Gel patterned surfaces

Abstract

To provide systems used for sequencing.SOLUTION: Provided is an array including: a solid support having a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells from the gel material in other wells; and a library of target nucleic acids in the gel material, where the gel material in each of the wells comprises a single species of the target nucleic acids of the library. Methods for making and using the array are also provided.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 61 / 769,289, filed February 26, 2013, and This application claims the benefit of U.S. Application No. 13 / 787,396, filed March 6, 2013. No. 60 / 639,333 filed on Oct. 23, 2007, and all such applications are incorporated herein by reference in their entirety.

[0002] The present disclosure relates generally to solid-phase analytical chemistry and methods for high-throughput analysis of genomics. It has particular applicability to nucleic acid arrays. [Background technology]

[0003] The task of classifying human genetic variants and correlating these variants with susceptibility to disease is This classification effort is well positioned to benefit from advances in genome-wide sequencing. This will hopefully identify markers in each individual's genome that will help determine a person's susceptibility to disease and prescriptions. Responsiveness to specific therapies such as medications, dangerous drug side effects and other medically relevant conditions This will aid medical professionals in determining susceptibility to potential traits. The study has made considerable progress, mainly due to the fact that it has been evaluated in a research setting with a sufficiently high cost-effectiveness. This is due to the availability of commercial genome sequencing methods that allow testing of subjects with Improvements in sequencing methods are also needed to accelerate the process. Sequencing is a technology that goes beyond research centers to allow doctors to obtain sequences from patients in the general population. This is preventing the transition to clinics where this is possible.

[0004] Sequencing methods and the systems used to perform them utilize a complex collection of techniques. Improvements that result in substantial cost savings have been demonstrated in some of these techniques. and estimate which techniques, if any, have the potential to improve and lead to cost savings. It is difficult to determine which technique is the most important in a sequencing system, given the dependencies between the techniques. It is difficult to estimate how the problem can be fixed without adversely affecting the overall performance of the system. Therefore, the potential of genomics research to improve lives and in many cases There is a need to identify improvements that can be transferred to clinics where they can be rescued. , fulfills this need and provides related advantages as well. Summary of the Invention [Problem to be solved by the invention]

[0005] The present disclosure relates to a solid support having a surface, the surface having a plurality of wells, contains a gel material, the wells being separated from one another by gap areas on a surface thereof, the gap areas a solid support that isolates the gel material in each well from the gel material in the other wells; a library of target nucleic acids in a gel material, wherein each well of gel material represents a single species of the library; and a library comprising the target nucleic acid. [Means for solving the problem]

[0006] In some embodiments, the substrate is arranged as an array of wells and the analytes are nucleic acids. Accordingly, the present disclosure provides a solid support having a surface, the surface having a plurality of wells. the wells contain a gel material, the wells being separated from one another by interstitial areas on a surface; The interstitial regions separate the gel material in each well from the gel material in the other wells. and a library of target nucleic acids in a gel material, wherein the gel material in each well contains a library. and a library containing a single species of target nucleic acid.

[0007] The present disclosure also provides a method of making a substrate, the method comprising: (a) providing a solid support having a planar surface; providing a support, the plane being interrupted by one or more concave features; , one or more concave features are adjacent to one or more gap regions on the planar surface. and (b) coating at least a portion of the solid support with a gel material. The portion includes at least one recessed feature and at least one gap region. (c) polishing the flat surface to remove the gel material from at least one gap region; and and retaining the gel material in the at least one recessed feature. Cut.

[0008] A method for making an array includes: (a) providing a solid support having a surface containing a plurality of wells; a step of: the wells containing a gel material, the wells being defined by interstitial regions on a surface thereof; each well having a gap area separating the gel material from the gel material of the other wells. (b) delivering a library of target nucleic acids to the wells of the solid support and A process for producing an array of wells having a single type of target nucleic acid attached to the gel material of the well. wherein different wells in the array have different target nucleic acid species from a library. (c) amplifying target nucleic acids attached to the gel material in the wells of the array to and generating a clonal population of individual target nucleic acids in each well of the array. Cut.

[0009] The present disclosure further provides a method for detecting an analyte, the method comprising: (a) detecting a solid having a planar surface; Providing a support, the plane being interrupted by one or more recessed features. the recessed features contain a gel material, and one or more recessed features are adjacent to one or more gap regions, the gap regions being substantially free of gel material, and the gel material being substantially free of the target (b) attaching the target analyte to or containing the target analyte; and contacting the solid support with the probe under conditions that interact with the probe; ) detecting the solid support to identify at least a subset of the target analytes that interact with one or more of the probes. and identifying the set.

[0010] In certain embodiments, the nucleic acid is the analyte to be detected and the recessed features are wells. For example, a method for detecting nucleic acids includes (a) detecting a surface and a solid support having a library of nucleic acids. providing a support, the surface having a plurality of wells, the wells containing a gel material; the wells being separated from one another by gap areas on a surface thereof, the gap areas being adjacent to each other, The gel material of the wells is isolated from the gel material of the other wells, and a single target nucleic acid of the library is (b) a solid support attached to the gel material in each well; and (b) a solid support attached to the gel material that binds the target nucleic acid. (c) contacting the solid support with at least one probe; and (b) detecting the solid support. and identifying wells having a target nucleic acid species that binds to at least one probe. It is possible to do so.

[0011] The compositions, devices, and methods of the present disclosure are demonstrated herein in the context of gel materials. Gel materials are representative and can, for example, form a surface coating and, by themselves, The body can be replaced with other organic materials, such as polymers, that are not necessarily considered gels. It should be understood that the arrays produced can be used in analytical or preparative methods, etc. Applying a gel material to the surface, removing the gel material from the interstitial areas, and attaching the analyte to the gel material. The methods described herein are easily adapted by substituting a non-gel material for the gel material. It can be done. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows a schematic of a method for making and using patterned arrays of DNA features, where each feature is a well having a gel material with DNA clusters attached to it, and using the arrays in a sequencing method. [Diagram 2] Images are shown from a BeadChip substrate modified to have gel material instead of beads in the wells. Panel A: brightfield image taken before polishing. Panels B-C: fluorescent images taken after polishing and hybridization to fluorescently labeled oligonucleotides. [Figure 3-1] Panel A shows a schematic process flow utilizing photolithography and a Cr hard mask along with reactive ion etching to fabricate recessed features in a substrate, and panel B shows example SEM images of wells and features in a glass substrate. [Figure 3-2] In panel C, an image of the wafer, an image of a portion of the wafer containing the array of references and wells, and an image from a portion of the array containing wells are shown. [Figure 4] 1 shows high-resolution fluorescence microscopy images of nanowell substrates showing patterned gel features on the nanowell substrates after coating the substrates with PAZAM and polishing with silica bead slurry. For visualization, the PAZAM is labeled with a dye. [Diagram 5]Multicolor fused images obtained from a HiSeq sequencing cycle of a 1.5 μm pitch nanowell substrate with patterned clusters. Panel A: Image showing the extent of the patterned cluster in a gel-containing well alongside four bullseye fiducials. Panel B: High-resolution image showing the mixing of colors (due to a mixed population of amplicons) in a single bullseye fiducial. [Figure 6A] Panel A shows multicolor fusion of patterned clusters in Hiseq sequencing performed on a 750 nm pitch nanowell substrate. [Figure 6B] In panel B, the arrays are aligned and nearest neighbor curves are shown showing that the clusters pass the quality filter. [Figure 6C] Panel C shows sequencing quality metrics indicating successful passage through the quality filter at a density of 1.6 million clusters / mm2. [Figure 7] Graph of clonality versus occupancy where the curve is a Poisson distribution, the line is the ideal clonality and occupancy, and the crosses are the average measures obtained from sequencing using substrates with patterns of gel-containing nanowells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] The present disclosure provides structured substrates, methods of making structured substrates, and methods of using structured substrates. In certain embodiments, the substrate contains a gel material (e.g., is coated with a gel material). The gel material then comprises a solid support having recessed areas, such as recessed (recessed) wells. In certain embodiments, the gel-containing region can be They are discrete and separated by interstitial regions that lack the ability to attach analytes of interest. For example, The gap regions may be devoid of gel material. Alternatively, the gel material in the gap regions may be inactive. The activity or properties of the gel material in the recessed regions, e.g., analyte attachment, may be detected or otherwise altered. The resulting isolation of the gel area provides a barrier to the analyte. These provide advantages when performing reactions and / or detecting analytes. Typical advantages are: This can be demonstrated with the example of an array of target nucleic acids distributed within a gel-containing well. The paper describes a method for carrying out amplification reactions on a structured substrate using nucleic acids as templates, and performing the amplification reactions in a gel or gel. The nucleic acid colonies that grow on the substrate (e.g., nucleic acid features of an array) can be formed. The interstitial regions serve to limit the area over which colonies can grow. Each feature is relatively distinct due to the distinct patterns created by the gel-containing wells. The pattern increases the density of features and makes them easier to distinguish from random arrays of nucleic acids. It may also provide the advantage of reducing the processing requirements of image registration in comparison.

[0014] A representative process for making patterned arrays of nucleic acids is shown in Figure 1. Well-patterned substrate In the example, the wells are spaced at a pitch of 1.5 μm (center-to-center spacing). The well-patterned substrate has a well-shaped groove (single) and each well has a diameter of 0.5 μm. A gel material can be coated so that it enters the wells and covers the interstitial areas. The resulting gel-coated substrate is polished to leave the gel material in the wells. By removing the gel material from the gap areas, a gel-patterned substrate can be formed. Gels can serve to capture DNA templates and support amplification of the templates. For example, before surface coating, after surface coating and before polishing, or after polishing. The gel can be grafted with an oligonucleotide primer. Capable of capturing a DNA template and functioning for primary amplification using the captured template The resulting DNA patterned substrates can be analyzed, for example, in a sequencing procedure.

[0015] Patterned arrays of nucleic acids in gel-containing wells offer several advantages for DNA sequencing. Random arrays (i.e., arrays with random feature patterns) and Comparative advantages include dense feature packing, concentration-independent mold seeding, etc. Improved control and tuning of feature density using reduced image registers These include the processing required for signal processing and simplified signal extraction. Further advantages may result from the spatial confinement of the nucleic acid population that the pattern of the present disclosure provides. The features of the quantified array are the sites where nucleic acid colonies grow (e.g., by cluster amplification). It can act to limit the area or volume of a structure without limiting its area or volume. Some nucleic acid colonies may be amplified to a larger size than others because of the group in their sequence. Due to the difference in the percent content of anine and cytosine (i.e., GC content), this In the methods and compositions described herein, the individual features Alternatively, the volume or area of ​​the tube may be determined by the difference in GC content between the amplified template species. Prevent or minimize differences in the size of nucleic acid colonies that may result from amplification reactions of different factors. For example, the volume or area of ​​a feature can be selected to be the feature that can be grown at the fastest rate. It can be made small enough to limit the growth of colonies that grow slowly, while still allowing To ensure that growing colonies effectively fill in the features upon completion of the amplification reaction can be done.

[0016] In certain embodiments, the present disclosure provides covalently bonded patterned gels, such as poly(N- (5-Azidoacetamidylpentyl)acrylamide-co-acrylamide) (PAZ AM, see, e.g., U.S. Provisional Patent Application No. 61 / 753,833, incorporated herein by reference. 3. A glass, silicon, plastic or other suitable solid support having a The present invention provides for the manufacture of wells (e.g., microwells or nanowells) on a substrate, the process comprising: The gel pads used for sequencing were prepared, which were then used to perform multiple cycles of sequencing. Covalent attachment of the polymer to the wells can be used in a variety of ways. This helps to retain the gel in the structured features throughout the life of the structured substrate while it is being However, in many embodiments, the gel does not necessarily have to be covalently attached to the wells. For example, under some conditions, silanes that are not covalently bonded to any part of the structured substrate may be Non-sulfuric acid-containing acrylamides (SFAs, e.g., U.S. Pat. No. 5,393,311, which are incorporated herein by reference) are disclosed in US Pat. See published application 2011 / 0059865) may be used as the gel material.

[0017] In certain embodiments, a solid having wells (e.g., microwells or nanowells) The support material is patterned and the patterned support is coated with a gel material (e.g., PAZAM, SFA or We have developed these chemically modified variants, such as the azidolyzed version of SFA. coated with azide-SFA) and gel-coated The body is polished, for example by chemical or mechanical polishing, so that the well The method comprises the steps of: removing substantially all of the gel from the interstitial areas on the surface of the structured substrate between the wells while retaining the gel; By removing or passivating the ply, a structured substrate can be created. The target nucleic acid (e.g., fragmented human IgG) can then be attached to the gel material. A solution of target nucleic acids (DNA) can be contacted with the polished substrate, resulting in the formation of individual target nucleic acids. are seeded into individual wells by interacting with primers attached to the gel material. However, the gap regions are free of gel material or are inactive, so the target nucleic acid occupies the gap regions. The absence or inactivity of gel in the interstitial regions prevents the growth of nucleic acid colonies. Since migration out of the knee is prevented, amplification of the target nucleic acid is confined to the confines of the well. The process is conveniently manufacturable, scalable, and compatible with conventional micro- or utilizes nanofabrication techniques.

[0018] In certain embodiments, the fiducial markers are located at individual features (e.g., wells or The gel-containing recessed features (and other gel-containing recessed features) are included in the structured substrate for identification and localization. A quasi-marker is a reference marker because it provides a reference point for the relative position of other features. This is particularly useful for structured substrates having spatially aligned pattern features. Fiducial markers can also be used to register images of random arrays, but HiSeq and Genom, manufactured by Lumina, Inc. (San Diego, CA) Commercially available sequencing platforms such as the eAnalyzer or MiSeq platforms When used with random arrays generated on a cluster, the inherent cluster perturbation is used instead. The fiducial markers may be used to repeatedly detect the structured substrate and time-lapse the individual features. Fiducial markers are particularly useful in applications where multiple sequencing Sequential images obtained over a fixed cycle allow tracking of individual nucleic acid clusters. As a result, the sequence of each cluster can be determined individually.

[0019] The present disclosure provides a fiducial marker having a pattern of recessed and gap regions. Representative designs of markers include: recessed rings, gap rings, and wells or other recessed frames. A feature that has two or more alternate patterns of rings (e.g., "bull's eye") In some embodiments, the recessed area of ​​the fiducial marker comprises a gel material. The specific location of the gel on this surface is described herein. This can be accomplished using the gel coating and polishing techniques described above. Typically, the gel-containing area In some cases, the discrimination is based on the presence of a detection method that can distinguish the interstitial regions from the interstitial regions. It may also be based on the presence of a particular analyte in a region of the gel that is not detected by the ELISA. , the gel-containing regions of fiducial markers are used to label target nucleic acids on the array The reference markers can contain nucleic acids that are labeled by the same methods as those described above. The stencils are conveniently fabricated using the same methods used to fabricate the specimen features. Accordingly, fiducial markers and specimen features can be created, if necessary. The structured substrates and the substrates may be fabricated simultaneously in one or more steps. Another useful fiducial marker that can be used in the method is one that has subregions, A pattern of wells (or other recessed features) in one subregion overlaps with a pattern in another subregion. Such a reference grid is incorporated herein by reference. Image resists such as those described in U.S. Patent Application Serial No. 13 / 267,565, incorporated herein by reference. It can be configured and used for

[0020] As a further example, beads may be used as fiducials. The beads may be labeled with a fluorophore or other In this case, the surface contains at least two wells (or other The relatively large wells may contain one or more reference beads. while the smaller wells are too small to contain beads and contain only gel material. Therefore, the smaller wells act as analytical features for the analysis and The large, bead-filled well serves as a reference. The char may be a channel such as that present in the bullseye configuration exemplified above. The channel can be sized to accommodate beads. Several beads are positioned in the channel, e.g., a distinct series of bead shapes A standard can be created.

[0021] Patterned arrays, methods of making and using same are provided for attaching analytes of interest. The gel material used is described herein in relation to the gel material. and other techniques that can be used to mediate localization of analytes to features on a surface. It should be understood that organic materials may be substituted. Such organic materials may include, for example, Polyesters that can form coatings and are not necessarily considered gels in themselves Examples include polymers that can be polymerized by ATRP (atom transfer radical polymerization) or surface initiated polymerization. The polymer is formed by the above-mentioned method. The method described herein includes applying a gel material to a surface and removing the gel material from the interstitial regions. , and can be easily adapted for use with non-gel materials.

[0022] Terms used herein are to be taken as given their ordinary meaning in the relevant art unless otherwise specified. It should be understood that certain terms and their meanings used herein are understood to be within the scope of the present invention. The details are described below.

[0023] As used herein, the term "attached" means that two things are connected or fixed to one another. For example, analytes such as nucleic acids may It can be attached to materials such as gels or solid supports by covalent or non-covalent bonds. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are characterized by the sharing of electron pairs between atoms. These are chemical bonds that do not require any chemical reaction, such as hydrogen bonds, ionic bonds, van der Waals forces, Mention may be made of hydrophilic and hydrophobic interactions.

[0024] As used herein, the term "clonal population" refers to a population of cells that are homogeneous for a particular nucleotide sequence. A uniform sequence is usually at least 10 nucleotides in length, but It may be longer, for example, at least 50, 100, 250, 500, 1000. A clonal population is a set of clonal sequences that contain a single target or template nucleic acid. A clonal population may be derived from a population of clones that contains at least two copies of a target nucleotide sequence. , 5, 10, 100, 1000 or more copies. The copies may be present in a single nucleic acid molecule, or the copies may be present on a single nucleic acid molecule. (i.e., a clonal population may be a set of nucleic acid molecules having the same target nucleotide sequence. (It may contain at least 2, 5, 10, 100, 1000 or more). Typically, All nucleic acids in a clonal population will have the same nucleotide sequence. without departing from the principles of the present invention, a very small number of contaminating nucleic acids or mutations (e.g., amplification artifacts) Amplification artifacts (caused by It should be understood that the population may therefore be at least 80%, 90%, 95% In some cases, 100% pure clonal populations exist. This may also occur.

[0025] As used herein, the term "coating" when used as a verb means to describe a layer or coating on a surface. At least a portion of the surface is covered with a layer or coating. In some cases, the entire surface may be provided with a layer or coating. In an alternative embodiment, only a portion of the surface may be provided with a layer or coating. When the term "material" is used to describe the relationship between a surface and a material, The term "material" is intended to mean that the material is present as a layer or coating on a surface. , the surface may be sealed, for example to prevent liquids or gases from contacting the surface. However, a material does not necessarily have to form a seal. For example, a material may be a liquid, a gas, or Alternatively, the material may be permeable to one or more components carried in the liquid or gas. Exemplary materials that can coat the surface include, but are not limited to, gels, polymers, , organic polymers, liquids, metals, a second surface, plastics, silica, or gases. can be.

[0026] As used herein, the term "recessed features" is used in reference to a solid support. In the present case, the term "recess" refers to a depression or indentation in a solid support. These include, but are not limited to, wells, pitches, holes, depressions, channels, or troughs. The concave features may optionally have a curved cross section (a dimension perpendicular to the surface of the solid support). ), but cross sections with one or more linear cross sections, angles or corners are also possible. Cross sections having a combination of curved and linear cross sections are also possible. The features do not necessarily need to pass completely through the solid support, but rather, for example, The plate has a bottom surface or base.

[0027] As used herein, the term "different" when used in reference to nucleic acids means that the nucleic acids are different from each other. It means that two or more nucleic acids have different nucleotide sequences that are not the same. Alternatively, two or more nucleic acids may have different nucleotide sequences within the entire length of the nucleic acid. can have different nucleotide sequences within a substantial portion of their length. For example, the two or more nucleic acids may have target nucleotide sequence portions that differ in the two or more molecules. However, two or more molecules may have the same universal sequence portion.

[0028] As used herein, the term "each" when used in reference to a collection of items: It is intended to identify individual items in a collection, but not necessarily all items in the collection. Unless clear disclosure or the context clearly indicates otherwise, It is also possible.

[0029] As used herein, the term "fluidic access" means When used in reference to molecules and fluid-contacting locations in the body, it refers to the location of the molecule in or near the fluid. refers to the ability of a molecule to move through a fluid to contact or enter a location. It can also refer to the ability of a molecule to separate or leave its location and enter solution. Fluid access allows molecules to enter, contact, and leave the location. , and / or may occur when there is no barrier to prevent the fluid from leaving its location. Unless access is absolutely prevented, proliferation may halt, reduce or change. It is understood that fluid access exists.

[0030] As used herein, the term "gel material" refers to a semi-rigid material that is permeable to liquids and gases. Typically, gel materials expand when liquid is introduced and dry out. They may shrink when the liquid is removed by drying. Exemplary gels include, but are not limited to: However, colloidal structures such as agarose, polymer mesh structures such as gelatin, or Polyacrylamide, SFA (see, e.g., U.S. Pat. No. 6,393,326, which is incorporated herein by reference) See Publication No. 2011 / 0059865) or PAZAM (see, for example, this reference). Crosslinked polymers such as those described herein (see U.S. Provisional Patent Application No. 61 / 753,833, incorporated herein by reference). Particularly useful gel materials include those having a well or other structure. Some useful gel materials conform to the shape of the well or recessed feature in which they are located. (b) can conform to the shape of other concave features and (c) can conform to the shape of the well or concave feature in which it resides The mixture may also have a volume that does not substantially exceed the volume of the char.

[0031] As used herein, the term "interstitial region" refers to a region of a substrate that separates other areas of the substrate or surface. For example, an interstitial area refers to the area within or on a surface of a recessed feature of an array. The two regions that separate from each other can be separated by a concave feature in the ray. In another example, the gap region may separate the first feature portion from the second feature portion. In many embodiments, the interstitial region is continuous. While this is a one-dimensional array of wells, it is possible to separate each filter, for example, as in the case of an array of wells in a contiguous surface. The separation provided by the interstitial regions is not complete even though some of the separation is complete. The interstitial regions are typically of a different surface material than the surface material of the features on the surface. For example, the features of the array may have an amount or concentration of In some embodiments, the gel material or analyte may have an amount or concentration greater than Alternatively, no gel material or analyte may be present in the interstitial regions.

[0032] As used herein, the term "library" when used in reference to a sample refers to a collection of different A library refers to a collection of analytes that have a chemical composition consistent with the given set of analytes. Typically, the analytes in a library have the common characteristics or properties of a genus or class, but differ in some way in another part For example, libraries may differ in nucleotide sequence but may also differ in sugar-phosphoryl It is possible to include nucleic acid species that are similar in that they have an acid backbone.

[0033] As used herein, the terms "nucleic acid" and "nucleotide" are used in accordance with their meanings in the art. Consistent with the use of the term "naturally occurring species" in the present specification, the term is intended to include naturally occurring species or functional analogs thereof. Useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific manner. or can be used as a template for the replication of a particular nucleotide sequence. Naturally occurring nucleic acids generally have backbones containing phosphodiester bonds. The structure can have alternative backbone linkages, including any of those known in the art. Naturally occurring nucleic acids generally consist of deoxyribose sugars (e.g., deoxyribonucleic acid (DN A) or ribose sugars (e.g., found in ribonucleic acid (RNA)). Nucleic acids may contain any of a variety of analogs of these sugar moieties known in the art. Nucleic acids can contain natural or non-natural nucleotides. In this context, natural deoxyribonucleic acid is composed of adenine, thymine, cytosine or can have one or more bases selected from the group consisting of guanine, and the ribonucleic acid can be One or more bases selected from the group consisting of uracil, adenine, cytosine, and guanine Useful non-natural bases that can be included in nucleic acids or nucleotides include those described herein. The terms "probe" and "target" are used in reference to nucleic acids as known in the art. In the context of the methods or compositions described herein, a semantic identifier for a nucleic acid may be used. The term "generic identifier" is intended to refer to a nucleic acid structure or function that is not necessarily otherwise clearly defined. The terms "probe" and "target" are used interchangeably herein without limitation. It can be applied to other analytes such as proteins, small molecules, and cells as well.

[0034] As used herein, the term "random pattern" refers to wells on a surface. where the relative positions of a subset of wells in a region of a surface are not known, or This means that the location of a subset of wells in another region of the surface is not predictable. The subset used in the routine generally contains at least three wells, but at least A random pattern can contain 4, 5, 6, 10 or more wells. , does not include multiple repetitions of any subpattern. This term does not include other concave features in addition to the wells. Can be applied to char.

[0035] As used herein, the term "repeating pattern" refers to wells on a surface. When the relative positions of a subset of wells in a region of the surface are determined by at least one This means that the relative positions of a subset of wells in another region of the Therefore, the relative positions of the wells in one area of ​​the repeating pattern are generally The relative positions of the wells in different regions of the cell can be predicted. A plate generally contains at least three wells, but may contain at least 4, 5, 6, 10 or more wells. Typical repeating patterns include linear and and hexagonal patterns. Repeating patterns may include multiple repetitions of a subpattern. The term can be applied to other recessed features besides wells.

[0036] As used herein, the term "isolated" refers to the separation of two wells (or two other features). When used in reference to the gel material in a gel plate, one of the wells (or one of the features) Separating or isolating the gel material in one well from the gel material in other wells (or other features) Therefore, in the first well (or first feature) The gel material does not directly contact the gel material in other wells (or other features). In some embodiments, the gel material in the two wells (or two features) can be, e.g., Indirect contact occurs through the solution contacting the two wells (or features). Or the gel material in the two wells (or two features) is in indirect contact. The gap areas on the surface are separated by two wells ( or two features). In certain embodiments, The gel material may be discontinuous on the surface and may be present in recessed features such as wells. but not in the interstitial regions between the features.

[0037] As used herein, the term "surface" refers to the exterior or outer layer of a solid support or gel material. The surface may be any of a gas, liquid, gel, polymer, organic polymer, and the like. By contacting it with another material, such as a second surface of a similar or different material, metal, or coating The surface or region thereof may be substantially flat. The surface may include wells, pitches, , having surface features such as channels, ridges, raised areas, pegs, posts, etc. It may be possible.

[0038] As used herein, the term "monospecies" refers to a particular genus that is essentially one and only one species. This term is not necessarily intended to be a limiting number representing a single species present. For example, a population of nucleic acid molecules, each of which has the same nucleotide sequence, represents a single species of nucleic acid. The term "single" in this context refers to the presence of other things not within the genus concerned. For example, the present invention is not intended to exclude the use of a single target nucleic acid from a library. The wells containing the nucleic acids may contain multiple nucleic acids with the same sequence, It will exclude other target nucleic acids, but not necessarily exclude any other non-nucleic acid components. A single species population may be characterized by a level of impurities or artifacts that one of skill in the art would consider negligible for a particular application of the population. It is understood that there may be small amounts of other species present at levels that are considered artifacts. For example, a nucleic acid cluster derived from a single template having a first sequence can be used to detect the first sequence. When the nucleic acid molecule is subjected to the second sequence, the amount of any nucleic acid molecule having the second sequence is undetectable or negligible. If the concentration is sufficiently low, it may be considered as having a single species. A single-species population will have one, and only one, species.

[0039] As used herein, the term "solid support" refers to a rigid material that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate may optionally be capable of absorbing liquid. Although it is possible for the substrate to be porous (e.g., due to its porosity), typically the substrate will not be substantially effective in taking up liquid. and sufficiently so as not to expand substantially when the liquid is removed by drying. A non-porous solid support will generally be impermeable to liquids or gases. The solid support is optionally inert to the chemistry used to modify the gel. For example, the solid support may be used to support a sample, such as a nucleic acid, in the methods described herein. The solid may be inert to the chemical reaction used to attach the solid to the gel. Supports include, but are not limited to, glass and modified or functionalized glass, plus Plastics (acrylic resin, polystyrene and styrene and other materials, polypropylene, Polyethylene, Polybutylene, Polyurethane, Teflon®, Cyclic Olefins , copolymers with polyimide, etc.), nylon, ceramic, resin, Zeonoa, Silica or silica-based materials such as silicon and modified silicon, carbon, metals, inorganic glasses , fiber optic bundles, and polymers. The support is placed within a flow cell apparatus. A representative flow cell is described in more detail below. Describe.

[0040] As used herein, the term "well" refers to a surface opening that is completely surrounded by the interstitial area of ​​the surface. A well refers to a discrete recessed feature in a solid support having an opening in a surface. The portion may have any of a variety of shapes, including but not limited to, circular, elliptical, or the like. These include circles, squares, polygons, stars (with any number of vertices), etc. The cross section of the well may be curved, square, polygonal, hyperbolic, conical, angular, etc. good.

[0041] The embodiments described and claimed below are to be understood in light of the above definitions. It is possible.

[0042] The present disclosure provides a substrate comprising a solid support having a surface, the surface comprising at least at least one recessed feature, the at least one recessed feature being made of a gel material and the at least one recessed feature comprises at least one interstitial region on the surface. The present disclosure also provides a library of analytes in a gel material, The gel material in the tube contains a single species of analyte from the library.

[0043] In some embodiments, the substrate is configured as an array of wells and the analytes are nucleic acids. In response to the above, the present disclosure provides an array comprising a solid support having a surface, the surface comprising a plurality of The well has a gel material therein, the gel material being spaced apart by interstitial areas on a surface of the well. The gap regions separate the gel material in each well from the gel material in the other wells. The present disclosure provides a library of target nucleic acids in a gel material. However, the gel material in each well contains a single species of target nucleic acid of the library.

[0044] The solid supports used in the structured substrates described herein can be any of the solid supports described herein, e.g. , made from any of the various materials defined above, in the examples below, or immediately below. A particularly useful material is glass. Other suitable substrate materials are polymeric materials, plastics, Plastic, silicon, quartz (fused silica), borofloat glass, silica, silica-based materials Materials, carbon, metal, optical fiber or optical fiber bundle, sapphire, or COC and The material may include plastic materials such as epoxy resins. The material can be selected based on the desired properties. For example, a material that is transparent to radiation of the desired wavelength can be used. The material can be subjected to an analytical method utilizing radiation of a desired wavelength, such as one or more of the techniques described herein. Conversely, materials that do not transmit certain wavelengths of radiation (e.g., are opaque, absorbing, or reflective) are It may be desirable to select a material that is reflective. or for use in the manufacture of a structured substrate, such as those described herein. The present invention may be useful in forming masks for use in chemical reactions or analytical detection carried out using the same. Other properties of the materials that can be exploited include those used in downstream processes, such as those described herein. Inertness or reactivity to certain reagents used or those described herein The structured substrate or method of the present disclosure may be advantageous in that it is easy to operate or inexpensive to fabricate. Further examples of materials that can be used in the method include those disclosed in US Pat. US Patent Application No. 13 / 661524 and US Patent Application No. 2012 / 0316086 It is written.

[0045] In certain embodiments, sol-gel based substrates can be prepared and used. -Gel-based patterning can be done by, for example, spin-coating, dipping or spraying. The adhesive coating can be applied to rigid or flexible substrates such as glass, silicon, plastics and metals. This can be achieved by coating a flexible substrate with a sol-gel coating. The gel may be provided in a liquid state when applied to a substrate, and the sol-gel may be exposed to light or heat to form a A photoinitiator or After coating the substrate with the sol-gel, the substrate may contain either a thermal initiator or a thermal initiator. The sol-gel is then coated with a substrate having single or multiple raised features prior to hardening the material. The mold can be made of, for example, silicon, Glass (e.g. quartz), metal (e.g. nickel), plastic or polymer (e.g. PDMS) The stamping of the sol-gel can be performed by placing the template on the sol-gel. This can be achieved by placing the template in contact with the sol-gel. The sol-gel redistributes and conformally surrounds the template structures. and by external forces applied to the template or substrate, or inherent to the nature of the patterned template. Capillary forces can facilitate the redistribution of the sol-gel. When the template comes into contact with the sol-gel, The substrate + sol-gel + mold stack is exposed to light or heat to cure the sol-gel, transforming the original The pattern in the mold can be fixed in the sol-gel. This pattern forming method is This technique is called nanoimprint lithography. After the sol-gel hardens, the mold is then applied to the substrate. + The mold can be separated from the sol-gel stack and discarded, but the uncured sol-gel can be removed. The gel-coated substrate may be reused for patterning. The substrate with the patterned sol-gel can then be subjected to chemical vapor deposition, or If a pure glass-like surface is desired, the substrate + patterned sol-gel stack can be replaced by a sol-gel. It can be subjected to a thermal process (sintering) that removes the organic material originally present in the gel. This is not a requirement, but the substrate may be pure S, which may be beneficial in certain chemical addition schemes. It can be made into iO2 material.

[0046] Another approach to fabricate patterned substrates is COC or COP (Zeonor or To A plastic material such as pas is used and hot embossing is performed to create an indentation array. The method is similar to nanoimprint lithography. The plastic substrate may then be mounted on a temperature-controlled chuck. The substrate can be heated to a temperature above the glass transition temperature of the plastic skin. While the substrate is at high temperature, the mold (e.g., quartz, silicon, polymeric, or metallic) is The mold is typically formed by coating the plastic conformally with the structured mold. During contact at high temperature, an external force is applied to ensure that the plastic The chromosome redistributes itself and becomes a negative replica of the template, e.g., when the template is post-arrayed. When the mold is pressed against the plastic, the embossed plastic becomes an array of wells. During contact with the substrate, the temperature of the substrate is reduced, so that the embossed pattern adheres to the substrate. is fixed inside.

[0047] The recessed features on the substrate can have any of a variety of shapes. In terms of the shape on the substrate, a feature may have curved sides, linear sides, corners, or a combination of these. For example, features may be circular, oval, square, polygonal, or any combination of shapes. A wafer having openings on its surface that are shaped like a star (with any number of vertices) or irregularly shaped. The features may be channels, and the channels on the surface may be The shape can include sides that are curved, linear, angular, or a combination thereof. Other channel features include lines, snakes, rectangles, squares, triangles, circles, and ellipses. The channel can be circular, hyperbolic, or a combination of these. A channel can connect two points on a surface. 3B and 3C show a cross-sectional view of a substrate, one or both of which may be at the edge of the substrate. Representative wells in and around the bullseye are included. Indicates the channel feature.

[0048] The cross-sectional shape of a concave feature viewed perpendicular to the surface can be curved, linear, or a combination of both. The cross-sectional shape may therefore be circular or elliptical. It may be part of a shape (e.g., U-shaped) or it may have two or more linear sides that meet at a corner ( For example, the cross-sectional shape may be V-shaped, square, polygonal, or star-shaped. From this point, the base of the concave feature may be narrower or wider, and the opening on the surface may be The cross-sectional shapes of these may be approximately the same as the recessed features being wells, When the well has a cylindrical cross section, the opening at the surface is generally the same area as the bottom of the well, On the other hand, when the well has a conical cross section, the bottom of the well has a different area than the opening on the surface. Of course, the cross section is I have explained this in terms of shells, but it could also apply to channels.

[0049] In embodiments in which the recessed features form wells, each well may be capable of containing a liquid. The minimum or maximum volume may be, for example, The throughput (e.g., multiplexing), resolution, sample composition, or sample reaction characteristics that are suitable for downstream use For example, the volume may be selected to correspond to the responsiveness of the -3 μm 3 , 1×10 -2 μm 3 , 0.1 μm 3 , 1 μm 3 , 10μm 3 , 100μm 3 End Alternatively, or in addition, the volume may be up to 1×10 4 μm 3 , 1×10 3 μm3 , 100μm 3 , 10μm 3 , 1 μm 3 , 0.1 μm 3 The gel material may be It should be understood that the entire volume or a portion of the wells may be filled. The volume of gel in the well may be greater or less than the values ​​specified above, or may vary from these values. It may be between values.

[0050] The area occupied by each well opening on the surface is determined by the same criteria as above for well volume. For example, the area of ​​each well opening on the surface may be selected based on at least 1× 10 -3 μm 2 , 1×10 -2 μm 2 , 0.1 μm 2 , 1 μm 2 , 10μm 2 , 100μ m 2 Alternatively, or in addition, the area may be up to 1×10 3 μm 2 , 1 00μm 2 , 10μm 2 , 1 μm 2 , 0.1 μm 2 , 1×10 -2 μm 2 The following is also fine. The depth of each well may be at least 0.1 μm, 1 μm, 10 μm, 100 μm or more. Alternatively, or in addition, this depth may be up to 1×10 3 μm, 100μm, 10μ m, 1 μm, or 0.1 μm or less.

[0051] Many different patterns are available, including regular, repeating, and irregular patterns. Any layout of wells or other recessed features may be envisaged. For example, wells may be close-packed. They may be arranged in a hexagonal grid for improved packing and density. Other layouts include, e.g. For example, there are rectilinear (i.e. rectangular) layouts, triangular layouts, etc. The differences between a particular layout and different layouts in different domains (if used) are No. 7,813,013 and / or U.S. Pat. No. 6,213,633, which are incorporated herein by reference. The various crystal patterns or Either the crystal or amorphous patterns can be useful.

[0052] The well pattern is determined in terms of the average pitch (i.e. center-to-center spacing) of the wells. The pattern is regulated so that the coefficient of variation around the average pitch is small. The pattern may be regular, or the pattern may be irregular with a relatively large coefficient of variation. In either case, the average pitch is, for example, at least 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 100 μm or more. or additionally, the average pitch may be, for example, up to 100 μm, 10 μm, 5 μm, 1 Of course, the specific pattern of wells may be smaller than 0.1 μm, 0.5 μm, or even smaller than 0.1 μm. The average pitch may be between one of the minimum and one of the maximum values ​​selected from the ranges above. good.

[0053] The well pattern also determines the density of wells (i.e., the number of wells) within a defined area. For example, wells may be characterized on the basis of approximately 2 million / mm 2 It exists at a density of According to the manufacturing methods described herein, the density may be, for example, at least 100 / mm 2 , 1000 / mm 2, 100000 / mm 2 , 1000000 / mm 2 , 2000 000 / mm 2 , 5000000 / mm 2 Easily adjustable to different densities, such as 100% or more Alternatively, or in addition, the density is 5,000,000 / mm 2 Below, 2000 000 / mm 2 , 1000000 / mm 2 , 100000 / mm 2 , 1000 / mm 2 , 100 / mm 2 Of course, the density of wells on the substrate may be adjusted to within the above range. It may be between one of the minimum values ​​and one of the maximum values ​​selected from the range.

[0054] In certain embodiments, a gel material is used. In some cases, a gel-forming (e.g., polymerizable) The material is applied to the solid support in a liquid state and then converted into a gel. Examples of polymerizable materials include Examples of suitable amides include, but are not limited to, acrylamide, methacrylamide, hydroxyethyl methacrylate, and the like. acrylate, N-vinylpyrrolidinone or derivatives thereof. The polymerizable material is useful in preparing hydrogels. The compound may contain two or more different species of compounds that form a polymer. For example, amide, methacrylamide, hydroxyethyl methacrylate, N-vinylpyrrolidinone or or two or more different species of these derivatives can be polymerized to form copolymer hydrogels. Useful hydrogels include, but are not limited to, However, silane-free acrylamide (SFA) polymers (incorporated herein by reference) are also available. See U.S. Patent Application Publication No. 2011 / 0059865, which is incorporated herein by reference), poly(N-(5-azide) (PAZAM, see) acrylamide-co-acrylamide See U.S. Provisional Patent Application No. 61 / 753,833, incorporated herein by reference), e.g. , acrylamides as described in WO00 / 31148, which is incorporated herein by reference. and polyacrylic acid formed from acrylic acid or acrylic acid containing vinyl groups. mide polymers, e.g. those described in WO01 / 01143 or WO03 / 014392 (respectively and the like, which is incorporated herein by reference, to form a [2+2] photocycloaddition reaction. Polyacrylamide polymers formed from monomers such as those described in U.S. Pat. No. 6,465,177. No. 8, WO01 / 62982 or WO00 / 53812 (each of which is incorporated herein by reference). These gel materials include the polyacrylamide copolymers described in React with a chemically engineered variant of, for example, an oligonucleotide having a corresponding reactive group. Chemically treated SFA (e.g., 5'- or 3'-alkynyl-modified oligonucleotides) Azidolysis of SFA to produce azido-SFA which reacts with azide is also useful. Suitable hydrogels and polymerizable materials that can be used to form hydrogels include, for example, No. 61 / 753,833 or U.S. Pat. No. 6,493,633, each of which is incorporated herein by reference. Other useful gels are described in US Patent Application Publication No. 2011 / 0059865. , which is formed by a temperature-dependent change from a liquid to a gelatinous state. Examples of suitable substrates include, but are not limited to, agar, agarose, or gelatin.

[0055] The gel material in the wells or other recessed features on the surface of the structured substrate is For example, PAZAM can be used as a surface material and is incorporated herein by reference. No. 61 / 753,833, incorporated herein by reference, and described in the Examples section herein. However, the gel material is not covalently bonded to the surface using other reagents such as cellulose acetate, cellulose acetate phosphate ... As demonstrated for the SFA in the Examples section, be sure to include a well or other concave feature. It is not necessarily covalently bonded.

[0056] One or more analytes are present in or on a gel material that is present on a structured substrate. The gel-containing substrates of the present disclosure are particularly useful for detecting analytes or for performing synthetic reactions with analytes. Therefore, any of a variety of analytes to be detected, characterized, modified, synthesized, etc. , may be present in or on the gel material of the substrate described herein. Suitable analytes include, but are not limited to, nucleic acids (e.g., DNA, RNA, or similar). body), proteins, polysaccharides, cells, antibodies, epitopes, receptors, ligands, enzymes (e.g. These include enzymes, such as kinases, phosphatases, or polymerases, and small molecule drug candidates. The structured substrate can include a plurality of different species from a library of analytes. For example, The species may be a variety of antibodies from an antibody library, a variety of antibodies with different sequences from a library of nucleic acids, Proteins with different structures and / or functions from a library of nucleic acids and proteins The therapeutic candidates may be drug candidates from combinatorial libraries of proteins, small molecules, and the like.

[0057] In some embodiments, the analytes are dispensed onto a structured substrate such that they can be individually dissolved. For example, a single molecule of each analyte can be present in each gel-containing well of a structured substrate. Alternatively, the specimen may be present as a colony or population, such that individual Molecules are not necessarily dissolved. Colonies or populations contain only a single species (albeit multiple copies). For example, each nucleic acid on the structured substrate can be uniform in content of the analyte. The wells can contain colonies or populations of nucleic acids, and all of the nucleic acids in the colonies or populations can be All of the nucleic acids can have the same nucleotide sequence (either single-stranded or double-stranded). Such colonies are subjected to cluster amplification or amplification procedures as described in more detail elsewhere herein. Multiple repeats of a target sequence, such as rolling circle amplification, can be generated by bridge amplification. Concatemers generated using multiple amplification techniques can exist in a single nucleic acid molecule. Thus, the gel material in each well on the structured substrate may contain multiple copies of a single analyte. Alternatively, the colonies or populations of specimens in a well may contain two or more different species. For example, one or more wells on a structured substrate can each contain two or more different nucleic acids. A colony may contain mixed species (i.e., nucleic acid molecules with different sequences). Two or more nucleic acid species in a mixed colony may be present in non-negligible amounts, e.g. This allows multiple nucleic acids to be detected in a colony.

[0058] The analyte may be attached to the gel material, either covalently or non-covalently. Representative methods and reactants for attaching nucleic acids to gels are, for example, No. 2011 / 0059865, also incorporated herein by reference. The method is described in U.S. Provisional Patent Application No. 61 / 753,833. The analyte may be a nucleic acid. Instead, a nucleic acid may be linked via its 3' oxygen, 5' oxygen, or any other position along its length. For example, the base portion of the 3' terminal nucleotide, the base portion of the 5' nucleotide, and / or It can be attached to the gel via one or more base moieties elsewhere in the molecule. Covalent attachment can be achieved, for example, by ionic interactions between the nucleic acid and the gel, by the ionic interactions between the nucleic acid and the gel inside the pores of the gel, and by the ionic interactions between the nucleic acid and the gel inside the pores of the gel. Uptake, protein-protein interactions, receptors and ligands on gels and / or nucleic acids This includes bonds between bonds between adjacent bonds, as well as other known patterns.

[0059] In some embodiments, the gel coating applied to the surface contains one or more analytes; The gel material is then removed from the gap area. The gel material in the interstitial regions can adhere to one or more different analytes. adding an analyte to the gel material in the recessed features after removing the gel material from the gap areas. .

[0060] The structured substrates of the present disclosure can be incorporated into flow cells. Exemplary Flow Cells and Methods of Manufacturing Them The method and methods of use thereof are described in U.S. Pat. No. 6,393,341, each of which is incorporated herein by reference. No. 2010 / 0111768 or No. 2012 / 0270305, or WO05 The flow cell is described in US Pat. No. 4,954,434. A convenient format for storing the sigma-based ... Other techniques that require repeated delivery of reagents (e.g., repeated or cyclic steps) Representative detection methods are described in more detail below. do.

[0061] In some embodiments, a flow cell or other vessel with multiple surfaces is used. Having multiple surfaces, where only one surface has gel-containing recessed features (e.g., wells) Alternatively, a container having two or more surfaces with gel-containing concave fillers may be used. One or more surfaces of the flow cell may be selectively detected. For example, opposing surfaces inside the flow cell can be imaged using techniques known in the art, such as confocal imaging. Using the above method, the target can be selectively addressed by focused radiation. and devices for selectively directing radiation to multiple surfaces of a vessel (e.g., a flow cell), e.g. U.S. Patent Application Publication No. 2009 / 027291, each of which is incorporated herein by reference. 4 or U.S. Pat. No. 8,039,817.

[0062] The present disclosure provides a method of making a substrate, the method comprising: (a) providing a solid support having a planar surface; providing a body, the plane being interrupted by one or more concave features; the one or more concave features being adjacent to one or more gap regions on a planar surface; b) coating at least a portion of the solid support with a gel material, the portion includes at least one recessed feature and at least one gap area. and (c) polishing the planar surface to remove the gel material from at least one of the gap regions. and retaining a gel material in one of the recessed features.

[0063] The substrate is then etched into the recessed features using any of a variety of techniques known in the art. In many embodiments, the recessed features can be fabricated to have nanometer-sized It will be miniature, on the order of meters or micrometers in size. In such cases, nano- or micro-fabrication techniques can be used. Examples of these techniques are: Further exemplary nanofabrication methods are described elsewhere herein, such as in Example 2 of the same application. and microfabrication techniques are described in U.S. Pat. Nos. 5,393,321, 5,393, 5,393, 6,450, 6,5 ... No. 13 / 661524 and U.S. Patent Application Publication No. 2012 / 0316086. It has been done.

[0064] One or more recessed features, e.g., wells, may be formed by depositing preformed gel material and / or The gel can then be coated with a liquid to produce a gel material. Spin-coating, dipping, flowing gel under positive or negative pressure, or by reference The technique described in U.S. Provisional Patent Application No. 61 / 753,833, which is incorporated herein by reference, was used. , coating of the substrate with preformed PAZAM. Coating of an array of wells with PAZAM is demonstrated in Example 3 below. As an example of the application of a liquid that forms a gel material in a liquid state, a silane-free acrylamide and N-[5-(2-bromoacetyl)aminopentyl]acrylamide (BRAP A) Coating of the well array with a reagent by polymerizing the reagent on the surface. Coating of the array in this manner is shown in Example 1 below. and U.S. Patent Application Publication No. 2011 / 0059865, which is incorporated herein by reference. In some embodiments, the chemical reagents and techniques described in, for example, When the well-containing substrate is immersed in the preformed gel material, the gel material selectively fills the wells. This eliminates the need for polishing.

[0065] The sample may be added to the gel material either before or after contact with the solid support. Adding the sample to the gel (i.e., after the gel has formed from its precursor reagents) does not allow the sample to be transferred to the gel. It may be added to the forming reagent solution (i.e., before gel formation). Various analytes may be added before gel formation and others after gel formation. A primer nucleic acid is added to the gel-forming solution, and the solution is then allowed to gel (e.g., SF A and PAZAM). Gel formation may occur on a solid support. Alternatively, the gel may be preformed and then coated onto the solid support. In this method, the primers are attached to the gel present in recessed features such as wells. A target nucleic acid complementary to the primer is then added to the primer-containing gel, As a result, after the gel material is coated onto the solid support, The target nucleic acid can be attached to the gel via hybridization of the target nucleic acid. may optionally occur after a polishing step has been performed (polishing is described below). The preceding examples demonstrate the ability to deposit nucleic acids (plasma) at different stages in the manufacture of the structured substrate. We describe several cases in which a stimulant (which acts as either an inhibitor or a target) is added to the gel. is.

[0066] In some embodiments, the gel is attached to (or otherwise in or on) the gel. A primer nucleic acid (present in the nucleic acid sequence) is used for capturing and / or amplifying a template nucleic acid. The primers are universal primers that attach to the various target nucleic acids in the library. It may also be a universal primer that hybridizes to the adapter sequence (i.e. Each target nucleic acid comprises a target region that is distinct from other target nucleic acids in the library, Some target nucleic acids in the sequence have the same universal adapter sequence. In this embodiment, the target nucleic acid can be attached to a gel material and the primer (either in solution or (either attached to a gel) can be used to amplify the attached target nucleic acid. (ie, the target nucleic acid is capable of serving as a template for amplification).

[0067] The methods described herein may use any of a variety of amplification techniques. Representative techniques that may be used include: Suitable techniques include, but are not limited to, polymerase chain reaction (PCR), rolling Random prime amplification (RCA), multiple displacement amplification (MDA), or random prime amplification (R In certain embodiments, one or more of the primers used for amplification can be genomic DNA (e.g., DNA fragments). In PCR embodiments, one or both of the polymeric materials used for amplification can be attached to the polymeric material. Either primer can be attached to the gel material. Two types of adhesion primers are used. The format is often called bridge amplification because it uses a double-stranded amplicon. The conjugate forms a bridge-like structure between the two attached primers flanking the copied template sequence. Representative reagents and conditions that can be used for bridge amplification include, for example, No. 5,641,658, U.S. Pat. No. 5,641,658, each of which is incorporated herein by reference. US Patent Publication No. 2002 / 0055100, US Patent No. 7115400, US Patent Publication No. 4 / 0096853, U.S. Patent Publication No. 2004 / 0002090, U.S. Patent Publication No. No. 2007 / 0128624, and U.S. Patent Publication No. 2008 / 0009420. PCR amplification is carried out using amplification primers attached to the gel material and a second primer in solution. It is also possible to carry out the procedure using one of the solid-phase attached primer and the liquid-phase primer. Representative formats using combinations with timers are, for example, Dressman et al., Proc. Natl. Acad. Sci., incorporated herein by reference. USA100:8817-8822(2003), WO05 / 010145, or US Described in Patent Publication No. 2005 / 0130173 or No. 2005 / 0064460 Emulsion PCR is an example of a format and is For purposes of the methods described herein, the use of emulsions is optional and in practice It should be understood that in some embodiments, no emulsion is used. The primers do not need to be attached directly to a solid support as described in the ePCR reference. Rather, it can be attached to the gel materials described herein. In the ridge amplification format, the target nucleic acid is allowed to attach to the gel material and serve as a template for amplification. It is possible.

[0068] The RCA technique may be modified for use in the methods of the present disclosure. Representative components that can be used in the RCA reaction and principles for generating ricons include: See, for example, Lizardi et al., Nat. Gen. et. al. 19:225-232 (1998) and U.S. Patent Application Publication No. 2007 / 0099 The primer used in RCA is a gel material even in solution. It may be attached to the material.

[0069] MDA techniques may be modified for use in the methods of the present disclosure. Some basic principles and useful conditions are described in, for example, each of which is incorporated herein by reference. Dean et al., Proc Natl. Acad. Sci. USA 99:5261-66 (2002); Lage et al., Genome Research 13:294-307( 2003);Walker et al., Molecular Methods for Viru s Detection, Academic Press, Inc., 1995; Wal ker et al., Nucl. Acids Res. 20:1691-96 (1992); The patents listed in Patent Nos. 5455166, 5130238, and 6214587 are The primer used for MDA can be in solution or attached to the gel material. stomach.

[0070] In certain embodiments, a combination of the above exemplified amplification methods may be used. For example, RCA and MDA may be used in combination, and RCA may be used in combination with other processes (e.g., liquid phase processes). This is used to generate concatemeric amplicons in solution (using primers). The amplicons are then templated for MDA using primers attached to the gel material. In this example, the RCA and MDA steps are combined. The amplicons generated after the amplification will adhere to the gel material. Generally, it will contain concatemeric repeats of the target nucleotide sequence.

[0071] The amplification methods exemplified above produce gel-containing features with multiple copies of the target nucleic acid. Individual features such as wells can be created by RCA. in the form of single molecule concatemers such as those generated by A collection of nucleotide sequences in the form of multiple nucleic acid molecules with the same sequence, such as those Generally, a nucleic acid containing several copies of an amplified target is It will adhere to the material.

[0072] In some applications, the individual gel-containing wells (or other recessed features) are is occupied by amplicons derived from a first target nucleic acid and is occupied by amplicons derived from a second target nucleic acid or Contaminating amplicons from naturally occurring mutations can be present at low levels. One or more amplification sites may have a contaminating amplicon of different sizes, which This can have an unacceptable impact on subsequent use of the array. For example, For use in such applications, the acceptable level of contamination must be within the signal-to-noise or resolution limits of the detection technique. This is considered a level that will not shock the user in an unacceptable way. Loanability generally refers to a particular use or utility of an array produced by the methods described herein. Individual wells or other features for specific applications. Typical acceptable contamination levels include, but are not limited to, 0.1%, 0.5% maximum. , 1%, 5%, 10% or 25% contaminating amplicons. The arrays were Contain one or more wells or other features with a contaminating level of amplicons. For example, up to 5%, 10%, 25%, 50%, 75%, or 1% of the array can be 00% of the features may have some contaminating amplicons.

[0073] The gel material coated on the surface of the solid support may be covalently attached to the support. As described above, the step of attaching analytes, such as nucleic acids, to a gel material can be performed by using various different types of gels on a structured substrate. Thus, the method can be carried out at any stage of manufacture, before or after the specimen is attached to the gel material. The gel material can then be attached to the solid support. Attachment can be by any useful chemical reaction, including but not limited to, any of the methods described herein that are incorporated by reference. No. 61 / 753,833, which is incorporated herein by reference, or as demonstrated in Example 3 below. Covalent attachment of the gel material to the solid support can be carried out using methods known in the art. It should be understood that this is not necessarily the case in all embodiments. Polishing the subsequent gel-coated support or using the polished substrate. Optionally, but not required, the groove may be shared by a concave feature such as a well. It can be performed on a substrate having a gel material attached thereto.

[0074] The methods described herein may include removing the gel material from the surface of the solid support. The gel material that is coated onto the solid support can be prepared using any of a variety of techniques. For example, the gel material can be selectively removed from the gap regions using mechanical The recessed features and interstitial areas can be removed from the solid support by polishing. Mechanical polishing can be carried out by applying an abrasive force to the surface of the solid support. Typical methods include polishing with a slurry of beads, wiping with a sheet or cloth, and rubbing. An example of polishing is filtration with 3 μm silica bead slurry (10% w / v in water). Remove the gel between the lattices using a coated, lint-free (cleanroom grade) wipe. Use a polishing wheel / grinder with this slurry to remove Mechanical polishing can also be accomplished using a fluid or air jet, Remove the gel from the area.

[0075] Polishing is based on hydrolysis or acrylamide groups (e.g., Kuren kov et al., Russian Journal of Applied Chemistry y, 75:1039~1050 (2002); Caulfield et al. Polym.44 :1331-1337 (2003); and Caulfield et al., Chem. Rev. 102:3067-3083 (2002) Chemical polishing, such as through exposure to oxygen, may be required.

[0076] Polishing may also involve a combination of chemical and mechanical polishing techniques, which reduces particle dispersion. The material is mechanically stripped using a chemical slurry containing a suspension of iodides, and then removed from the gap area. Other methods of abrading or cleaning the gap areas include: Adhesive-based techniques, for example, coating a rigid, flat adhesive film onto the surface that is compatible with the gel material; This includes techniques for intimately contacting the gel material in the interstitial regions (e.g., via chemical bonding). Mechanical removal / peeling of this adhesive film mechanically removes the gel material from the gap area. but will at the same time leave gel material in the recessed features.

[0077] In another example, thiophosphate-grafted SFA can be grafted from the interstitial regions on the surface as follows: The aluminum oxide (approximately 100 ml) can be removed by wiping a Whatman wipe moistened with water. g, 0.3 um) or steel beads. Then, using uniform pressure, The resulting slurry may then be rubbed into small concentric circles on the surface of the solid support. Use a Whatman wipe dampened with clean water at 25°C to remove any slurry on the surface. The mechanical and chemical methods exemplified herein for removing the gel material from the gap regions can be used. A selective polishing method is used to remove gel material in the gap area, whether or not the gel material has been removed. For example, the gel material may be inactivated for its ability to adhere to analytes such as nucleic acids. Alternatively, the nucleic acid may be inactivated for its ability to facilitate amplification of nucleic acids.

[0078] A method for making an array includes: (a) providing a solid support having a surface with a plurality of wells; The wells contain a gel material, the wells being separated from one another by interstitial areas on a surface thereof. The interstitial regions isolate the gel material in each well from the gel material in the other wells. and (b) delivering a library of target nucleic acids to the wells of the solid support and gelling the target nucleic acids in each well. creating an array of wells having a single type of target nucleic acid attached to a substrate material; whereby different wells in the array have different target nucleic acid species from the library. and (c) amplifying target nucleic acids attached to the gel material in the wells of the array to generate a nucleic acid sequence for each well of the array. and generating clonal populations of individual target nucleic acids in the wells.

[0079] In some embodiments, the structured substrates described herein are prepared by subjecting a mixture to a number of different The advantage of constructing an array is that the analytes can be conveniently delivered to individual locations on the substrate. The structured substrate defines each individual gel-containing well (or other recessed feature). (i) facilitating the selective capture of a single analyte from a mixture of analytes in contact with the substrate. Patterning and filling gel-containing wells (or other recessed features) on a structured substrate Efficiency is based on the analyte density and the number of features that have a single analyte. The purity of the resulting array can be adjusted to obtain desired characteristics, such as high density. Wells with high density are used to obtain a high density of analytes on the array, whereas wells with low density are used to obtain a high density of analytes on the array. is used to obtain a low density of analytes on the array. The concentration or amount of analyte in the liquid may be increased to obtain a high density of analyte on the array, or decreased. Each gel-containing well (or other recessed field) may be filled with a small amount of analyte to obtain a low density of analytes on the array. The average purity of the samples (char) is described in more detail below and demonstrated in the Examples section. The properties of the substrate or the conditions for analyte delivery can be adjusted. Cut.

[0080] In certain embodiments, the size or volume of the well (or other recessed feature) is adjusted. For example, wells can be adjusted to accommodate a particular type of 3. The gel material may have an area or volume that accommodates only a single analyte of interest, and thus Prevent multiple analyte molecules from being captured by steric exclusion or from seeding the wells. Steric exclusion can be particularly useful for large analytes such as nucleic acids. More specifically, The wells (or other recessed features) are each a portion of the excluded volume of the target nucleic acid to be seeded onto the substrate. There can be a gel surface having an area equal to or less than the diameter of the target nucleic acid. The diameter can be determined, for example, from the length of the target nucleic acid. Methods for determining the diameter of the excluded volume can be found, for example, in US Pat. No. 7785790; Rybenkov et al., Proc. Natl. Acad. Sc iUSA 90:5307-5311(1993);Zimmerman et al., J. Mol. Biol. 222:599-620 (1991); or Sobel et al., Bio Polymers 31: 1559-1564 (1991). The conditions are described in U.S. Patent Application No. 13 / 661,524 and incorporated herein by reference. No. 7,785,790 and can be readily used with the structured substrates of the present disclosure. .

[0081] In some embodiments, the well (or other recessed feature) is transported to an amplification location. It is possible to provide a gel surface having an area substantially larger than the diameter of the excluded volume of the target nucleic acid. It should be understood that the area of ​​the features should therefore be sufficient so that steric exclusion does not occur. There is a great possibility that

[0082] In some embodiments, such as the steric exclusion embodiments described above, a library of target nucleic acids The gel-containing wells (or other recessed features) of the solid support are filled with the nuclei before the amplification process begins. For example, the target nucleic acid may be delivered to a gel material in a substrate under conditions in which the target nucleic acid is seeded. The substrate can be optionally washed to remove the gel. The target nucleic acid that is not seeded onto the substrate as well as any other material that is not required for subsequent steps or uses of the substrate. Amplification may include one or more of the techniques described herein above. can.

[0083] In an alternative embodiment, the library of target nucleic acids is placed in a gel-containing well (or other concave features) and the amplification process can occur simultaneously with the seeding event. For example, seeding can be carried out using methods such as those described in U.S. Patent Application Serial No. 61 / 71, which is incorporated herein by reference. Utilizing the kinetic exclusion described in No. 5478 Kinetic exclusion can occur in a variety of ways. This can occur when the action occurs at a rate fast enough to cause a reaction to occur. In the case of an array of gel-containing wells The wells are randomly seeded with target nucleic acids from a solution, and copies of the target nucleic acid are generated by amplification. Conditions under which the amplification rate exceeds the seeding rate The seeding and amplification steps can be carried out simultaneously under the same conditions. At the location where the target nucleic acid was seeded, copies are made at a relatively high rate, thereby forming a second nucleic acid. kinetic exclusion effectively blocks subsequent target The first copy of the target nucleic acid is generated relative to the relatively fast rate of making the copy or first copy of the nucleic acid. For example, kinetic exclusion can be used to exploit the relatively slow rate at which a gene can make copies of itself. , the relatively fast rate at which subsequent copies are made to fill that location, and the gel-containing well A delay (e.g., a delay or slow activity) in the formation of the first copy of the target nucleic acid seeded on the target In this example, each gel-containing well can contain a number of different targets. The target nucleic acid may be seeded with a target nucleic acid (e.g., a number of target nucleic acids may be However, any target nucleic acid can be present at any position in the first copy. Copy formation can be activated randomly, so that the average rate of first copy formation is faster than that of subsequent copies. In this case, the individual gel-containing wells are filled with Several different target nucleic acids could be seeded, but kinetic exclusion would allow the cytoplasmic separation of these target nucleic acids. Typically, the gel-containing well (or other recessed feature) is No. 61 / 715,478, which is incorporated herein by reference. These can serve as sites of amplification and array construction in the

[0084] As an alternative to delivering multiple different analytes from a mixture to individual gel-containing recessed features Analytes may be delivered discretely to individual features from pure sources. Analytes may be synthesized at individual features by discrete delivery of constituent elements (e.g., nucleases). (The nucleic acid can be synthesized by continuously delivering nucleotide precursors.) Exemplary delivery methods of building blocks for in situ synthesis of analytes include, but are not limited to, Although there are no known methods, inkjet array positioning and photolithographic array synthesis are proposed. A useful photolithography method is the GeneChip® microarray. The assay was performed using a system developed by Affymetrix (Santa Clare, CA) to manufacture the ELISA kit. or any other related art which is incorporated herein by reference. U.S. Patent Nos. 5,324,633, 5,744,305, 5,624,711, and 60 22963, 6291183, and 6416949. Also includes inkjet positioning methods, such as the SurePrint® algorithm. A commercially available 3D printer for printing 3D printers is available from Agilent (Santa Clara, CA). or U.S. Pat. No. 6,333,633, each of which is incorporated herein by reference. No. 37393, No. 6419883, No. 6420180 or No. 6689319 Such methods are also useful for the gel-containing features of the present disclosure. The antibodies can be easily modified to direct delivery to the target area.

[0085] The gel material in a particular recessed feature must contain only a single analyte Rather, in some embodiments, the recessed features are of several different types. For example, the bull's eye fiducial markers of FIG. The fiducial marker contains two "bright" ring-shaped channels. Each of the two channels contains a gel material, and the gel material in each light channel The dye adheres to multiple different nucleic acid colonies. The nucleic acid colonies in the bright channel are Each ring is seeded with several different species of target nucleic acid that serve as templates in an amplification procedure. The fiducial marker was also formed by two "dark" ring-shaped areas. The dark rings are made up of an interspaced surface pattern. A typical bull's eye is , formed by alternating dark and light rings in a concentric pattern. In five examples, the structured substrates typically contain clones each derived from a single nucleic acid target. The well also contains a gel-containing well that contains the population. The well is between the light and dark rings. The criteria are therefore an alternating pattern of gap rings, The wells contain bands and channel rings. Using the same amplification technique, A mixed population of clonal nucleic acid colonies and fiducial markers was grown simultaneously (see below). (See Example 3.) Another example of a fiducial having an alternating pattern of rings is shown in Figures 3B and 3C. vinegar.

[0086] The present disclosure further provides a method for detecting an analyte, the method comprising: (a) a solid support having a planar surface; Providing a body support, the plane being interrupted by one or more concave features. wherein the recessed features contain a gel material, and wherein one or more recessed features are disposed on a planar surface. adjacent to one or more gap regions, the gap regions being substantially free of gel material, and the gel material being substantially free of the target (b) attaching to or containing an analyte; and (b) detecting a target analyte specifically probed. (c) contacting the solid support with the probe under conditions that interact with the solid support; and detecting the analytes to identify at least a subset of the target analytes that interact with one or more of the probes. and separating the first and second electrodes from the second electrodes.

[0087] In certain embodiments, a nucleic acid is the analyte to be detected and the recessed feature is a well. For example, a method for detecting nucleic acids includes (a) detecting a solid support having a surface and a nucleic acid library; providing a body, the surface having a plurality of wells, the wells containing a gel material; the wells are separated from one another by interstitial areas on the surface, the interstitial areas defining a gel in each well. The material is isolated from the gel material of the other wells, and a single target nucleic acid in the library is isolated. (b) attaching the solid support to the gel material in each well; and (c) contacting the solid support with at least one probe; and (b) detecting the solid support. and identifying wells having target nucleic acid species that bind to one probe at a time. This can be done.

[0088] The structured substrates of the present disclosure containing nucleic acid arrays can be used for any of a variety of purposes. A particularly desirable use of the nucleic acid is to hybridize to a target nucleic acid having a complementary sequence. Once hybridized to the capture probe, The captured target nucleic acid can be detected, for example, by a label incorporated into the capture probe. Methods for detecting a target nucleic acid via hybridization to a capture probe are known in the art. No. 7,558,313, each of which is incorporated herein by reference. No. 2420, No. 6890741, No. 6913884 or No. 6355431 No. 2005 / 0053980 and U.S. Patent Application Publication No. 2009 / 018634. 9 or 2005 / 0181440. The capture probe is hybridized to a target probe carrying a label, thereby capturing the label. In another example, the target probe can be hybridized to the capture probe. by ligating to a labeled oligonucleotide ( by ligase activity) or by adding labeled nucleotides (e.g. by polymerase activity) to allow the capture probe to be extended, attaching the label to the capture probe. can be replenished.

[0089] Nucleic acid arrays can also be used in sequencing techniques, such as sequencing by synthesis (SBS). Briefly, SBS involves the cleavage of a target nucleic acid with one or more labeled nucleotides, DNA The target nucleic acid can be used as a template, for example by contacting the target nucleic acid with a polymerase. The feature where the primer is extended introduces a detectably labeled nucleotide. By selection, the labeled nucleotide may be a reversible terminator. The primer may further include a nucleotide addition feature, which is When the primer reaches a certain position, it terminates further primer extension. For example, a primer having a reversible termination portion A nucleotide analog is added to the primer, which delivers a deblocking agent to the moiety. Therefore, it is possible to prevent further elongation from occurring until the removal of the In embodiments utilizing this technique, a deblocking agent may be delivered to the flow cell (detection may be performed before Washing may be performed between the various delivery steps. The cycle is then repeated. Repeated times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Representative SBS techniques, fluidics systems, and detection platforms are described in the present paper. It can be readily adapted for use with arrays fabricated by the methods shown, e.g. See, for example, Bentley et al., Nature 45, each of which is incorporated herein by reference. 6:53~59(2008), WO04 / 018497, WO91 / 06678, WO0 7 / 123744, U.S. Patent No. 7,057,026, U.S. Patent No. 7,329,492, U.S. Patent No. 7,211 414, 7315019 or 7405281, and U.S. Patent Application Publication No. This is described in US Pat. No. 2008 / 0108082.

[0090] Other sequencing methods that use cycling reactions, such as pyrosequencing, Pyrosequencing is a method for determining when a specific nucleotide is introduced into a nascent nucleic acid strand. Detect the release of inorganic pyrophosphate (PPi) (see, e.g., US Pat. No. 6,393,621, each of which is incorporated herein by reference). Ronaghi et al., Analytical Biochemistry 242(1 ), 84~9(1996); Ronaghi, Genome Res.11(1), 3~ 11(2001);Ronaghi et al., Science 281(5375), 363( 1998); U.S. Patent Nos. 6,210,891; 6,258,568 and 6,2743 In pyrosequencing, the released PPi is converted to ATP sulfurylase. It can be detected by converting it to adenosine triphosphate (ATP) using can be detected via luciferase light emission. It can be monitored via a luminescence detection system. It can also be used for a fluorescence-based detection system. The excitation radiation source is not required for the pyrosequencing method. The apparatus and methods can be used to apply pyrosequencing to the arrays of the present disclosure. See, for example, WIPO patent applications PCT / US No. 11 / 57111, U.S. Patent Application Publication No. 2005 / 0191698, U.S. Pat. No. 595,883, and U.S. Pat. No. 7,244,559.

[0091] Decoding during ligation is also useful, see, for example, US Pat. No. 6,393,361, each of which is incorporated herein by reference. Shendure et al., Science 309:1728-1732 (2005); No. 5,599,675; and those described in U.S. Pat. No. 5,750,341. Some embodiments include, for example, Bai ns et al., Journal of Theoretical Biology 135(3 ), 303-7 (1988); Drmanac et al., Nature Biotechnol. ogy 16, 54-58 (1998); Fodor et al., Science 251 (499 5), 767-773 (1995); and the hybrid Decoding upon ligation and hybridization may be included. The gel-containing wells (or other recessed features) are used for both detection and reading. The nucleic acid present is subjected to repeated cycles of oligonucleotide delivery and detection. The fluid systems for the SBS methods described in the literature or cited herein are The method can be easily adapted for reagent delivery for either hybridization or hybridization readings. Typically, the oligonucleotides are fluorescently labeled and can be used in the methods described herein or in the present specification. The assay was performed using a fluorescence detector similar to that described for the SBS method in the literature cited in this document. It can be put out.

[0092] Some embodiments involve methods that require real-time monitoring of DNA polymerase activity. For example, the incorporation of nucleotides can be achieved using fluorophore-bearing polynucleotides. Fluorescence resonance energy transfer (FRE) between γ-phosphate-labeled nucleotides and T) by interaction or by zeromode waveguide techniques FRET-based sequencing techniques and reagents are described, for example, in Levene et al., Science 299, 682-686, incorporated herein by reference. (2003);Lundquist et al., Opt.Lett.33, 1026~1028( 2008); Korlach et al., Proc. Natl. Acad. Sci. USA105 , 1176-1181 (2008).

[0093] Some SBS embodiments include a method for determining the amount of photons emitted upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of emitted photons can be achieved using Ion Torr ent (Guilford, CT, a subsidiary of Life Technologies) No. 6,393,962, and each of which is incorporated herein by reference. US Patent Application Publication Nos. 2009 / 0026082 and 2009 / 01275 89; No. 2010 / 0137143; or No. 2010 / 0282617 The above described sequencing methods and systems may be used. In certain embodiments, the emitted photons The electrical detector used to detect the present invention may be modified to include a well. It may contain a gel material as described herein.

[0094] Another application for which the arrays of the present disclosure are useful is gene expression analysis. Detection or quantification using sequencing techniques, such as digital RNA sequencing RNA sequencing can be performed by any method known in the art. Gene expression can be measured by hybridization assays, for example, using the methods described above. Alternatively, the antibodies can be detected or quantified using a hybridization method, which involves hybridizing the antibodies directly to the array. or using multiplex assays, The products are detected on an array. The arrays of the present disclosure are comprised of arrays that are arrayed to measure genomic DNA from one or more individuals. It can also be used to determine the genotype of a sample. Representative methods for array-based expression and genotyping analysis that can be used are Nos. 7,582,420 and 6,890,741, which are incorporated herein by reference. No. 6,913,884 or 6,355,431, or U.S. Patent Application Publication No. 005 / 0053980, 2009 / 0186349 or 2005 / 0 It is described in No. 181440.

[0095] Some applications of the arrays of the present disclosure are exemplified above in the context of ensemble detection. However, here multiple copies of the target nucleic acid are present in each feature and are detected together. In an alternative embodiment, a single nucleic acid, either the target nucleic acid or an amplicon thereof, is included in each filter. For example, a gel-containing well (or other recessed feature) can be detected by a Constructed to contain a single nucleic acid molecule having a target nucleotide sequence to be detected. Any of a variety of single molecule detection techniques can be used, for example, high resolution The above amplification methods have been modified to detect the marker at elevated positions or to use more sensitive labels. Examples of other single molecule detection methods that can be used include modifications of the sample detection method. No. 2011 / 0312529, which is incorporated herein by reference. No. 61 / 578684 and U.S. Patent Application No. 61 / 540714. It has been done.

[0096] For example, the gel-containing substrate of the present disclosure produced by the methods described herein does not necessarily It should be understood that the structured substrate is not intended to be used in any detection method. Rather, the structured substrate is intended to be used in any detection method. The structured substrate can then be used to store the For example, the nucleic acid can be stored in a gel-containing well that adheres to the nucleic acid. The substrate may be dried, frozen (e.g., in liquid nitrogen), or in a solution that protects the nucleic acid. Alternatively, or in addition, the structured substrate may store the nucleic acid library in a For example, a gel containing wells that attach to nucleic acids can be used to replicate the The substrate may be used to generate replicate amplicons from one or more wells on the array. good.

[0097] The following examples are intended to illustrate, but not limit, the present invention. do not have. EXAMPLES

[0098] Silane-free acrylamide-coated multiwell substrate This example shows the coating of a nanowell substrate with silane-free acrylamide (SFA). The gel was then grafted with a thiophosphate primer. The mers were hybridized to complementary fluorescent oligonucleotides to verify the success of the functionalization method. This demonstrates that it is possible to

[0099] The chip substrates commonly used in the manufacture of BeadChips are manufactured by Illumina (Sa The chips were obtained from Sigma-Aldrich Co., Ltd. (St. Diego, Calif.). on Corp., Tokyo, Japan) arranged in a hexagonal pattern with a pitch of 1.5 μm. The wells had 0.5 μm wells, but did not contain beads. The chip was patterned with gel pads as described and shown in FIG.

[0100] The chip is placed into a gasket-sealed chamber and filled with fluidic reagents for SFA generation. Oxygen was removed by polymerization of SFA on the chip in the chamber. The reagents and polymerization conditions are described elsewhere in U.S. Pat. App. No. 6,393,341, which is incorporated herein by reference. The sealed chamber was then inserted into the chip to introduce the polymerization mixture. In direct contact with the polymer, the free radical polymerization of SFA is an air-insensitive process. Therefore, the air was thoroughly removed. After polymerization, U.S. Patent Application Publication No. 2011 / 0059 The primer is glued to the SFA polymer in a sealed chamber as described in 865 and below. The BeadChip was polymer-coated with a solution containing a primer. The mixture was then incubated at 65 °C for 1.25 h. (The entire sealed assembly was placed in a large oven).

[0101] This technique results in a uniformly coated substrate. A sample image is shown in panel A of Fig. 2. In order to create discontinuous polymer regions, the “ The excess polymer was dissolved in a slurry of aluminum oxide nanoparticles (diameter 300 nm) in distilled water. The surface was removed by mechanical polishing using a 10 wt% slurry of 3 micron silica particles. Lee (Kisker Biotech GmbH, Steinfurt, Germany) also used You can manually rub the surface with the nanoparticle slurry using a lint-free optical tissue. After washing to remove slurry and polymer debris, the fluorescently labeled probe was The solution was hybridized to the chip. Images taken using a fluorescent microscope showed that this technique The gap regions showed that empty polymer features could be created (Figure 2, panels B-C). .

[0102] These results indicate that the fluorescence intensity of the gel-filled wells is significantly higher than that of the gel-filled wells, with no signal coming from the interstitial regions. These results also indicate that the nanofabricated Gel patterning can be achieved using non-covalently bonded gel material on a substrate with wells. It was also demonstrated that this is possible. EXAMPLES

[0103] Fabrication of substrates with gel-containing nanowells A structured array that can then be loaded with gel material using multiple techniques. can be manufactured.

[0104] The process starts with a blank substrate / wafer and then fabricates a The substrate / wafer material is ordinary silicon. , glass, plastic, COC or any of a variety of materials that can be constructed. The representative techniques for introducing patterning into a substrate are photolithography, nanoimprinting, and Print lithography, embossing structures into plastic / COC based materials, and Injection molding of plastic or COC onto a master mold with patterned structures Photolithography-based techniques typically involve the use of photoresists. , which is patterned with a stepper or mask aligner and exposed to radiation; This transfers the pattern present on the reticle / photomask to the photoresist, which then The resist is developed with a lithography machine to obtain a structured film (photoresist) on top of the substrate. The substrate is optionally the final substrate that can be used for subsequent gel coating or resist. The pattern in the mask may be transferred to a substrate by subsequent processing. is typically performed using reactive etching (plasma-based etching) or wet etching. The transfer of the pattern to a substrate involves patterned photolithography. The resist is then removed to provide a patterned substrate for subsequent gel coating. It is recommended to use a sacrificial material film such as aluminum or titanium (metal) under the photoresist. In some cases, a photoresist pattern is first transferred to a metal film, and then this film is then hard-masked. The pattern is transferred to the substrate by using the film as a mask. The nanoimprinting process removes the When lithography is used, imprinted photoresist can be the sacrificial material, and patterned laser can be used. It can also be used as an intermediate tool to transfer resist to a substrate, or for subsequent imprinting of resist. Resist variations can be used to act as input to the next coating step. The following examples of resists that may leave a pattern are sol-gel based materials. cormorant.

[0105] A diagram showing how a structured substrate can be manufactured is shown in FIG. 3 and is described below. Images of the patterned substrate are shown in Figures 3B and 3C at various magnification levels.

[0106] Creating chemically specific gel pads on the sequencing substrate / flow cell This may involve one or more of the nano-fabrication techniques described earlier in this example. The silane may then be optionally subjected to one or more chemical treatment steps, such as gel polysilane. This can include a silanization treatment to bond the polymer to the substrate. P) removes any interstitial polymer on the surface of the substrate. The structured features in the substrate are effectively removed from the plane of the interstitial regions of the array. The structured feature is effectively offset, so grinding removes the porosity from the gap before removing the feature. If the polishing process is stopped after an optimal time, the structure will be left with no polymer coating. The gel pad is then patterned to hold the gel in place and the gap areas are left free of polymer. is grafted onto a primer, a target nucleic acid is seeded on a gel pad, and the target nucleic acid is is used as a template to generate a nucleic acid cluster. EXAMPLES

[0107] Multiwell substrate coated with PAZAM This example illustrates the fabrication of an array of gel-containing wells, the amplification of nucleic acid clusters in the wells, and and sequencing of the nucleic acids in the cluster.

[0108] The substrate was fabricated as follows: Nanowell substrate (diameter 400 nm, pitch 1.5 μm) The wells (300 nm deep) were fabricated using nanoimprint lithography. Aminosilane (APTES or APTMS) single / multilayers are deposited on the substrate using chemical vapor deposition. Then, 1 ml of NHS acrylate solution was added to the surface and The concentration was measured by covering with a thin glass coverslip and allowing the reaction to proceed for 1 hour at room temperature. 100 mM acrylic acid N-hydroxysuccinimide ester (Aldrich PN 8060) in phosphate buffered saline (pH 7.4) was reacted with the aminosilane surface. Then, 500 μl of 2 wt% PAZAM aqueous solution was added to the newly formed acrylamide functionalized The polymer (PAZAM) is applied to the surface by spin-coating onto the photocatalytic surface. PAZAM is disclosed in U.S. Provisional Patent Application No. 61 / 753, which is incorporated herein by reference. The PAZAM-coated substrate was then heated at 60° C. for 1 h. Heating for 10 h resulted in covalent bonds between the polymer and the surface. The polymer surface was polished with a slurry of 3 μm SiO2 particles dissolved in 10 wt% water. The Janeway surface (acryloyl chloride + DIPEA(Me (in CN) was used in place of the aminosilane-coated surface in the above procedure. may be used.

[0109] Then, in U.S. Provisional Patent Application No. 61 / 753,833, which is incorporated herein by reference, The patterned polymer substrate was grafted to the primer as described. Then, the dye label ( Reverse complement of Cy5 grafted primer t) is exposed to a surface in 1X PBS buffer at a concentration of 20 μM of the complementary sequence, and then the surface is The plate was washed with 50 ml of PBS buffer using a squirt bottle. FLA9500Typh set to y5 scan channel and using PMT setting 450 The labeled complement on the substrate was also visualized by high-resolution microscopy. Imaged and patterned with no polymer / primer remaining in the gaps The substrate was then seeded with phiX DNA, which is incorporated herein by reference. As described in U.S. Patent Application No. 61 / 715,478, which is incorporated herein by reference, clusters are grown. Ta.

[0110] The flow cell containing the cluster-containing substrate was placed on a HiSeq2000 (Illumina, In c., San Diego, CA). Using an algorithm to extract the raster position (rigid registration), High quality sequencing metrics were obtained ( Sequencing results show that the occupancy rate for clonality is similar to that of standard pores. The results show that the results are surprisingly higher than those predicted by the Son distribution. The average occupancy for clonality measured for the sequencing runs shown is: It lies on the boundary of the Poisson curve and approaches the straight line of the ideal clone rate.

[0111] Throughout this application, various publications, patents, or patent applications have been referenced. The disclosures of these publications, all of which are incorporated herein by reference in their entireties, are incorporated herein by reference in their entireties. Describe the new technology more fully.

[0112] The term "comprising" is used herein to mean open-ended and includes only the listed elements. It is intended to encompass any additional elements without departing from the spirit or scope of the invention.

[0113] Although the present invention has been described with reference to the above embodiments, various modifications may be made without departing from the invention. It should be understood that modifications and variations are possible. The present invention is therefore limited only by the following claims. Limited to.

Claims

1. 1. A solid support comprising a surface, the surface comprising a plurality of wells, the wells being made of a gel material. the wells being separated from one another by interstitial areas on a surface thereof, the interstitial areas comprising: a solid support isolating the gel material of each well from the gel material of the other wells; A library of target nucleic acids in a gel material, wherein the gel material in each well contains A library containing a distinct single target nucleic acid. An array containing:

2. Each well has a maximum volume of 1000 μm ３ 2. The array of claim 1,

3. The gel volume in each well is up to 1000 μm ３ 2. The array of claim 1,

4. Each well is up to 100 μm ２ The array of claim 1 , comprising an opening in a surface thereof.

5. 5. The method according to claim 1, wherein the plurality of wells form an array having a repeating pattern. The array of any one of claims 1 to 4.

6. 6. The method of claim 5, wherein the wells in the pattern have a pitch of 5 micrometers or less. array.

7. 5. Any one of claims 1 to 4, wherein the plurality of wells constitute an array having a random pattern. An array according to any one of claims 1 to 4.

8. The array of claim 1 , wherein the gel comprises a hydrogel.

9. The array according to any one of claims 1 to 7, wherein the gel comprises SFA or PAZAM. stomach.

10. 10. The gel material in each well contains a single species of target nucleic acid in the library. The array of any one of claims 1 to 5.

11. 10. The method of claim 1, wherein multiple copies of a single species constitute a clonal population of nucleic acid molecules in each well. Array according to claim 0.

12. 10. The method of claim 10, wherein multiple copies of a single species are present as concatemeric repeats in the nucleic acid molecule. The array described in

13. 11. Any of claims 1 to 10, wherein a single species is present in each well as just a single nucleic acid molecule. An array according to any one of claims 1 to 4.

14. 14. The array of claim 13, wherein the single nucleic acid molecule comprises two complementary strands.

15. The surface has at least 1000 wells / mm ２ Any of claims 1 to 13, including a density of An array according to any one of claims 1 to 4.

16. 16. The method of claim 1 , wherein the gel material is covalently attached to the surface of the well. array.

17. (a) providing a solid support comprising a flat surface, said flat surface having one or more concave flaps; the one or more concave features being interrupted by one or more recessed features on a plane adjacent the gap region; (b) coating at least a portion of the solid support with a gel material, The portion includes at least one recessed feature and at least one gap region. Tep and (c) polishing the planar surface to remove the gel material from at least one interstitial region; retaining a gel material in the recessed features; A method for making a substrate, comprising:

18. 20. The method of claim 17, wherein the recessed feature comprises a well surrounded by a gap region. 。

19. Each well has a maximum volume of 1000 μm ３ 20. The method of claim 18, wherein:

20. The gel volume in each well is up to 1000 μm ３ 20. The method of claim 18, wherein:

21. Each well has a maximum of 100 μm ２ 19. The method of claim 18, further comprising: The method described.

22. 22. Any of claims 18 to 21, wherein the wells form an array having a repeating pattern.

3. The method according to claim 1 .

23. 23. The method of claim 22, wherein the wells in the pattern have a pitch of 5 μm or less.

24. 23. The method according to claim 18, wherein the plurality of wells are arranged in an array having a random pattern.

2. The method according to claim 1 .

25. The surface has at least 1000 wells / mm ２ 25. The method of claim 18, further comprising the step of:

2. The method according to any one of claims 1 to 11.

26. Coating involves contacting the solid support with a preformed gel material.

25. The method according to any one of claims 17 to 24.

27. Coating involves contacting the solid support with a liquid which then produces a gel material.

25. The method of any one of claims 17 to 24, comprising:

28. 28. The method of any one of claims 17 to 27, wherein the gel material comprises a hydrogel.

29. 28. The gel material according to claim 17, wherein the gel material comprises SFA or PAZAM. Method of posting.

30. 30. The method according to any one of claims 17 to 29, wherein the gel material further comprises attaching nucleic acid thereto. How to.

31. 31. The method of claim 30, wherein the nucleic acid comprises an amplification primer.

32. The nucleic acid comprises a template nucleic acid, and the method amplifies the template nucleic acid in the gel material in the recessed features.

32. The method of claim 30 or 31, further comprising:

33. Amplification produces multiple copies of a template nucleic acid, forming a clonal population of nucleic acid molecules.

32. The method according to claim 32.

34. Amplification produces multiple copies of the template nucleic acid to form concatemeric repeats of the nucleic acid molecule.

33. The method of claim 32.

35. 35. Any of claims 18 to 34, further comprising covalently bonding the gel material to a surface of the well. The method according to any one of claims 1 to 5.

36. 36. Any of claims 17 to 35, wherein the polishing comprises mechanical abrasion with a slurry of beads.

13. The method according to claim 1.

37. The polishing comprises mechanical abrasion by wiping or rubbing the surface of the solid support.

37. The method of any one of claims 17 to 36.

38. 38. The method of any one of claims 17 to 37, wherein polishing comprises chemical polishing.

39. 39. The method of claim 17, further comprising attaching the gel material to a protein. How to.

40. (a) providing a solid support comprising a surface, said surface comprising a plurality of wells; wherein the wells contain a gel material and the wells are separated from one another by interstitial areas on a surface thereof. the gap regions separating the gel material in each well from the gel material in the other wells. Steps (b) delivering a library of target nucleic acids to the wells of the solid support and depositing the gel material in each well; producing an array of wells having a single type of target nucleic acid attached to the substrate; wherein different wells in the array contain different target nucleic acid species from a library; (c) amplifying the target nucleic acid attached to the gel material in the wells of the array to form a nucleic acid sequence for each well of the array; generating clonal populations of individual target nucleic acids in wells; A method for producing an array comprising:

41. The gel volume in each well is up to 1000 μm ３ 41. The method of claim 40, wherein:

42. Each well has a maximum of 100 μm ２ 41. The method of claim 40, further comprising: The method described.

43. 41. The method of claim 40, wherein the plurality of wells form a repeating pattern on the surface.

44. 44. The method of claim 43, wherein the wells in the pattern have a pitch of 5 μm or less.

45. 41. The method of claim 40, wherein the plurality of wells are arranged in a random pattern on the surface.

46. 41. The method of claim 40, wherein the gel comprises a hydrogel.

47. 41. The method of claim 40, wherein the gel comprises SFA or PAZAM.

48. The surface has at least 1000 wells / mm ２ The method of claim 40, comprising a density of Law.

49. 41. The method of claim 40, wherein the gel material is covalently attached to the surface of the well.

50. Providing a solid support in step (a) (i) providing a solid support comprising a planar surface, said surface comprising a plurality of wells, the wells being separated from one another by interstitial areas on a surface thereof; (ii) coating the wells and the interstitial areas with a gel material; (iii) polishing the flat surface to remove gel material from at least one interstitial region and and holding the gel material.

50. The method of any one of claims 40 to 49, comprising:

51. Coating involves contacting the solid support with a preformed gel material.

51. The method of claim 50.

52. Coating involves contacting the solid support with a liquid which then produces a gel material.

52. The method of claim 50 or 51.

53. 53. Any of claims 50 to 52, wherein the polishing comprises mechanical abrasion with a slurry of beads.

13. The method according to claim 1.

54. The polishing comprises mechanical abrasion by wiping or rubbing the surface of the solid support.

54. The method of any one of claims 50 to 53.

55. 55. The method of any one of claims 50 to 54, wherein polishing comprises chemical polishing.

56. 56. Any one of claims 40 to 55, wherein step (b) is performed before step (c). The method described above.

57. 56. Any of claims 40 to 55, wherein steps (b) and (c) are performed simultaneously.

3. The method according to claim 1 .

58. The delivery of step (b) provides the library as a mixture of target nucleic acids in a fluid, The fluid is contacted with the solid support, thereby causing the target nucleic acid to be transferred to the wells and interstitial regions.

58. The method of any one of claims 40 to 57, comprising having fluid access.

59. The amplification may be by solid-phase polymerase chain reaction (PCR), multiple displacement amplification (MDA) or rolling.

59. The method of any one of claims 40 to 58, comprising repeat circle amplification (RCA).

60. Amplification is achieved by polymerizing at least one primer species attached to the gel material in the well.

60. The method of claim 59, comprising gene extension.

61. The amplification used at least two primer species that were attached to the gel material in the wells.

59. The method of any one of claims 40 to 58, comprising bridge amplification.

62. Each clonal population contains at least 1000 copies of the target nucleic acid from the library.

62. The method of any one of claims 40 to 61, comprising:

63. (a) providing a solid support comprising a surface and a nucleic acid library, The surface includes a plurality of wells, the wells containing a gel material, the wells being spaced apart from one another on the surface. The wells are separated from one another by gap areas, the gap areas allowing the gel material in each well to flow through the other multiple wells. The gel material of each well is then isolated, and a single target nucleic acid from the library is bound to the gel material of each well. Attached, steps and (b) contacting the solid support with at least one probe that binds to the target nucleic acid; Top and (c) detecting the solid support to have a target nucleic acid species that binds to at least one of the probes; identifying a well to be sampled; A method for detecting a nucleic acid comprising:

64. The gel volume in each well is up to 1000 μm ３ 64. The method of claim 63, wherein:

65. Each well has a maximum of 100 μm ２ 64. The method of claim 63, further comprising: The method described.

66. 64. The method of claim 63, wherein the plurality of wells form a repeating pattern on the surface.

67. 67. The method of claim 66, wherein the wells in the pattern have a pitch of 5 micrometers or less. How to.

68. 64. The method of claim 63, wherein the plurality of wells are arranged in a random pattern on the surface.

69. 64. The method of claim 63, wherein the gel comprises a hydrogel.

70. 64. The method of claim 63, wherein the gel comprises SFA or PAZAM.

71. The surface has at least 1000 wells / mm ２ The method of claim 63, comprising a density of Law.

72. 64. The method of claim 63, wherein the gel material is covalently bonded to the surface of the well.

73. Providing a solid support in step (a) (i) providing a solid support comprising a planar surface, said surface comprising a plurality of wells, the wells being separated from one another by interstitial areas on a surface thereof; (ii) coating the wells and the interstitial areas with a gel material; (iii) polishing the flat surface to remove gel material from at least one interstitial region and and holding the gel material.

73. The method of any one of claims 63 to 72, comprising:

74. Coating involves contacting the solid support with a preformed gel material.

74. The method of claim 73.

75. Coating involves contacting the solid support with a liquid which then produces a gel material.

75. The method of claim 73 or 74.

76. 76. Any of claims 73 to 75, wherein the polishing comprises mechanical abrasion with a slurry of beads.

13. The method according to claim 1.

77. The polishing comprises mechanical abrasion by wiping or rubbing the surface of the solid support.

77. The method of any one of claims 73 to 76.

78. 78. The method of any one of claims 73 to 77, wherein polishing comprises chemical polishing.

79. The providing in step (a) comprises (iv) under conditions that allow a single species of target nucleic acid of the library to adhere to the gel material of each well. delivering a library of target nucleic acids to wells of a solid support; (v) amplifying target nucleic acids attached to the gel material in the wells of the array to generate a nucleic acid sequence for each well of the array; To generate clonal populations of individual target nucleic acids in wells 78. The method of any one of claims 73 to 77, further comprising:

80. 80. The method of claim 79, wherein step (iv) is performed before step (v).

81. 80. The method of claim 79, wherein steps (iv) and (v) are performed simultaneously.

82. The delivery in step (iv) comprises preparing the library as a mixture of target nucleic acids in a fluid. and contacting the fluid with a solid support, whereby the target nucleic acid is distributed within the wells and gaps.

82. The method of claim 79, further comprising having fluid access to the region. method.

83. The amplification may be by solid-phase polymerase chain reaction (PCR), multiple displacement amplification (MDA) or rolling.

83. The method of any one of claims 79 to 82, comprising repeat circle amplification (RCA).

84. The amplification uses at least one primer species that is attached to the gel material in the well.

84. The method of claim 83.

85. The amplification used at least two primer species that were attached to the gel material in the wells.

83. The method of any one of claims 79 to 82, comprising bridge amplification.

86. Each clonal population contains at least 1000 copies of the target nucleic acid from the library.

86. The method of any one of claims 79 to 85, comprising:

87. At least one probe is complementary to at least a portion of at least one target nucleic acid.

87. The method of any one of claims 63 to 86, comprising at least one nucleic acid.

88. 9. The method of claim 8, wherein at least one probe comprises a fluorescent label that is detected in step (c).

7. The method according to claim 7.

89. At least one of the probes further comprises a polymerase and nucleotides.

87. The method according to claim 87.

90. The detection in step (c) involves the addition of a nucleotide into the probe or into the target nucleic acid.

90. The method of claim 89, comprising detecting incorporation of.

91. 90. The method of claim 89, wherein the detecting in step (c) comprises a decoding-by-synthesis method.

92. 7. The method of claim 6, wherein steps (b) and (c) are repeated several times.

92. The method of any one of claims 3 to 91.

93. 92. The method of claim 63, wherein the probe comprises a nucleic acid that binds to the protein. The method described.

94. 7. The method of claim 6, wherein the detection is selected from the group consisting of optical detection, electrical detection, and thermal detection.

94. The method of any one of claims 3 to 93.

95. Optical detection includes fluorescence, luminescence, chemiluminescence, absorbance, and fluorescence resonance energy.

95. The method of claim 94, wherein the at least one of the groups is selected from the group consisting of energy transfer.

96. (a) providing a solid support comprising a flat surface, said flat surface having one or more concave surfaces; the recessed features are interrupted by a gel material, the recessed features containing a gel material, and one or more The recessed feature is adjacent to one or more gap regions on the planar surface, and the gap regions are substantially a gel material comprising a target analyte; (b) exposing the solid support to a probe under conditions in which the target analyte specifically interacts with the probe. contacting the contact hole with a contact hole; (c) detecting the solid support to identify a small number of target analytes that interact with one or more of the probes. identifying at least one subset of the A method for detecting an analyte, comprising:

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