Gel-patterned surface

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 of related applications This application is in accordance with U.S. Provisional Patent Application No. 61 / 769289, filed on February 26, 2013, and We claim the benefit of U.S. application No. 13 / 787396, filed on March 6, 2013. All of these requests are incorporated herein by reference in their entirety.

[0002] This disclosure generally relates to solid-phase analytical chemistry and is intended for high-throughput analysis of genomics. It has specific applicability to nucleic acid arrays. [Background technology]

[0003] The task of classifying human genetic variations and relating these variations to disease susceptibility is This classification approach is in a position to benefit from advances in genome-scale sequencing methods. This technology is expected to identify markers in each individual's genome, which can help determine their susceptibility to disease and their treatment options. Responsiveness to specific therapies such as medications, side effects of dangerous drugs, and other medically relevant factors. This will help medical professionals in determining sensitivity to certain traits. Classification approach. This has made considerable progress. This is mainly because it is highly cost-effective and well-received in research settings. This is due to commercial genome sequencing methods that make it possible to examine the target. Improvements in sequencing methods are needed to accelerate this process. Moreover, relatively expensive Sequencing is a technology that allows physicians to obtain sequences of patients from the general population, beyond just research centers. This is hindering the transition to a clinic that can perform these procedures.

[0004] Sequencing methods and the systems used to implement them utilize a complex collection of techniques. . Improvements that result in substantial cost reduction are shown by some of these techniques. However even if there are any, it is difficult to estimate which technique leads to improvements in cost reduction. Judging from the dependency state among techniques in the sequencing system, it is even more difficult to estimate which technique can be modified without adversely affecting the overall performance of the method or system. Therefore, it is necessary to identify improvement points that can transfer the potential of genomic research to hospitals that can improve and, in many cases, save lives. The present invention meets this need and provides related advantages as well.

SUMMARY OF THE INVENTION

PROBLEMS TO BE SOLVED BY THE INVENTION

[0005] The present disclosure provides 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 a gap region on the surface, the gap region isolating the gel material of each well from the gel materials of other wells, and a library of target nucleic acids in the gel material, wherein the gel material of each well contains a single species of the target nucleic acid of the library.

MEANS FOR SOLVING THE PROBLEM

[0006] In some embodiments, a substrate is arranged as an array of wells and the sample is a nucleic acid. Accordingly, the present disclosure provides 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 a gap region on the surface, the gap region isolating the gel A body and a library of target nucleic acids in a gel material, wherein the gel material in each well is a library The present invention provides an array containing a library and a single target nucleic acid.

[0007] This disclosure also provides a method for fabricating a substrate. The method involves (a) a solid support having a plane A step of providing a body, wherein the plane is interrupted by one or more concave features The step in which one or more concave features are adjacent to one or more gap regions on a plane. (b) a step of coating at least a portion of the solid support with a gel material. , a portion of which includes at least one concave feature and at least one gap region (c) Polish the step and the plane to remove the gel material from at least one gap region, The step may include the step of holding the gel material in at least one concave feature. Cut.

[0008] A method for fabricating an array involves (a) providing a solid support having a surface containing multiple wells. A step wherein the well contains a gel material and the well is formed by the gap region on the surface They are separated from each other, and the gap region separates the gel material of each well from the gel material of the other wells. (b) a separation step, and (b) a delivery of the target nucleic acid library to the wells of the solid support, each This process involves creating an array of wells containing a single type of target nucleic acid attached to the gel material of the wells. A step in which different wells in the array contain different target nucleic acid species derived from the library. (c) amplifying the target nucleic acid attached to the gel material in the wells of the array. The process may include the step of creating a clonal population of individual target nucleic acids in each well of the array. Cut.

[0009] This disclosure further provides a method for detecting a sample. The method involves (a) a solid having a plane A step of providing a support, wherein the plane is interrupted by one or more concave features Furthermore, the concave feature contains a gel material, and one or more concave features are on a plane. Adjacent to one or more gap regions, the gap regions substantially do not contain gel material, and the gel material is (b) The step of having the target sample attached to or containing the target sample The steps include bringing a solid support into contact with the probe under conditions of interaction with the probe, and (c ) Detect a solid support and identify at least one sub-substrate of the target sample that interacts with one or more probes. This may include a step of identifying a set.

[0010] In a particular embodiment, nucleic acids are detected in the sample, and the concave feature is a well. For example, a method for detecting nucleic acids is (a) a solid having a surface and a nucleic acid library. A step of providing a body, wherein the surface has a plurality of wells, and the wells contain a gel material. The wells are separated from each other by gaps on the surface, and these gaps are located in the gutter of each well. The material is isolated from the gel material in the other wells, and a single target nucleic acid in the library is detected. The steps attached to the gel material in each well, and (b) the solid support, are used to bind the target nucleic acid. (c) the step of making contact with at least one probe, and the step of detecting a solid support, The process includes the step of identifying a well having a target nucleic acid species that binds to at least one probe. It is possible.

[0011] The compositions, apparatus, and methods of this disclosure are demonstrated herein in relation to gel materials. Gel materials are a typical example; for instance, they can form surface coatings, and they themselves The body can be replaced with other organic materials such as polymers that are not necessarily considered gels. Please understand the following: Using the array prepared in the analytical method or preparation method, A gel material is applied to the surface, the gel material is removed from the gap area, and the sample is attached to the gel material. The methods described herein can be easily adapted by replacing the gel material with a non-gel material. It can be made to happen. [Brief explanation of the drawing]

[0012] [Figure 1] A diagram illustrates a method for preparing and using a patterned array of DNA features, wherein each feature is a well containing a gel material attached to a DNA cluster, and the array is used in a sequencing method. [Figure 2] Images from a BeadChip substrate modified to have gel material instead of beads in the wells are shown. Panel A: Bright-field images obtained before polishing. Panels B-C: Fluorescence images obtained after polishing and hybridization with fluorescently labeled oligonucleotides. [Figure 3-1] Panel A shows a schematic process flow for manufacturing concave features in a substrate using photolithography and a Cr hard mask in conjunction with reactive ion etching, while Panel B shows examples of SEM images of wells and reference areas in a glass substrate. [Figure 3-2] Panel C shows an image of the wafer, an image of a portion of the wafer including the reference and well arrays, and an image from a portion of the array including the wells. [Figure 4] This image shows a high-resolution fluorescence microscope image of a nanowell substrate, illustrating the patterned gel features on the nanowell substrate after coating with PAZAM and polishing with a silica bead slurry. PAZAM is labeled with a dye for visualization. [Figure 5]This panel shows multicolor fused images obtained from HiSeq sequencing cycles of a 1.5 μm pitch nanowell substrate with patterned clusters. Panel A: Image showing the extent of patterned clusters in a gel-containing well alongside four bullseye references. Panel B: High-resolution image showing color mixing in a single bullseye reference (due to a mixed population of amplicons). [Figure 6A] Panel A shows multicolor fusion of patterned clusters in Hiseq sequencing performed on a 750nm pitch nanowell substrate. [Figure 6B] Panel B shows the nearest neighbor curves, indicating that the arrays are aligned and the clusters pass through the quality filter. [Figure 6C] Panel C shows the sequencing quality metric, indicating that the samples successfully passed the quality filter at a density of 1.6 million clusters / mm². [Figure 7] This graph shows the cloning rate versus occupancy rate, where the curve represents the Poisson distribution, the straight line represents the ideal cloning rate and occupancy rate, and the × symbol represents the average scale obtained from sequencing using a substrate with a pattern of gel-containing nanowells. [Modes for carrying out the invention]

[0013] This disclosure provides a structured substrate, a method for fabricating a structured substrate, and a method for using a structured substrate. In certain embodiments, the substrate contains a gel material (for example, coated with a gel material). The gel material includes a solid support having concave regions such as wells. It can be attached to elephant specimens, for example, nucleic acids. In certain embodiments, the gel-containing region is It is discrete and separated by interstitial regions that lack the ability to adhere to the target sample. For example, The interstitial region may lack gel material. Alternatively, the gel material in the interstitial region may be inert. It may be possible, or otherwise the activity or properties of the gel material in the concave region, for example, with a sample attached. It may be modified so that it does not have the ability to help with adhesion. The resulting isolation of the gel region to the specimen It offers advantages when performing the reaction and / or detecting the sample. Typical advantages are This can be demonstrated with an example of an array of target nucleic acids distributed within a gel-containing well. The book describes how to perform amplification reactions on a structured substrate using nucleic acids as templates, in a gel or gel. It is possible to form nucleic acid colonies (e.g., nucleic acid features on an array) that grow on the surface. The interstitial regions limit the area in which colonies can grow. Each feature is relative due to the individual patterns created by the gel-containing wells. They can be easily distinguished. The pattern increases the density of features and the random array of nucleic acids. This can also offer the advantage of reducing the processing requirements for image registration.

[0014] A typical process for fabricating nucleic acid patterned arrays is shown in Figure 1. (Well patterned substrate) A cross-sectional view is shown in the figure. In the example, the wells have a pitch of 1.5 μm (center-to-center spacing) The well-patterned substrate has a single well with a diameter of 0.5 μm. The gel material can be used to coat the wells and cover the interstitial regions. The resulting gel-coated substrate is polished, leaving the gel material in the wells. By removing the gel material from the gap region, a gel-patterned substrate can be formed. The gel can support the capture and amplification of DNA templates. For example, before surface coating, after surface coating, and before or after polishing. The gel can be grafted with oligonucleotide primers. The primers are It can capture a DNA template and use the captured template to perform primary amplification. The resulting DNA patterned substrate can be analyzed, for example, by sequencing.

[0015] Patterned arrays of nucleic acids in gel-containing wells offer several advantages to DNA sequencing methods. Random arrays (i.e., arrays with random feature patterns) and Examples of advantages when compared include high-density feature packing and concentration-independent template seeding. Improved control and tuning of feature density using this method, reduced image resistance These include the processing required for transcription and simplified signal extraction. Further advantages may arise from the spatial confinement of nucleic acid populations provided by this disclosure. The features of the ionization array include the growth of nucleic acid colonies (for example, through cluster amplification). (This can have the effect of limiting area or volume.) Nucleic acid colonies can amplify to a larger size than others, because of the globulin in their sequence. Due to the difference in the percentage content of anine and cytosine (i.e., GC content), this It affects the relative amplification factor. In the case of the methods and compositions described herein, individual feet Alternatively, the volume or area of ​​the char can be thought of as being caused by the difference in GC content between the amplifying template types. To prevent or minimize differences in nucleic acid colony size that are thought to arise from the amplification reaction of the causal agent. You can choose to do so. For example, the volume or area of ​​a feature can be set to the fastest speed. It can be made small enough to limit the growth of the colony, while slowly To ensure that the growing colony effectively fills in the features when the amplification reaction is complete. It is possible.

[0016] In certain embodiments, the disclosure relates to a covalently bonded patterned gel, for example, poly(N- (5-azidoacetamidylpentyl)acrylamide-co-acrylamide)(PAZ AM, for example, U.S. Provisional Patent Application No. 61 / 753833, incorporated herein by reference. Glass, silicon, plastic or other suitable solid supports having (see number) The process provides the manufacturing of upper wells (e.g., microwells or nanowells). A gel pad used for sequencing is fabricated, which performs sequencing cycles multiple times. It can remain stable throughout the process. Covalent bonding of polymers to wells is used in a variety of ways. In this context, it helps to retain the gel within the structured feature throughout the lifespan of the structured substrate. However, in many embodiments, the gel does not necessarily have to be covalently bonded to the well. For example, under certain conditions, a sila that is not covalently bonded to any part of the structured substrate Acrylamide-free (SFA), for example, a U.S. patent incorporated herein by reference. (See Patent Publication No. 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 made of a gel material (e.g., PAZAM, SFA or These are chemically modified mutants, for example, the azidolyzed type of SFA. Coated with (ed version) (Azide-SFA) and supported by gel coating. The body is polished, for example, by chemical or mechanical polishing, thereby allowing the wells to While retaining the gel, substantially all of the gel escapes from the gap regions on the surface of the structured substrate between the wells. A structured substrate can be fabricated by removing or inactivating the ply. The nucleic acid can then be attached to the gel material. Next, the target nucleic acid (for example, fragmented nucleic acid) The solution of the genome can be brought into contact with the polished substrate, and as a result, individual target nucleic acids can be brought into contact with each other. However, by interacting with the primers attached to the gel material, they are seeded into individual wells. However, since the interstitial region is either devoid of or inactive gel material, the target nucleic acid occupies the interstitial region. This does not happen. Due to the absence or inactivity of the gel in the interstitial region, the grown nucleic acid colon Because movement outside the well is prevented, the amplification of the target nucleic acid is kept within the range of the well. It is likely. This process is conveniently manufacturable, scalable, and conventional micro- This utilizes nanofabrication techniques.

[0018] In certain embodiments, the reference marker is an individual feature (e.g., a well or It is included in the structured substrate for the identification and localization of other gel-containing concave features. A quasi-marker provides a reference point for the relative position of other features, so the reference marker - is particularly useful for structured substrates having spatially aligned pattern features. Reference markers can also be used for registration of random array images, but Il HiSeq, Genom, manufactured by Lumina, Inc. (San Diego, CA) Commercial sequencing platforms such as e Analyzer or MiSeq platform When used with random arrays generated on a network, use the inherent cluster disorder instead. This may be done. The reference marker repeatedly detects the structured substrate and measures the time course of each feature. This is particularly useful for applications that track changes that occur sequentially. Reference markers are used in multiple sequencing applications. The continuous images obtained through a fixed cycle enable the tracking of individual nucleic acid clusters, As a result, the sequences of individual clusters can be determined individually.

[0019] This disclosure provides a reference marker having a pattern of concave and interstitial regions. Typical marker designs include: concave rings, gap rings, and wells or other concave rings. The same as having two or more alternative patterns among the rings of the catcher (e.g., "Bullseye") It is a set of core circles. In some embodiments, the concave region of the reference marker contains gel material. On the other hand, it does not contain interstitial regions. The specific locations of the gel on this surface are described herein. This can be achieved using the gel coating and polishing methods described. Typically, the gel-containing region A detection method is used that can distinguish it from the gap region. In some cases, the identification is that the gap region is not present. This may also be based on the presence of a specific sample within a gel region. For example, in the case of nucleic acid arrays. The gel-containing region of the reference marker is used to label the target nucleic acid on the array. It can contain nucleic acids labeled by the same method as the reference marker. - Conveniently manufactured using the same method used to manufacture the sample feature It can be manufactured accordingly. Accordingly, if necessary, reference markers and sample features can be manufactured. - may be manufactured simultaneously through one or more steps. Structured substrates and Another useful reference marker that can be used in the method is one that has a subregion, The pattern of wells (or other concave features) in one subregion is in another subregion. This rotates based on the pattern. Such a reference grid is shown in the details below. Image resists such as those described in U.S. Patent Application No. 13 / 267565 incorporated in the book It can be configured and used for rations.

[0020] As a further example, beads may be used as a reference. The beads are fluorophosphoric Any of the following labels may be included. In this case, the surface has at least two types of wells (or other It may have a concave feature. Relatively large wells can accommodate one or more reference beads. While it can accommodate beads, the smaller wells are too small to contain beads and only contain gel material. Therefore, the smaller well acts as an analytical feature for the analysis, and A large, bead-filled well serves as the reference. As an alternative to the well, a reference fee The char may be a channel that exists in the bullseye configuration exemplified above. The channel may have dimensions to accommodate beads. Some beads are positioned within the channel, for example, forming a distinct series of bead shapes. It is possible to create standards for this.

[0021] A patterned array, its manufacturing method, and its usage method are provided so that it adheres to the target sample. The gel material used is demonstrated herein. The gel material is typically It is such that other methods can be used to intervene in localization to features on the surface of the specimen. It should be understood that organic materials can be used as substitutes. Such organic materials are, for example, surface materials. A coating can be formed, and it does not necessarily have to be considered a gel itself. Includes rimers. Specific examples include ATRP (atomic transfer radical polymerization) or surface-initiated polymerization. This is a polymer formed by using an array obtained by an analytical method or preparation method. The method described herein, which involves applying a gel material to a surface and removing the gel material from the gap region, is Furthermore, it can be easily adapted to the use of non-gel materials.

[0022] Unless otherwise specified, terms used herein have the common meaning in the relevant art. Please understand that some terms used in this specification and their meanings are as follows. The details are listed below.

[0023] In this specification, the term “attached” means that two things are connected or fixed to each other. It means a state of being attached, connected, or bound. For example, a sample such as nucleic acid is bound together. It can adhere to materials such as gels or solid supports through bonded 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. These are unnecessary chemical bonds, such as hydrogen bonds, ionic bonds, and van der Waals forces. Hydrophilic and hydrophobic interactions can be cited as examples.

[0024] In this specification, the term “clonal population” means “uniform with respect to a particular nucleotide sequence.” It refers to a collection of nucleic acids. A uniform sequence is usually at least 10 nucleotides long, They can be even longer, for example, at least 50, 100, 250, 500, 1000 It contains a length of 2500 nucleotides. Clonal populations are single target nucleic acids or template nucleic acids. It may be derived from another organism. The clonal population has at least two copies of the target nucleotide sequence. It may include 5, 10, 100, 1000 or more. A copy is, for example, a concatemer. They may exist within a single nucleic acid molecule, or copies may exist on a single nucleic acid molecule. It may be (i.e., a clonal population may have nucleic acid molecules with the same target nucleotide sequence) (This can include at least 2, 5, 10, 100, or 1000 or more.) Typically, All nucleic acids in a clonal population will have the same nucleotide sequence. Without deviating from the standard, a very small number of impure nucleic acids or mutants (e.g., amplified artificial production (caused by amplification artifacts) Please understand that this is possible. Therefore, the group is at least 80%, 90%, 95% They could be 99% clones. In some cases, 100% pure clone populations exist. Sometimes that happens.

[0025] In this specification, the term "coating," when used as a verb, means to create a layer on a surface. It is intended to mean that a coating is applied. At least a portion of the surface is layered. Alternatively, it may have a covering. In some examples, the entire surface may have a layer or covering. This is also possible. In an alternative example, only a portion of the surface would have a layer or coating. The term "finishing" is used to describe the relationship between surfaces and materials. This is intended to mean that the material exists on the surface as a layer or coating. The surface may be sealed, for example, to prevent a liquid or gas from coming into contact with the surface. However, the material does not necessarily have to form a seal. For example, the material can be a liquid, a gas, or Alternatively, it may be permeable to one or more components supported in a liquid or gas. Typical materials that can coat surfaces include, but are not limited to, gels and polymers. Examples include organic polymers, liquids, metals, second surfaces, plastics, silica, or gases. It can be done.

[0026] In this specification, the term “concave feature” is used in relation to a solid support. In this case, it means a depression or indentation in a solid support. As a typical concave feature, Examples include, but are not limited to, wells, pitches, holes, depressions, channels, or troughs. The concave feature can be optionally configured to have a curved cross-section (a dimension perpendicular to the surface of the solid support). ) may have, but cross-sections with one or more linear sections, corners or edges are also possible. Cross-sections having a combination of curved and linear sections are also possible. Generally, concave The feature does not necessarily have to pass through the solid support completely; rather, for example, the base It has a bottom surface or base within the plate.

[0027] In this specification, the term “different” means, when used in relation to nucleic acids, that nucleic acids are mutually exclusive. This means that two or more nucleic acids have nucleotide sequences that are not identical. It can have different nucleotide sequences within its entire length. Or, two or more nucleic acids. These can have different nucleotide sequences within the essential parts of their length. For example, two or more nucleic acids have different target nucleotide sequence regions in two or more molecules. On the other hand, two or more molecules can also have the same universal sequence region.

[0028] In this specification, the term “each” is used in reference to a collection of items. Intended to identify individual items within a collection, but not necessarily all items within the collection. This does not refer to specific items. Exceptions apply unless clearly indicated by explicit disclosure or context. That's also possible.

[0029] In this specification, the term "fluidic access" refers to flow When used in relation to the location of contact with molecules and fluids throughout the body, the molecules are also in the fluid. This refers to the ability of molecules to move through a fluid and to come into contact with or enter a particular location. It can also refer to the ability to separate from or leave its position and enter the solution. Fluid access is when molecules enter their position, come into contact with that position, and separate from that position. This can occur when there is no barrier to prevent it from leaving that position. However, the fluid Unless access is absolutely prevented, even if the spread stagnates, decreases, or changes, Fluid access is understood to exist.

[0030] In this specification, the term "gel material" refers to a semi-rigid material that is permeable to liquids and gases. It is intended to have a taste. Typically, gel materials expand when they absorb liquid and dry. It can shrink when the liquid is removed by drying. Typical gels are not limited to these. However, colloidal structures such as agarose, polymer mesh structures such as gelatin, or Polyacrylamide, SFA (e.g., U.S. Patent Application incorporated herein by reference) See Publication No. 2011 / 0059865) or PAZAM (for example, by reference this Crosslinked polymers such as (see U.S. Provisional Patent Application No. 61 / 753833 incorporated in the specification) —Examples include those having a structure. Particularly useful gel materials are those in which it is found in wells or other structures. It conforms to the shape of concave features. Some useful gel materials (a) have wells or (b) it can be adapted to the shape of other concave features, and also (b) it has a well or concave feature It may also have a volume that does not substantially exceed the volume of the container.

[0031] In this specification, the term “gap region” refers to a substrate or a surface that separates other areas of a substrate. It refers to an area on the inside or surface. For example, a gap region is a concave feature of an array. It can be separated from another concave feature of the ray. The two regions that separate from each other are, They can be discrete and not in contact with each other. In another example, the gap region is the first feature portion, the second It can be separated from feature portion 2. In many embodiments, the gap region is continuous On the other hand, for example, in the case of an array of wells in a separate continuous surface, each phi The elements are discrete. The separation brought about by the gap region is not complete even if it is only partial separation. Separation is also acceptable. The gap region is usually made of a surface material different from the surface material of the feature on the surface. It will have, for example, the array feature may have the amount or concentration present in the gap region. It is possible to have a gel material or sample exceeding a certain amount or concentration. In some embodiments, The gel material or sample does not need to be present in the interstitial region.

[0032] In this specification, the term "library" is used in relation to specimens, and may differ in meaning. This refers to a collection of specimens having a certain chemical composition. Typically, this includes specimens in a library. They share common characteristics or traits with a genus or class, but differ in some other way. They are likely different species. For example, the libraries have different nucleotide sequences, but the sugar-phosphorus This may include nucleic acid species that are similar in that they have an acidic backbone.

[0033] In this specification, the terms “nucleic acid” and “nucleotide” have the same meaning in the context of these terms in the art. In accordance with the use of, it is intended to include species of natural origin or their functional analogues. Functional analogues of useful nucleic acids can hybridize to nucleic acids in a sequence-specific manner. It can be used to replicate or as a template for specific nucleotide sequences. Naturally occurring nucleic acids generally have a skeleton containing phosphate diester bonds. (Analogous structure) The structure may have alternative skeletal connections, any of which are known in the art. Nucleic acids of natural origin are generally derived from deoxyribose sugars (e.g., deoxyribonucleic acid (DN)). A) containing (or ribose sugar, for example, found in ribonucleic acid (RNA)) Nucleic acids are those having any of the various analogues of these sugar moieties known in the art. It may contain creotides. Nucleic acids may contain natural or unnatural nucleotides. This can be done. In relation to this, natural deoxyribonucleic acid contains adenine, thymine, cytosine, and It can have one or more bases selected from the group consisting of guanine, and ribonucleic acid is One or more bases selected from the group consisting of uracil, adenine, cytosine, or guanine. It may have useful non-natural bases that may be contained in nucleic acids or nucleotides. Known in the technical field. The terms "probe" or "target" are used in relation to nucleic acids. If applicable, the semantic identifier (SEMAN) of nucleic acids in relation to the methods or compositions described herein. (Tic identifier) ​​is intended to indicate the structure or function of nucleic acids, not necessarily otherwise clearly. It does not need to be limited beyond what is indicated. The terms "probe" and "target" are used in a way that is appropriate for a target. It can be similarly applied to other samples such as proteins, small molecules, and cells.

[0034] In this specification, the term "random pattern" is used in relation to wells on a surface. In such cases, the relative positions of a subset of wells within a region of the surface are unknown, or This means that the location of a subset of wells in a different region of the surface is unpredictable. The subset used in standard methods generally contains at least 3 wells, but at least It can also contain 4, 5, 6, or 10 or more wells. Random patterns are generally , does not include multiple repetitions of any subpattern. This term refers to a well with another concave fee Applicable to Char.

[0035] In this specification, the term “repeating pattern” is used in relation to wells on a surface. If so, the relative positions of a subset of wells within a region of the surface are such that at least one of the surfaces This means that the relative position of the subset of wells in other regions is the same. Therefore The relative position of the wells within one region of the repeating pattern is generally the same as the repeating pattern. The relative position of the well in another region of the circle can be predicted. Subsequent measurements used A set generally contains at least 3 wells, but at least 4, 5, 6, or 10 wells. The upper well can be included. Typical repeating patterns are linear patterns. This includes hexagonal patterns. Repeating patterns may include multiple repetitions of subpatterns. This term can be applied to other concave features besides wells.

[0036] In this specification, the term “isolation” refers to two wells (or two other features). ) When used in relation to gel material in a well (or feature) To separate the gel material in one well from the gel material in other wells (or other features) This means to isolate. Therefore, in the first well (or first feature) The gel material does not come into direct contact with the gel material in other wells (or other features). In one embodiment of the part, the gel material in two wells (or two features) is, for example, or indirectly through a solution that comes into contact with two wells (or features). In other words, the gel material in the two wells (or two features) is in indirect contact. It does not. The gap region on the surface does not contain gel material, thus creating two wells. Alternatively, the gel material can be isolated within two features. In certain embodiments, The gel material may be discontinuous on the surface and present in concave features such as wells. However, it does not exist in the gaps between each feature.

[0037] In this specification, the term "surface" refers to the outer or outer layer of a solid support or gel material. It is intended to mean: The surface is gas, liquid, gel, polymer, organic polymer, etc. By bringing it into contact with another material such as a second surface of a similar or different material, metal, or coating. The surface or area may be substantially flat. The surface may have wells and pitches. It has surface features such as channels, raised areas, raised regions, pegs, and posts. It's fine if you do that.

[0038] In this specification, the term “single species” refers to substantially one, or only one species of a particular genus. This term does not necessarily mean limiting the number of existing single species. For example, a group of nucleic acid molecules, each having the same nucleotide sequence, is a single type of nucleic acid. Includes. The term “single” in this context implies the presence of other things that are not within the scope of the related genus. This is not intended to exclude single target nucleic acids from a library. A well containing a nucleic acid may contain multiple nucleic acids having the same sequence, from a library. This excludes other target nucleic acids, but does not necessarily exclude any other non-nucleic acid components. (Clear) A single species of population contains impurities or human-derived substances that are negligible to a person skilled in the art for the specific use of the population. Please understand that it is possible to have small amounts of other species present at a level that can be considered as crafts. For example, a nucleic acid cluster derived from a single template having the first sequence can detect the first sequence. When this is done, the amount of any nucleic acid molecule having the second sequence is undetectable or negligible. If the level is sufficiently low, it would clearly be considered to be a single species. Or, absolute A single-species population will have only one, and only one species.

[0039] In this specification, the term "solid support" refers to a highly rigid material that is insoluble in aqueous liquids. The substrate may be non-porous or porous. The substrate may optionally incorporate liquid. It is possible (for example, due to porosity), but usually the substrate is substantial when it absorbs liquid. It does not expand and does not shrink substantially when the liquid is removed by drying. It would be rigidity. Non-porous solid supports are generally impermeable to liquids or gases. The solid support is optionally inert to the chemical reactions used to modify the gel. For example, the solid support may be a sample such as nucleic acid in the method described herein. It may be inert to the chemical reaction used to attach it to the gel. Typical solid As a support, but not limited to these, glass and modified or functional glass, plus Plastic (acrylic resin, polystyrene and styrene and other materials, polypropylene, por Polyethylene, polybutylene, polyurethane, Teflon®, cyclic olefin (Copolymers with polyimide, etc.), nylon, ceramic, resin, Zeonoa, Silica or silica-based materials such as silicon and modified silicon, carbon, metals, inorganic glass Examples include optical fiber bundles and polymers. Solid supports are particularly useful in some embodiments. The device is placed inside the flow cell apparatus. Typical flow cells are described in more detail below. To describe.

[0040] In this specification, the term "well" refers to a surface opening that is completely enclosed by a gap region on the surface. This refers to discrete concave features in a solid support having wells. A well is its opening in the surface. The part may have any of the following shapes, but is not limited to these: circular, elliptical, etc. Examples include circles, squares, polygons, and star shapes (with any number of vertices). The surfaces are perpendicular to each other. The cross-section of the well, when viewed in this way, may have curves, squares, polygons, hyperbolas, cones, angles, etc. good.

[0041] The embodiments described below and enumerated in the claims shall be understood in consideration of the above definitions. It is possible.

[0042] This disclosure provides a substrate including a solid support having a surface, the surface being at least It has at least one concave feature, and the at least one concave feature is made of gel material. It contains, and the at least one concave feature is at least one gap region on the surface Adjacent to, and also this disclosure provides a sample library in gel material, each we The gel material in the library contains a single type of sample.

[0043] In some embodiments, the substrate is configured as an array of wells, and the sample is nucleic acid. Accordingly, this disclosure provides an array including a solid support having a surface, the surface being a plurality It has wells, the wells contain a gel material, and the wells are formed by gaps on the surface They are separated from each other, and the gap region allows the gel material in each well to flow into the gel material in the other wells. This disclosure isolates the target nucleic acid from the gel material, and also provides a library of target nucleic acids in the gel material. However, the gel material in each well contains a single target nucleic acid from the library.

[0044] The solid support used in the structured substrate described herein is, for example, as described herein. , made from one of the above definitions, the following examples, or the various materials described immediately afterward. Glass is a particularly useful material. Other suitable substrate materials include polymer materials, etc. Plastic, silicon, quartz (fused silica), boroflote glass, silica, silica-based materials Materials, carbon, metals, optical fibers or optical fiber bundles, sapphire, or COC and It may include plastic materials such as epoxy resin. Specific materials may be used for specific purposes. It can be selected based on the desired properties. For example, a transparent material to radiation of a desired wavelength. The material is used in analytical methods that utilize radiation of a desired wavelength, such as one or more techniques described herein. Useful. Conversely, it does not allow radiation of certain wavelengths to pass through (e.g., opaque, absorbing, or reflective). It may also be desirable to select a material that is projectile. For example, structures used in the manufacture of structured substrates, or those described herein, etc. It may be useful for forming masks used in chemical reactions or analytical detections. Other properties of the materials that can be used are those described herein, etc., and can be used in downstream processes. Inert or reactive to the specific reagent used, or as described herein. The advantages are ease of operation during the manufacturing process or low cost. Structured substrate or method of the present disclosure Further examples of materials that can be used in this context are incorporated herein by reference. National Patent Application No. 13 / 661524 and U.S. Patent Application No. 2012 / 0316086 It is stated.

[0045] In certain embodiments, a sol-gel based substrate can be fabricated and used. - Gel-based pattern molding can be done by, for example, spin coating, dipping, or blowing. The coating provides rigidity or flexibility to materials such as glass, silicone, plastic, and metal. This can be achieved by coating a flexible substrate with a sol-gel coating agent. - The gel may be supplied in a liquid state when applied to the substrate, and the sol-gel may be exposed to light or heat. A photoinitiator that can be cured (turned into a gel) by doing so. It may contain any of the thermal initiators after coating the substrate with a sol-gel. Next, before the material hardens, the sol-gel has one or more raised features. It can be imprinted using a mold (three-dimensional stamp). The mold can be made of, for example, silicone. Glass (such as quartz), metals (such as nickel), plastics or polymers (such as PDMS) It is fine if it is made using (do). Stamping onto sol-gel is done by casting a mold into the sol- This can be achieved by positioning the template so that it is in contact with the gel. The sol-gel then redistributes and surrounds the template structure isometrically. The template then comes into contact with the sol-gel. This is due to external forces being applied to the mold or substrate, or to properties inherent in the patterned mold. Capillary forces can facilitate the redistribution of sol-gel. When the mold comes into contact with the sol-gel, The substrate + sol-gel + mold stack is exposed to light or heat, which hardens the sol-gel, originally The pattern that was in the mold can be fixed in the sol-gel. This pattern molding method is more advanced than conventional methods. This is called nanoimprint lithography. After the sol-gel hardens, the template is the substrate. + It can be separated from the sol-gel stack, and this mold can be discarded, but another uncured sol- The gel-coated substrate may be reused for patterning and molding. A substrate having a patterned sol-gel can then be subjected to chemical vapor deposition, or If a pure glass-like surface is desired, a stack of substrate + patterned sol-gel is used. The gel can be subjected to a thermal process (sintering) to remove any organic materials originally present within it. This is not a requirement, but the substrate has advantages in certain chemical addition schemes, such as pure S It can be used as an iO2 material.

[0046] Another method for manufacturing patterned substrates is COC or COP (Zeonor or To Using plastic materials such as PAS, a heat embossing process is performed to create an indentation array. It is something that is made. This method is similar to nanoimprint lithography. Plastic The circuit board may be mounted on a temperature-controlled chuck. Then, plastic The substrate can be heated to a temperature above the glass transition temperature of the plastic outer casing. While the substrate is at a high temperature, the mold (e.g., quartz, silicon, polymer, or metal) To make it closely attached to the object. The mold is typically coated with plastic to create an isometrically structured mold. External force is applied to ensure that it adheres. During contact at high temperatures, the plastic The element itself redistributes itself and becomes a negative replica of the template, for example, if the template is a post-array. Having this, the embossed plastic becomes an array of wells. The mold is plastic While in contact with the substrate, the temperature of the substrate decreases, and therefore the embossed pattern on the substrate It will be secured inside.

[0047] The concave features on the substrate can have any of the following shapes. From the perspective of the shape on the substrate, the feature is a curved side, a linear side, a corner, or a combination thereof. Features can be circular, elliptical, square, polygonal. We have openings on the surface that are shaped like a star (with any number of vertices) or irregular in shape. It may also be a feature, and the surface channel The shape may include curved, linear, angular, or a combination thereof. Other channel features include linear, snake-like, rectangular, square, triangular, circular, and elliptical shapes. The shape can be circular, hyperbolic, or a combination of these. There is one or more channels. It may have an upper branch or corner. A channel can connect two points on a surface. This is possible, and one or both of them may be the edges of the substrate. Figures 3B and 3C show Along with wells within and around the bullseye standard, representative teeth within the bullseye standard. Show channel features.

[0048] The shape of the cross-section of the concave feature viewed perpendicular to the surface may have curved, linear, or walls of a combination of these two . Thus, the shape of the cross-section may be part of a circular or elliptical shape (e.g., U-shaped), or may have two or more linear sides that meet at corners ( e.g., V-shaped, square, polygonal, or star-shaped). From the perspective of the shape of the cross-section, the bottom of the concave feature may be narrow, wide, or approximately the same as the opening on the surface . These cross-sectional shapes can be explained in the case where the concave feature is a well and the well has a cylindrical cross-section, the opening on the surface is generally the same area as the bottom of the well, while when the well has a conical cross-section, the bottom of the well is different from the area of the opening on the surface (usually smaller). Of course, the cross-section is described for a well, but it can also apply to a channel. When the concave feature forms a well, each well can have any volume that can trap liquid. The minimum or maximum volume can be selected, for example, to correspond to throughput (e.g., multiplicity), resolution, analyte composition, or analyte reaction reactivity suitable for downstream use of the substrate. For example, the volume can be at least 1×10 μm , 1×10

[0049] μm , 0.1 μm , 1 μm , 10 μm -3 μm 3 , 1×10 -2 μm 3 , 0.1 μm 3 , 1 μm 3 , 10 μm 3 , 100 μm 3 or more . Alternatively, or additionally, this volume can be up to 1×10 4 μm 3 , 1×10 3 μm3 , 100 μm 3 , 10 μm 3 , 1 μm 3 , 0.1 μm 3 The following is also acceptable. The gel material is It should be understood that the wells can fill the total volume or a portion of the volume. The volume of the gel in the gel may be greater than or less than the value specified above, or these may be different. The values ​​can be in between.

[0050] The area occupied by each well opening on the surface is determined using the same criteria as above for the well volume. The selection may be based on the following: For example, the area of ​​each well opening on the surface is at least 1 × 10 -3 μm 2 , 1 x 10 -2 μm 2 , 0.1 μm 2 , 1 μm 2 , 10 μm 2 , 100μ m 2 That's all. Alternatively, or additionally, this area can be up to 1 × 10 3 μm 2 , 1 00 μm 2 , 10 μm 2 , 1 μm 2 , 0.1 μm 2 , 1 x 10 -2 μm 2 The following is also acceptable. The depth of each well may be at least 0.1 μm, 1 μm, 10 μm, or 100 μm or more. Alternatively, or additionally, this depth is up to 1 × 10 3 μm, 100μm, 10μ m, 1 μm, or 0.1 μm or less are also acceptable.

[0051] Many different patterns, including regular patterns, repeating patterns, and irregular patterns. A layout of a well or other concave feature may be conceivable. For example, a well may be densely packed. They may be arranged in a hexagonal grid to improve filling and density. Other layouts include, for example... For example, we can cite linear (i.e., rectangular) layouts, triangular layouts, and so on. The differences between layouts of different domains (if used) are as follows: , U.S. Patent No. 7813013 and / or U.S. Patent as incorporated herein by reference This may also be done in accordance with the teachings in Application No. 13 / 267565. Various crystal patterns and Any of the amorphous patterns may be useful.

[0052] The well pattern is determined in terms of the average pitch of the wells (i.e., the spacing between centers). It can be characterized. The pattern is designed so that the coefficient of variation around the average pitch is small. The pattern may be regular, or it may be irregular in that the coefficient of variation is relatively large. It may be present. In either case, the average pitch should be, for example, at least 10 nm, 0.1 The size may be μm, 0.5 μm, 1 μm, 5 μm, 10 μm, or 100 μm or more. Alternatively, the average pitch can be, for example, up to 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, or 0.1 μm or less are also acceptable. Naturally, this depends on the specific pattern of the wells. The average pitch is between one of the minimum and one of the maximum values ​​selected from the above range. good.

[0053] Furthermore, the pattern of wells is determined by the density of wells (i.e., the number of wells) within the defined area. It can also be characterized based on the following criteria. For example, a well is approximately 2 million / mm³. 2 It exists at this density It may be present. According to the manufacturing method described herein, the density is, for example, at least 100 / mm 2 , 1000 / mm 2, 100,000 / mm 2 , 1,000,000 / mm 2 , 2000 000 / mm 2 , 5,000,000 / mm 2 The density can be easily adjusted to different densities, such as those mentioned above. Alternatively, or additionally, this density is 5,000,000 / mm². 2 The following is from 2000 000 / mm 2 , 1,000,000 / mm 2 , 100,000 / mm 2 , 1000 / mm 2 , 100 / mm 2 The following adjustments may be made. Naturally, the density of wells on the substrate should be 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, gel materials are used. In some examples, gel formation (e.g., polymerizable) The material is supplied to a solid support in a liquid state and then converted into a gel. Examples of polymerizable materials include... , but not limited to, acrylamide, methacrylamide, hydroxyethyl meth Examples include acrylates, N-vinylpyrrolidinone, or derivatives thereof. The material is useful in preparing hydrogels. In some embodiments, the polymerizable material is copolymer. It may contain two or more different types of compounds that form a rimer. For example, acryla Mid, methacrylamide, hydroxyethyl methacrylate, N-vinylpyrrolidinone Alternatively, two or more different species of these derivatives can polymerize to form copolymer hydrogels. They can function as comonomers. Useful hydrogels are not limited to these. However, silane-free acrylamide (SFA) polymers (incorporated herein by reference) See U.S. Patent Application Publication No. 2011 / 0059865, poly(N-(5-Aji) (PAZAM, see reference) See U.S. Provisional Patent Application No. 61 / 753833 incorporated herein, for example. , the acrylamide described in WO00 / 31148 (incorporated herein by reference) and polyacrylamine formed from acrylic acid or acrylic acid containing vinyl groups. Midpolymers, for example, WO01 / 01143 or WO03 / 014392 (each The [2+2] photoaddition cyclization reaction described herein (as incorporated herein by reference) Polyacrylamide polymers formed from monomers, or U.S. Patent No. 646517 No. 8, WO01 / 62982 or WO00 / 53812 (refer to the original source for reference) These gel materials include polyacrylamide copolymers as described in the detailed specifications. Chemically treated mutants, for example, react with oligonucleotides having the corresponding reactive group. Chemically treated SFA (e.g., 5'- or 3'-alkynyl modified oligonucleotides) Azidrisis of SFAs (which generates azide-SFAs that react with ozide) is also useful. Examples of typical hydrogels and polymerizable materials that can be used to form hydrogels include, for example, U.S. Patent Application No. 61 / 753833 or, respectively, are incorporated herein by reference. It is described in National Patent Application Publication No. 2011 / 0059865. Other useful gels are It is formed by a temperature-dependent change from a liquid to a gelatinous state. For example, this Examples include, but are not limited to, agar, agarose, or gelatin.

[0055] Gel material in wells or other concave features on the surface of a structured substrate is on the surface Covalent bonding is possible. For example, PAZAM is a surface material and, by reference, specified herein. As incorporated in U.S. Patent Application No. 61 / 753833 and described in the Examples section of this Specification. It can be covalently bonded to the surface using other reagents. However, gel materials are subject to the following practical requirements. As demonstrated in the section on SFA in the examples, it is not always necessary to include wells or other concave features. Covalent bonding is not necessary.

[0056] One or more samples are present in or on the gel material present on the structured substrate. The gel-containing substrate of this disclosure is particularly useful for detecting a sample or carrying out a synthesis reaction with a sample. Therefore, various arbitrary samples on which detection, characterization, modification, synthesis, etc. are performed are These can be present in or on the gel material of the substrate described herein. The sample may contain, but is not limited to, nucleic acids (e.g., DNA, RNA, or similar substances). Body, protein, polysaccharide, cell, antibody, epitope, receptor, ligand, enzyme (for example) Examples include kinases, phosphatases, or polymerases, and small molecule drug candidates. The structured substrate can contain multiple different species from the sample library. For example, The species have various antibodies from an antibody library and different sequences from a nucleic acid library. Nucleic acids, proteins with different structures and / or functions from a library of proteins Drug candidates may also be derived from a library of combinations of molecules, small molecules, etc.

[0057] In some embodiments, the samples are distributed onto a structured substrate so that they can be dissolved individually. This is possible. For example, a single molecule from each sample can be present in each gel-containing well of the structured substrate. This is possible. Alternatively, the specimen may exist as a colony or group, and as a result, individual Molecules do not necessarily dissolve. Colonies or groups are of a single species (though multiple copies). It can be uniform in terms of the content of the sample. Taking nucleic acids as an example, each of the components on the structured substrate A cellulose can contain colonies or populations of nucleic acids, and the nucleic acids within the colonies or populations are All of them can have the same nucleotide sequence (either single-stranded or double-stranded). Such colonies undergo cluster amplification, which is described in more detail elsewhere in this specification. Alternatively, it can be fabricated by bridge amplification. Multiple repeats of the target sequence, for example, rolling circles Concatemers produced using amplification techniques can exist within a single nucleic acid molecule. Therefore, Furthermore, even if the gel material in each well on the structured substrate contains multiple copies of a single type of sample... Good. Alternatively, the colonies or populations of specimens in the well may contain two or more different species. This is possible. For example, one or more wells on a structured substrate can each contain two or more different nucleic acids. It can contain mixed colonies having different species (i.e., nucleic acid molecules with different sequences). It is possible. Two or more nucleic acid species may be present in a mixed colony in amounts that cannot be ignored, for example, mixed This makes it possible to detect multiple nucleic acids within a colony.

[0058] The sample may be attached to the gel material. This attachment may be shared or not shared. Good. Typical methods and reactants for attaching nucleic acids to gels are, for example, U.S. Patent Application Publication No. 2011 / 0059865, incorporated herein by reference, This is described in U.S. Provisional Patent Application No. 61 / 753833. The sample may be nucleic acid. Nucleic acids, via their 3' oxygen, 5' oxygen, or other positions along their length, For example, the base portion of the 3' terminal nucleotide, the base portion of the 5' nucleotide, and / or It can adhere to the gel via one or more base moieties located elsewhere in the molecule. Co-modal adhesion includes, for example, ionic interactions between nucleic acids and gels, and nucleic acids within the pores of gels. Uptake, protein-protein interactions, receptors and ligands on gels and / or nucleic acids This includes connections between dots, and other known forms.

[0059] In some embodiments, the gel coating applied to the surface contains one or more samples. Subsequently, the gel material is removed from the gap region. Therefore, the gel material remains in the gap region. The gel material in the interstitial region can adhere to one or more different specimens. After removing the gel material from the gap region, the sample is added to the gel material in the concave feature. .

[0060] The structured substrates described herein can occur in a flow cell. Typical flow cells and their manufacturing methods are also described. The law and its use are incorporated herein by reference in the U.S. Patent Application Publications. Document No. 2010 / 0111768 or No. 2012 / 0270305, or WO05 It is described in / 065814. The flow cell is prepared by the method of this disclosure. A format suitable for containing I is provided, and during synthesis-time decoding (SBS) or during the cycle. Other techniques requiring repeated delivery of reagents (e.g., iterative or cyclical steps) The sample is subjected to a synthesis or detection method (which includes [specific method / method]). Typical detection methods are described in more detail below. ru.

[0061] In some embodiments, a flow cell or other container having multiple surfaces is used. It has multiple surfaces, with gel-containing concave features (e.g., wells) on only one surface. A container may be used. Alternatively, two or more gel-containing recessed particles may be placed on the surface inside the container. A turbulence may be present. It is possible to selectively detect one or more surfaces of the flow cell. For example, opposing surfaces inside a flow cell are known in the art, such as confocal methods. This method allows for selective treatment by focused radiation. Useful confocal methods and A device for selectively directing radiation to multiple surfaces of a container (e.g., a flow cell) is, for example, U.S. Patent Application Publication 2009 / 027291, each incorporated herein by reference. It is described in U.S. Patent No. 4 or U.S. Patent No. 8039817.

[0062] This disclosure provides a method for manufacturing a substrate. This method involves (a) a solid support having a plane A step of providing a body, wherein the plane is interrupted by one or more concave features, The steps include: ( the one or more concave features being adjacent to one or more gap regions on a plane, b) A step of coating at least a portion of a solid support with a gel material, wherein A step in which the part includes at least one concave feature and at least one gap region. (c) Polish the plane to remove the gel material from at least one gap region, and at least The process may also include the step of holding a gel material within another concave feature.

[0063] Using any of the various techniques known in the art, the substrate is made into a concave feature It can be manufactured to have a nano It would be a small object that lies on an array of meter or micrometer dimensions. In such cases, nanofabrication or microfabrication techniques can be used. Examples of these techniques are as follows: Further representative nanofabrication methods are described elsewhere in this specification, such as in Example 2. The techniques of the microfabrication method are incorporated herein by reference in the respective U.S. Patent Publications. As described in Patent Application No. 13 / 661524 and U.S. Patent Publication No. 2012 / 0316086 It is being done.

[0064] One or more concave features, such as wells, are formed with a pre-generated gel material, and It can then be coated with a liquid that generates a gel material. An example of a conventional method is: Spin coating, dipping, gel flow under positive or negative pressure, or by reference Using the technique described in U.S. Provisional Patent Application No. 61 / 753833 incorporated herein This is the coating of the substrate using PAZAM, which has been generated in advance. The coating of well arrays with PAZAM is demonstrated in Example 3 below. As an example of applying a liquid to generate gel material, silane-free acrylamine in liquid state is used. and N-[5-(2-bromoacetyl)aminopentyl]acrylamide (BRAP A) is a coating of the well array, and by polymerizing the reagent on the surface A gel is generated. Coating of the array by this method is shown in Example 1 below. U.S. Patent Application Publication No. 2011 / 0059865, incorporated herein by reference. The chemical reagents and methods described in [reference] can be used. In some embodiments, for example, [reference] When a gel-containing substrate is immersed in a pre-prepared gel material, the gel material selectively fills the wells. This eliminates the need for polishing.

[0065] The sample can be added to the gel material either before or after contact with the solid support. Even after adding the body to the gel (i.e., after the gel has been generated from its precursor), the sample will not form a gel. It may be added to the generating reagent solution (i.e., before gel formation). In some embodiments, gel formation Various samples may be added before formation, and others may be added after gel formation. In one example... Then, the primer nucleic acid is added to the gel-forming solution, and the solution is allowed to form a gel (for example, SF (By polymerization that occurred in A and PAZAM). Gel formation may also occur on a solid support. Alternatively, a gel may be produced beforehand and then coated onto a solid support. In this method, the primer adheres to the gel present in concave features such as wells. It is likely. Next, a target nucleic acid complementary to the primer is added to the primer-containing gel, and As a result, after the gel material is coated onto the solid support, (hybridization occurs) (Through this process) the target nucleic acid can be attached to the gel. Hybridization of target nucleic acid This can occur optionally after the polishing step has been performed (polishing is discussed below). (Further details will be provided.) In previous examples, nucleic acids (plastic) were produced at different stages of the manufacturing process of the structured substrate. This document describes several examples of adding a substance (which functions as either an absorber or a target) to a gel. It is.

[0066] In some embodiments, the gel adheres to (or otherwise is in or on) the gel. The primer nucleic acid (present in the map) is used for capturing and / or amplifying the template nucleic acid. This can be done. Primers are universal primers that attach to 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 contains a target region that is different from other target nucleic acids in the library. Some of the target nucleic acids have the same universal adapter sequence. In this state, the target nucleic acid can be attached to the gel material, and the primer (in solution, also (Any of the substances attached to the gel) can be used to amplify the attached target nucleic acid. (That is, the target nucleic acid can act as a template for amplification.)

[0067] The methods described herein may use any of the various amplification methods. Techniques include, but are not limited to, polymerase chain reaction (PCR) and rolling saturation. RCA amplification, multi-substitution amplification (MDA), or random prime amplification (R Examples include PA). In certain embodiments, one or more primers used for amplification are used. It can be attached to the material. In the PCR embodiment, one or both used for amplification The 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 amplifier. The cone forms a bridge-like structure between two adhesive primers located on the sides of the copied template array. This is to form a structure. Typical reagents and conditions that can be used for bridge amplification are, for example, U.S. Patent No. 5,641,658, and U.S. Patent Publication No. 5,641,658, respectively, incorporated herein by reference. U.S. Patent Publication No. 2002 / 0055100, U.S. Patent No. 7115400, U.S. Patent Publication No. 200 No. 4 / 0096853, US Patent Publication No. 2004 / 0002090, US Patent Publication No. 2 007 / 0128624, and those described in US Patent Publication No. 2008 / 0009420 are described. PCR amplification can also be carried out using one of the amplification primers attached to the gel material and the second primer in the solution. A typical format using a combination of a solid-phase attached primer and a liquid-phase primer is, for example, as described in Dressman et al., Proc. Natl. Acad. Sci. USA 100:8817 - 8822 (2003), WO05 / 010145, or US Patent Publication No. 2005 / 0130173 or No. 2005 / 0064460, which are incorporated herein by reference respectively and is emulsion PCR. Emulsion PCR is an example of a format, and for the methods described herein, the use of an emulsion is optional, and it should be understood that in fact in some embodiments, no emulsion is used. Furthermore the primer does not have to be directly attached to the solid support described in the ePCR reference, but rather can be attached to the gel material described herein. In some solid-phase PCR or bridge amplification formats, the target nucleic acid can be attached to the gel material and used as a template for amplification . 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 principle that generate amplicon are , for example, Lizardi et al., Nat. Genet. 19:225 - 232 (1998) and US Patent Application Publication No. 2007 / 0099, which are incorporated herein by reference respectively , and the primer does not have to be directly attached to the solid support described in the ePCR reference, but rather can be attached to the gel material described herein. In some solid-phase PCR or bridge amplification formats, the target nucleic acid can be attached to the gel material and used as a template for amplification . Rather, it can be attached to the gel material described herein. In some solid-phase PCR or bridge amplification formats, the target nucleic acid can be attached to the gel material and used as a template for amplification . In some solid-phase PCR or bridge amplification formats, the target nucleic acid can be attached to the gel material and used as a template for amplification .

[0068] The RCA technique may be modified for use in the methods of the present disclosure. The RCA reaction and principle that generate amplicon can be used. Representative components that can be used in the RCA reaction and principle that generate amplicon are , for example, Lizardi et al., Nat. Genet. 19:225 - 232 (1998) and US Patent Application Publication No. 2007 / 0099 which are incorporated herein by reference respectively It is described in No. 208. The primers used in RCA may adhere to the gel material even in solution. It may adhere to the gel material even in solution.

[0069] The MDA technique may be modified for use in the method of the present disclosure. Some basic principles and useful conditions for MDA are incorporated herein by reference, for example, 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 (2003); Walker et al., Molecular Methods for Viru s Detection, Academic Press, Inc., 1995; Wal s Detection, Academic Press, Inc., 1995; Wal ker et al., Nucl.Acids Res.20:1691 - 96(1992); U.S. Pat. No. 5455166; No. 5130238; and No. 6214587. The primers used in MDA may adhere to the gel material even in solution. It may adhere to the gel material even in solution. It may adhere to the gel material even in solution.

[0070] In certain embodiments, combinations of the amplification methods exemplified above can be used. For example, RCA and MDA can be used in combination. RCA is used to generate concatemeric amplicons in solution (e.g., using a liquid - phase primer). Next, the amplicons can be used as templates for MDA using primers attached to the gel material. In this example, the amplicons generated after the combined steps of RCA and MDA will adhere to the gel material. The amplicons will generally contain concatemeric repeats of the target nucleotide sequence. the amplicons can be used as templates for MDA using primers attached to the gel material. In this example, the amplicons generated after the combined steps of RCA and MDA will adhere to the gel material. The amplicons will generally contain concatemeric repeats of the target nucleotide sequence. the amplicons can be used as templates for MDA using primers attached to the gel material. In this example, the amplicons generated after the combined steps of RCA and MDA will adhere to the gel material. The amplicons will generally contain concatemeric repeats of the target nucleotide sequence. the amplicons can be used as templates for MDA using primers attached to the gel material. In this example, the amplicons generated after the combined steps of RCA and MDA will adhere to the gel material. The amplicons will generally contain concatemeric repeats of the target nucleotide sequence. the amplicons can be used as templates for MDA using primers attached to the gel material. In this example, the amplicons generated after the combined steps of RCA and MDA will adhere to the gel material. The amplicons will generally contain concatemeric repeats of the target nucleotide sequence.

[0071] The amplification method exemplified above produces a gel-containing feature having multiple copies of the target nucleic acid. It can be used to achieve this. Individual features such as wells are provided by RCA. These are produced in the form of single-molecule concatemers or by bridge PCR. A collection of clones of nucleotide sequences in the form of multiple nucleic acid molecules that have the same sequence, such as those that are used. It can have a group. Generally, nucleic acids that have several copies of the amplified target are It will adhere to the material.

[0072] In some applications, individual gel-containing wells (or other concave features) are mostly It is occupied by an amplicon derived from the first target nucleic acid, and also the second target nucleic acid or generated during amplification. Contaminating amplicons derived from spontaneous mutations may occupy the array at low levels. It may have one or more amplification positions that have a Bell contamination amplicon, and this This can have an unacceptable impact on the subsequent use of the array. For example, if the array is used for detection... When used in applications, an acceptable level of contamination is permissible for the signal-to-noise ratio or resolution of the detection technique. It seems that this will not cause any shock in an unacceptable way. Accordingly, Loan properties generally refer to the specific use of arrays prepared by the method described herein. Or it will be related to the application. Individual wells or other features for specific applications Typical acceptable levels of contamination are not limited to these, but include a maximum of 0.1% and 0.5%. , containing 1%, 5%, 10%, or 25% contaminated amplicons. The array is representative of these. Including one or more wells or other features having an amplicon with a typical level of contamination. This is possible. For example, up to 5%, 10%, 25%, 50%, 75%, or 1% of the array. A 00% feature may have some contamination amplicons.

[0073] The gel material coated on the surface of the solid support may be covalently bonded to the support. As described above, the step of attaching samples such as nucleic acids to the gel material involves various differences in the structured substrate. This can be carried out during the manufacturing stage. Therefore, before the sample is attached to the gel material, The gel material can then be attached to the solid support. Adhesion is a useful chemical reaction, for example, but not limited to, incorporated herein by reference. As described in U.S. Provisional Patent Application No. 61 / 753833, or as demonstrated in Example 3 below. This can be carried out using the materials provided. The covalent bonding of the gel material to the solid support is Please understand that this does not necessarily apply to all embodiments. Therefore, The following steps involve polishing the gel-coated support or using the polished substrate. The `P` is optional and not required, but it can be shared with concave features such as wells. This can be performed on a substrate having a bonded gel material.

[0074] The method described herein includes the step of removing the gel material from the surface of a solid support. This can be done. The gel material coated on the solid support can be one of various techniques. It can be selectively removed from the gap region using a mechanical method. For example, gel material can be mechanically removed. By polishing, concave features and void regions can be removed from solid supports. Mechanical polishing can be performed by applying abrasive force to the surface of a solid support. Representative methods include polishing with a slurry of beads, wiping with a sheet or cloth, scraping, etc. An example of polishing involves using a lint-free (cleanroom grade) wipe coated with a 3 μm silica bead slurry (10% w / v in water) to remove the gel between the grids. A polishing wheel / grinder may also be used with this slurry. Mechanical polishing can also be achieved using a fluid jet or an air jet to remove the gel from the gap region. Polishing may require chemical polishing, such as exposure to benzoyl peroxide or diluted hydrogen peroxide (e.g., as described in Kurenkov et al., Russian Journal of Applied Chemistry, 75:1039 - 1050 (2002); Caulfield et al., Polym. 44:1331 - 1337 (2003); and Caulfield et al., Chem. Rev. 102:3067 - 3083 (2002)) through hydrolysis or decomposition based on acrylamide groups. Polishing may also require a combination of chemical and mechanical polishing methods. This involves mechanically peeling using a chemical slurry containing a colloidal suspension of particles, and then chemically dissolving the gel material in the separated portion from the gap region. Other methods for polishing or cleaning the gap region include adhesion - based techniques, for example, coating a rigid planar adhesive film with an affinity for the gel material on the surface and bringing it into close contact with the gel material in the gap region (e.g., through chemical bonding). The mechanical removal / peeling of this adhesive film will mechanically remove the gel material from the gap region while leaving the gel material in the concave features.

[0075]

[0076] [[ID=;21]]

[0077] <000100;1>

[0078]

[0077] In another example, thiophosphate-grafted SFA is used in the interstitial regions on the surface as follows. It can be removed by using a water-dampened Whatman wipe to remove aluminum oxide (approximately 100ml). It may be applied to g (0.3um) or steel beads. Then, using uniform pressure... The formed slurry may then be rubbed into small concentric circles on the surface of the solid support. Then, use a Whatman wipe dampened with clean water to remove the slurry from the surface. This can be done. Mechanical and chemical methods illustrated herein for removing the gel material from the gap region. Using a polishing method, the gel material is removed from the gap region, whether or not the gel material has been removed. It can also be inactivated. For example, gel materials have the ability to adhere to samples such as nucleic acids. It can be inactivated in terms of its ability to promote nucleic acid amplification.

[0078] A method for fabricating an array is to (a) provide a solid support having a surface with multiple wells, The well contains a gel material, and the wells are separated from each other by interstitial regions on the surface. The gap region isolates the gel material in each well from the gel material in the other wells. (b) deliver the library of target nucleic acids into wells of a solid support, and in each well, This step involves creating an array of wells containing a single type of target nucleic acid attached to a material. This is a step in which different wells in the array have different target nucleic acid species derived from the library. (c) The target nucleic acid attached to the gel material in the array wells is amplified, and each well of the array This may include a step of creating a clonal population of individual target nucleic acids.

[0079] In some embodiments, the structured substrate described herein is made from a mixture of multiple different By conveniently delivering the samples to individual locations on the substrate, the advantages of constructing an array are realized. The structured substrate brings about each individual gel-containing well (or other concave features). -) This facilitates the selective capture of a single sample from the sample mixture in contact with the substrate. Patterning and filling of gel-containing wells (or other concave features) on a structured substrate. Efficiency is measured based on sample density and the presence of a single type of sample for each feature. It can be adjusted to obtain an array with desired properties such as purity. For example, high density High-density wells are used to obtain a high-density sample on the array, while low-density wells are used. This is used to obtain a low-density sample on the array. Alternatively, or additionally, The concentration or volume of the sample in the liquid may be increased to obtain a high-density sample on the array, or it may be decreased. A low-density sample may be obtained on the array by removing it from each gel-containing well (or other concave form). The average purity of the Char samples is described in more detail below and demonstrated in the Examples section. This can be adjusted by changing the characteristics of the conditions for delivering the substrate or sample. Cut.

[0080] In certain embodiments, the size or volume of the well (or other concave feature) is adjusted. This can affect the purity of the captured sample. For example, the well can be a specific type It may have an area or volume of gel material that accommodates only a single sample of the p, therefore Steri-exclusion prevents multiple sample molecules from being captured or seeded into the wells. This prevents steric exclusion. Stereoexclusion can be particularly useful for large samples such as nucleic acids. More specifically, The well (or other concave feature) is the excluded volume of the target nucleic acid seeded on the substrate. A gel surface having an area less than or equal to its diameter can be created. The exclusion volume of the target nucleic acid and Its diameter can be determined, for example, from the length of the target nucleic acid. The excluded volume of the nucleic acid and Methods for determining the diameter of the excluded volume are, for example, incorporated herein by reference. National Patent No. 7785790; Rybenkov et al., Proc.Natl.Acad.Sc iUSA90:5307~5311(1993); Zimmerman et al., J. Mol.Biol.222:599~620(1991); or Sobel et al., Bio It is described in polymers 31:1559~1564 (1991). Steric exclusion The conditions are incorporated herein by reference in U.S. Patent Application No. 13 / 661524 and As described in U.S. Patent No. 7,785,790, it can be easily used in the structured substrate of the present disclosure. .

[0081] In some embodiments, the well (or other concave feature) is transported to the amplification position. This can provide a gel surface having a substantially larger area than the diameter of the excluded volume of the target nucleic acid. Please understand this. Therefore, the area of ​​the feature is sufficient so that 3D exclusion does not occur. There is a great possibility of this.

[0082] In some embodiments, for example, in the steric exclusion embodiment described above, a library of target nucleic acids Before the amplification process begins, the gel-containing wells (or other concave features) of the solid support are... ) may be delivered to a substrate. For example, the target nucleic acid may be seeded onto the gel material in the substrate. Under these conditions, it can be delivered to a structured substrate. The substrate can be optionally cleaned and gel Target nucleic acids that are not seeded, as well as any other materials that are not needed for subsequent processes or substrate use. It can be removed. Amplification may include one or more of the techniques previously described herein. can.

[0083] In an alternative embodiment, the library of target nucleic acids is placed in the gel-containing wells of a solid support (or It may also be delivered to other concave features, and the amplification process can occur simultaneously with the seeding event. For example, sowing is incorporated herein by reference, for example, U.S. Patent Application No. 61 / 71 Utilize the kinetic exclusion described in No. 5478. This can occur under certain conditions. Kinetic exclusion effectively blocks the occurrence of another event or action. This can occur when the action takes place at a speed fast enough to cause it. In the case of an array of gel-containing wells... In the wells, target nucleic acids are randomly seeded from the solution, and copies of the target nucleic acids are produced through amplification. This allows each seeding position to be filled to its full capacity. Conditions under which amplification rate exceeds seeding rate The sowing and amplification processes can be carried out simultaneously below. As such, the first target At the location where the target nucleic acid is seeded, copies are made at a relatively fast rate, thereby creating a second nucleic acid. This effectively blocks the seeding of the subsequent target. Similarly, kinetic exclusion prevents the subsequent target from being seeded at the amplification site. Relatively fast rate for making copies or first copies of nucleic acids, compared to the first copy of the target nucleic acid The relatively slow speed at which copies are made can be utilized. For example, kinetic exclusion is For subsequent copies to be made and fill their positions at a relatively fast rate, the gel-containing wells Delay in the formation of the first copy of the seeded target nucleic acid (e.g., delayed or slow activity) This can occur due to (sexualization). In this example, each gel-containing well contains several different standards. It may be seeded with target nucleic acids (for example, several target nucleic acids before amplification). (They can be present at each position). However, no matter what the target nucleic acid is, the first copy - Formation can be activated randomly, and as a result, the average rate of the first copy formation is lower than that of subsequent copies. This is relatively slower than the rate at which P is generated. In this case, in each gel-containing well, Although several different target nucleic acids could have been seeded, kinetic exclusion determined that these target nucleic acids were... Only one type is amplified. Generally, gel-containing wells (or other concave features) The method described in U.S. Patent Application No. 61 / 715478, which is incorporated herein by reference. It can function as a location for amplification and array configurations.

[0084] As an alternative to delivering multiple different samples from a mixture to individual gel-containing concave features The sample may be delivered discretely from the pure raw material to individual features. Similarly, the synthetic composition Samples may be synthesized in individual features by discrete delivery of components (for example, (Creotide precursors can be continuously delivered to synthesize nucleic acids). Pure sample or Typical delivery methods for components for in-situ synthesis of specimens are limited to these. However, inkjet array positioning and photolithography array synthesis methods are mentioned. It can be done. Useful photolithography methods are available from GeneChip® MicroA Ray is manufactured by Affymetrix (Santa Clare, CA) Those used on a commercial basis, or incorporated herein by reference, respectively U.S. Patent No. 5,324633, No. 5744305, No. 5624711, No. 60 The following are also described in No. 22963, No. 6291183, and No. 6416949. This includes inkjet positioning methods, such as SurePrint®. Commercially produced by Agilent (Santa Clara, CA) for printing ray designs. Products that have been commercialized, or those incorporated herein by reference as U.S. 63 No. 37393, No. 6419883, No. 6420180, or No. 6689319 The methods described in issue number are also useful. Such methods are the gel-containing features of the disclosure. It can be easily modified to direct delivery to -.

[0085] The gel material in a specific concave feature must contain only one type of sample. That's not the case. Rather, in some embodiments, the concave feature is several different types The sample may be contained within the gel. For example, by the bullseye reference marker in Figure 5 This is demonstrated. The reference marker contains two "bright" ring-shaped channels, and these Each of the two channels contains gel material, and the gel material in each bright channel The material attaches to multiple different nucleic acid colonies. The nucleic acid colonies in the bright channel are In the amplification method, several different species of target nucleic acids that function as templates are seeded into each ring. It was formed by this process. Additionally, the reference marker also has two "dark" ring-shaped regions. Includes. The dark ring is composed of a spaced surface pattern. Typical bullseye is This is formed by alternating dark and bright rings within a concentric pattern. (See diagram) In example 5, the structured substrates are clones that generally originate from a single nucleic acid target. It also includes gel-containing wells containing clusters. The wells are between the light ring and the dark ring. It exists within a ring-shaped band. Therefore, the reference is the alternating pattern of gap rings. It has a well-containing band and channel ring. Using the same amplification technique, in the well Clone nucleic acid colonies and mixed populations in reference markers were grown simultaneously (see below for details) (See Example 3). Other examples of standards with alternating ring patterns are shown in Figures 3B and 3C. vinegar.

[0086] This disclosure further provides a method for detecting a sample. This method involves (a) a solid having a plane A step of providing a body support, wherein the plane is intermittently connected by one or more concave features The concave feature contains a gel material, and one or more concave features are on a plane. Adjacent to one or more gap regions, the gap regions substantially do not contain gel material, and the gel material is targeted (b) the step of adhering to or containing the sample, and (b) the target sample specifically probes (c) The steps of bringing a solid support into contact with the probe under conditions of interaction with the solid support The system detects the body and identifies at least a subset of target specimens that interact with one or more probes. It may include separate steps.

[0087] In a particular embodiment, nucleic acid is the sample to be detected, and the concave feature is a well. For example, a method for detecting nucleic acids is (a) a solid support having a surface and a nucleic acid library. A step of forming a body, wherein the surface has a plurality of wells, and the wells contain a gel material. The wells are separated from each other by gaps on the surface, and these gaps form the gel of each well. The material is isolated from the gel material in the other wells, and a single target nucleic acid from the library is selected. (b) The step of attaching to the gel material in each well, and the step of binding the solid support to the target nucleic acid. (c) The step of making contact with at least one probe, and the step of detecting a solid support, The procedure includes the step of identifying a well having a target nucleic acid species that binds to one probe. It is possible.

[0088] The structured substrates of this disclosure containing nucleic acid arrays can be used for any of the following purposes. It is possible. A particularly desirable use of nucleic acids is to hybridize them with target nucleic acids having complementary sequences. It acts as a capture probe for soybeans. Once the capture probe hybridizes... The targeted nucleic acid can be detected, for example, by a label added to the capture probe. Methods for detecting target nucleic acids via hybridization of capture probes are relevant to this technology. Known as, for example, U.S. 758, which is incorporated herein by reference. No. 2420, No. 6890741, No. 6913884, or No. 6355431 U.S. Patent Application Publication No. 2005 / 0053980, or U.S. Patent Application Publication No. 2009 / 018634 This includes items listed in item 9 or item 2005 / 0181440. For example, The label is captured by hybridizing the capturing probe with a target probe that has a label. The probe can be replenished. In another example, the target probe can be hybridized with the capture probe. By soybean fermentation, and as a result ligation to labeled oligonucleotides, ( (for example, by ligase activity) or by adding labeled nucleotides (for example) (By polymerase activity) the capture probe can be extended, and the label can be captured by the capture probe. It can be refilled.

[0089] Nucleic acid arrays can also be used in sequencing methods, such as sequencing at synthetic time (SBS). Yes, it is possible. To put it simply, SBS uses one or more labeled nucleotides to target nucleic acids, DNA. It can be initiated by contact with polymerase, etc. Using the target nucleic acid as a template The feature to which the primer is extended introduces a detectable labeled nucleotide. Optional By selection, the labeled nucleotide undergoes a reversible termination. It can further include properties, which means that the nucleotide is added to the primer. This property terminates further primer elongation. For example, it has a reversible termination portion. A nucleotide analog is added to the primer, thereby delivering the deblocking agent to the portion. Until the term is removed, subsequent extensions cannot occur. Therefore, a reversible termination method. In embodiments utilizing this method, the deblocking agent may be delivered to the flow cell (detection is performed beforehand). (This will be done later). Cleaning may be performed between the various delivery steps. Then the cycle is n The process is repeated several times to extend the primer by n nucleotides, thereby detecting a sequence of length n. This is possible. Typical SBS methods, fluid systems, and detection platforms are described in this document. It can be easily adapted for use with arrays manufactured by the method shown, for example. For example, Bentley et al., Nature 45, which are incorporated herein by reference. 6:53~59(2008), WO04 / 018497, WO91 / 06678, WO0 7 / 123744, U.S. Patent No. 7057026, U.S. Patent No. 7329492, U.S. Patent No. 7211 Patent No. 414, No. 7315019, or No. 7405281, and U.S. Patent Application Publications It is listed in issue number 2008 / 0108082.

[0090] Using other sequencing methods that utilize cycle reactions, such as pyrosequencing It may also be possible. Pyrosequencing is a method in which, when a specific nucleotide is introduced into a nascent nucleic acid chain, Detecting the release of inorganic pyrophosphate (PPi) (as 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 No. 6210891; U.S. Patent Nos. 6258568 and 62743 (No. 20). In pyrosequencing, the released PPi is transmitted to ATP sulfurylase. It can be detected by converting it to adenosine triphosphate (ATP), and the resulting ATP It can be detected via luciferase-emitting photons. Therefore, the sequencing reaction is It can be monitored via a mineescence detection system. It is used in fluorescence-based detection systems. The excitation radiation source is not necessary for pyrosequencing techniques. Useful fluid systems, detection The apparatus and method are used to apply pyrosequencing to the array of this disclosure. For example, the WIPO patent applications PCT / US incorporated herein by reference, respectively. Patent No. 11 / 57111, U.S. Patent Application Publication No. 2005 / 0191698, U.S. 7 This is described in U.S. Patent No. 595883 and U.S. Patent No. 7,244,559.

[0091] Decoding during ligation is also useful, for example, as each is incorporated herein by reference. Shendure et al., Science 309:1728~1732 (2005); The items described in U.S. Patent No. 5,599,675 and U.S. Patent No. 5,750,341 This includes, for example, the Bai incorporated herein by reference. 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 described in WO1989 / 10977 This may include decoding at ligation time. In both decoding and auto-decoding, the gel-containing well (or other concave feature) The nucleic acids present are subjected to a repeated cycle of oligonucleotide delivery and detection. The fluid systems for the SBS method described in this book or listed in the literature herein are ligase To easily adapt to reagent delivery for decoding during fusion or during hybridization. This can be done. Typically, oligonucleotides are fluorescently labeled, as described herein or in this specification. The same type of fluorescence detector used in the SBS method described in the literature listed in this book is used for detection. It can be released.

[0092] Some embodiments involve methods requiring real-time monitoring of DNA polymerase activity. This can be used. For example, nucleotide incorporation can be achieved by fluorophore-supported poly Fluorescence resonance energy transfer (FRE) between melanase and γ-phosphate-labeled nucleotides. Detected by T) interaction or by zeromode waveguides method Yes, it is possible. FRET-based sequencing techniques and reagents are disclosed, for example, by reference. Levene et al., Science 299, 682-686, which are more incorporated herein. (2003);Lundquist et al., Opt.Lett.33, 1026~1028( 2008); Korlach et al., Proc.Natl.Acad.Sci.USA105 It is described in 1176-1181 (2008).

[0093] Some SBS embodiments involve the emission of photons when nucleotides are incorporated into the extension product. Includes detection. For example, sequencing based on the detection of emitted photons is Ion Torr ent (Guilford, CT, a subsidiary of Life Technologies) From commercially available electrical detectors and related technologies or each incorporated herein by reference U.S. Patent Application Publication No. 2009 / 0026082; 2009 / 01275 No. 89; No. 2010 / 0137143; or No. 2010 / 0282617 The sequencing methods and systems described herein may be used. In certain embodiments, emitted photons The electrical detector used to detect this may be modified to include a well, and the well may be this The gel material described in the specification may be included.

[0094] Another useful application of the arrays described herein is gene expression analysis. Gene expression is determined by RNA Detection or quantification can be performed using sequencing methods, such as digital RNA sequencing. RNA sequencing is a sequencing method known in the art. For example, this can be carried out using the above. Gene expression is hybridized Detection or quantification can also be performed using the hybridization method, which involves directly hybridizing the array. This can be done by using a multiplex assay, The product is detected on the array. The array of this disclosure is genomic DNA from one or more individuals. It can also be used to determine the genotype of a sample. This can be done on the array of this disclosure. Representative methods for array-based expression and genotype analysis are as follows: U.S. Patent No. 7,582,420 and No. 6,890,741 are incorporated herein by reference. No. 6913884 or No. 6355431, or U.S. Patent Application Publication No. 2 Issue No. 005 / 0053980, No. 2009 / 0186349, or No. 2005 / 0 It is listed in issue 181440.

[0095] Some applications of the arrays of this disclosure are exemplified above in relation to ensemble detection. However, multiple copies of the target nucleic acid are present in each feature and are detected together. In an alternative embodiment, a single nucleic acid is either the target nucleic acid or its amplicon, each phi It can be detected by a curve. For example, a gel-containing well (or other concave feature) It is configured to contain a single nucleic acid molecule having the target nucleotide sequence to be detected. Yes, it is possible. Any of the various single-molecule detection methods can be used, for example, resolution To detect at a raised position, or to use a more sensitive marker, the above AN This includes modified sample detection methods. Examples of other available single-molecule detection methods are listed below. U.S. Patent Application Publication No. 2011 / 0312529, incorporated herein by reference. As described in National Patent Application No. 61 / 578684 and U.S. Patent Application No. 61 / 540714 It is being done.

[0096] For example, the gel-containing substrate of this disclosure prepared by the method described herein does not necessarily It should be understood that this is not used in the detection method. Rather, the structured substrate is used for nucleic acid It can be used to store braley. Accordingly, the structured substrate has The nucleic acids can be stored in a state where they are contained within a gel. For example, a gel-containing well attached to the nucleic acids. The substrate having the following conditions: dry, frozen (e.g., in liquid nitrogen), or in a solution that protects nucleic acids. It may be stored inside. Alternatively, or additionally, the structured substrate may contain the nucleic acid library. It can be used for replication. For example, it has a gel-containing well that adheres to nucleic acids. The substrate can also be used to fabricate duplicate amplicons from one or more wells on the array. good.

[0097] The following examples are for illustrative purposes only and are not intended to limit the present invention. do not have. [Examples]

[0098] Multiwell substrate coated with silane-free acrylamide This example involves coating a nanowell substrate with silane-free acrylamide (SFA). Then, grafting is performed using a thiophosphate primer, followed by gel grafting. Hybridizing the MAR with complementary fluorescent oligonucleotides confirmed the success of the functionalization method. This demonstrates that it is possible.

[0099] The chip substrates commonly used in the manufacture of BeadChips are made by Illumina (Sa The chip was obtained from Diego, CA. The chip is made of silicon or Zeonor (Ze Composed of (on Corp., Tokyo, Japan), arranged in a hexagonal pattern with a pitch of 1.5 μm. It had a 0.5 μm well, but the well did not contain any beads. As described and as shown in Figure 1, the chip was patterned with a gel pad.

[0100] Place the tip in a gasket-sealed chamber and replace it with the fluid reagent for SFA generation. Oxygen was removed by [method]. SFA was polymerized on a tip in the chamber. SFA was produced. The reagents and polymerization conditions are separately referred to in the U.S. Patent Application Publication No. It was described in issue 2011 / 0059865. A sealed chamber is used to place the polymerization mixture into chips. Used for direct contact, the free radical polymerization of SFA is an air-instability process. Therefore, complete removal of air was thoroughly carried out. After polymerization, U.S. Patent Application Publication No. 2011 / 0059 As described in No. 865 and below, the primer is mixed with SFA polymer in a sealed chamber. Rafted. BeadChip polymer coated with a primer-containing solution. Spread the mixture over the entire surface of s, then incubate the mixture at 65°C for 1.25 hours. (The entire sealed assembly was placed in a large oven.)

[0101] This method results in a uniformly coated substrate. See the panel in Figure 2 for a sample image. As shown in A, in order to create discontinuous polymer regions, the following are located between the wells on the substrate. Excess polymer in a slurry of aluminum oxide nanoparticles (300 nm in diameter) in distilled water. It was removed by a mechanical polishing method using 3-micron silica particles at 10 wt%. Lee (Kisker Biotech GmbH, Steinfurt, Germany) also uses it. Yes, you can. Use lint-free optical tissue and rub the surface with nanoparticle slurry by hand. After washing to remove slurry and polymer residue, the fluorescently labeled probe was... The solution was hybridized to the chip. Images taken using a fluorescence microscope show that this method... This demonstrates that we were able to fabricate polymer features with empty void regions (Figure 2, panels B-C). .

[0102] These results indicate that the fluorescence intensity of the gel-filled wells is such that there is no signal from the interstitial region. In contrast, this indicates that they were spatially discrete. Furthermore, these results suggest that nanofabrication Gel patterning can be achieved using non-covalently bonded gel material on a substrate having wells. It also demonstrated that it is possible. [Examples]

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

[0104] This process starts with a blank substrate / wafer and is performed using micro or nano fabrication methods. This pattern can be introduced onto the substrate. The substrate / wafer material is ordinary silicon. It may be any of glass, plastic, COC, or any of the various constructable materials. Typical techniques for incorporating patterns into substrates include photolithography and nanoin. Print lithography, embossing structures onto plastic / COC-based materials, and Injection molding of plastic or COC into a master mold having a patterned structure Includes. Photolithography-based techniques typically involve the use of photoresist. It requires patterning with a stepper or mask aligner and exposure to radiation. This transfers the pattern present on the reticle / photomask to the photoresist, and then The resist is developed to obtain a structured film (photoresist) on the top of the substrate. The substrate is, in some cases, the final substrate that can be used for subsequent gel coating, or a resin The pattern in the image may be transferred to the substrate by a subsequent process. Subsequent processing step These are typically reactive etching (plasma-based etching) or wet etching. This includes chemically based methods. When transferring a pattern to a substrate, the patterning photoresist is used. The chlorine is then removed to obtain a patterned substrate for subsequent gel coating. It is desirable to use a sacrificial material film such as lum or titanium (metal) under the photoresist. In some cases, the photoresist pattern is first transferred to a metal film, and then this film is hard mass By using it as a tool, the pattern is transferred to the substrate. After the pattern is transferred to the substrate, the film It removes and is therefore considered sacrificial to the processing process. Nanoimprint When using photography, the imprinted photoresist can become a sacrificial material, and the patterned area It can be used similarly as an intermediate tool to transfer the resist to the substrate, or the imprinting resist follows The resist variation can be used as input to the coating step. The following examples of resists that are likely to retain the pattern are sol-gel based materials. cormorant.

[0105] Figure 3 shows a diagram illustrating how structured substrates can be manufactured, and this is explained below. Images of the patterned substrate are shown in Figures 3B and 3C at various magnification levels.

[0106] The fabrication of chemically specific gel pads on sequencing substrates / flow cells is This embodiment may require one or more of the nanofabrication methods described earlier. The process involves, by choice, one or more chemical treatment steps, for example, then gel via silane. This may include a silanization treatment to bond the rimer to the substrate. This is followed by chemical / mechanical polishing (CM). P) removes all gap polymer from the substrate surface. The polishing process is performed using top dow. The material is removed in a specific manner, and the structured features in the substrate are effectively removed from the plane of the gap region of the array. Since it is effectively offset, polishing removes the gap from the structured feature. The rimer is removed. If the polishing process is stopped after the optimal time has elapsed, the structure will have a polymer coating. The gel is retained, and the polymer is no longer present in the gap region. Next, the patterned gel pad substrate is formed. Graft the target nucleic acid onto the primer, seed the target nucleic acid on the gel pad, and then place the target nucleic acid on the gel pad It is used as a template for creating nucleic acid clusters. [Examples]

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

[0108] The substrate was manufactured as follows: Nanowell substrate (diameter 400 nm, pitch 1.5 μm) Wells with a depth of 300 nm were fabricated using nanoimprint lithography. Minosylane (APTES or APTMS) monolayer / multilayer is applied to a substrate using chemical vapor deposition. The entire surface was vapor-deposited. Next, 1 ml of NHS acrylate solution was added to the surface, and By covering it with a thin glass coverslip and allowing the reaction to proceed at room temperature for 1 hour, the concentration 100 mM N-hydroxysuccinimide acrylate (Aldrich PN The phosphate-buffered saline (pH 7.4) from 8060) was reacted with the aminosilane surface. Next, 500 μl of 2 wt% PAZAM aqueous solution was used to create a newly formed acrylamide functional The polymer (PAZAM) is applied to the surface by spin coating. PAZAM did so. PAZAM is incorporated herein by reference in U.S. Provisional Patent Application No. 61 / 753. The synthesis was carried out as described in issue 833. Then, the PAZAM-coated substrate was heated at 60°C for 1 As a result of heating for a certain amount of time, covalent bonds were formed between the polymer and the surface. The polymer surface was polished with a slurry of 3 μm SiO2 particles dissolved in 10 wt% water. It was removed by the following: Janeway surface (acryloyl chloride + DIPEA(Me In the above method, use CN)) instead of the aminosilane-coated surface. It may be used.

[0109] Next, U.S. Provisional Patent Application No. 61 / 753833, incorporated herein by reference As described, the patterned polymer substrate was grafted onto the primer. Next, dye labeling ( The reverse complementary sequence of the grafted primer (Cy5) t) is exposed to the surface in 1XPBS buffer at a complementary sequence concentration of 20 μM, and then the surface is... The substrate was washed with 50 ml of 1 PBS buffer using a spray bottle. The labeled complement on the substrate was removed by C Set to Y5 scan channel and use PMT setting 450 with FLA9500Typh The image was created using oon Imager. The labeled complementary material on the substrate was also visible using a high-resolution microscope. Image the pattern or polymer / plastic so that no polymer / primer remains in the gaps. The image was shown (Figure 4). Next, phiX DNA was seeded onto the substrate, and according to this specification by reference. As described in U.S. Patent Application No. 61 / 715478 incorporated therein, the cluster grows Ta.

[0110] Flow cells containing cluster-containing substrates are processed using HiSeq2000 (Illumina, In Sequence determination was performed on c. San Diego, CA. Patterned sequencing Using an algorithm (rigid body registration) to extract the raster position, successfully High-quality sequencing metrics were obtained. (Figures 5 and 6). The sequencing results showed that the occupancy rate relative to clonality was the same as that of a standard pore. This shows that the results were surprisingly higher than predicted by the Son distribution. In particular, as shown in Figure 7 with an "×" The average occupancy rate relative to clonality measured for the sequenced sequences shown was: It lies on the boundary of the Poisson curve and approaches the straight line of the ideal cloning rate.

[0111] Throughout this application, various publications, patents, or patent applications have been referenced. The disclosure of the publication is incorporated into this application by reference in its entirety, and the most relevant part of the present invention. To explain the new technology more completely.

[0112] The term "includes" in this specification means that there are no restrictions and that it includes only the listed elements. It is not intended to be limited to, but to also include any additional elements.

[0113] Although the present invention has been described with reference to the above embodiments, various methods will be described without departing from the present invention. It should be understood that modifications are possible. Therefore, the present invention is limited to the claims only. It is limited to.

Claims

1. (a) A step of providing a solid support including a planar surface, wherein the planar surface is interrupted by one or more wells, the wells are formed by indentations within the solid support, the one or more wells are bounded by one or more void regions on the planar surface, and the depth of each well is at least 0.1 μm. (b) The step of depositing a silane layer on at least a portion of the solid support, (c) A step of bonding the gel material to the solid support via a silane layer, (d) The step of removing the gel material from the gap region, (e) The step of grafting a primer onto the gel material in the well, A method for preparing an array of primer nucleic acids, including [the specified component].

2. The method according to claim 1, wherein the plurality of wells form an array having a repeating pattern.

3. The volume of each of the aforementioned wells is a maximum of 1000 μm ３ Each of the aforementioned wells has a maximum thickness of 100 μm. ２ The method according to claim 2, wherein the surface includes an opening, or the wells in the pattern have a pitch of 5 micrometers or less, or a combination thereof.

4. The method according to claim 1 or 2, wherein the gel material is a hydrogel.

5. The method according to claim 4, wherein the gel material is a silane-free acrylamide or poly(N-(5-azidoacetamidylpentyl)acrylamide-coacrylamide).

6. The method according to claim 1 or 2, wherein the removal of the gel material includes polishing the planar surface to remove the gel material from the gap region.

7. The method according to claim 4, wherein the removal of the gel material includes polishing the planar surface to remove the gel material from the gap region.

8. The method according to claim 6, wherein the polishing includes mechanical abrasion by wiping or rubbing the surface of the solid support, or chemical polishing.

9. The method according to claim 7, wherein the polishing includes mechanical abrasion by wiping or rubbing the surface of the solid support, or chemical polishing.

10. (a) A step of providing a solid support including a planar surface, wherein the planar surface is interrupted by one or more wells covered with a gel material, the wells are formed by indentations within the solid support, the one or more wells are bounded by one or more void regions on the planar surface, the void regions lack gel material, the depth of each well is at least 0.1 μm, and the primer nucleic acid is bound to the gel material in the wells. (b) The step of contacting the primer nucleic acid with the target nucleic acid, thereby hybridizing the target nucleic acid with the primer nucleic acid, and seeding the target nucleic acid onto the gel material in the well, (c) The step of extending the primer nucleic acid with a nucleotide labeled using the target nucleic acid as a template, thereby incorporating the labeled nucleotide, (d) A step of detecting each of the incorporated labeled nucleotides and thereby determining the sequence of the target nucleic acid, A method for determining the sequence of a target nucleic acid, including [the specified element].

11. The method according to claim 10, wherein the plurality of wells constitute an array having a repeating pattern.

12. The volume of each of the aforementioned wells is a maximum of 1000 μm ３ Each of the aforementioned wells has a maximum thickness of 100 μm. ２ The method according to claim 11, wherein the openings of the surface are included, or the wells in the pattern have a pitch of 5 micrometers or less, or a combination thereof.

13. The method according to claim 10, wherein the gel material is bonded to the solid support via silane.

14. The method according to any one of claims 10 to 13, wherein the gel material is a hydrogel.

15. The method according to claim 14, wherein the gel material is a silane-free acrylamide or poly(N-(5-azidoacetamidylpentyl)acrylamide-coacrylamide).

16. The method according to any one of claims 10 to 13, wherein the primer nucleic acid is configured for capturing and / or amplifying a target nucleic acid.

17. The method according to claim 14, wherein the primer nucleic acid is a universal primer configured to hybridize to a universal adapter sequence that attaches to different target nucleic acids in the library.

18. The method according to claim 15, wherein the primer nucleic acid is a universal primer configured to hybridize to a universal adapter sequence that attaches to different target nucleic acids in a library.

19. The method according to claim 17 or 18, wherein the primer nucleic acid comprises two primer species bound to the gel material in the well.

20. The method according to claim 10, wherein the detection is selected from the group consisting of optical detection and electrical detection.

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