acids: when stretched nucleic acids are placed on a surface, some enzymatic reactions may not occur on the stretched substrate, or some enzymatic reactions may occur less efficiently compared to nucleic acids suspended in solution.

[0006] On one hand, this article provides a method for treating at least one nucleic acid molecule on a substrate surface, comprising: (a) stretching the nucleic acid molecule on the surface of the substrate; and (b) forming a plurality of discrete features on the surface of the substrate by removing a plurality of subparts from the surface of the substrate; wherein the nucleic acid molecule contacts two members of the plurality of discrete features, thereby suspending a portion of the nucleic acid between the two members of the plurality of discrete features.

[0007] In some embodiments of the aspects provided herein, the plurality of discrete features form a topological pattern. In some embodiments of the aspects provided herein, the nucleic acid molecule is in contact with two other members of the plurality of discrete features. In some embodiments of the aspects provided herein, an additional portion of the nucleic acid molecule is suspended between the two other members of the plurality of discrete features. In some embodiments of the aspects provided herein, the method further includes: (c) forming a hydrogel on the surface of the substrate, the hydrogel being in contact with some or all of the members of the plurality of discrete features. In some embodiments of the aspects provided herein, in (c) at least a portion of the nucleic acid molecule is enclosed in the hydrogel. In some embodiments of the aspects provided herein, the method further includes: (d) removing the hydrogel from the surface of the substrate.

[0008] In some embodiments of the aspects provided herein, the method further includes, in (a), stretching additional nucleic acid molecules onto the surface of the substrate. In some embodiments of the aspects provided herein, the additional nucleic acid molecules contact two other members of the plurality of discrete features, thereby suspending the additional nucleic acid molecules between the other two members of the plurality of discrete features. In some embodiments of the aspects provided herein, each of the plurality of discrete features is independently a pit, pore, groove, channel, well, pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, each of the two members of the plurality of discrete features is independently a pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, each of the two other members of the plurality of discrete features is independently a pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, at least a portion of the nucleic acid molecules is enclosed in the hydrogel.

[0009] In some embodiments of the aspects provided herein, each of the two other members of the plurality of discrete features is independently a pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, the nucleic acid molecule is deoxyribonucleic acid (DNA). In some embodiments of the aspects provided herein, the DNA is double-stranded or single-stranded. In some embodiments of the aspects provided herein, the nucleic acid molecule is active in an enzymatic reaction. In some embodiments of the aspects provided herein, the method further includes performing an enzymatic reaction on the nucleic acid molecule. In some embodiments of the aspects provided herein, the method further includes performing an enzymatic reaction on the additional nucleic acid molecule. In some embodiments of the aspects provided herein, the method further includes performing a protein-binding reaction, a hybridization reaction, a primer extension reaction catalyzed by a polymerase, a nick translation reaction, or a nick extension reaction on the nucleic acid molecule. In some embodiments of the aspects provided herein, the method further includes performing a protein-binding reaction, a hybridization reaction, a primer extension reaction catalyzed by a polymerase, a nick translation reaction, or a nick extension reaction on the additional nucleic acid molecule. In some embodiments of the aspects provided herein, each of the two members of the plurality of discrete features includes a photoresist.

[0010] In some embodiments of the aspects provided herein, the method further includes: before (a), applying a photoresist layer to the top of the substrate. In some embodiments of the aspects provided herein, the method further includes: before (a), irradiating the photoresist layer with ultraviolet light through a mask. In some embodiments of the aspects provided herein, the removal in (b) includes developing the photoresist layer. In some embodiments of the aspects provided herein, each of two members of the plurality of discrete features contains the photoresist. In some embodiments of the aspects provided herein, each of two other members of the plurality of discrete features contains the photoresist. In some embodiments of the aspects provided herein, the photoresist is a positive photoresist or a negative photoresist. In some embodiments of the aspects provided herein, before (a), the surface includes a topological pattern containing the plurality of features and the plurality of cavities, wherein each of the plurality of features is independently a pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, each of the plurality of cavities is independently a pit, hole, groove, channel, or aperture. In some embodiments of the aspects provided herein, the method further includes: prior to (a), filling each of the plurality of cavities with the photoresist. In some embodiments of the aspects provided herein, the removal in (b) includes developing the photoresist layer, wherein the photoresist is a negative photoresist. In some embodiments of the aspects provided herein, the method further includes: prior to (a), irradiating the surface of the photoresist with ultraviolet light, wherein the photoresist is a positive photoresist. In some embodiments of the aspects provided herein, the removal in (b) includes developing the positive photoresist layer.

[0011] On the other hand, this paper provides a system comprising: (a) a substrate having a surface comprising a plurality of discrete features; and (b) a nucleic acid molecule in contact with two members of the plurality of discrete features.

[0012] In some embodiments of the aspects provided herein, a portion of the nucleic acid molecule is suspended between two members of the plurality of discrete features. In some embodiments of the aspects provided herein, the nucleic acid molecule is in contact with two other members of the plurality of discrete features. In some embodiments of the aspects provided herein, an additional portion of the nucleic acid molecule is suspended between the two other members of the plurality of discrete features. In some embodiments of the aspects provided herein, the plurality of discrete features form a topological pattern.

[0013] In some embodiments of the aspects provided herein, the system further comprises a hydrogel on a surface, the hydrogel being in contact with some or all of the members of the plurality of discrete features. In some embodiments of the aspects provided herein, at least a portion of the nucleic acid molecule is enclosed in the hydrogel. In some embodiments of the aspects provided herein, the nucleic acid molecule is stretched. In some embodiments of the aspects provided herein, each of the plurality of discrete features is independently a pit, hole, groove, channel, pore, pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, each of two members of the plurality of discrete features is independently a pillar, bump, protrusion, ridge, or rod. In some embodiments of the aspects provided herein, each of two other members of the plurality of discrete features is independently a pillar, bump, protrusion, ridge, or rod.

[0014] In some embodiments of the aspects provided herein, the nucleic acid molecule is deoxyribonucleic acid (DNA). In some embodiments of the aspects provided herein, the DNA is double-stranded or single-stranded. In some embodiments of the aspects provided herein, the nucleic acid molecule is active in enzymatic reactions. In some embodiments of the aspects provided herein, the nucleic acid molecule is active in protein binding reactions, hybridization reactions, primer extension reactions catalyzed by polymerases, nick translation reactions, or nick extension reactions. In some embodiments of the aspects provided herein, each of the two members of the plurality of discrete features comprises a photoresist. In some embodiments of the aspects provided herein, the photoresist is a positive photoresist or a negative photoresist. In some embodiments of the aspects provided herein, each of the two other members of the plurality of discrete features comprises a photoresist. In some embodiments of the aspects provided herein, the photoresist is a positive photoresist or a negative photoresist. In some embodiments of the aspects provided herein, the system further comprises additional nucleic acids in contact with the two other members of the plurality of discrete features. In some embodiments of the aspects provided herein, each of the two members of the plurality of discrete features does not contain a photoresist.

[0015] Other aspects and advantages of this disclosure will become readily apparent to those skilled in the art based on the following detailed description, which only shows and describes illustrative embodiments of the disclosure. It should be understood that this disclosure is applicable to other and different embodiments, and that certain details thereof can be modified in several obvious ways without departing from this disclosure. Therefore, the accompanying drawings and descriptions should be considered illustrative rather than restrictive in nature.

[0016] Incorporation

[0017] All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the extent that each individual publication, patent or patent application is specifically and individually cited and incorporated herein by reference. Attached Figure Description

[0018] The novel features of the invention are specifically set forth in the appended claims. A better understanding of the features and advantages of the invention will be obtained by referring to the following detailed description and accompanying drawings, which illustrate illustrative embodiments utilizing the principles of the invention, in which:

[0019] Figure 1 An image of an SU-8 column (SU-8 bar) showing DNA molecules stretched through a surface feature.

[0020] Figure 2A-2D This diagram illustrates the generation of DNA molecules suspended on a surface feature using a DNA cross-linking method.

[0021] Figures 3A-3D A schematic diagram showing the generation of DNA molecules suspended on surface features, with the DNA placed on a photoresist-exposed surface.

[0022] Figures 4A-4D A schematic diagram shows DNA molecules suspended on surface features by filling gaps in a patterned surface with a second material.

[0023] Figure 5 Another image of an SU-8 bar is shown, in which stretched DNA molecules run through the bar.

[0024] Figure 6 Another image of an SU-8 bar is shown, in which stretched DNA molecules run through the bar.

[0025] Figure 7 The image shows a column / capture rod with suspended DNA molecules in a checkerboard conformation.

[0026] Figure 8 Images of both the extension product and the template DNA molecule are shown. The template DNA molecule was suspended across the SU-8 line before the extension reaction.

[0027] Figure 9 Genomic DNA suspended throughout the patterned rods of SU-8 was shown.

[0028] Figure 10 Genomic DNA fragments transferred to the hydrogel are shown.

[0029] Figure 11A-11F A schematic diagram showing the generation of stretched DNA molecules in a hydrogel is shown.

[0030] Figure 12 This shows the vertically suspended DNA extending through the SU-8 horizontal line in an aqueous buffer by incorporating labeled and unlabeled nucleotides. Labeled nucleotides are represented by green dots (or multiple green dots) appearing along the stained DNA (red lines or multiple red lines).

[0031] Figure 13 The image shows DNA molecules captured in a hydrogel extended during a sequencing reaction using a labeled reversible terminator via DNA polymerase. The labeled nucleotides are red, green, and blue dots (multiple dots) appearing across a surface feature (bar). Detailed Implementation

[0032] Capturing and comparing enzymatically reactive DNA molecules is invaluable. For example, captured DNA molecules can serve as templates for DNA polymerase. Such capture methods can form the basis for a variety of research and diagnostic approaches, including DNA sequencing. Although various methods for combining and stretching DNA molecules have been disclosed, in general, these methods make the captured DNA molecules less manipulable for further enzymatic reactions. Stretching DNA across a surface can be an efficient method for studying the genome. For example, DNA molecules located on a solid surface can restrict reversible interactions with nearby enzymes to only one side of the DNA molecule, as the other side is blocked by the surface.

[0033] This invention provides a method for suspending DNA molecules using discontinuous "pillars" or capture rods, wherein the DNA molecules can be suspended on multiple pillars or capture rods. Segments of the suspended DNA molecules, such as DNA molecules not placed on pillars or capture rods, can be immersed in a buffer solution, within a hydrogel, or used for enzymatic reactions. Therefore, enzymes can access these segments from both ends of the DNA molecule compared to DNA molecules on a solid surface. In one embodiment, the DNA can be stretched across a photoresist surface. Within a certain spatial interval, such as, for example, from about 1 μM to about 200 μM, UV light can be applied to construct capture rods or "pillars" beneath the cross-linked DNA molecules. The combined result allows some DNA molecule segments to be suspended on the capture rods or pillars. In another embodiment, a photoresist (such as a SU-8-based or polymethyl methacrylate (PMMA) photoresist) can be exposed to light in a specific pattern. The DNA can then be stretched on the exposed photoresist. The photoresist is then developed, and the exposed and unexposed portions can react differently in a process such as chemical processing, so that capture rods or columns containing DNA molecules are suspended on multiple capture rods or columns. In another embodiment, a patterned surface with columns, such as a PMMA- or SU-8-based surface, can be used. The gaps between the columns can be filled with a second material. DNA can be stretched on the surface and subsequently on the second material, such as a removable hydrogel, to suspend DNA fragments. This platform can be used for DNA sequencing, mapping, and other applications. In one embodiment, suspendable DNA molecules can be transferred to a hydrogel and hybridized with primers. Single-molecule sequencing can be performed from multiple extension sites via primer extension using, for example, a reversible terminator.

[0034] As used herein, the term "oligonucleotide" generally refers to a nucleotide chain. In some cases, oligonucleotides are less than 200 residues in length, for example, 15-100 nucleotides in length. Oligonucleotides may contain at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 bases. Oligonucleotides may be about 3 to about 5 bases, about 1 to about 50 bases, about 8 to about 12 bases, about 15 to about 25 bases, about 25 to about 35 bases, about 35 to about 45 bases, or about 45 to about 55 bases. Oligonucleotides (also called "oligomers") can be any type of oligonucleotide (e.g., primers). Oligonucleotides may contain natural nucleotides, non-natural nucleotides, or combinations thereof.

[0035] As used herein, the term "about" typically refers to a specified amount of + / - 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%.

[0036] As used in this article, the term "basically" when describing the relative value, relative quantity, or relative degree of two individuals typically means that the value, quantity, or degree is within 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, or 110% of each other.

[0037] As used herein, open-ended terms such as “include,” “containing,” “including,” “have,” and “having” refer to inclusion unless otherwise stated.

[0038] As used herein, the term "fixation" generally refers to the formation of a covalent bond between two reactive groups. For example, the polymerization of reactive groups is a form of fixation. The formation of a covalent bond between two atoms, such as carbon atom and carbon atom, carbon atom and heteroatom, and heteroatom and heteroatom, when the two atoms are derived from two different reactive groups, are examples of fixation.

[0039] As used herein, the term "substrate" or "solid substrate" generally refers to a substance, structure, surface, material, manner, or composition that includes non-biological, synthetic, non-living, planar, spherical, or flat materials. Substrates may include, for example, but not limited to, semiconductors, synthetic metals, synthetic semiconductors, insulators, and dopants; metals, alloys, elements, compounds, and minerals; synthetically produced, cut, etched, photolithographically formed, printed, mechanized, and microfabricated slides, devices, structures, and surfaces; industrial polymers, plastics, and films; silicon, silicates, glass, metals, and ceramics; wood, paper, cardboard, cotton, wool, cloth, woven and nonwoven fibers, materials, and fabrics; nanostructures and microstructures. Substrates may include fixed substrates, such as, but not limited to, insoluble substances, solid phases, surfaces, layers, coatings, woven or nonwoven fibers, substrates, crystals, films, insoluble polymers, plastics, glass, biological or biocompatible or biodegradable polymers or substrates, microparticles, or nanoparticles. Other examples may include, but are not limited to, monolayers, bilayers, commercial membranes, resins, substrates, fibers, separation media, chromatographic supports, polymers, plastics, glass, mica, gold, beads, microspheres, nanospheres, silicon, gallium arsenide, organic and inorganic metals, semiconductors, insulators, microstructures, and nanostructures. Microstructures and nanostructures may include, but are not limited to, miniaturized, nanoscale, and supramolecular probes, tips, rods, nails, pins, sticks, sleeves, wires, filaments, and tubes. Substrates may be, for example, in the form of one or more particles, strands, precipitates, gels, sheets, tubes, spheres, containers, capillaries, pads, slices, thin films, plates, glass slides, or semiconductor integrated chips. Substrates may be planar or may employ other surface constructions. For example, a substrate may contain raised or recessed regions on which synthesis or deposition occurs. In some examples, a substrate may contain raised or recessed regions comprising different 3-D shapes and / or heights. In some examples, a substrate may contain multiple features. In some instances, the substrate may contain a topological pattern, and the topological pattern may include a set of grooves, a set of rods, a set of pillars, a set of holes, or combinations thereof. In some instances, the topological pattern is formed from at least two different materials, such as a silicon chip covered with a layer of photoresist, or a quartz sheet covered with hydrogel. In some instances, the substrate may contain raised or recessed regions of substantially the same 3D shape and / or height. In some instances, the substrate may be selected to provide suitable light absorption properties. For example, the substrate may be a polymerized Langmuir Blodgett film, a functionalized glass (e.g., a controlled-aperture glass), silica, titanium dioxide, alumina, indium tin oxide (ITO), Si, Ge, GaAs, GaP, SiO2, SiN4, modified silicon, a top dielectric layer of a semiconductor integrated circuit (IC) chip, or any of a variety of gels or polymers, such as (poly)tetrafluoroethylene, (poly)vinylidene fluoride, polystyrene, polycarbonate, polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polycyclic olefins, or combinations thereof.

[0040] The substrate may comprise a polymer coating or gel, such as a polyacrylamide gel or a PDMS gel. The gel and coating may additionally contain components that alter their physicochemical properties, such as hydrophobicity. For example, the polyacrylamide gel or coating may contain modified acrylamide monomers in its polymer structure, such as ethoxylated acrylamide monomers, phosphorylcholine acrylamide monomers, betaine acrylamide monomers, and combinations thereof.

[0041] As used herein, the term "nucleic acid" generally refers to a polymer containing one or more nucleic acid subunits or nucleotides. Nucleic acids may contain one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. Nucleotides may include A, C, G, T, or U, or variants thereof. Nucleotides may include any subunit that can be incorporated into a growing nucleic acid chain. Such subunits may be A, C, G, T, or U, or any other subunit complementary to one or more of A, C, G, T, or U, or complementary to purines (i.e., A or G, or variants thereof) or pyrimidines (i.e., C, T, or U, or variants thereof). Subunits may be resolved by a single nucleic acid base or base group (e.g., AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil counterparts). In some instances, nucleic acids are deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or derivatives thereof. Nucleic acids may be single-stranded or double-stranded.

[0042] As used herein, the terms "nearby" or "adjoining" include "next to," "adjoining," and "abutting." In one instance, a first location is adjacent to a second location when the first location is in direct contact with the second location and shares a common boundary with the second location, and there is no space between the two locations. In some cases, proximity is not diagonal proximity.

[0043] As used herein, the term "sequencing" generally refers to methods and techniques used to determine the sequence of nucleotide bases in one or more polynucleotides. Polynucleotides can be, for example, nucleic acid molecules, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), including their variants or derivatives (e.g., single-stranded DNA). Sequencing can be performed using a variety of currently available systems, including, but not limited to, those described above. Pacific Or Life The sequencing system described herein. Alternatively or additionally, sequencing can be performed using nucleic acid amplification, polymerase chain reaction (PCR) (e.g., digital PCR, quantitative PCR, or real-time PCR), or isothermal amplification. Such a system can provide multiple raw genetic data corresponding to the genetic information of a subject (e.g., a human), such as those generated by the system from a sample provided by the subject. In some instances, such a system provides sequencing reads (also referred to herein as “reads”). Reads may include a string of nucleic acid bases corresponding to the sequenced nucleic acid molecule sequence. In some cases, the systems and methods described herein can be used in conjunction with proteomic information.

[0044] As used herein, the term "sample" generally refers to a biological sample from a subject. A biological sample can contain any number of macromolecules, such as cellular macromolecules. A sample can be a cell sample. A sample can be a cell line or cell culture sample. A sample can include one or more cells. A sample can include one or more microorganisms. A biological sample can be a nucleic acid sample or a protein sample. A biological sample can also be a carbohydrate sample or a lipid sample. The biological sample can be derived from another sample. A sample can be a tissue sample, such as a biopsy, core biopsy, needle aspiration, or fine needle aspiration sample. A sample can be a fluid sample, such as a blood sample, urine sample, or saliva sample. A sample can be a skin sample. A sample may be a buccal swab. A sample can be a plasma or serum sample. A sample can be cell-free or cell-free. Cell-free samples can include extracellular polynucleotides. Extracellular polynucleotides can be isolated from a body sample, which may be selected from blood, plasma, serum, urine, saliva, mucosal secretions, sputum, feces, and tears.

[0045] As used herein, the term "nucleic acid sequence" or "nucleotide sequence" generally refers to a nucleic acid molecule having a given nucleotide sequence, for which it may be desirable to know the presence or number of nucleotides. A nucleotide sequence can contain ribonucleic acid (RNA) or DNA, or a sequence derived from RNA or DNA. Examples of nucleotide sequences are sequences corresponding to natural or synthetic RNA or DNA, including genomic DNA and messenger RNA. The sequence length can be any length that can be amplified into a nucleic acid amplification product or amplicon, for example, up to about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 or more 10,000 nucleotides, or at least about 20, 50, 100, 200, 300, 400, 500, 600, 700, 800, 1,000, 1,200, 1,500, 2,000, 5,000, 10,000 or 10,000 nucleotides.

[0046] As used herein, the term "template" generally refers to a single polynucleotide molecule from which another nucleic acid, comprising a complementary nucleic acid chain, can be synthesized by a nucleic acid polymerase. Alternatively, a template can be one or both chains of a polynucleotide capable of serving as a template for template-dependent nucleic acid polymerization catalyzed by a nucleic acid polymerase. The use of this term should not be construed as limiting the scope of this disclosure to polynucleotides that are actually used as templates in subsequent enzyme-catalyzed polymerization reactions. The template can be RNA or DNA. The template can be cDNA corresponding to an RNA sequence. The template can be DNA.

[0047] As used herein, “amplification” of a template nucleic acid generally refers to the process of generating (e.g., in vitro) a nucleic acid chain that is at least partially identical to or complementary to the template nucleic acid sequence, or a universal or tag sequence used as a substitute for the template nucleic acid sequence, which can only be generated if the template nucleic acid is present in the sample. Typically, nucleic acid amplification uses one or more nucleic acid polymerases and / or transcriptases to generate multiple copies of the template nucleic acid or fragments thereof, or multiple copies of a sequence complementary to the template nucleic acid or fragments thereof. In vitro nucleic acid amplification techniques can include transcription-related amplification methods, such as transcription-mediated amplification (TMA) or nucleic acid sequence-based amplification (NASBA), as well as other methods such as polymerase chain reaction (PCR), reverse transcriptase-PCR (RT-PCR), replicase-mediated amplification, and ligase chain reaction (LCR).

[0048] As used herein, the term "hydrogel" generally refers to a gel in which water is the swelling agent. The term "gel" refers to a non-fluid colloidal or polymeric network that expands in its volume by a fluid. The term "swelling agent" is a fluid used to swell a gel or network. For example, water can be a swelling agent for a hydrogel. The hydrogels of this disclosure can be prepared by polymerization of one or more acrylamide-functionalized monomers. For example, an acrylamide tail can also be bonded to the surface of a substrate (e.g., a quartz sheet). A solution containing acrylamide monomers can then be contacted with the surface bonded to the acrylamide tail. The poured-out solution can then be polymerized with the acrylamide monomers and the acrylamide tail, thereby forming a hydrogel. In some cases, the hydrogels of the present invention contain polyacrylamide. In some cases, the hydrogels of the present invention contain cross-linked polyacrylamide. In some cases, the hydrogels disclosed herein comprise about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% polyacrylamide by weight. In some cases, the hydrogels can be obtained by combining acrylamide and methylenebisacrylamide. The polymerization reaction can be initiated by an initiator free radical. The hydrogels can be obtained by combining acrylamide and methylenebisacrylamide in a molar ratio of 150:1 to 1000:1 in the presence of a free radical initiator. Methylenebisacrylamide can provide crosslinking between polymer chains, and the molar ratio can be varied to provide a variety of crosslinking densities for the hydrogel. The conditions for obtaining the hydrogel can be varied. Ammonium persulfate (AMPS) can be used as an initiator for the polymerization reaction.

[0049] Surface combed DNA

[0050] In some cases, DNA molecules can be combed or stretched on a surface. However, the ability of previously reported combed or stretched DNA molecules on surfaces to serve as templates for polymerase reactions may be limited. In one aspect of the invention, a method is provided for placing stretched DNA molecules on a surface and for placing stretched DNA molecules on patterned rods or pillars. Figure 1As shown, DNA molecules can be stretched onto multiple patterned rods made of a photoresist such as SU-8. Various implementations can be used to suspend DNA using intermittent pillars or trapping rods or patterns of materials made of photoresist. DNA molecules on the substrate surface can contain two types of DNA fragments: a first type (hereinafter referred to as "anchored fragments") in contact with the top of the pillars or trapping rods; and a second type (hereinafter referred to as "suspended fragments") located between two adjacent anchored fragments, such that the DNA of the suspended fragment is suspended between two nearby pillars or trapping rods. Anchored fragments and suspended fragments can be tandem. Suspended fragments of DNA molecules are more readily enzymatically reacted with enzymes and primers compared to DNA molecules on a surface without pillars or trapping rods. Suspended fragments of DNA can be immersed in buffers or hydrogels for enzymatic reactions.

[0051] This disclosure provides several methods for constructing suspended DNA on surfaces having pillars, trapping rods, or other features. The stretching of DNA on the surface can be achieved through several methods: meniscus contraction, flow fields, mechanical trampling, etc. Various techniques can be used to pattern the surface, and a variety of materials can be used.

[0052] DNA molecules (such as genomic DNA molecules) can be stretched in various ways, including but not limited to using alternating current (AC) electric fields (Kaji, N., "Molecular stretching of long DNA in agarose gel using alternating current electric fields," Biophys. J., 82(1Pt 1):335-44, 2002), using electric field gradients in hyperbolic shrinkage microchannels (Randall, GC, et al., "Methods toelectrophoretically stretch DNA: microconstractions, gels, and hybrid gel-microconstraction devices," Lab. Chip, 6(4):516-25, 2006), using optical tweezers for uniform flow (Smith, SB, et al., "Overstretching B-DNA: the elastic response of individual double-stranded and single-stranded DNA molecules," Science, 271:795-9, 1996), and using flow homogenization (Perkins, TT, et al., "Stretching of a single tethered polymer in..."). auniform flow, Science, 268:83-7, 1995; using a uniform electric field (Ferree, S., et al., "Electrokinetic stretching of tethered DNA," Biophys. J., 85(4):2539-46, 2003); using acoustic force spectroscopy (AFS) (Sitters, G., et al., "Acoustic force spectroscopy," Nat. Methods, 12(1):47-50, 2015); forcing DNA into nanochannels (Tegenfeldt, JO, et al., "The dynamics of genomic-length DNA molecules in 100-nm channels," Proc. Natl. Acad. Sci. USA, 101(30):10979-83, 2004); and multi-stream water focusing (Wong, PK, et al.).(e.g., "Deformation of DNA molecules by hydrodynamic focusing," J. Fluid. Mech., 497:55-65, 2003) and dynamic combing to the surface (Dimalanta, ET, et al., "A microfluidic system for large DNA molecule arrays," Anal. Chem., 76(18):5293-301, 2004).

[0053] Figure 1 This demonstrates how genomic DNA can be stretched across SU-8 photoresist patterned rods. In this case, the SU-8 rods are shown as horizontal rods separated by grooves. Figure 1 As shown, the width of the bars is much smaller than the width of the grooves. The stretched DNA can be seen suspended between adjacent bars, similar to the threads of fabric running through the drying rack. Because combed or stretched DNA can be suspended on intermittent columns or trapping bars or other patterns on the substrate surface, the suspended portions of the DNA (e.g., Figure 1 The portion above the midslot is more likely to react with enzymes and / or primers or other biomolecules. For example, the suspended portion can be immersed in a buffer or hydrogel.

[0054] Figure 2A-2D An example scheme of the disclosed DNA suspension method is shown. In this example, nucleic acids are captured by cross-linking to a column or rod via ultraviolet (UV) light. The capture points, for example, from approximately 10 μM to approximately 100 μM, can be created by a photomask pattern. Figure 2A A photoresist layer 204 on a substrate 202 is shown. Figure 2B Nucleic acid 206 stretched on photoresist layer 204 is shown. Figure 2C A patterned mask 208 is shown, which can be placed on a stretched nucleic acid 206 and a photoresist layer 204. The photoresist layer 204 can then be exposed to ultraviolet light 210 through the mask 208. The exposed portions of the nucleic acid 206 can form covalent bonds with the exposed portions 212 of the photoresist. Figure 2D This illustrates that after developing an exposed photoresist (e.g., a negative photoresist), a portion of the photoresist can be removed, thereby suspending the stretched nucleic acid 206 between the remaining photoresist features (e.g., pillars or strips 212) on the surface of the substrate 202.

[0055] Alternatively, Figures 3A-3D Another example scheme of the disclosed DNA suspension method is shown. In this example, the photoresist layer is exposed before the nucleic acid is stretched on it. Figure 3AA photoresist layer 304 on a substrate 302 is shown. Figure 3B A patterned mask 308 is shown, which can be placed over a photoresist layer 304. The photoresist layer 304 can then be exposed to ultraviolet light 310 through the mask 308. Exposed portions 312 of the photoresist can be formed within the photoresist layer 304. Figure 3C A stretched nucleic acid 306 is shown on a photoresist layer 304 including an exposed portion 312. Figure 3D It is shown that after developing an exposed photoresist (e.g., a negative photoresist), a portion of the photoresist can be removed, thereby suspending the stretched nucleic acid 306 between the remaining photoresist features (e.g., pillars or strips 312) on the surface of the substrate 302.

[0056] As shown in this article, nucleic acids can be stretched onto a photoresist, such as SU-8, that has already been exposed to UV light. Stretching the nucleic acid onto the photoresist allows the photoresist to be developed without excessive loss of the nucleic acid. The size of the pillars, trapping rods, or surface features used to suspend and stretch the nucleic acid can influence the requirements for capturing DNA and the methods for fabricating or generating features on the substrate surface. For example, the size of the pillars, trapping rods, or surface features can range from nanometers to micrometers, and in some cases, the size of the capture region (e.g., on the pillar, rod, or surface feature) in contact with the suspended nucleic acid is minimized. In some cases, the DNA on the capture region is less responsive to enzymatic reactions (e.g., polymerase-catalyzed chain elongation, nick extension, endonuclease reactions, etc.) or other biological reactions (e.g., hybridization).

[0057] Figures 4A-4D Another example of the disclosed method for suspending DNA is shown. In this example, the surface of substrate 402 may include surface feature 412 as shown in FIG4. Figure 4B A filler material 404 is shown that can be used to fill the cavity between surface features 412 on a substrate 402 and is covered by a coverslip 408. Depending on the properties of the filler material 404, it can be treated to form a semi-solid or solid form, such that the treated filler material 404 and the surface features 412 together can form a substantially flat surface for placing stretched nucleic acid 406. The filler material 404 can then be removed by another treatment. For example, the filler material 404 can be a negative photoresist. Figure 4D The resulting stretched nucleic acid 406 is shown suspended between surface features 412 on substrate 402.

[0058] Stretched nucleic acids suspended between surface features can be further processed with hydrogels to seal the stretched nucleic acids. For example, hydrogels (e.g., 6% polyacrylamide gel) can be used to cover the space between surface features (or pillars or rods) 212, 312, or 412. Figure 2D , 3D And the cavity of the surface shown in 4D. The hydrogel can then be separated from the substrate (and surface features) together with the nucleic acid. Nucleic acid molecules can then be trapped inside or on the gel.

[0059] Figure 5 This shows another example of nucleic acid molecules (e.g., DNA molecules) suspended between surface features (e.g., SU-8 bars). The suspended DNA molecules can relax and be prepared to react with enzymes. Figure 6 This demonstrates that when suspended between surface features (such as SU-8 bars), suspended DNA molecules do not need to be perfectly straight, as the shape of the DNA molecules can be traced under a microscope or other visual / chemical / biological methods. The shape of the surface features can be selected to facilitate further reactions. Figure 7 A checkerboard pattern for surface features is shown. Those skilled in the art will understand that many different patterns can be used, as long as there are at least two capture regions in the surface feature that can suspend at least a portion of combed or stretched nucleic acids. Figure 8 This shows nucleic acids (e.g., DNA molecules, Figure 8 (shown as a thin line in the image) can be stretched vertically and suspended on the SU-8 horizontal bar (in... Figure 8 (The text is incomplete and appears to be a fragment from a larger document. A more accurate translation would require the full context.) Additionally, by incorporating labeled and / or unlabeled nucleotides into the extension product, stretched and suspended nucleic acids can be used as templates to extend primers. Labeled nucleotides can be inserted along the suspended DNA (shown as nearby red lines or...). Figure 8 The lines in the diagram appear in color (e.g., green dots or...). Figure 8 (points in the middle).

[0060] like Figure 9 As shown, DNA is suspended on SU-8 photoresist patterned rods. Various implementations can be used to suspend DNA using intermittent capture rods (or other structures) made of materials such as photoresist. The suspended portion of DNA is more readily reacted with enzymes and primers. The suspended portion of DNA can be immersed in buffer or hydrogel.

[0061] Figure 10The image shows DNA fragments that have been transferred into a hydrogel. A hydrogel can be formed by pouring reagents onto a surface feature suspended above the DNA before polymerization, and the reagents can be covered with a treated cover slip. After polymerization, the gel can be separated from the surface feature of the substrate, and the DNA fragments can be retained in the gel in a substantially linear manner, similar to how they were before gel formation. Figure 10 In the image, DNA stained with an intercalation dye is shown in green (or as a thin line).

[0062] Figures 11A to 11F It shows in Figure 9 and 10 A schematic process for obtaining suspended DNA from surface features. See also Figure 9 and 10 In some cases, DNA can be stretched on photoresist (such as SU-8) that has been exposed to ultraviolet light. (Figure) Figure 11A A photoresist layer 1104 on a substrate 1102 is shown. Figure 11B A patterned mask 1108 is shown, which can be placed over a photoresist layer 1104. The photoresist layer 1104 can then be exposed to ultraviolet light 1110 through the mask 1108. Exposed portions 1112 of the photoresist can be formed within the photoresist layer 1104. Figure 11C A stretched nucleic acid 1106 is shown on a photoresist layer 1104 including an exposed portion 1112. Figure 11D It is shown that after developing an exposed photoresist (e.g., a negative photoresist), a portion of the photoresist can be removed, thereby leaving the stretched nucleic acid 1106 suspended between the remaining photoresist features (e.g., pillars or rods 1112) on the surface of the substrate 1102. Figure 11E The diagram illustrates that a reagent for hydrogel 1116 can be poured onto the surface of substrate 1102, filling the surface cavity between surface features (or pillars or rods) 1112 and sealing suspended, stretched DNA, before covering the reagent with a coverslip 1114. A subsequent polymerization reaction can form hydrogel 1116, thereby trapping suspended, stretched DNA 1106 within the hydrogel. Figure 11F This demonstrates that after the substrate (along with surface feature 1112) is separated from the hydrogel, suspended, stretched DNA 1106 is trapped within the hydrogel 1116.

[0063] Figure 12 Genomic DNA molecules suspended in aqueous buffer and running through an SU-8 rod are shown. The DNA can be stained with an intercalation dye, in which case the DNA can... Figure 12The DNA is presented in red (or shown as a thin line). In the presence of DNA polymerase, a mixture of labeled and unlabeled nucleotides can be used to extend random primers. Labeled nucleotides may appear green and as dots along one or more red lines suspended and stretched in the DNA.

[0064] exist Figure 13 In this process, fragments of genomic DNA can be captured in a hydrogel. The DNA may not be visible because it may not be stained. Random primers can be extended in the presence of DNA polymerase using a mixture of three reversibly terminated labeled nucleotides and one unlabeled nucleotide. The three labeled nucleotides can be displayed in three colors: Figure 13 The colors are red, green, and blue (or displayed as dots).

[0065] Many reactions can be performed on the captured DNA obtained by the methods disclosed herein, such as hybridization, protein binding, and primer or nick extension reactions.

[0066] Example

[0067] Optical sequencing of DNA fragments trapped in hydrogels by incorporating reversibly terminated labeled nucleotides.

[0068] DNA stretching is useful in all applications related to molecular topology. DNA can be stretched on surfaces using a variety of methods (meniscus contraction, flow fields, mechanical trampling, etc.). However, once the molecule is stretched, surface effects can limit the ability of various enzymes or reagents to access and manipulate the DNA. This paper discloses a novel method for anchoring DNA to intermittent capture rods, or similar structures called surface features. DNA portions suspended between surface features are more readily subjected to enzymatic reactions and other manipulation methods compared to DNA portions in direct contact with the surface (or surface feature). For example, the method disclosed herein can be used for DNA sequencing or DNA mapping. Furthermore, it provides a simple platform for protein-DNA binding assays.

[0069] The substrate was fabricated by spin-coating (approximately 1000 rpm) the photoresist polymer SU-8 (formulation 2002) onto a 22 × 22 mm silicon substrate to obtain a 1.75 μm thick SU-8 layer. The coated substrate was baked on a hot plate at approximately 95 °C for 1 minute. The substrate was then placed in a vacuum and brought into contact with a photomask featuring alternating patterns (10 μm apart) with 10 μm wide lines. The substrate was then exposed to 365 nm UV light at 80 mJ / cm² and finally baked at 95 °C for 2 minutes.

[0070] After heating at 94°C for 1 minute, anneal the DNA to random primers. Immerse the undeveloped substrate in a cuvette containing 50 ng of annealed DNA suspended in 1.2 mL of MES buffer (pH 5.5). Incubate for 1 hour. Then, pull the substrate out of the cuvette at a speed of 67 μm / sec. The pulling direction must be substantially perpendicular to the pattern centerline.

[0071] Substrate development followed by development of the substrate (with stretched DNA on top) in propylene glycol methyl ether acetate (PGMEA). The polymer portions not yet exposed to UV light dissolved under the DNA, suspending the DNA between anchor points to form “DNA bridges.” Excess PGMEA was removed by rinsing the substrate with isopropanol. Finally, the sample was rinsed with purified water. The surface was incubated for 15 minutes in a solution of magnesium chloride and Tween 20.

[0072] The bonded silane coating on a quartz substrate involved immersing a 22×22 mm quartz slide in a solution containing ethanol, acetic acid, and a bonded silane (γ-methacryloyloxypropyltrimethoxysilane). The slide was stirred in the solution for 1 hour, followed by rinsing with water and ethanol.

[0073] Acrylamide gel formulation: 6% acrylamide gel was mixed with tert-methyltetraethylenediamine (TEMED) and ammonium persulfate (APS) to initiate polymerization. 15 μL of the mixture was dispersed onto a substrate and sealed with a quartz slide pre-treated with a bonding silane solution. After one hour, the substrate was separated, and the DNA embedded in the gel was transferred onto the quartz slide. The gel was washed to remove excess unpolymerized monomers.

[0074] First nucleotide incorporation involves applying a solution containing DNA polymerase, appropriate buffer, three reversibly terminator-labeled nucleotides, and one reversibly terminator-labeled but unlabeled nucleotide to the sample and incubating in a 50°C oven for 20 minutes. The substrate is then washed with buffer to remove unincorporated nucleotides. DNA can be stained by applying a solution containing intercalation dye (YOYO) to the sample surface, followed by rinsing with water.

[0075] The imaged sample was imaged in four different channels under a TIRF microscope (three for labeling nucleotides and one for DNA staining dye). Exposure time: 0.3 seconds, camera gain: 300, laser power of the light source: 100mW.

[0076] The subsequent incorporation involves incubating a solution containing TCPE (a cleavage agent for incorporating reversible terminators) on the sample surface at 55°C for 15 minutes, followed by washing with water. A solution containing DNA polymerase and three reversible terminator-labeled nucleotides and one reversible terminator-labeled but unlabeled nucleotide is applied to the sample and incubated at 50°C for 20 minutes. The substrate is washed with buffer and then imaged. The last two steps are repeated to obtain the desired incorporation amount.

[0077] While preferred embodiments of the invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided merely as examples. Numerous variations, modifications, and substitutions will occur to those skilled in the art without departing from the scope of the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be used in the practice of the invention. The following claims are intended to define the scope of the invention, thereby covering the methods and structures and their equivalents within the scope of these claims.

Claims

1. A method for treating at least one nucleic acid molecule on a substrate surface, comprising: (a) Stretching nucleic acid molecules onto a layer on the surface of the substrate; and (b) Following (a), multiple discrete features are formed by removing multiple subparts from the layer on the surface of the substrate while the nucleic acid molecule is in contact with the layer on the surface of the substrate; Following (b), the nucleic acid molecule comes into contact with at least two members of the plurality of discrete features, thereby suspending at least a portion of the nucleic acid molecule between the at least two members of the plurality of discrete features, wherein: (i) Each of the plurality of discrete features is independently a pit, hole, groove, channel, pillar, bump, ridge, or rod, and (ii) Each of the at least two members of the plurality of discrete features is independently a pillar, a bump, a ridge, or a rod.

2. The method according to claim 1, wherein the plurality of discrete features form a topological pattern.

3. The method of claim 1, wherein the nucleic acid molecule is in contact with two other members of the plurality of discrete features.

4. The method of claim 3, wherein the additional portion of the nucleic acid molecule is suspended between the two other members of the plurality of discrete features.

5. The method of claim 1, further comprising: (c) A hydrogel is formed on the surface of the substrate, the hydrogel being in contact with some or all of the members of the plurality of discrete features.

6. The method of claim 5, further comprising: (d) Remove the hydrogel from the surface of the substrate.

7. The method of claim 1, further comprising, in (a), stretching additional nucleic acid molecules onto the layer on the surface of the substrate.

8. The method of claim 7, wherein after (b), the additional nucleic acid molecule contacts at least two other members of the plurality of discrete features, thereby suspending the additional nucleic acid molecule between the at least two other members of the plurality of discrete features.

9. The method according to claim 3 or 4, wherein each of the two other members of the plurality of discrete features is independently a pillar, a bump, a ridge, or a rod.

10. The method according to any one of claims 5-6, wherein at least a portion of the nucleic acid molecule is encapsulated in the hydrogel.

11. The method of claim 8, wherein each of the at least two other members of the plurality of discrete features is independently a pillar, a bump, a ridge, or a rod.

12. The method according to any one of claims 1-8, wherein the nucleic acid molecule is DNA.

13. The method of claim 12, wherein the DNA is double-stranded or single-stranded.

14. The method according to any one of claims 1-8, wherein the nucleic acid molecule is active in the enzyme reaction.

15. The method according to any one of claims 1-8, further comprising: An enzymatic reaction is performed on the nucleic acid molecule, wherein the nucleic acid molecule is suspended between at least two members of the plurality of discrete features.

16. The method according to claim 7 or 8, further comprising: An enzymatic reaction is performed on the additional nucleic acid molecules.

17. The method according to any one of claims 1-8, further comprising: Protein binding, hybridization, nick translation, or nick extension reactions are performed on the nucleic acid molecules suspended between at least two members of the plurality of discrete features.

18. The method according to any one of claims 1-8, further comprising: A primer extension reaction catalyzed by a polymerase is performed on the nucleic acid molecule suspended between at least two members of the plurality of discrete features.

19. The method of claim 7 or 8, further comprising performing a protein binding reaction, hybridization reaction, nick translation reaction, or nick extension reaction on the additional nucleic acid molecule.

20. The method of claim 7 or 8, further comprising performing a primer extension reaction catalyzed by a polymerase on the additional nucleic acid molecule.

21. The method of any one of claims 1-8, wherein each of the at least two members of the plurality of discrete features comprises a photoresist.

22. The method of any one of claims 1-8, further comprising: Before (a), a photoresist layer is applied to the top of the substrate.

23. The method of claim 22, further comprising: Before (a), ultraviolet light is irradiated onto the photoresist layer through a mask.

24. The method of claim 23, wherein the removal in (b) comprises developing the photoresist layer.

25. The method of claim 24, wherein each of the at least two members of the plurality of discrete features comprises a photoresist.

26. The method of claim 24, wherein each of the two other members of the plurality of discrete features comprises a photoresist.

27. The method of claim 22, wherein the photoresist is a positive photoresist or a negative photoresist.

28. The method according to any one of claims 1 or 3-8, wherein prior to (a), the surface comprises a topological pattern containing the plurality of discrete features and the plurality of cavities, wherein each of the plurality of discrete features is independently a pillar, bump, ridge, or rod.

29. The method of claim 28, wherein each of the plurality of cavities is independently a pit, hole, groove, or channel.

30. The method of claim 28, further comprising: Prior to (a), each of the plurality of cavities is filled with a photoresist.

31. The method of claim 30, wherein the removal in (b) comprises developing a photoresist layer, wherein the photoresist is a negative photoresist.

32. The method of claim 30, further comprising: Before (a), ultraviolet light is irradiated onto the surface of the photoresist, wherein the photoresist is a positive photoresist.

33. The method of claim 32, wherein the removal in (b) comprises developing a positive photoresist layer.

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