Gel patterned surfaces

Cross-reference to related applications This application claims the interests of US Provisional Patent Application No. 61/769289 filed February 26, 2013 and US Application No. 13/787396 filed March 6, 2013. All of the above applications are incorporated herein by reference in their entirety.

The present disclosure generally has specific applicability to nucleic acid arrays for high-throughput analysis of genomics with respect to solid phase analytical chemistry.

The task of classifying human genetic mutations and correlating them with disease susceptibility is in a position to benefit from advances in genome-wide sequencing. This classification effort can be expected to identify markers in each person's genome, their susceptibility to disease, their responsiveness to specific therapies such as prescription drugs, dangerous drug side effects and other medical effects. It will help medical professionals in determining susceptibility to possible properties. Classification efforts have made considerable progress. This is primarily due to commercial genome decoding methods that are reasonably cost effective and allow the subject to be evaluated in the field of study to be examined. Improvements in sequencing methods are also needed to accelerate classification efforts. Moreover, relatively expensive sequencing has prevented technology from moving beyond research centers to clinics where physicians can obtain sequences of patients in the general population.

Sequencing methods and the systems used to implement them utilize a complex set of techniques. Improvements that result in substantial cost savings have been demonstrated in some of these techniques. However, it is difficult to estimate, if any, which techniques have improvements that lead to cost savings. Judging from the dependency between 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 technique or system. Therefore, it is necessary to identify improvements that can transfer the potential of genomics research to clinics that can improve life and, in many cases, be rescued. The present invention meets this requirement and provides related benefits as well.

<p> The present disclosure is a solid support having a surface, the surface having a plurality of wells, the wells containing a gel material, the wells separated from each other by a gap region on the surface, each of which is a gap region. A solid support that isolates the gel material in the wells from the gel material in multiple other wells, and a library of target nucleic acids in the gel material, where the gel material in each well provides a single target nucleic acid in the library. Provides an array containing, including, libraries.</p>

<p> In some embodiments, the substrate is arranged as an array of wells and the sample is nucleic acid. Accordingly, the present disclosure is 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 interstitial region is a solid support that separates the gel material in each well from the gel material in multiple other wells and a library of target nucleic acids in the gel material, where the gel material in each well is a single library. An array containing a library containing a type of target nucleic acid is provided.</p><p> The disclosure also provides a method of making a substrate. The method is (a) a step of providing a solid support with a plane, the plane being interrupted by one or more concave features, with one or more concave features being one or more gap regions on the plane. A step adjacent to, and (b) a step of coating at least a portion of the solid support with a gel material, the portion of which comprises at least one concave feature and at least one interstitial region. c) It can include the step of polishing the flat surface to remove the gel material from at least one interstitial region and holding the gel material in at least one concave feature.</p><p> The method of making an array is (a) a step of providing a solid support with a surface containing a plurality of wells, wherein the wells contain a gel material and the wells are separated from each other by a gap region on the surface. The interstitial region separates the gel material in each well from the gel material in multiple other wells, and (b) delivers the library of target nucleic acids to the wells of the solid support and adheres to the gel material in each well. Steps to make an array of wells with a single target nucleic acid species, in which different wells in the array have different target nucleic acid species from the library, and (c) the gel material in the wells of the array. A step of amplifying the target nucleic acid attached to the cell to create a clone population of an individual target nucleic acid in each well of the array can be included.</p><p> The present disclosure further provides a method of detecting a specimen. The method is (a) a step of providing a solid support with a plane, the plane being interrupted by one or more concave features, the concave features containing a gel material, and one or more concave features. Adjacent to one or more interstitial regions on a plane, the interstitial regions are substantially free of gel material, and the gel material is attached to or contains a target specimen, and (b). The step of bringing the solid support into contact with the probe under conditions where the target specimen specifically interacts with the probe, and (c) at least one of the target specimens that detects the solid support and interacts with one or more of the probes. It can include a step of identifying a subset.</p><p> In certain embodiments, the nucleic acid is the specimen detected and the concave feature is the well. For example, a method of detecting nucleic acid is (a) a step of providing a solid support having a surface and a nucleic acid library, wherein the surface has a plurality of wells, the wells contain a gel material, and the wells. Are separated from each other by interstitial regions on the surface, which sequester the gel material in each well from the gel material in multiple other wells, and a single target nucleic acid in the library adheres to the gel material in each well. Steps to contact (b) the solid support with at least one probe bound to the target nucleic acid, and (c) the target nucleic acid species that detects the solid support and binds to at least one probe. Can include a step of identifying wells having.</p><p> The compositions, devices, and methods of the present disclosure are demonstrated herein in the context of gel materials. It should be understood that gel materials are representative and can be replaced, for example, with other organic materials such as polymers that can form surface coatings and are not necessarily considered gels by themselves. The method described herein, wherein the gel material is applied to the surface, the gel material is removed from the interstitial region, and the sample is attached to the gel material using an array prepared in an analytical method, a preparation method, or the like. It can be easily adapted by replacing the material with a non-gel material.</p>

<figref num="1">Schematic representation of a method of making and using a patterned array of DNA features, where each feature is a well with a gel material attached to a DNA cluster and the array is used in a sequencing method.</figref><figref num="2">An image from a BeadChip substrate modified to have a gel material instead of beads in the well is shown. Panel A: Brightfield image obtained before polishing. Panels B-C: Fluorescent images obtained after polishing and hybridization to fluorescently labeled oligonucleotides.</figref><figref num="3-1">Panel A shows a schematic process flow for producing concave features in a substrate using photolithography and Cr hardmasks with reactive ion etching, and panel B shows the SEM of wells and reference portions in a glass substrate. An example of the image is shown.</figref><figref num="3-2">Panel C shows an image of the wafer, an image of a portion of the wafer containing a reference and an array of wells, and an image from a portion of the array containing the wells.</figref><figref num="4">High resolution fluorescence microscopic images of nanowell substrates showing patterned gel features on nanowell substrates after the substrate is coated with PAZAM and polished with silica bead slurry. For visualization, PAZAM is pigment labeled.</figref><figref num="5">A multicolor fusion image obtained from a HiSeq sequencing cycle of a 1.5 μm pitch nanowell substrate with patterned clusters is shown. Panel A: Image showing the extent of patterned clusters in a gel-containing well alongside four bullseye criteria. Panel B: High resolution image showing a mixture of colors in a single bullseye reference (due to a mixed population of amplicon).</figref><figref num="6A">Panel A shows multicolor fusion of patterned clusters in Hiseq sequencing performed on a 750 nm pitch nanowell substrate.</figref><figref num="6B">Panel B shows the closest curve indicating that the arrays are arranged and the clusters pass through the quality filter.</figref><figref num="6C">Panel C presents a sequencing quality metric that indicates successful passage through the quality filter at a density of 1.6 million clusters / mm2.</figref><figref num="7">It is a graph of clonality vs. occupancy, in which the curve is a Poisson distribution, the straight line is the ideal clonality and occupancy, and x is an average measure obtained from sequencing using a substrate having a gel-containing nanowell pattern.</figref>

The present disclosure provides a structured substrate, a method for producing a structured substrate, and a method for using a structured substrate. In certain embodiments, the substrate comprises a solid support having a concave region, such as a well containing a gel material (eg, coated with a gel material). The gel material can then be attached to a sample of interest, such as nucleic acid. In certain embodiments, the gel-containing regions are discrete and separated by interstitial regions that lack the ability to adhere the specimen of interest. For example, the interstitial region may lack gel material. Alternatively, the gel material in the interstitial region may be inactive, or otherwise modified so that the activity or properties of the gel material in the concave region, eg, the inability to aid specimen attachment. Isolation of the resulting gel region provides advantages when reacting to the specimen and / or when detecting the specimen. Representative advantages can be demonstrated in the example of an array of target nucleic acids distributed within the gel-containing wells. As used herein, nucleic acids as templates can be used to perform amplification reactions on structured substrates to form nucleic acid colonies that grow in or on gels (eg, nucleic acid features in arrays). The interstitial region serves to limit the area in which the colony grows. The individual features of the resulting array are relatively easily distinguishable due to the individual patterns created by the gel-containing wells. Patterns can also provide the advantage of increasing the density of features and reducing the processing requirements for image registration compared to random arrays of nucleic acids.

Figure 1 shows a representative process for making a patterned array of nucleic acids. A cross-sectional view of the well-patterned substrate is shown graphically. In the example, the wells have a pitch of 1.5 μm (intercenter spacing) and the diameter of each well is 0.5 μm. The well-patterned substrate can be coated with a gel material such that the material enters the wells and covers the interstitial areas. A gel-patterned substrate can be formed by polishing the resulting gel-coated substrate and removing the gel material from the interstices while leaving the gel material in the wells. The gel can serve to support the capture of the DNA template and the amplification of the template. For example, the gel can be grafted with oligonucleotide primers before surface coating, after surface coating and before polishing, or after polishing. Primers can capture the DNA template and use the captured template to function for major amplification. The resulting DNA patterned substrate can be analyzed, for example, in a sequencing method.

The patterned array of nucleic acids in the gel-containing wells provides multiple advantages for DNA sequencing. Examples of advantages when compared to random arrays (ie, arrays with random feature patterns) are high density feature packing, improved control and tuning of feature density using concentration-independent template seeding, and reduced feature density. Examples include processing required for image registration and simplified signal extraction. Further benefits can be provided by the spatial confinement of the nucleic acid population provided by each feature. The patterned array features of the present disclosure can serve to limit the area or volume in which nucleic acid colonies grow (eg, by amplification of clusters). Without area or volume restrictions, some nucleic acid colonies can be amplified to a larger size than others because of differences in the percent content of guanine and cytosine (ie, GC content) in their sequences. , This affects the relative amplification factor. In the case of the methods and compositions described herein, the volume or area of the individual features may otherwise result from an amplification reaction due to differences in GC content between the template species to be amplified. Can be selected to prevent or minimize size differences in. For example, the volume or area of the feature can be small enough to limit the growth of the fastest growing colonies, while the slow growing colonies effectively fill the feature when the amplification reaction is complete. Can be done.

In certain embodiments, the present disclosure describes covalently bonded patterned gels such as poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM, eg, reference herein). Provided is the manufacture of wells (eg, microwells or nanowells) on glass, silicon, plastic or other suitable solid supports with incorporated US Provisional Patent Application No. 61/753833). The process produces a gel pad used for sequencing, which can be stable throughout multiple cycles of sequencing. The covalent bond of the polymer to the wells helps to retain the gel in the structured features throughout the life of the structured substrate during various uses. However, in many embodiments, the gel does not necessarily have to be covalently attached to the well. For example, see US Patent Application Publication No. 2011/0059865 (SFA, eg, incorporated herein by reference) that is not covalently attached to any portion of the structured substrate under some conditions. ) May be used as the gel material.

In certain embodiments, a solid support material with wells (eg, microwells or nanowells) is patterned and the patterned support is made into a gel material (eg, PAZAM, SFA or chemically modified variants thereof, such as SFA azides. Type (azidolyzed) Version) (Azide-SFA)) and the gel-coated support is polished, eg, by chemically or mechanically polishing, thereby retaining the gel in the wells while retaining the structured substrate between the wells. A structured substrate can be made by removing or inactivating virtually all gels from the interstitial regions on the surface. Primer nucleic acid can be attached to the gel material. A solution of the target nucleic acid (eg, the fragmented human genome) can then be contacted with the polished substrate so that the individual target nucleic acids interact with the primers attached to the gel material. The wells are seeded, but the interstitial region is not occupied by the target nucleic acid because the gel material is absent or inactive. The absence or inactivity of the gel in the interstitial region prevents the growth of nucleic acid colonies from migrating out, so amplification of the target nucleic acid will remain within the wells. The process is conveniently manufacturable, expandable and utilizes conventional micro or nano-processing methods.

In certain embodiments, reference markers are included on the structured substrate for identification and localization of individual features (eg, wells or other gel-containing concave features). Reference markers are particularly useful for structured substrates that have features with spatially aligned patterns, as they provide a reference point for the relative position of other features. Reference markers can also be used to register images in random arrays, but with random arrays generated on commercially available sequence platforms such as the Illumina, Inc. (San Diego, CA) HiSeq, Genome Analyzer, or MiSeq platforms. If used, the unique cluster turbulence may be used instead. Reference markers are particularly useful in applications where the structured substrate is repeatedly detected to track changes that occur over time in individual features. The reference marker allows tracing of individual nucleic acid clusters by continuous images obtained through multiple sequencing cycles, so that individual clusters can be individually sequenced.

The present disclosure provides reference markers with patterns of concave and interstitial regions. A typical design of a reference marker is the following: a set of concentric circles with two or more alternative patterns of concave rings, interstitial rings and rings of wells or other concave features (eg, "bullseye"). In some embodiments, the concave region of the reference marker contains the gel material, while the interstitial region does not. The specific location of the gel on this surface can be achieved using the gel coating and polishing methods described herein. Typically, a detection method is used that can distinguish the gel-containing region from the interstitial region. In some examples, the identification may be based on the presence of a particular specimen in the gel region that is not present in the interstitial region. For example, in the case of a nucleic acid array, the gel-containing region of the reference marker can contain nucleic acid labeled by the same method used to label the target nucleic acid on the array. Therefore, the reference marker can be conveniently produced using the same method used to produce the specimen feature. Accordingly, if desired, reference markers and sample features may be produced simultaneously through one or more steps. Another useful reference marker that can be used in the structured substrates and methods described herein is that it has sub-regions, where the pattern of wells (or other concave features) within one sub-region is another. It rotates with respect to the pattern in the sub-region. Such a reference grid can be configured and used for image registration as described in US Patent Application No. 13/267565, which is incorporated herein by reference.

As a further example, beads may be used as a reference. The beads can include a label such as a fluorophore. In this case, the surface may have at least two types of wells (or other concave features). Relatively large wells can accommodate one or more reference beads, while smaller wells are too small to contain beads and contain only gel material. Therefore, the smaller well serves as an analytical feature for analysis and the larger, bead-filled well serves as a reference. As an alternative to the wells, the reference feature may be a channel such as that present in the bullseye configuration illustrated above, which channel can have dimensions to accommodate the beads. As such, some beads can be located in the channel to create, for example, a distinct set of bead shape criteria.

Patterned arrays, methods of manufacture thereof, and methods of use thereof are demonstrated herein in relation to the gel material used to adhere to the specimen of interest. It should be understood that the gel material is typical and may be replaced with other organic materials that can be used to mediate localization to features on the surface of the specimen. Such organic materials include, for example, polymers that can form surface coatings and do not necessarily have to be considered gels in their own right. A specific example is a polymer formed by the ATRP (Atom Transfer Radical Polymerization) method or the surface initiation polymerization method. The method described herein, which applies the gel material to the surface and removes the gel material from the interstitial region using an array obtained, such as by analytical or preparative methods, also facilitates the use of non-gel materials. Can be adapted.

It should be understood that the terms used herein are taken in their usual sense in the art, unless otherwise specified. Some terms used herein and their meanings are described below.

As used herein, the term "attached" means a state in which two things are connected, fixed, bonded, connected, or bonded to each other. For example, a sample such as nucleic acid can adhere to a material such as a gel or solid support by covalent or non-covalent bond. Covalent bonds are characterized by the sharing of electron pairs between atoms. Non-covalent bonds are chemical bonds that do not require the sharing of electron pairs and may include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions.

As used herein, the term "cloned population" means a population of nucleic acids that is homogeneous for a particular nucleotide sequence. Uniform sequences are usually at least 10 nucleotides in length, but can be even longer, including, for example, at least 50, 100, 250, 500, 1000 or 2500 nucleotides in length. The clonal population may be derived from a single target nucleic acid or template nucleic acid. The clonal population may contain at least 2, 5, 10, 100, 1000 or more copies of the target nucleotide sequence. The copy may be present in a single nucleic acid molecule, eg, as a concatemer, or the copy may be present on a single nucleic acid molecule (ie, the clonal population has the same target nucleotide sequence. Can contain at least 2, 5, 10, 100, 1000 or more nucleic acid molecules). Typically, all nucleic acids in the clonal population will have the same nucleotide sequence. Very few impure nucleic acids or mutants (eg, amplification) without departing from clonality It should be understood that artifacts) can occur in the clonal population. Therefore, the population can be at least 80%, 90%, 95% or 99% clones. In some cases, there may be a 100% pure clonal population.

As used herein, the term "coating" is intended to mean applying a layer or coating on a surface when used as a verb. At least a portion of the surface may be provided with a layer or coating. In some examples, the entire surface may include a layer or coating. In an alternative example, only part of the surface will have a layer or coating. The term "coating" is intended to mean that a material is present on a surface as a layer or coating when used to describe the relationship between the surface and the material. The material may have a surface sealed, eg, preventing liquids or gases from coming into contact with the surface. However, the material does not necessarily have to form a seal. For example, the material may be permeable to a liquid, gas, or one or more components carried in a liquid or gas. Typical materials that can coat a surface include, but are not limited to, gels, polymers, organic polymers, liquids, metals, second surfaces, plastics, silica, or gases.

As used herein, the term "concave feature", when used in connection with a solid support, means a depression or indentation in the solid support. Typical concave features include, but are not limited to, wells, pitches, holes, dents, channels, or troughs. The concave feature may optionally have a curved cross section (dimensions orthogonal to the surface of the solid support), but a cross section with one or more linear cross sections, corners or corners is also possible. Cross sections with a combination of curved and linear sections are also possible. In general, the concave feature does not necessarily have to pass completely through the solid support, but rather has a bottom or bottom in the substrate, for example.

As used herein, the term "different" means that nucleic acids, when used in connection with nucleic acids, have nucleotide sequences that are not identical to each other. Two or more nucleic acids can have different nucleotide sequences within their full length. Alternatively, two or more nucleic acids can have different nucleotide sequences within an essential portion of their length. For example, two or more nucleic acids can have different target nucleotide sequence moieties in two or more molecules, while two or more molecules can also have the same universal sequence moiety.

As used herein, the term "each" is intended to identify an individual item in a collection when used in connection with a collection of items, but does not necessarily refer to all items in the collection. There can be exceptions if the clear disclosure or context dictates otherwise.

As used herein, the term "fluidic access" is used in connection with a molecule in a fluid and its location in contact with the fluid, as the molecule moves into or through the fluid. Refers to the ability to touch or enter the position. The term can also refer to the ability of a molecule to separate from or exit its position and enter a solution. Fluid access can occur when there is no barrier that prevents the molecule from entering the position, contacting the position, separating from the position, and / or leaving the position. However, unless fluid access is absolutely prevented, it is understood that fluid access exists even if diffusion is stagnant, reduced or altered.

As used herein, the term "gel material" is intended to mean a semi-rigid material that is permeable to liquids and gases. Generally, the gel material can expand when the liquid is taken in and shrink when the liquid is removed by drying. Typical gels include, but are not limited to, colloidal structures such as agarose, polymer mesh structures such as gelatin, or polyacrylamide, SFA (eg, US Patent Application Publication No. 2011/0059865, which is incorporated herein by reference). (See No.) or PAZAM (see, eg, US Provisional Patent Application No. 61/753833, incorporated herein by reference), which has a crosslinked polymer structure. A particularly useful gel material will fit the shape of the well or other concave feature it is in. Some useful gel materials are (a) adaptable to the shape of the well or other concave feature it is in and (b) have a volume that does not substantially exceed the volume of the well or concave feature it is in. You can also.

As used herein, the term "gap region" refers to an area in or on a substrate that separates the substrate or other areas of the surface. For example, the interstitial region can separate one concave feature of the array from another concave feature of the array. The two regions that separate from each other can be discrete and have no contact with each other. In another example, the interstitial region can separate the first feature portion from the second feature portion. In many embodiments, the interstitial regions are continuous, while each feature is discrete, as in the case of an array of wells in a separate continuous surface, for example. The separation provided by the interstitial region may be partial or complete. The interstitial region will usually have a different surface material than the surface material of the features on the surface. For example, array features can have an amount or concentration of gel material or sample that exceeds the amount or concentration present in the interstitial region. In some embodiments, the gel material or specimen may not be present in the interstitial region.

As used herein, the term "library", when used in connection with a sample, means a collection of samples with different chemical compositions. Typically, the specimens in the library will be different species, with common characteristics or characteristics of the genus or class, but with some differences elsewhere. For example, a library can contain nucleic acid species that differ in nucleotide sequence but are similar in terms of having a sugar-phosphate backbone.

As used herein, the terms "nucleic acid" and "nucleotide" are consistent with their use in the art and are intended to include species of natural origin or functional analogs thereof. Functional analogs of particularly useful nucleic acids can be hybridized to nucleic acids in a sequence-specific manner or can be used as a template for replication of a particular nucleotide sequence. Nucleic acids of natural origin generally have a backbone containing phosphodiester bonds. The analog structure can have an alternative skeletal connection having any of those known in the art. Nucleic acids of natural origin generally have deoxyribose sugars (eg, those found in deoxyribonucleic acid (DNA)) or ribose sugars (eg, those found in ribonucleic acid (RNA)). Nucleic acids can contain nucleotides having any of the various analogs of these sugar moieties known in the art. Nucleic acids can include natural or non-natural nucleotides. In this regard, the native deoxyribonucleic acid can have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine, and the ribonucleic acid from the group consisting of uracil, adenine, cytosine or guanine. It can have one or more bases of choice. Useful non-natural bases that can be contained in nucleic acids or nucleotides are known in the art. The term "probe" or "target", when used in connection with a nucleic acid, is intended as a semantic identifier of the nucleic acid in the context of the methods or compositions described herein, and the structure or structure of the nucleic acid. The function does not necessarily have to be limited beyond what is specifically indicated. The terms "probe" and "target" can be applied similarly to other specimens such as proteins, small molecules, cells and the like.

As used herein, the term "random pattern" is used in connection with wells on a surface where the relative position of a subset of wells within one region of the surface is unknown or another region of the surface. Means unpredictable from the location of a subset of the wells inside. The subset used in the measurement method generally contains at least 3 wells, but can include at least 4, 5, 6 and 10 or more wells. Random patterns generally do not include multiple iterations of any subpattern. The term applies to other concave features in addition to wells.

As used herein, the term "repetitive pattern" is used in connection with wells on a surface where the relative position of a subset of wells in one region of the surface is within at least one other region of the surface. It means that it is the same as the relative position of a subset of wells. Therefore, the relative position of the wells in one region of the repeating pattern is generally predictable from the relative positions of the wells in another region of the repeating pattern. The subset used in the measurement method generally contains at least 3 wells, but could include at least 4, 5, 6, 10 or more wells. Representative repeating patterns include linear and hexagonal patterns. The repeating pattern may include multiple iterations of the subpattern. The term applies to other concave features in addition to wells.

As used herein, the term "isolation" is used in connection with the gel material in two wells (or two other features), the gel in one of the wells (or one of the features). Means separating or isolating the material from the gel material in other wells (or other features). Therefore, the gel material in the first well (or first feature) does not come into direct contact with the gel material in the other well (or other feature). In some embodiments, the gel material in the two wells (or two features) is indirect contact, for example, through a solution that contacts the two wells (or features). Alternatively, the gel material in the two wells (or two features) is not even indirect contact. The interstitial region on the surface can isolate the gel material in the two wells (or two features) by having no gel material. In certain embodiments, the gel material may be discontinuous on the surface and is present in concave features such as wells, but not in the interstitial regions between each feature.

As used herein, the term "surface" is intended to mean the outer or outer layer of a solid support or gel material. The surface may be in contact with another material such as a gas, liquid, gel, polymer, organic polymer, a second surface of a similar or different material, a metal, or a coat. The surface or its area may be substantially flat. The surface may have surface features such as wells, pitches, channels, ridges, raised areas, pegs, posts and the like.

As used herein, the term "single species" means substantially one, or only one, specific genus. The term is not necessarily intended to limit the number of single species that exist. For example, a population of nucleic acid molecules, each having the same nucleotide sequence, comprises a single species of nucleic acid. The term "single" in this context is not intended to exclude the existence of others that are not within the scope of the relevant genus. For example, a well containing a single target nucleic acid from a library may contain multiple nucleic acids having the same sequence, excluding other target nucleic acids from the library, but any other non-nucleic acid component. It will not necessarily be excluded. It should be appreciated that a clear single species population can have a small amount of another species present at a level that one of ordinary skill in the art would consider to be a negligible level of impurities or man-made objects for the particular use of the population. For example, in a nucleic acid cluster derived from a single template with the first sequence, the amount of any nucleic acid molecule with the second sequence is undetectable or ignored when the first sequence is detected. If it is reasonably low, it will clearly be considered to have a single species. Alternatively, an absolutely single population will have one or only one species.

As used herein, 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 can optionally take up the liquid (eg, because of its porosity), but usually the substrate does not substantially expand when taking in the liquid and is substantially when the liquid is removed by drying. It will be sufficiently rigid that it will not contract. Non-porous solid supports are generally impermeable to liquids or gases. The solid support may optionally be inert to the chemical reactions used to modify the gel. For example, the solid support may be inert to the chemical reactions used to attach a sample, such as nucleic acid, to the gel in the methods described herein. Typical solid supports include, but are not limited to, glass and modified or functional glass, plastics (acrylic resin, polystyrene and styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethane, Teflon®, annular. Examples include silica or silica-based materials such as olefins, copolymers with polyimide, etc.), nylons, ceramics, resins, Zeonoa, silicon and modified silicon, carbon, metals, inorganic glass, optical fiber bundles, and polymers. A solid support that is particularly useful in some embodiments is placed inside the flow cell device. A typical flow cell is described in more detail below.

As used herein, the term "well" refers to discrete concave features in a solid support having a surface opening completely enclosed by a surface interstitial region. The well can have any of a variety of shapes in its opening in the surface, including, but not limited to, circular, elliptical, square, polygonal, star-shaped (having any number of vertices). And so on. The cross section of the well viewed orthogonal to the surface may be curved, square, polygonal, hyperbolic, conical, angled or the like.

The embodiments described below and listed in the claims can be understood in consideration of the above definitions.

The present disclosure provides a substrate comprising a solid support having a surface, the surface having at least one concave feature, the at least one concave feature containing a gel material, said at least one of which. The concave feature is adjacent to at least one interstitial region on the surface, and the present disclosure provides a sample library in a gel material, where the gel material in each well is a single species of sample in the library. Including.

In some embodiments, the substrate is configured as an array of wells and the sample is nucleic acid. Accordingly, the present disclosure provides an array containing a solid support having a surface, the surface having a plurality of wells, the well containing a gel material, the well being on the surface. Separated from each other by interstitial regions, the interstitial regions isolate the gel material in each well from the gel material in a plurality of other wells, and the present disclosure provides a library of target nucleic acids in the gel material. Provided, but the gel material in each well contains a single species of target nucleic acid in the library.

Solid-state supports used in the structured substrates described herein are made of any of the various materials described herein, eg, the definitions above, the examples below, or just described below. Can be done. A particularly useful material is glass. Other suitable substrate materials are polymer materials, plastics, silicon, quartz (molten silica), borofloat glass, silica, silica-based materials, carbon, metals, optical fibers or bundles of optical fibers, sapphire, or plastics such as COC and epoxy resins. Materials can be included. Specific materials can be selected based on the properties desired for a particular use. For example, materials that are transparent to radiation of the desired wavelength are useful for analytical methods that utilize radiation of the desired wavelength, such as one or more techniques described herein. Conversely, it may be desirable to select a material that does not pass radiation of a particular wavelength (eg, opaque, absorbent or reflective). It is used during the manufacture of structured substrates, such as the methods described herein, or for chemical reactions or analytical detections performed using structured substrates, such as those described herein. , May be useful for mask formation. Other properties of the material that can be utilized are inactivity or reactivity with certain reagents used in downstream processes, such as those described herein, or operations during the manufacturing process, such as those described herein. Easy or low cost. Further examples of materials that can be used in the structured substrates or methods of the present disclosure are described in U.S. Patent Application No. 13/661524 and U.S. Patent Application No. 2012/0316086, respectively incorporated herein by reference.

In certain embodiments, sol-gel based substrates can be made and used. Sol-gel-based patterning can be achieved by coating rigid or flexible substrates such as glass, silicon, plastics, and metals with a sol-gel coating, for example by spin coating, dipping or spray coating. The sol-gel can be supplied in a liquid state when applied to a substrate and can be cured by exposing the sol-gel to light or heat (the liquid can be a gel) photoinitiator or heat. It can contain any of the initiators. Following coating of the substrate with sol-gel and before curing the material, the sol-gel can be stamped with a mold (three-dimensional stamp) having a single or multiple raised features. The mold may be made of, for example, silicon, glass (such as quartz), metal (such as nickel), plastic or polymer (such as PDMS). Imprinting the stamp on the sol-gel can be achieved by arranging the mold in contact with the sol-gel. When the mold comes into contact with the sol-gel, the sol-gel redistributes and equiangularly surrounds the structure of the mold. When the mold comes into contact with the sol-gel, redistribution of the sol-gel can be facilitated by external forces exerting on the mold or substrate, or by capillary forces inherent in the nature of the patterned mold. When the mold comes into contact with the sol-gel, the substrate + sol-gel + mold stack is exposed to light or heat to cure the sol-gel and fix the pattern originally in the mold into the sol-gel. This pattern forming method has conventionally been called nanoimprint lithography. After curing of the sol-gel, the mold can be separated from the substrate + sol-gel stack and this mold can be discarded but re-patterned for another uncured sol-gel coated substrate. You may use it. Substrates with a cured patterned sol-gel can then be subjected to chemical vapor deposition, or if a pure glass-like surface is desired, the substrate + The patterned sol-gel stack can be subjected to a thermal process (sintering) to remove the organic material originally present in the sol-gel. This is not a requirement, but a pure SiO that benefits the substrate in a particular chemical addition scheme.₂Can be used as a material.

Another method of manufacturing patterned substrates is to use a plastic material such as COC or COP (such as Zeonor or Topas) and perform thermal embossing to create an indentation array. This method is similar to nanoimprint lithography. A plastic substrate may be mounted on a temperature-controlled chuck. The plastic substrate can then be heated to a temperature at which the plastic skin is above the glass transition temperature. The mold is brought into close contact with the mold (eg, quartz, silicon, polymer, or metal) while the substrate is hot. The mold is typically subjected to external forces to ensure that the plastic isometrically coated on the structured mold. During high temperature contact, the plastic redistributes itself and becomes a negative replica of the mold, for example if the mold has a post array, the embossed plastic becomes an array of wells. While the mold is in contact with the plastic substrate, the temperature of the substrate is reduced and thus the embossed pattern is fixed in the substrate.

The concave feature on the substrate can have any of a variety of shapes. From the point of view of the shape on the substrate, the feature can have curved sides, linear sides, corners or a combination thereof. For example, the feature may be a well with a circular, elliptical, square, polygonal, star-shaped (with any number of vertices), or irregularly shaped openings on the surface. The feature may be a channel, and the shape of the channel on the surface can include curved, linear, angular, or a combination of these sides. Other channel features can be linear, snake-like, rectangular, square, triangular, circular, oval, hyperbolic, or a combination thereof. The channel can have one or more branches or corners. The channel can connect two points on the surface, one or both of which may be the edges of the substrate. Figures 3B and 3C show typical channel features in the bullseye reference, along with wells within and around the bullseye reference.

The cross-sectional shape of the concave feature viewed orthogonal to the surface may have curved, linear, or a combination of the two walls. Thus, the shape of the cross section may be part of a circle or ellipse (eg U-shape), or two or more linear sides (eg V-shape, square, polygon, or) that meet at the corners. It may have a star shape). From the point of view of the shape of the cross section, the bottom of the concave feature may be narrowed, widened, or roughly the same as the opening on the surface. The shape of these cross sections, when 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 the well has a conic section. The case where the bottom of the surface is different (usually smaller) than the area of the opening on the surface can be described. Of course, the cross section described wells, but can also apply to channels.

In embodiments where the concave features form wells, each well can have any volume in which the liquid can be trapped. The minimum or maximum volume can be selected, for example, to accommodate throughput (eg, multiplicity), resolution, sample composition, or sample reactivity, suitable for downstream use of the substrate. For example, the volume is at least 1x10^-3μm³、1×10^-2μm³, 0.1 μm³, 1 μm³, 10 μm³, 100 μm³That's all. Alternatively, or additionally, this volume can be up to 1x10.⁴μm³、1×10³μm³, 100 μm³, 10 μm³, 1 μm³, 0.1 μm³It may be as follows. It should be understood that the gel material can fill wells of total volume or partial volume. The volume of gel in the individual wells may be greater than, less than, or between these values specified above.

The area occupied by each well opening on the surface may be selected based on the same criteria as above for well volume. For example, the area of each well opening on the surface is at least 1x10^-3μm²、1×10^-2μm², 0.1 μm², 1 μm², 10 μm², 100 μm²That's all. Alternatively, or additionally, this area can be up to 1x10³μm², 100 μm², 10 μm², 1 μm², 0.1 μm²、1×10^-2μm²It may be as follows. The depth of each well may be at least 0.1 μm, 1 μm, 10 μm, 100 μm or more. Alternatively, or additionally, this depth can be up to 1x10³It may be μm, 100 μm, 10 μm, 1 μm, 0.1 μm or less.

Layouts of many different wells or other concave features, such as regular patterns, repeating patterns, and irregular patterns, can be envisioned. For example, the wells may be placed in a hexagonal grid for close packing and improved density. Other layouts include, for example, straight (ie, rectangular) layouts, triangular layouts, and the like. Differences between specific layouts and layouts with different domains (if used) are in accordance with the teachings of U.S. Pat. No. 7813013 and / or U.S. Patent Application No. 13/267565, which are incorporated herein by reference. It may be. Either a variety of crystalline or non-crystalline patterns can be useful.

The pattern of wells can be characterized in terms of the average pitch of wells (ie, spacing between centers). The pattern may be regular so that the coefficient of variation before and after the average pitch is small, or the pattern may be irregular so that the coefficient of variation is relatively large. In either case, the average pitch may be, for example, at least 10 nm, 0.1 μm, 0.5 μm, 1 μm, 5 μm, 10 μm, 100 μm or more. Alternatively, or additionally, the average pitch may be, for example, up to 100 μm, 10 μm, 5 μm, 1 μm, 0.5 μm, 0.1 μm or less. Of course, the average pitch of the wells in a particular pattern may be between one of the minimum and one of the maximum values selected from the above range.

Well patterns can also be characterized on the basis of the density of wells (ie, the number of wells) within a defined area. For example, the well is about 2 million / mm²May be present at a density of. According to the manufacturing method described herein, the density is, for example, at least 100 / mm.², 1000 / mm², 100000 / mm², 1000000 / mm², 2000000 / mm², 5000000 / mm²It can be easily adjusted to different densities such as the above densities. Alternatively, or additionally, this density is 5000000 / mm.²Below, 2000000 / mm², 1000000 / mm², 100000 / mm², 1000 / mm², 100 / mm²It may be adjusted as follows. Of course, the density of wells on the substrate may be between one of the minimum values and one of the maximum values selected from the above range.

In certain embodiments, a gel material is used. In some examples, a gel-forming (eg, polymerizable) material is fed to a solid support in a liquid state and then converted to a gel. Examples of polymerizable materials include, but are not limited to, acrylamide, methacrylamide, hydroxyethyl methacrylate, N-vinylpyrrolidinone or derivatives thereof. Such materials are useful in preparing hydrogels. In some embodiments, the polymerizable material can include two or more different species of compounds forming a copolymer. For example, acrylamide, methacrylamide, hydroxyethyl methacrylate, N-vinylpyrrolidinone or two or more different species of these derivatives can function as comonomer to form a copolymer hydrogel by polymerization. Useful hydrogels are, but are not limited to, silane-free acrylamide (SFA) polymers (see US Patent Application Publication No. 2011/0059865, incorporated herein by reference), poly (N- (5-azidoacet). Amidylpentyl) acrylamide-co-acrylamide) (PAZAM, see US Provisional Patent Application No. 61/753833 incorporated herein by reference), eg WO 00/31148 (incorporated herein by reference). [2+] as described in acrylamide and polyacrylamide polymers formed from acrylates or acrylic acids containing vinyl groups, such as WO01 / 01143 or WO03 / 014392, respectively, incorporated herein by reference. 2] A polyacrylamide polymer formed from a monomer that forms a photoaddition cyclization reaction, or the polyacrylamide described in US Pat. No. 6,465,178, WO 01/62982 or WO 00/53812, each incorporated herein by reference. Contains copolymers. Chemically treated variants of these gel materials, such as chemically treated SFA (eg, 5', reacted with oligonucleotides with corresponding reactive groups). -Or azido that reacts with 3'-alkynyl-modified oligonucleotides-Azidosis of SFA to produce SFA) is also useful. Typical hydrogels and polymerizable materials that can be used to form hydrogels are described, for example, in U.S. Patent Application No. 61/753833 or U.S. Patent Application Publication No. 2011/0059865, which are incorporated herein by reference, respectively. ing. Other useful gels are formed by a temperature-dependent change from liquid to gelatinous. Examples include, but are not limited to, agar, agarose, or gelatin.

The gel material in the wells or other concave features on the surface of the structured substrate can be covalently bonded to the surface. For example, PAZAM may be covalently attached to a surface using surface material and other reagents described herein in US Patent Application No. 61/753833 and Examples section herein. it can. However, the gel material does not necessarily have to be covalently attached to a well or other concave feature, as demonstrated for SFA in the Examples section below.

One or more specimens may be present in or on the gel material present on the structured substrate. The gel-containing substrate of the present disclosure is particularly useful for detecting a sample or performing a synthetic reaction with the sample. Thus, any variety of specimens to be detected, characterized, modified, synthesized, etc. can be present in or on the gel material of the substrates described herein. Representative analytes include, but are not limited to, nucleic acids (e.g. DNA, RNA or analogues thereof), proteins, polysaccharides, cells, antibodies, epi taupe, receptors, ligands, enzymes (e.g. kinases, phosphatases or polymerase) , Small molecule drug candidates and the like. The structured substrate can contain several different species from the library of specimens. For example, species are from a combination library of various antibodies from an antibody library, nucleic acids with different sequences from a library of nucleic acids, proteins with different structures and / or functions from a library of proteins, small molecules, and the like. It may be a drug candidate of.

In some embodiments, the specimens can be dispensed on a structured substrate so that they can be dissolved individually. For example, a single molecule of each sample can be present in each gel-containing well of the structured substrate. Alternatively, the specimen can exist as a colony or population, so that individual molecules are not necessarily lysed. The colony or population can be uniform for the inclusion of a single species sample (although multiple copies). Taking nucleic acids as an example, each well on a structured substrate can contain a colony or population of nucleic acids, and all nucleic acids within the colony or population have the same nucleotide sequence (either single-stranded or double-stranded). Can have. Such colonies can be produced by cluster amplification or bridge amplification described in more detail elsewhere herein. Concatemers made using multiple iterations of the target sequence, such as rolling circle amplification techniques, can be present in a single molecule of nucleic acid. Therefore, the gel material in each well on the structured substrate may contain multiple copies of a single species of sample. Alternatively, the colony or population of specimens in the well can contain two or more different species. For example, one or more wells on a structured substrate can each contain mixed colonies with two or more different nucleic acid species (ie, nucleic acid molecules with different sequences). Two or more nucleic acid species in a mixed colony can be present in non-negligible amounts, for example making it possible to detect multiple nucleic acids in a mixed colony.

The sample may be attached to the gel material. This attachment may be shared or non-shared. Representative methods and reactants for attaching nucleic acids to gels are described, for example, in US Patent Application Publication No. 2011/0059865 or US Provisional Patent Application No. 61/553833, which are incorporated herein by reference, respectively. ing. The specimen may be a nucleic acid, which is a base portion of, for example, a 3'terminal nucleotide, a base of a 5'nucleotide, via its 3'oxygen, 5'oxygen, or other position on its length. It can adhere to the gel via a moiety and / or one or more base moieties at different locations in the molecule. Non-covalent attachments include, for example, ionic interactions between nucleic acids and gels, uptake of nucleic acids inside the pores of gels, protein-protein interactions, binding between receptors and ligands on gels and / or nucleic acids, and other known. Including the style of.

In some embodiments, the gel coating applied to the surface contains one or more specimens, after which the gel material is removed from the interstitial region. Therefore, the gel material is present in the interstitial region, and the gel material in the interstitial region can adhere to one or more different specimens. Alternatively, after removing the gel material from the interstitial region, the specimen is added to the gel material in the concave feature.

The structured substrate of the present disclosure can occur in a flow cell. Representative flow cells, methods of manufacture thereof, and methods of use thereof are described in US Patent Application Publication Nos. 2010/0111768 or 2012/0270305, or WO 05/065814, which are incorporated herein by reference, respectively. The flow cell provides a convenient format for accommodating the array produced by the methods of the present disclosure and requires decoding at the time of synthesis (SBS) or repeated delivery of reagents during the cycle (eg, repeated steps or circulating steps). (Synthesis method or detection method). Typical detection methods are described in more detail below.

In some embodiments, flow cells or other containers with multiple surfaces are used. Containers with multiple surfaces may be used such that there are gel-containing concave features (eg wells) on only a single surface. Alternatively, there may be gel-containing concave features on two or more surfaces present in the container. One or more surfaces of the flow cell can be selectively detected. For example, the opposing surfaces inside the flow cell can be selectively addressed by focused radiation using methods known in the art such as the confocal method. Useful confocal methods and devices that selectively direct radiation to multiple surfaces of a container (eg, flow cell) are, for example, US Patent Application Publication No. 2009/0272914 or US Pat. No. 8039817, respectively incorporated herein by reference. It is described in the issue.

The present disclosure provides a method of making a substrate. This method is (a) a step of providing a solid support with a plane, the plane being interrupted by one or more concave features, and the one or more concave features being one or more interstitial regions on the plane. Adjacent to (b) a step of coating at least a portion of the solid support with a gel material, the portion of which comprises at least one concave feature and at least one interstitial region, and (c). It can include a step of polishing the flat surface to remove the gel material from at least one interstitial region and holding the gel material in at least one concave feature.

The substrate can be manufactured to have concave features using any of a variety of techniques known in the art. In many embodiments, the concave feature will be a small one in an array of nanometer or micrometer dimensions. In such cases, a nano-processing method or a micro-processing method can be used. Examples of these techniques are described elsewhere herein, such as Example 2 below. Further representative nano- and micro-processing techniques are described in US Patent Application No. 13/661524 and US Patent Application Publication No. 2012/0316086, which are incorporated herein by reference, respectively.

One or more concave features, such as wells, can be coated with a pre-generated gel material or a liquid that then produces the gel material. Examples of conventional techniques are pre-generated using the techniques described in US Provisional Patent Application No. 61 / 75833, which is incorporated herein by spin coating, dipping, gel flow under positive or negative pressure, or by reference. It is the coating of the substrate by PAZAM that has been saved. The coating of the well array with the pre-generated PAZAM is demonstrated in Example 3 below. An example of applying a liquid that produces a gel material is then a well array coating with liquid silane-free acrylamide and N- [5- (2-bromoacetyl) aminopentyl] acrylamide (BRAPA), with the reagent surfaced. A gel is produced by polymerizing on top. Coating the array in such a manner can use the chemical reagents and techniques described in US Patent Application Publication No. 2011/0059865, which is shown in Example 1 below and incorporated herein by reference. In some embodiments, for example, immersing the well-containing substrate in a pre-generated gel material allows the gel material to selectively fill the wells, eliminating the need for polishing.

The sample may be added to the gel material before or after contact with the solid support. In addition, the sample may be added to the gel (ie, after the gel has been generated from its precursor reagent) or the sample may be added to the gel-forming reagent solution (ie, before gel formation). In some embodiments, various specimens may be added prior to gel formation and others may be added after gel formation. In one example, the primer nucleic acid is added to the gel-forming solution and then the solution is gel-generated (eg, by polymerization occurring in SFA and PAZAM). The gel formation may occur on a solid support, or the gel may be preformed and then coated on a solid support. With either method, the primer will adhere to the gel present in the concave features such as wells. A target nucleic acid complementary to the primer is then added to the primer-containing gel, so that the target nucleic acid is attached to the gel (via hybridization) after the gel material has been coated onto the solid support. Can be done. Hybridization of the target nucleic acid can occur optionally after the polishing step has been performed (polishing will be described in more detail below). The previous examples describe some examples of adding nucleic acid (which acts as either a primer or a target) to a gel at different manufacturing stages of a structured substrate.

In some embodiments, the primer nucleic acid that adheres to the gel (or is otherwise present in or on the gel) can be used for capture and / or amplification of the template nucleic acid. The primer may be a universal primer that hybridizes to a universal adapter sequence that attaches to various target nucleic acids in the library (ie, each target nucleic acid has a different target region than the other target nucleic acids in the library. Including, some target nucleic acids in the library have the same universal adapter sequence). In some embodiments, the target nucleic acid can be attached to the gel material and primers (either in solution or on the gel) can be used to amplify the attached target nucleic acid (either in solution or on the gel). That is, the target nucleic acid can serve as a template for amplification).

The methods described herein may use any of the various amplification methods. Representative techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), polysubstituted amplification (MDA), or random prime amplification (RPA). In certain embodiments, one or more primers used for amplification can be attached to the gel material. In the PCR embodiment, one or both primers used for amplification can be attached to the gel material. The format that utilizes two adherent primers is often referred to as bridge amplification, because the double-stranded amplicon is bridge-like between the two adherent primers located on the sides of the copied template sequence. This is to form a structure. Representative reagents and conditions that can be used for bridge amplification include, for example, U.S. Pat. Nos. 5641658, U.S. Patent Publication Nos. 2002/0055100, U.S. Patent No. 7115400, U.S. Patent Publication No. 2004, respectively incorporated herein by reference. It is described in / 0096853, US Patent Publication No. 2004/0002090, US Patent Publication No. 2007/0128624, and US Patent Publication No. 2008/0009420. PCR amplification can also be performed using one of the amplification primers attached to the gel material and the second primer in solution. Typical formats using combinations of monosolid phase adherent primers and liquid phase primers are described, for example, by Dressman et al., Proc. Natl. Acad. Sci. USA 100, which are incorporated herein by reference, respectively. Emulsion PCR according to 8817-8822 (2003), WO 05/010145, or US Patent Publication No. 2005/0130173 or 2005/0064460. It is understood that emulsion PCR is an example of a format, the use of emulsions is optional for the purposes described herein, and in fact emulsions are not used in some embodiments. I want to. In addition, the primers need not be attached directly 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 adhere to the gel material and can be used as a template for amplification.

RCA techniques may be modified for use in the methods of the present disclosure. Representative components that can be used in the RCA reaction and principles by which RCA produces amplicon are, for example, Lizardi et al., Nat. Genet. 19: 225-232 (1998) and, respectively, incorporated herein by reference. It is described in US Patent Application Publication No. 2007/0099208. The primers used for RCA may be in solution or attached to the gel material.

MDA techniques may be modified for use in the methods of the present disclosure. Some basic principles and useful conditions for MDA are incorporated herein by reference, eg, 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 Virus Detection, Academic Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20: 1691 ~ 96 (1992); US Pat. No. 5,455,166; It is described in No. 5130238; and No. 6214587. The primers used for MDA may be in solution or attached to the gel material.

In certain embodiments, a combination of amplification methods exemplified above can be used. For example, RCA and MDA may be used in combination, which is used to produce concatemer amplicon in solution (eg, using liquid phase primers). An amplicon can then be used as a template for MDA using primers attached to the gel material. In this example, the amplicon produced after the combined RCA and MDA step will adhere to the gel material. Amplicons will generally contain concatemer repeats of the target nucleotide sequence.

The amplification methods exemplified above can be used to generate gel-containing features with multiple copies of the target nucleic acid. Individual features, such as wells, have a clonal population of nucleotide sequences in the form of single molecule concatemers, such as those produced by RCA, or in the form of multiple nucleic acid molecules with the same sequence, such as those produced by bridge PCR. be able to. In general, nucleic acids with some copies of the amplified target will adhere to the gel material.

In some application examples, the individual gel-containing wells (or other concave features) are largely occupied by amplicon from the first target nucleic acid and naturally occur during the second target nucleic acid or amplification. Contaminated amplicon from the mutation can occupy low levels. The array may have one or more amplification positions with sufficiently low levels of contaminated amplicon, which may give unacceptable impact to subsequent use of the array. For example, when an array is used for detection applications, acceptable levels of contamination appear to be levels that do not impact the signal-to-noise or resolution of the detection technique in an unacceptable manner. Correspondingly, the apparent clonality will generally be associated with the particular use or use of the array made by the methods described herein. Typical contamination levels that can be tolerated for individual wells or other features for a particular application are not limited to these, but up to 0.1%, 0.5%, 1%, 5%, 10% or 25% contamination amplifiers. Including recon. The array can include one or more wells or other features with these representative pollution levels of amplicon. For example, up to 5%, 10%, 25%, 50%, 75%, or 100% features in an array can have some contaminated amplicon.

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 a sample such as nucleic acid to the gel material can be performed at various different manufacturing steps of the structured substrate. Therefore, the gel material can be attached to the solid support before or after the sample is attached to the gel material. Adhesion of the gel material to a solid support is any useful chemical reaction, such as that described in US Provisional Patent Application No. 61 / 75833, which is incorporated herein by reference, such as, but not limited to, or the following examples. It can be carried out using what has been demonstrated in 3. It should be understood that the covalent bond of the gel material to the solid support is not necessarily in all embodiments. Therefore, subsequent steps of polishing the gel-coated support or using the polished substrate are optional and not essential, but on substrates with gel material that are covalently bonded to concave features such as wells. Can be implemented against.

The method described herein can include removing the gel material from the surface of the solid support. The gel material coated on the solid support can be selectively removed from the interstitial region using any of a variety of techniques. For example, the gel material can be removed from a solid support with concave features and interstitial areas by mechanical polishing. Mechanical polishing can be performed by applying a polishing force to the surface of the solid support. Typical methods include polishing with a slurry of beads, wiping with a sheet or cloth, scraping and the like. An example of polishing involves removing interstitial gel using a lint-free (clean room grade) wipe coated with a 3 μm silica bead slurry (10% w / v in water). A polishing wheel / grinder may be used with this slurry. Mechanical polishing can also be achieved using fluid or air jets to remove the gel from the interstitial areas.

Polishing is hydrolysis or decomposition based on acrylamide groups (eg, Kurenkov et al., Russian Journal of Applied Chemistry, 75: 1039-1050 (2002); Caulfield et al., Polym. 44: 1331-1337 (2003); and Chemical polishing such as Caulfield et al. (via exposure to benzoyl peroxide or diluted hydrogen peroxide as described in Chem. Rev. 102: 3067-3083 (2002)) may be required.

Polishing also requires a combination of chemical and mechanical polishing methods, which mechanically exfoliate using a chemical slurry containing a colloidal suspension of particles, and then the portion away from the interstitial region. Chemically dissolve the gel material. Another method of polishing or cleaning the interstitial area is to coat the surface with an adhesive-based technique, eg, a rigid planar adhesive film that is compatible with the gel material, thereby (eg, via chemical bonds). Techniques include close contact with the gel material inside. Mechanical removal / peeling of this adhesive film will mechanically remove the gel material from the interstices, but at the same time leave the gel material in the concave feature.

In another example, thiophosphate grafted SFA can be removed from the interstitial region on the surface as follows. Water-moistened Whatman wipes may be applied to aluminum oxide (approximately 100 mg, 0.3 um) or steel beads. The resulting slurry may then be rubbed into small concentric circles on the surface of the solid support using uniform pressure. A Whatman wipe moistened with clean water can then be used to remove the slurry on the surface. The mechanical and chemical polishing methods exemplified herein for removing the gel material from the gap region are used to inactivate the gel material in the gap region, whether or not the gel material has been removed. You can also let it. For example, the gel material can be inactivated for its ability to attach to specimens such as nucleic acids, or to facilitate the amplification of nucleic acids.

The method of making an array is as follows: (a) a solid support having a surface with a plurality of wells is provided, the wells contain a gel material, the wells are separated from each other by a gap region on the surface, and the gap region Separates the gel material in each well from the gel material in multiple other wells, and (b) delivers the library of target nucleic acids to the wells of the solid support and attaches to the gel material in each well. In the steps of making an array of wells with a single target nucleic acid, different wells in the array have different target nucleic acid species from the library, and (c) the gel material in the wells of the array. A step of amplifying the attached target nucleic acid and creating a clone population of individual target nucleic acids in each well of the array can be included.

In some embodiments, the structured substrates described herein provide the advantage of constructing an array by conveniently delivering a plurality of different specimens from the mixture to individual locations on the substrate. The structured substrate facilitates the selective capture of a single sample from the sample mixture in contact with the substrate at each individual gel-containing well (or other concave feature). The pattern and filling efficiency of the gel-containing wells (or other concave features) on the structured substrate have the desired properties such as sample density and purity of each feature relative to having a single species of sample. Can be adjusted to obtain. For example, high density wells are used to obtain high density samples on the array, while low density wells are used to obtain low density samples on the array. Alternatively, or additionally, the concentration or amount of the sample in the solution may be increased to obtain a high density sample on the array, or decreased to obtain a low density sample on the array. The average purity of the sample in each gel-containing well (or other concave feature) is described in more detail below and also changes the characteristics of the conditions for delivering the substrate or sample demonstrated in the Examples section. Can be adjusted by.

In certain embodiments, the size or volume of the well (or other concave feature) can be adjusted to affect the purity of the captured specimen. For example, the well can have an area or volume of gel material that accommodates only a single sample of a particular type, thus preventing trapping of multiple sample molecules by steric exclusion, or seeding the well. To prevent. Three-dimensional exclusion can be particularly useful for large specimens such as nucleic acids. More specifically, the well (or other concave feature) can have a gel surface having an area less than or equal to the diameter of the exclusion volume of the target nucleic acid seeded on the substrate. The excluded volume of the target nucleic acid and its diameter can be determined, for example, from the length of the target nucleic acid. Methods for determining the exclusion volume of nucleic acids and the diameter of the exclusion volume are described, for example, in US Pat. No. 7,785,790, respectively incorporated herein by reference; Rybenkov et al., Proc. Natl. Acad. Sci. USA 90: 5307-531 (1993). Zimmerman et al., J. Mol. Biol. 222: 599-620 (1991); or Sobel et al., Biopolymers 31: 1559-1564 (1991). The conditions for steric exclusion are described in U.S. Patent Application No. 13/661524 and U.S. Pat. No. 7,785,790, which are incorporated herein by reference, and are readily available for the structured substrates of the present disclosure.

It should be appreciated that in some embodiments, the wells (or other concave features) may provide a gel surface with an area substantially larger than the diameter of the exclusion volume of the target nucleic acid transported to the amplification position. Therefore, the area of the feature may be large enough that stereoscopic exclusion does not occur.

In some embodiments, eg, steric exclusion embodiments described above, the library of target nucleic acids may be delivered to the gel-containing wells (or other concave features) of the solid support before the amplification step begins. .. For example, the target nucleic acid can be delivered to the structured substrate under the condition that the target nucleic acid is seeded on the gel material in the substrate. The substrate can optionally be washed to remove target nucleic acids that are not seeded in the gel and any other material that is not required for subsequent steps or use of the substrate. Amplification can include one or more of the techniques described above herein.

In an alternative embodiment, the library of target nucleic acids may be delivered to the gel-containing wells (or other concave features) of the solid support, and the amplification process can occur at the same time as the seeding event. For example, seeding is kinetic as described in US Patent Application No. 61/715478, eg, incorporated herein by reference. It can occur under a method that utilizes exclusion). Kinetic exclusion can occur when an action occurs at a rate fast enough to effectively block the occurrence of another event or action. In the case of an array of gel-containing wells, the wells are randomly seeded with the target nucleic acid from the solution, and a copy of the target nucleic acid is amplified to fill each seeding position to full capacity. The sowing and amplification steps can be carried out simultaneously under conditions where the amplification rate exceeds the sowing rate. As such, a copy is made at a relatively high rate at the position where the first target nucleic acid is seeded, which effectively blocks the seeding of the second nucleic acid at the amplified position. Similarly, kinetic exclusion utilizes the relatively slow rate of making a first copy of a target nucleic acid, as opposed to the relatively fast rate of making a subsequent copy or first copy of the target nucleic acid. be able to. For example, kinetic exclusion is a delay (eg, delayed or slow) in the formation of the first copy of the target nucleic acid seeded in the gel-containing wells relative to the relatively fast rate at which subsequent copies are made and fill their position. Can occur by activation). In this example, the individual gel-containing wells may be seeded with a number of different target nucleic acids (eg, a number of target nucleic acids can be present at each position prior to amplification). However, the first copy formation can be randomly activated for any target nucleic acid, so that the average rate of first copy formation is relative to the rate at which subsequent copies are produced. Become slow. In this case, individual gel-containing wells could be seeded with several different target nucleic acids, but kinetic exclusion results in amplification of only one of these target nucleic acids. In general, gel-containing wells (or other concave features) can serve as a location for amplification and array configuration in the method described in US Patent Application No. 61/715478, which is incorporated herein by reference.

As an alternative to delivering a plurality of different specimens from the mixture to the individual gel-containing concave features, the specimens may be delivered discretely from the pure raw material to the individual features. Similarly, discrete delivery of synthetic components may be used to synthesize specimens with individual features (eg, nucleotide precursors can be delivered continuously to synthesize nucleic acids). Typical delivery methods of components for in situ synthesis of pure specimens or specimens include, but are not limited to, inkjet array positioning methods and photolithography array synthesis methods. A useful photolithography method is Affymetrix (Santa) for manufacturing GeneChip® microarrays. US Pat. Nos. 5324633, 5744305, 5624711, 6022963, 6291183, which are used on a commercial basis by Clare, CA) or incorporated herein by reference, respectively. , And those described in No. 6416949. Also commercialized by Agilent (Santa Clara, CA) for printing inkjet positioning methods, such as SurePrint® arrays, or US Pat. No. 6,337,393, each incorporated herein by reference. No., No. 6419883, No. 6420180 or No. 6689319 are also useful. Such a method can be readily modified to direct delivery to the gel-containing features of the present disclosure.

The gel material in a particular concave feature does not have to contain only a single specimen. Rather, in some embodiments, the concave feature may contain several different species of specimen in the gel therein. For example, demonstrated by the Bullseye reference marker in Figure 5. The reference marker contains two "bright" ring-shaped channels, each of which contains a gel material, and the gel material in each bright channel attaches to a number of different nucleic acid colonies. Nucleic acid colonies in bright channels were formed by seeding each ring with several different species of target nucleic acids that acted as templates in amplification techniques. The reference marker also includes two "dark" ring-shaped areas. The dark ring is composed of spaced surface patterns. A typical bull's eye is formed by alternating dark and bright rings in a concentric pattern. In the example of FIG. 5, the structured substrate also includes a gel-containing well, each containing a population of clones, generally derived from a single nucleic acid target. Wells are present in a ring-shaped band between a bright ring and a dark ring. Therefore, the reference has alternating patterns of interstitial rings, well-containing bands and channel rings. The same amplification technique was used to simultaneously grow clonal nucleic acid colonies in the wells and mixed populations in the reference markers (see Example 3 below). Other examples of criteria with alternating patterns of rings are shown in Figures 3B and 3C.

The present disclosure further provides a method of detecting a specimen. This method is (a) a step of providing a solid support with a plane, the plane being interrupted by one or more concave features, the concave features containing a gel material, and one or more concave features. A step in which one or more interstitial regions are adjacent on a plane and the interstitial regions are substantially free of gel material and the gel material adheres to or contains the gel material, and (b) the target specimen is specific. The step of contacting the solid support with the probe under conditions that interact with the probe, and (c) the step of detecting the solid support and identifying at least a subset of target specimens that interact with one or more of the probes. And can be included.

In certain embodiments, the nucleic acid is the specimen to be detected and the concave feature is the well. For example, a method of detecting nucleic acid is (a) a step of providing a solid support with a surface and a nucleic acid library, the surface having a plurality of wells, the wells containing a gel material, the wells. Are separated from each other by interstitial regions on the surface, which sequester the gel material in each well from the gel material in multiple other wells, and a single target nucleic acid from the library adheres to the gel material in each well. It has a step of contacting the solid support with at least one probe that binds to the target nucleic acid, and (c) a target nucleic acid species that detects the solid support and binds to at least one probe. A step of identifying a well can be included.

The structured substrate of the present disclosure containing a nucleic acid array can be used for any of a variety of purposes. A particularly desirable use of nucleic acids is to act as a capture probe that hybridizes to a target nucleic acid having a complementary sequence. The target nucleic acid once hybridized to the capture probe can be detected, for example, by labeling supplemented with the capture probe. Methods for detecting a target nucleic acid via hybridization to a capture probe are known in the art, for example, US Pat. Nos. 7,528,420, 6890741, 6913884, respectively, which are incorporated herein by reference. Alternatively, it includes those described in No. 6355431 or US Patent Application Publication No. 2005/0053980, 2009/0186349 or 2005/0181440. For example, the capture probe can be replenished with the label by hybridizing the capture probe to the labeled target probe. In another example, the capture probe is hybridized to a capture probe, resulting in a capture probe by ligating to a labeled oligonucleotide (eg, by ligase activity) or by adding a labeled nucleotide (eg, by polymerase activity). Can be extended to replenish the capture probe with the label.

Nucleic acid arrays can also be used in sequencing techniques such as Synthetic Decoding (SBS). Briefly, SBS can be initiated by contacting the target nucleic acid with one or more labeled nucleotides, DNA polymerases, and the like. The feature in which the primer is extended using the target nucleic acid as a template introduces a detectable labeled nucleotide. Optionally, labeled nucleotides are reversible. It can further include a termination) property, which is a property that terminates further primer extension once the nucleotide has been added to the primer. For example, a nucleotide analog with a reversible termination moiety can be added to the primer, which can prevent subsequent elongation from occurring until the deblocking agent has been delivered to remove the moiety. Thus, in embodiments that utilize reversible termination, the deblocking agent may be delivered to the flow cell (detection may occur before or after). Washing may be performed between the various delivery steps. The cycle is then repeated n times to extend the primer by n nucleotides, which allows the detection of a sequence of length n. Representative SBS techniques, fluid systems, and detection platforms can be readily adapted for use with arrays manufactured by the methods disclosed, eg, Bentley et al., Each incorporated herein by reference. Nature456: 53-59 (2008), WO04 / 018497, WO91 / 06678, WO07 / 123744, US Pat. Nos. 7057026, 7329492, 7211414, 7315019 or 7405281, and US Pat. It is described in Application Publication No. 2008/0108082.

Other sequencing techniques that utilize cycle reactions, such as pyrosequencing, may be used. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) when certain nucleotides are introduced into the nascent nucleic acid strand (Ronaghi et al., Analytical Biochemistry 242 (1), respectively incorporated herein by reference), 84 ~ 9 (1996); Ronaghi, Genome Res.11 (1), 3 ~ 11 (2001); Ronaghi et al., Science 281 (5375), 363 (1998); US Pat. No. 6210891; No. 6258568 and No. 6274320). In pyrosequencing, released PPi can be detected by conversion to adenosine triphosphate (ATP) by ATP sulfylase, and the resulting ATP can be detected via luciferase luminescent photons. Therefore, the sequencing reaction can be monitored via the luminescence detection system. The excitation radiation source used in the fluorescence-based detection system is not required for pyrosequencing techniques. Useful fluid systems, detectors and techniques can be used to apply pyrosequencing to the arrays of the present disclosure, eg, WIPO patent application PCT / US 11/57111, respectively incorporated herein by reference. No., US Patent Application Publication No. 2005/0191698, US Pat. No. 7595883, and US Pat. No. 7244559.

Ligation decoding is also useful, for example, as described in Shendure et al., Science 309: 17281732 (2005); U.S. Pat. No. 5,599,675; and U.S. Pat. including. Some embodiments are, for example, Bains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology, respectively, which are incorporated herein by reference. 16, 54-58 (1998); Fodor et al., Science251 (4995), 767-773 (1995); and WO1989 / 10977 can be included for hybridization decoding. In both ligation and hybridization decoding, nucleic acids present in gel-containing wells (or other concave features) are subjected to repeated cycles of oligonucleotide delivery and detection. The fluid system for the SBS method described herein or in the literature listed herein can be readily adapted for reagent delivery for ligation decoding or hybridization decoding. Typically, oligonucleotides can be fluorescently labeled and detected using a fluorescent detector similar to that described for the SBS approach herein or in the literature listed herein.

Some embodiments can utilize methods that require real-time monitoring of DNA polymerase activity. For example, nucleotide uptake can be detected by fluorescence resonance energy transfer (FRET) interactions between fluorophore-supported polymerases and γ-phosphate labeled nucleotides, or by the zeromode waveguides method. FRET-based sequencing techniques and reagents are described, for example, in Levene et al., Science 299, 682-686 (2003); Lundquist et al., Opt. Lett.33, 1026-1028 (2008), the disclosure of which is incorporated herein by reference. ); Korlach et al., Proc.Natl.Acad.Sci.USA105, 1176-1181 (2008).

Some SBS embodiments include detection of photons emitted upon incorporation of the nucleotide into the extension product. For example, sequencing based on emission photon detection is an electrical detector and related technology commercially available from Ion Torrent (a subsidiary of Life Technologies, Guilford, CT) or the United States, each of which is incorporated herein by reference. Sequencing methods and systems described in Publication No. 2009/0026082; No. 2009/0127589; No. 2010/0137143; Or No. 2010/0282617 may be used. In certain embodiments, the electrical detector used to detect emitted photons may be modified to include wells, which wells can contain the gel materials described herein.

Another application useful for the arrays of the present disclosure is gene expression analysis. Gene expression can be detected or quantified using an RNA sequencing method, such as what is called a digital RNA sequencing method. The RNA sequencing method can be performed using a sequencing method known in the art, such as those described above. Gene expression can also be detected or quantified using hybridization methods, which can be performed by hybridizing directly to the array or using a multiplex assay, the product of which is on the array. Is detected by. The arrays of the present disclosure can also be used to genotype genomic DNA samples from one or more individuals. Representative methods for array-based expression and genotyping that can be performed on the arrays of the present disclosure are described in US Pat. Nos. 7,582,420 and 6890741, respectively, which are incorporated herein by reference. It is described in No. 6913884 or No. 6355431, or US Patent Application Publication No. 2005/0053980, 2009/0186349 or 2005/0181440.

Some uses of the arrays of the present disclosure have been exemplified above in the context of ensemble detection, where multiple copies of the target nucleic acid are present in each feature and are detected together. In an alternative embodiment, a single nucleic acid can be detected in each feature, either the target nucleic acid or its amplicon. For example, gel-containing wells (or other concave features) can be configured to contain a single nucleic acid molecule with the detected target nucleotide sequence. Any of a variety of single molecule detection methods can be used, eg, the ensemble detection method described above has been modified to detect at a higher resolution position or to use a more sensitive label. Including those that have been Examples of other single molecule detection methods available are U.S. Patent Application Publication No. 2011/0312529, U.S. Patent Application No. 61/578684, and U.S. Patent Application No. 61/540714, which are incorporated herein by reference, respectively. It is described in the issue.

For example, it should be understood that the gel-containing substrates of the present disclosure, made by the methods described herein, are not necessarily used in detection methods. Rather, the structured substrate can be used to store the nucleic acid library. Accordingly, the structured substrate can be stored in a state in which the nucleic acid is stored. For example, substrates with gel-containing wells that adhere to nucleic acids may be stored in a dry, frozen state (eg, in liquid nitrogen), or in a solution that protects the nucleic acids. Alternatively, or additionally, the structured substrate can be used to replicate the nucleic acid library. For example, a substrate with gel-containing wells that adhere to nucleic acids may be used to make replicating amplicon from one or more wells on the array.

The following examples are for purposes of illustration, but are not intended to limit the invention.

<p>Multiwell Substrate Coated with Silane-Free Acrylamide In this example, the nanowell substrate is coated with silane-free acrylamide (SFA), then grafted with thiophosphate primers and hybridized with gel-grafted primers to complementary fluorescent oligonucleotides. Soybeans have been demonstrated to support the success of functionalization methods.</p><p> The chip substrates commonly used in the manufacture of Bead Chips were obtained from Illumina, San Diego, CA. The chip was composed of silicon or Zeonor (Zeon Corp., Tokyo, Japan) and had 0.5 μm wells arranged in a hexagonal pattern with a pitch of 1.5 μm, which wells did not contain beads. Chips were patterned with gel pads as described below and as shown in FIG.</p><p> Oxygen was removed by placing the tip in a gasket sealed chamber and replacing it with a fluid reagent for SFA production. SFA was polymerized on the chips in the chamber. The reagents that produce the SFA and the conditions for polymerization were described separately in US Patent Application Publication No. 2011/0059865, which is incorporated herein by reference. A sealed chamber was used to place the polymerization mixture in direct contact with the chip, and since free radical polymerization of SFA is an air instability process, complete removal of air was performed. After polymerization, primers were grafted onto the SFA polymer in a sealed chamber as described in US Patent Application Publication No. 2011/0059865 and below. The primer-containing solution was spread over the entire surface of the polymer-coated Bead Chips, and then the mixture was incubated at 65 ° C. for 1.25 hours (the entire sealed assembly was placed in a large oven).</p><p> This technique results in a uniformly coated substrate. A sample image is shown in panel A of FIG. To create discontinuous polymer regions, the "excess" polymer located between the wells on the substrate is removed by mechanical polishing using a slurry of aluminum oxide nanoparticles (300 nm in diameter) in distilled water. did. A 10 wt% slurry of 3 micron silica particles (Kisker Biotech GmbH, Steinfurt, Germany) can also be used. The surface was rubbed by hand with a nanoparticle slurry using a lint-free optical tissue. After washing to remove slurry and polymer debris, a solution of the fluorescently labeled probe was hybridized to the chip. Images taken with a fluorescence microscope showed that this technique was able to produce polymer features with empty interstitial regions (Fig. 2, panels B to C).</p><p> These results indicate that the fluorescence intensities of the gel-filled wells were spatially discrete, as opposed to the absence of signals from the interstitial regions. These results also demonstrate that gel patterning can be achieved using non-covalent gel materials on substrates with nanoprocessed wells.</p>

<p>Manufacture of Substrates with Gel-Containing Nanowells Multiple techniques can then be used to make structured arrays that can then load gel material.</p><p> The process can start with a blank substrate / wafer and introduce the pattern onto the substrate by micro or nano processing. The substrate / wafer material may be either ordinary silicon, glass, plastic, COC or a variety of constructable materials. Typical techniques for incorporating patterning into substrates are photolithography, nanoimprint lithography, structural embossing on plastic / COC-based materials, and injection molding of plastic or COC into master molds with patterned structures. including. Photolithography-based techniques typically require the use of photoresists, which are patterned with a stepper or mask aligner and exposed to radiation, thereby resisting the patterns present on the reticle / photomask. Then, the resist is developed to obtain a structured film (photoresist) on the upper part of the substrate. The structured resist may optionally be the final substrate that can be used for subsequent gel coating, or the pattern in the resist may be transferred to the substrate by subsequent processing. Subsequent processing steps typically include reactive etching (plasma-based etching) or wet etching (chemically based) methods. When transferring the pattern to a substrate, the patterned photoresist is then removed to obtain a patterned substrate for subsequent gel coating. It may be desirable to use a sacrificial material film such as chromium or titanium (metal) under the photoresist, by first transferring the photoresist pattern to a metal film and then using this film as a hardmask to create the pattern. Transfer to the board. After transferring the pattern to the substrate, the film is removed and therefore considered sacrificial to the processing process. When using nanoimprint lithography, the imprinted photoresist can be a sacrifice and can be used as an intermediate tool to transfer the patterned resist to the substrate, or the imprinted resist can be used as an input to subsequent coating steps. You can use resist variability to work. An example of a resist that would leave the following patterning would be a sol-gel based material.</p><p> A drawing showing how a structured substrate can be manufactured is shown in FIG. 3 and will be described below. Images of the patterned substrate are shown in FIGS. 3B and 3C at various magnification levels.</p><p> Making chemically specific gel pads on a sequencing substrate / flow cell may require one or more nanomachining methods described earlier in this example. The process can then optionally include one or more chemical treatment steps, eg, a silaneization treatment in which the gel polymer is attached to the substrate via silane. Chemical / mechanical polishing (CMP) is then used to remove all interstitial polymers on the surface of the substrate. The polishing process removes the material in a top-down fashion, and the structured features in the substrate are effectively offset from the plane of the gap region of the array, so that the polymer is removed from the gaps by polishing before removing the structured features. Will be removed. If the polishing process is stopped after the optimum time, the structure retains the polymer coating and the interstitial regions are free of polymer. The patterned gel pad substrate is then grafted onto a primer, the target nucleic acid is seeded with the gel pad, and the target nucleic acid is used as a template for making nucleic acid clusters with the gel pad.</p>

<p>PAZAM-coated multi-well substrate This example demonstrates the production of an array of gel-containing wells, the amplification of nucleic acid clusters in the wells, and the sequencing of nucleic acids in the clusters.</p><p> The substrate was manufactured as follows. Nanowell substrates (wells with a diameter of 400 nm, a pitch of 1.5 μm, and a depth of 300 nm) were manufactured using nanoimprint lithography. Aminosilane (APTES or APTMS) single layer / multilayer was deposited over the entire surface of the substrate using a chemical vapor deposition method. Next, 1 ml of NHS acrylate solution was added to the surface, which was covered with a thin glass coverslip, and the reaction was allowed to proceed for 1 hour at room temperature to allow a concentration of 100 mM acrylic acid N-hydroxysuccinimide ester (Aldrich). Phosphate buffered saline (pH 7.4) of PN8060) was reacted with the surface of aminosilane. The polymer (PAZAM) was then applied to the surface by spin-coating 500 μl of a 2 wt% PAZAM aqueous solution onto the newly formed acrylamide functionalized surface. PAZAM was synthesized as described in US Provisional Patent Application No. 61/753833, which is incorporated herein by reference. The PAZAM-coated substrate was then heated at 60 ° C for 1 hour, resulting in a covalent bond between the polymer and the surface. The covalently bonded polymer in the gap is 3 μm SiO in 10 wt% water.₂It was removed by polishing the surface with a slurry in which fine particles were dissolved. A Janeway surface (acryloyl chloride + DIPEA (in MeCN)) may be used in place of the aminosilane coated surface in the above procedure.</p><p> The patterned polymer substrate was then grafted onto the primers as described in US Provisional Patent Application No. 61/753833, which is incorporated herein by reference. The reverse compliment of the dye-labeled (Cy5) grafted primer was then exposed to the surface in 1 XPBS buffer at a concentration of 20 μM of the complementary sequence, and then the surface was exposed to 50 ml using a jet bottle. Washed with 1 PBS buffer. The labeled complement on the substrate was set to the Cy5 scan channel and imaged with the FLA9500 Typhoon Imager using PMT setting 450. The labeled complement on the substrate was also imaged by a high resolution microscope, showing a patterned or polymer / primer with no polymer / primer left in the gap (Figure 4). The substrate was then seeded with phiX DNA and clusters grew as described in US Patent Application No. 61/715478, which is incorporated herein by reference.</p><p> Flow cells containing cluster-containing substrates were sequenced on HiSeq 2000 (Illumina, Inc., San Diego, CA). An algorithm (rigid registration) for locating patterned sequencing clusters was used to successfully obtain high quality sequencing metrics (Figures 5 and 6). Sequencing results show that the occupancy for clonality was surprisingly higher than predicted by the standard Poisson distribution. In particular, the average occupancy for clonality measured for the sequence performed, indicated by an x in FIG. 7, is above the boundaries of the Poisson curve and approaches the straight line of ideal clonality.</p><p> Various publications, patents, or patent applications have been referenced throughout this application. The disclosures of these publications, the entire contents of which are incorporated herein by reference, more fully describe the state of the art in which the invention is involved.</p><p> The term "contains" is intended herein to include not only the unconstrained and listed elements, but also any additional elements.</p><p> Although the present invention has been described with reference to the above embodiments, it should be understood that various modifications can be made without departing from the present invention. Therefore, the present invention is limited to the scope of claims.</p>