array, method of producing the same, method of producing a substrate and method of detecting nucleic

专利摘要:
ARRANGEMENT, METHOD TO PRODUCE THE SAME, METHOD TO PRODUCE A SUBSTRATE and METHOD OF DETECTING NUCLEIC ACIDS. An arrangement is provided including a solid support having a plurality of vessels, the vessels containing a gel material, the vessels being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the vessels of the material gel in the other vessels of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each vessel comprises a single species of the target nucleic acids in the library. Methods for making and using the arrangement are also provided.
公开号:BR112015014793B1
申请号:R112015014793-3
申请日:2014-02-21
公开日:2020-10-27
发明作者:Steven M. Barnard；M. Shane Bowen；Maria Candelaria Rogert Bacigalupo；Wayne N. George；Andrew A. Brown；James Tsay
申请人:Illumina, Inc.；
IPC主号:

专利说明:

CROSS REFERENCE WITH RELATED REQUESTS
[001] The present application claims the benefit of US Provisional Patent Application 61 / 769,289, filed on February 26, 2013, and US Application 13 / 787,396, filed on March 6, 2013. Each of the above applications is entirely incorporated herein by reference. FUNDAMENTALS
[002] The present disclosure refers in general to solid phase analytical chemistry, and has specific applicability for nucleic acid arrays for high-throughput genomic analysis.
[003] The task of cataloging variation in human genetics and correlating that variation with susceptibility to disease benefits from advances in broad genome sequencing methodologies. This cataloging effort promises to identify the markers in each person's genome which will help medical professionals determine that person's susceptibility to the disease, the response to specific therapies such as prescription drugs, susceptibility to dangerous drug side effects and other characteristics that can be brought to justice in the medical field. The cataloging effort is well underway. This is largely due to commercially available genome sequencing methodologies that are cost effective enough to allow guinea pigs to be evaluated in a research environment. Improvements in sequencing methodologies are needed to speed up the cataloging effort. In addition, the relatively high cost of sequencing has prevented the technology from going beyond research centers and to the clinic where doctors can obtain sequences for patients in the general population.
[004] The sequencing methodologies and the systems used to carry them out, explore a complex library of technologies. Improvements in some of these technologies have been shown to provide substantial cost savings. However, it is difficult to predict which, if any, is responsible for improvements in cost reduction. Due to the dependencies between technologies in the sequencing systems it is even more difficult to predict which can be modified without having an adverse impact on the overall performance of the methodology or system. Therefore, it is necessary to identify improvements that can bring the promise of genomic research to the clinic where lives can be improved and in many cases saved. The present invention addresses this need and also provides related advantages. SUMMARY
[005] The present disclosure provides an arrangement that includes a solid support that has a surface, the surface having a plurality of wells that contain a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each well of the gel material in other wells of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each well comprises a unique species of the target nucleic acids in the library.
[006] In some embodiments, the substrate is configured as an array of wells and the analytes are nucleic acids. Therefore, the present disclosure provides an arrangement that includes a solid support that has a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each well of the gel material in other wells of the plurality; and a library of target nucleic acids in the gel material, wherein the gel material in each well includes a single species of target nucleic acids from the library.
[007] The present disclosure also provides a method for producing a substrate. The method may include the steps of (a) providing a solid support that has a flat surface, where the flat surface is interrupted by one or more concave features and where one or more concave features are bounded by one or more interstitial regions on the flat surface; (b) coating at least part of the solid support with a gel material, the part comprising at least one of the concave features and at least one of the interstitial regions; and (c) polishing the flat surface to remove the gel material from at least one of the interstitial regions and to keep the gel material in at least one concave feature.
[008] A method for producing an arrangement may include the steps of (a) providing a solid support that has a surface with a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions in the surface, the interstitial regions segregating the gel material in each well of the gel material in other wells of the plurality; (b) releasing a library of target nucleic acids into the wells of the solid support to produce an array of wells that have unique species of target nucleic acid for the gel material in each well, where the different wells in the array have acid species target nucleic acid other than the library; and (c) amplifying the target nucleic acids attached to the gel material in the wells of the array to produce a population of clones of an individual target nucleic acid in each well of the array.
[009] The present disclosure also provides a method of detecting analytes. The method may include the steps of (a) providing a solid support that has a flat surface, where the flat surface is interrupted by one or more concave features, where the concave features contain gel material, where the one or more concave features are bounded by one or more interstitial regions on the flat surface, the interstitial regions being substantially devoid of the gel material, and where the gel material is attached to or contains target analytes; (b) contacting the solid support with probes under conditions where the target analytes specifically interact with the probes; and (c) detecting the solid support to distinguish at least a subset of the target analytes that interact with one or more of the probes.
[010] In specific embodiments, nucleic acids are the analytes that are detected and concave resources are wells. For example, a method of detecting nucleic acids may include the steps of (a) providing a solid support that has a surface and a library of nucleic acids, the surface having a plurality of wells, wells containing gel material, wells being separated from each other by other interstitial regions on the surface, the interstitial regions segregating the gel material in each well of the gel material in other wells of the plurality, a single species of target nucleic acids from the library being fixed to the gel material in each well; (b) detecting the solid support to distinguish wells that have a kind of target nucleic acid that binds to at least one probe.
[011] The compositions, apparatus, and methods of the present disclosure are exemplified here with respect to the gel material. It should be understood that the gel material is exemplary and can be replaced by other organic materials including, for example, polymers that can form a surface coating and not that may not necessarily be considered as gels in themselves. The methods disclosed here for applying gel material to a surface, removing the gel material from the interstitial regions, fixing the analytes to the gel material, using the resulting arrangements in the analytical or preparative methods, etc., can be readily adapted by replacing the material in gel for materials that are not gel. BRIEF DESCRIPTION OF THE DRAWINGS
[012] Figure 1 illustrates a diagrammatic representation of a method for producing and using a standardized array of DNA resources, where each resource is a well that has gel material that is attached to a group of DNA and the arrangement is used in a sequence technique.
[013] Figure 2 illustrates an image of a “BeadChip” substrate modified to have a gel material in the wells instead of granules. Panel A: bright field images, obtained before polishing. Panels B to C: fluorescent images obtained after polishing and hybridization for fluorescently labeled oligonucleotides.
[014] Figure 3 illustrates in Panel A: a schematic process flow that uses photolithography and a rigid Cr mask along with reactive ion print to manufacture concave features on a substrate; on Panel B, images of sample SEM wells and parts of a fiducial on a glass substrate; and on Panel C an image of a tablet that includes a fiducial and a well arrangement, and an image of a part of the arrangement that includes wells.
[015] Figure 4 illustrates high resolution fluorescent microscopic images of nanowell substrates that illustrate standardized gel features on the nanowell substrate after the substrate is coated with PAZAM and polished with silica granule paste. PAZAM is marked with a dye for visualization purposes.
[016] Figure 5 illustrates multicolored image casting obtained from a HiSeq sequence cycle of a 1.5 pm nanowell substrate that has standardized clusters. Panel A: image that illustrates a field of standardized groups in wells containing gel together with four fiducials from the center of sight. Panel B: a higher resolution image that illustrates the mixture of colors (due to a mixed population of amplicons) in a single fiducial sighting center.
[017] Figure 6 illustrates in Panel A: a multicolored cast of standard groups in a Hiseq sequencing assay that consumes a 750 nm step nanowell substrate; Panel B: the nearest adjacent curve that illustrates the arrangement is ordered and the groups are passing quality filters; and Panel C: sequence quality measures that illustrate that a density of 1.6 million clusters / mm2 quality filters were successfully passed.
[018] Figure 7 is a representation of Clonal Fraction versus Fraction occupied with the expected curve for a Poisson distribution, a straight line expected for clonality and ideal occupancy and an X for the average measurement obtained from a sequencing test that consumes a substrate which has nanowells containing gel. DETAILED DISCLOSURE
[019] The present disclosure provides structured substrates, methods for making structured substrates and methods for using structured substrates. In specific embodiments the substrates include a solid support that has concave regions, such as wells, that contain gel material (for example, being coated by the gel material). The gel material can in turn be attached to an analyte of interest, such as a nucleic acid. In specific embodiments, the gel-containing regions are separated, being separated by interstitial regions that do not have the ability to fix the analyte of interest. For example, interstitial regions may not have gel material. Alternatively, the gel material in the interstitial regions can be inactivated or modified in another way so as not to have an activity or characteristic of the gel material in the concave regions, such as the ability to support analyte fixation. The resulting segregation of the gel regions provides advantages when carrying out reactions on the analytes and / or detecting the analytes. An exemplary advantage can be demonstrated in the case of an array of target nucleic acids distributed between the wells containing gel. Here, an amplification reaction can be performed on the structured substrate using nucleic acids as standards to form nucleic acid colonies that grow on or over the gel (for example, nucleic acid resources in the array). Interstitial regions work to confine the growth area to the colony. The individual features of the resulting arrangement can be distinguished relatively easily due to the separate pattern created by the gel-containing wells. The standard can also provide the benefits of increasing resource density and reducing processing requirements for image registration as compared to random nucleic acid arrays.
[020] An exemplary process for making a standardized arrangement of nucleic acids is illustrated in Figure 1. A profile view of a standardized well substrate is illustrated diagrammatically. The wells have a pitch (spacing from center to center) of 1.5 pm and the diameter of each well is 0.5 pm in the example. The standardized well substrate can be coated with a gel material so that the material enters the wells and searches the interstitial regions. The resulting gel-coated substrate can be polished to remove the gel material from the interstitial regions, leaving the gel material in the wells, thereby forming a gel-patterned substrate. The gel can work to support the capture of a DNA model and amplification of the model. For example, the gel can be grafted with oligonucleotide primers before coating the surface, after coating the surface and before polishing, or after polishing. Primers can work to capture DNA models and to amplify primers using the captured patterns. The resulting DNA-standardized substrate can be analyzed, for example, in a sequencing technique.
[021] A standardized array of nucleic acids in wells containing gel provides many advantages for DNA sequencing. Examples of advantages compared to random arrangements (that is, arrangements that have a random resource pattern) include increasing resource packaging density, increasing control and tuning resource density using concentration-independent standard seeding, reducing processing requirements for image registration and increased ease of signal extraction. An additional advantage can be derived from the spatial confinement of nucleic acid populations by each resource. A feature of a standardized arrangement of the present disclosure may work to restrict the area or volume within which a nucleic acid colony will grow (for example, through cluster amplification). In the absence of area or volume restrictions, some nucleic acid colonies may amplify to a larger size than others due to differences in the perennial content of guanine and cytosine in their sequences (ie, GC content) that influences relative amplification rates . For the methods and compositions described here, the volume or area of individual resources can be selected to prevent or minimize differences in nucleic acid colony sizes that would otherwise occur from the amplification reaction due to differences in the GC content between the standard species being amplified. For example, the volume or area of the resources may be small enough to limit the growth of the fastest growing colonies while allowing the slowest growing colonies to effectively fill the resource upon completion of the amplification reaction.
[022] In specific embodiments, the present disclosure provides manufacture of wells (for example, microwells or nanowells) in glass, silicon, plastic or other suitable solid supports with covalently bonded and standardized gel such as poly (N- (5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM, see, for example, Provisional Patent Application US 61 / 753,833, the disclosure of which is hereby incorporated by reference). The process creates gel pads used for sequence that can be stable during sequence runs with a large number of cycles. The covalent bonding of the polymer to the wells is useful for maintaining the gel in the structured resources for the life of the structured substrate during various uses. However, in many embodiments, the does not need to be covalently connected to the wells. For example, in some conditions, silane-free acrylamide (SFA, see, for example, US Patent Application Publication 2011/0059865 A1, the disclosure of which is hereby incorporated by reference) which is not covalently fixed no part of the structured substrate can be used as the gel material.
[023] In specific embodiments, a structured substrate can be prepared by standardizing a solid support material with the wells (for example, microprocessor wells or nanowells), coating the standardized support with a gel material (for example, PAZAN, SFA or variations chemically modified, such as the "azidolized" version of SFA (azido-SFA) and polishing the gel-coated support, for example, by chemical or mechanical polishing, thereby retaining the gel in the wells, but removing or substantially inactivating all gel from the interstitial regions on the surface of the structured substrate between the wells. The initiator nucleic acids can be attached to the gel material. A solution of target nucleic acids (for example, a fragmented human genome) can then be contacted with the polished substrate so that the individual target nucleic acids will seed individual wells through interactions with primers attached to the gel material, however, the acids Target nucleic acids will not occupy the interstitial regions due to the absence or inactivity of the gel material. The amplification of the target nucleic acids will be confined to the wells because the absence or inactivity of gel in the interstitial regions prevents the growth nucleic acid colony from leaving. The process can be manufactured conveniently, scalable and using conventional micro or nanofabrication methods.
[024] In specific embodiments, fiducial markers are included on a structured substrate to facilitate the identification and localization of individual resources (for example, wells or other concave resources containing gel). Fiducial markers are particularly useful for structured substrates that have spatially ordered resource patterns because fiducial markers provide a reference point for relative locations of other resources. Fiducial markers can also be used to record images of random arrays as well, but instead, the inherent clutter clutter with random arrays generated on commercial sequence platforms such as HiSeq, Genome Analyzer or MíSeq platforms can be used instead. Illumina, Inc. (SanDiego, CA). Fiducial markers are especially beneficial for applications where the structured substrate is detected repeatedly to follow changes that occur in individual resources over time. The fiducial markers allow the individual nucleic acid clusters to be tracked through sequential images obtained during multiple sequence cycles, so that the sequence of the individual clusters can be determined separately.
[025] Disclosure provides a fiducial marker that has a pattern of concave region (s) and interstitial region (s). An exemplary design for a fiducial marker is a set of concentric circles that have an alternating pattern of two or more of the following: a concave rim, an interstitial rim and a rim of wells or other concave features (for example, a “center aiming ”). In some embodiments, the concave region (s) of a fiducial marker contain (contain) gel material, while the interstitial regions do not. This differential location of the gel on the surface can be achieved using the gel coating and polishing methods reported here. Typically a detection method is used that can distinguish regions containing gel from interstitial regions. In some cases, the distinction may be based on the presence of a specific analyte in the gel regions that is not found in the interstitial regions. For example, in the case of nucleic acid arrays, the gel-containing region of a fiducial marker may contain nucleic acids that are tagged using the same methods that are used to tag target nucleic acids in the array. Therefore, fiducial markers can be manufactured conveniently using the same methods used to manufacture analyte resources. Therefore, if desired, fiducial markers and analyte resources can be manufactured simultaneously through one or more steps. Another useful fiducial marker that can be used on the structured substrates and methods described here is one that has subregions where the pattern of wells (or other concave resources) in one region is rotated with respect to the pattern in another sub-region. Such fiducial grids can be configured and used for image registration as described in Document US 13 / 267,565, the disclosure of which is incorporated herein by reference.
[026] As an additional example, granules can be used as a fiducial. The granules can include a label such as a fluorophore. In this case, a surface can be equipped with at least two types of wells (or other concave resources). Relatively large wells can accommodate one or more fiducial granules, while smaller wells, being too small to contain a granule, will have only gel material. Therefore, smaller wells function as analytical resources for analysis and larger wells filled with granules function as fiducials. As an alternative to the wells the fiducial resources can be channels, such as those present in the center sighting configuration exemplified above, and the channels can have dimensions that accommodate the granules. As such, several granules can be placed in the channel to create a fiducial, for example, in the apparent shape of a granule chain.
[027] The standardized arrangements, methods for their manufacture and methods for their use are exemplified here with respect to a gel material that is used to fix analytes of interest. It should be understood that the gel material is exemplary and can be replaced by another organic material that can be used for indirect location of analytes for resources on a surface. Such organic materials include, for example, polymers that can form a surface coating and cannot necessarily be considered as gels. A specific example is a polymer formed by ATRP (radical atom transfer polymerization) or polymerization processes initiated on the surface. The methods described here for applying gel material to a surface, removing gel material from interstitial regions, using the resulting arrangements in analytical or preparatory methods, etc., can be readily adapted for use with non-gel materials.
[028] The terms used here are to be understood in their common meaning in the relevant technique unless otherwise specified. Several terms used here and their meaning are described below.
[029] As used here, the term "fixed" refers to the state of two things that are joined, attached, attached, connected or linked together. For example, an analyte, such as a nucleic acid, can be attached to a material, such as a gel or solid support, by a covalent or non-covalent bond. A covalent bond is characterized by the sharing of electron pairs between atoms. A non-covalent bond is a chemical bond that does not involve sharing electron pairs and can include, for example, hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic interactions and hydrophobic interactions.
[030] As used here, the term "clonal population" refers to a population of nucleic acids that is homogeneous with respect to a specific nucleic sequence. The homogeneous sequence is typically at least 10 nucleotides in length, but can be even longer including, for example, at least 50, 100, 250, 500, 1,000 or 2500 nucleotides in length. A clonal population can be derived from a single target nucleic acid or model nucleic acid. A clonal population can include at least 2, 5, 10, 100, 1,000 or more copies of a target nucleotide sequence. The copies can be present in a single nucleic acid molecule, for example, as a concatamer or the copies can be present in separate nucleic acid molecules (that is, a clonal population can include at least 2, 5, 10, 100, 1,000 or more nucleic acid molecules that have the same target nucleotide sequence). Typically, all nucleic acids in a clonal population will have the same nucleotide sequence. It will be understood that an insignificant number of contaminating nucleic acids or mutations (for example, due to amplification artifacts) can occur in a clonal population without departing from clonality. Therefore, a population can be at least 80%, 90%, 95% or 99% clonal. In some cases, 100% pure clonal population may be present.
[031] As used here, the term “to coat”, when used as a verb, is meant to provide a layer or cover on a surface. At least part of the surface may be provided with a layer or cover. In some cases, the entire surface may be provided with a layer or covering. In alternative cases, only a part of the surface will be provided with a layer or covering. The term “coating”, when used to describe the relationship between a surface and a material, is intended to mean that the material is present as a layer or covering on the surface. The material can seal the surface, for example, preventing the contact of liquid or gas with the surface. However, the material does not have to form a seal. For example, the material can be porous for liquid, gas, or one or more components carried in a liquid or gas. Example materials that can coat a surface include, but are not limited to, a gel, polymer, organic polymer, liquid, metal, a second surface, plastic, silica, or gas.
[032] As used here, the term “concave feature”, when used with reference to a solid support, refers to a recess or notch in the solid support. Exemplary concave features include, but are not limited to, a well, pit, bore, depression, channel or pit. A concave feature can optionally have a curved cross section (in dimension orthogonal to the surface of the solid support); however, a cross section with one or more linear sections, angles or corners is also possible. Cross sections with combinations of curved and linear sections are also possible. Generally, a concave feature does not need to pass completely through the solid support, for example, instead of having a surface or bottom point on the substrate.
[033] As used here, the term "different", when used with reference to nucleic acids, means that nucleic acids have different nucleotide sequences that are not the same with each other. Two or more nucleic acids can have nucleotide sequences that are different along their length. Alternatively, two or more nucleic acids can have nucleotide sequences that are different over a substantial part of their length. For example, two or more nucleic acids can have nucleotide sequence parts that are different for two or more molecules while also having a universal sequence part that is the same in the two or more molecules.
[034] As used here, the term “each”, when used with reference to a collection of items, is intended to identify an individual item in the collection, but does not necessarily refer to each item in the collection. Exceptions may occur if the disclosure or context clearly dictates otherwise.
[035] As used here, the term “fluidic access”, when used with reference to a molecule and a location in contact with the fluid, refers to the molecule's ability to move in or through the fluid to contact or enter the local. The term can also refer to the molecule's ability to separate from or leave the site to enter the solution. Fluidic access can occur when there are no barriers that prevent the molecule from entering the site, contacting the site, separating from the site and / or leaving the site. However, it is understood that fluidic access exists even if diffusion is delayed, reduced or altered as long as access is not absolutely impeded.
[036] As used here, the term "gel material" is intended to mean a semi-rigid material that is permeable to liquids and gases. Typically the gel material can swell when the liquid is absorbed and can contract when the liquid is removed by drying. Exemplary gels include, but are not limited to, those with a colonial structure, such as agarose, polymer mesh structure, such as gelatin; or cross-linked polymer structure, such as polyacrylamide, SFA (see, for example, US Patent Application Publication 2011/0059865 A1, the disclosure of which is hereby incorporated by reference) or PAZAM (see, for example, Provisional Patent Application US 753,833, the disclosure of which is hereby incorporated by reference). The particularly useful gel material will conform to the shape of a well or other concave resource where it resides. Some useful gel materials may (a) conform to the shape of the well or other concave resource where you reside and (b) have a volume that does not substantially exceed the volume of the well or concave resource where you reside.
[037] As used here, the term "interstitial region" refers to an area on a substrate or on a surface that separates other areas from the substrate or surface. For example, an interstitial region can separate a concave feature from an arrangement from another concave feature from the arrangement. The two regions that are separated from each other can be separated without contact with each other. In another example, an interstitial region can separate a first part of an asset from a second part of an asset. In many embodiments, the interstitial region is continuous while the resources are separated, for example, as in the case of a well surface arrangement in other aspects. The separation provided by an interstitial region can be partial or total separation. Interstitial regions will typically have a surface material that differs from the surface material of the features on the surface. For example, the resources of an array may have an amount or concentration of gel material or analytes that exceeds the amount or concentration present in the interstitial regions. In some embodiments, the gel material or analytes may not be present in the interstitial regions.
[038] As used here, the term "library" ', when used with reference to analytes, refers to a collection of analytes that have different chemical compositions. Typically, the analytes in a library will be of different species that have a feature or characteristic of a genus or class, but in other ways different in some way. For example, a library may include species of nucleic acid that differ in the sequence of nucleotides, but which are similar with respect to having a sugar phosphate structure.
[039] As used here, the term "nucleic acid" and "nucleotide" are intended to be consistent with their use in the art and include naturally occurring species or functional analogues. Particularly useful functional analogs of nucleic acids are capable of hybridizing to a nucleic acid in a sequence-specific mode or capable of being used as a standard for replicating a specific nucleotide sequence. Naturally occurring nucleic acids generally have a structure that contains phosphodiester bonds. An analogous structure can have a structure link that includes any variety of those known in the art. Naturally occurring nucleic acids generally have a deoxyribose sugar (for example, found in deoxyribonucleic acid (DNA)) or a ribose sugar (for example, found in ribonucleic acid (RNA)). A nucleic acid can contain nucleotides that have any variety of analogs of these sugar moieties that are known in the art. A nucleic acid can include native or non-native nucleotides. In this regard, a deoxyribonucleic acid may have one or more bases selected from the group consisting of adenine, thymine, cytosine or guanine and a ribonucleic acid may have one or more bases selected from the group consisting of uracil, adenine, cytosine or guanine . Useful non-native bases that can be included in a nucleic acid or nucleotide are known in the art. The terms "probe" or "target", when used with reference to a nucleic acid, are intended to be semantic identifiers for the nucleic acid in the context of a method or composition disclosed herein and do not necessarily limit the structure or function of the nucleic acid beyond in other respects it is explicitly indicated. The terms "probe" and "target" can be similarly applied to other analytes such as proteins, small molecules, cells or the like.
[040] As used here, the term “random pattern”, when used in reference to wells on a surface, means that the relative locations of a subset of wells in a region of the surface are not known or predictable from the locations of a subset of wells in another region of the surface. The subset used for the measurement will generally include at least 3 wells, but can include at least 4, 5, 6 10 or more wells. A random pattern does not usually include multiple repetitions of any sub-pattern. The term can be applied to other concave resources besides wells.
[041] As used here, the term “repetition pattern”, when used with reference to wells on a surface, means that the relative locations of a subset of wells on a surface region are the same as the relative locations of a subset of wells. wells in at least another region of the surface. Therefore, the relative locations for wells in one region of a repeat pattern are generally predictable from the relative locations of wells in another region of the repeat pattern. The subset used for the measurement will generally include 3 wells, but can include at least 4, 5, 6, 10 or more wells. Repeat patterns can include multiple repetitions of a sub-pattern. The term can be applied to other concave resources besides wells.
[042] As used here, the term “segregate”, when used with reference to gel material in two wells (or in two other resources), means to separate or isolate the gel material in one of the wells (or in one of the resources ) of the gel material in the other well (or in the other resource). Therefore, the gel material in the first well (or in the first resource) is not in direct contact with the gel material in the other well (or in the other resource). In some embodiments, the gel material in two wells (or in two resources) is in direct contact, for example, through a solution that contacts the two wells (or resources). Alternatively, the gel material in the two wells (or in the two resources) is not leveled in direct contact. An interstitial region on a surface can segregate the gel material in two wells (or in two resources) being devoid of the gel material. In particular embodiments, a gel material may be discontinuous on a surface, being present in concave features, such as wells, but not present in interstitial regions between the features.
[043] As used here, the term "surface" is intended to mean an outer part 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, second surface of a different material, metal or coating. The surface or regions thereof can be substantially flat. The surface may have surface features such as wells, pits, channels, wrinkles, elevated regions, pegs, columns or the like.
[044] As used here, the term "unique species" means substantially one and only one species of a specific genus. The term is not necessarily intended to limit the number of representatives of a single species that is present. For example, a population of nucleic acid molecules each having the same nucleotide sequence comprises a single species of nucleic acid. The term “unique” in this context is not intended to exclude the presence of other things that are not within relevant genres. For example, a well that contains a single species of target nucleic acid from a library may include several nucleic acids that have the same sequence, it will exclude another target nucleic from the library, but it does not necessarily need to exclude any component other than nucleic acid. It will be understood that a population of a single apparent species may have a small amount of another species present at a level that is considered by those skilled in the art to be an insignificant level of contamination or artifact for the specific use of the population. For example, a nucleic acid cluster, derived from a single pattern that has a first sequence, will be considered to have a single apparent species if the number of nucleic acid molecules that have a second sequence is low enough to not be detected or ignored when the first sequence is detected. Alternatively, a population of a single absolute species will have one and only one species.
[045] As used here, the term "solid support" refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate may optionally be able to absorb a liquid (for example, due to porosity), but it will typically be rigid enough not to increase substantially when absorbing the liquid and not to contract substantially when the liquid is removed by drying. A solid non-porous support is generally impervious to liquids or gases. Solid supports can optionally be inert to a chemical that is used to modify a gel. For example, a solid support may be inert to a chemical used to attach analytes, such as nucleic acids, to gels in a method disclosed here. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, Teflon ™, cyclic olefins, polyimides, etc.), nylon, resins, Zeonor, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glass, fiber optic bundles, and polymers. Particularly useful for some embodiments, the solid supports are located within a flow cell apparatus. Exemplary flow cells are revealed in greater detail below.
[046] As used here, the term “well” refers to a separate concave feature on a solid support that has a surface opening that is completely surrounded by the interstitial region of the surface. Wells can have any of a variety of shapes in their opening on a surface that includes, but is not limited to, round, square, polygonal, star-shaped (with any number of vertices) etc. The cross section of a well taken orthogonally with the surface can be curved, square, polygonal, hyperbolic, conical, angular, etc.
[047] The embodiments disclosed below and reported in the claims can be understood in view of the definitions above.
[048] The present disclosure provides a substrate that includes a solid support that has a surface, the surface having at least one concave feature, the at least one concave feature containing a gel material, the at least one concave feature being limited by at least least one interstitial region on the surface; and a library of analytes in the gel material, wherein the gel material in each well includes a single species of analyte from the library.
[049] In some embodiments, the substrate is configured as an array of wells and the analytes are nucleic acids. Therefore, this disclosure provides an arrangement that includes a solid support that has a surface, the surface having a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each well of the gel material in other wells of the plurality, and the target nucleic acid library in the gel material, wherein the gel material in each well includes a single species of the target nucleic acids in the library.
[050] A solid support used on a structured substrate disclosed here can be made from any of a variety of materials disclosed here, for example, in the definitions above, below in the examples or immediately following. A particularly useful material is glass. Other suitable substrate materials may include polymeric materials, plastics, silicon, quartz (fused silica), BOROFLOAT® glass, silica, materials based on silica, carbon, metals, an optical fiber or bundles of optical fiber, sapphire, or plastic materials such as like COCs and epoxies. The specific material can be selected based on the properties desired for a specific use. For example, materials that are transparent to a desired wavelength of radiation are useful for analytical techniques that will use radiation of the desired wavelength, such as one or more of the techniques disclosed here. On the contrary, it may be desirable to select a material that does not pass radiation for a given wavelength (for example, being opaque, absorbable or flexible). This can be useful for forming a mask to be used during the manufacture of the structured substrate, such as a method disclosed here; or to be used for a chemical reaction or analytical detection performed using the structured substrate, such as those disclosed here. Other material properties that can be exploited are inertia or reactivity for certain reagents used in a downstream process, such as those disclosed here; or easy to manipulate or inexpensive during the manufacture of a manufacturing process, such as those disclosed here. Additional examples of materials that can be used in the structured substrates or methods of the present disclosure are described in Document US 13 / 661,524 and US Patent Application Publication 2012/0316086 A1, the descriptions of which are incorporated herein by reference.
[051] In a specific embodiment, a substrate based on Sol-Gel can be made and used. Standardization based on Sol-Gel can be carried out by coating a rigid or flexible substrate, such as glass, silicon, plastic, metal or the like, with a Sol-Gel coating can be carried out, for example, by centrifugal coating, coating by immersion or spraying. The Sol-Gel can be supplied in a liquid state when applied to the substrate and can contain either photo or thermal initiators that enable curing (made into a liquid in a gel) by exposing the Sol-Gel to light or heat. Subsequent to coating the substrate with Sol-Gel, and before curing the material, Sol-Gel can be printed with a pattern (three-dimensional print) that has or a plurality of protruding feature (s). The pattern can be made, for example, of silicon, glass (such as quartz), metal (such as nickel), plastic or polymer (such as PDMS). The printing of the pattern on the Sol-Gel can be done by placing the pattern in contact with the Sol-Gel. When the pattern is in contact with the Sol-Gel, the Sol-Gel redistributes to surround the pattern structure accordingly. When the pattern is in contact with the Sol-Gel, the redistribution of the Sol-Gel can be triggered either through an external force applied to the pattern or substantially, or through capillary forces intrinsic to the nature of the patterned pattern. When the pattern is in contact with the Sol-Gel, the stack of substrate + Sol-Gel + pattern can be exposed to light or heat to cure the Sol-Gel and block the pattern originally in the pattern in the Sol-Gel. This standardization process is conventionally referred to as nano-print lithography. Following the Sol-Gel cure, the pattern can be separated from the substrate + Sol-Gel stack, and the pattern can be discarded or reused to pattern another substrate coated with a Sol-Gel. The substrate with the cured standardized Sol-Gel can then be taken for a chemical deposition process, or if pure surface-type glass is desired, the substrate + standardized Sol-Gel stack can be taken through a thermal process (sintering) which will remove organic material that was originally present in Sol-Gel. This is not required, but the substrate can be made of a pure SiO2 material that has advantages in certain fixing schemes.
[052] Another approach to producing a standardized substrate is to use a plastic material such as COC or COP (such as Zeonor or Topas) and perform a thermal stamping process to create the notch arrangement. The process is similar to nano print lithography. A plastic substrate can be mounted on a mandrel that can be temperature controlled. The plastic substrate can then be heated to a temperature so that the outer crust of the plastic exceeds the glass transition temperature. Although the substrate is at elevated temperature, the pattern is placed in rigid contact with a pattern (for example, fourth, silicon, polymeric or metallic). The pattern typically has an external force applied to ensure that the plastic completely conforms to the structured pattern. Although in contact at elevated temperature, the plastic redistributes itself to become a negative replica of the pattern; for example, if the pattern has a column arrangement then the modeled plastic results in well arrangements. Although the pattern is in contact with the plastic substrate, the temperature of the substrate is reduced, thereby reducing the pattern modeled on the substrate.
[053] A concave feature that is on a substrate can have any of a variety of formats. In terms of the shape on the substrate, the feature can have curved sides, linear sides, corners or a combination of them. For example, resources can be wells that have openings on the surface that are circular, oval, square, polygonal, star-shaped (with any number of vertices), or irregular shapes. The features can be channels and the shape of the channels on the surface can include sides that are curved, linear, angled or a combination of them. Other channel features can be linear, serpentine, rectangular, square, triangular, circular, oval, hyperbolic or a combination of them. Channels can have one or more branches or corners. The channels can connect two points on a surface, one or both of which can be the edge of the substrate. Figure 3B and Figure 3C show exemplary channel features in sighting fiducials together with wells in and surrounding the sighting fiducials.
[054] The cross-sectional shape of a concave feature, taken orthogonal to the surface, can have walls that are curved, linear or a combination of the two. Therefore, the cross-sectional shape can be a part of a circle or oval (for example, U-shaped), or it can have two or more linear sides that meet at corners (for example, V-shaped, square, polygonal or star). In terms of cross-sectional shape, the bottom of the concave feature may be narrower, wider, or approximately the same as the opening in the surface. These cross-sectional shapes can be illustrated for the case where the concave feature is a well, in which case the opening in the surface will be approximately the same area as the bottom of the well when the well has a cylindrical cross section, while the bottom of the well will have a different area (typically smaller) than the opening area on the surface when the well has a conical cross section. Of course, cross sections, although illustrated for wells, can also apply to channels.
[055] For embodiments where concave resources form wells, each well can have any volume that can contain a liquid. The minimum or maximum volume can be selected, for example, to accommodate the yield (for example, multiplicity), resolution, analyte composition, or analyte reactivity expected for uses downstream of the substrate. For example, the volume can be at least 1 x 10'3 pm3, 1 x 10'2 pm3, 0.1 pm3, 1 pm3, 10 pm3, 100 pm3, or more. Alternatively or additionally, the volume can be a maximum of 1 x 104 pm3, 1 x 103 pm3, 100 pm3, 10 pm3, 1 pm3, 0.1 pm3 or less. It will be understood that the gel material can fill all or part of the volume of a well. The volume of the gel in an individual well may be greater than, less than or within the values specified above.
[056] The area occupied by each well opening in a surface can be selected based on criteria similar to those revealed for the well volume. For example, the area for each well opening on a surface can be at least 1 x 10'3 pm2, 1 x 10'2 pm2, 0.1 pm2, 1 pm2, 10 pm2, 100 pm2, or more. Alternatively or additionally, the area can be a maximum of 1 x 103 pm2, 100 pm2, 10 pm2, 1 pm2, 0.1 pm2, 1 x 10 '2 pm2, or less. The depth of each well can be at least 0.1 pm, 1 pm, 10 pm, 100 pm or more. Alternatively or additionally, the depth can be a maximum of 1 x 103 pm, 100 pm, 10 pm, 0.1 pm or less.
[057] Many different well positions and other concave resources can be designed, including regular, repetition, and non-regular patterns. For example, wells can be arranged in a hexagonal grid for compact packaging and improved density. Other mates can include, for example, straight mates (i.e. rectangular), triangular mates, and so on. Specific placements, and differences between placements in different domains, if used, may follow the teachings of US Patent 7,813,013, and / or US Patent Application 13 / 267,565, the descriptions of which are incorporated herein by reference . Any of a variety of crystalline or polycrystalline patterns can be useful.
[058] A well pattern can be characterized in terms of medium pitch (ie center-to-center spacing) for the wells. Again, the pattern can be regular so that the coefficient of variation around the middle step is small, or the pattern may not be regular, in which case the coefficient of variation can be relatively large. In any case, the average step can be, for example, at least 10 nm, 0.1 pm, 0.1 pm, 0.5 pm, 1 pm, 5 pm, 10 pm, 100 pm or more. Alternatively or in addition, the average step can be, for example, at most 100 pm, 5 pm, 1 pm, 0.5 pm, 0.1 pm or less. Of course, the average pitch for a specific well pattern can be between one of the lowest values and one of the highest values selected from the above variations.
[059] A well pattern can be characterized with respect to the well density (that is, the number of wells) in a defined area. For example, wells can be present at a density of approximately 2 million per mm2 According to the manufacturing methods disclosed here, the density can be easily tuned to different densities including, for example, a density of at least 100 per mm2, 1,000 per mm2, 0.1 million per mm2, 2 million per mm2, 2 million per mm2, 5 million per mm2 or more. Alternatively or additionally, the density can be tuned to not be greater than 5 million per mm2, 2 million per mm2, 1 million per mm2, 0.1 million per mm2, 1,000 per mm2, 100 per mm2, or less. Naturally, the density of the wells on a substrate can be between the lowest values and one of the highest values selected from the above variations.
[060] In specific embodiments, a gel material is used. In some cases, a gel-forming material (for example, polymerizable) is supplied to a solid support in a liquid state and subsequently converted to a gel. Examples of materials that can be polymerized include, without limitation, acrylamide, methacrylamide, hydroxylmethacrylate, N-vinyl pyrrolidone or derivatives thereof. Such materials are useful for making hydrogels. In some embodiments, the polymerizable material may include two or more different species of compounds that form a copolymer. For example, two or more different species of acrylamide, methacrylamide, hydroxylmethacrylate, N-vinyl pyrrolidone or derivatives thereof can function as polymerisers that polymerize to a copolymer hydrogel. Useful hydrogels include, but are not limited to, silane-free acrylamide polymer (SFA) (see US Patent Application Publication 2011/0059865 A1, the disclosure of which is hereby incorporated by reference), poly (N- ( 5-azidoacetamidylpentyl) acrylamide-co-acrylamide) (PAZAM, see, for example, Provisional Patent Application US 61 / 753,833, the disclosure of which is incorporated herein by reference), polyacrylamide polymers formed from acrylamide one acrylic acid or an acrylic acid containing a vinyl group as described, for example, in Document WO 00/31148 (the disclosure of which is hereby incorporated by reference); polyacrylamide polymers formed from monomers that form photo-cycloaddition reactions [2 + 2], for example, as described in WO 01/01143 or WO 03/014392 (the descriptions of which are incorporated herein by reference) ; or polyacrylamide copolymers described in US Patent 6,465,178, WO 01/62982 or WO 00/53812 (the descriptions of which are incorporated herein by reference). Chemically treated variations of these gel materials are also useful, such as chemically treated SFA made to react with oligonucleotides that have a corresponding reactive group (such as SFA azidolysis to produce SFA azido that is reactive with 5 '- or 3' - alkynyl modified oligonucleotides). Exemplary polymerizable hydrogels and materials that can be used to form hydrogels are described, for example, in Document US 753,833 or US Patent Application Publication 2011/0059865 A1, the descriptions of which are incorporated into the present by way of reference. Other useful gels are those formed by a change that depends on temperature in a liquid to gelatinous state. Examples include, but are not limited to, agar, agarose or gelatin.
[061] Gel material that is in a well or other concave feature on the surface of a structured substrate can be covalently attached to the surface. For example, PAZAM can be covalently attached to the surface using surface materials and other reagents disclosed in Document US 753,833, the disclosure of which is hereby incorporated by reference, as disclosed in the Examples section here. However, the gel material does not need to be covalently attached to wells or other concave resources as exemplified for SFA in the Examples section below.
[062] One or more analytes may be present in or on the gel material that is present on a structured substrate. The gel-containing substrates of the present disclosure are particularly useful for the detection of analytes, or for carrying out synthetic reactions with analytes. Therefore, any of a variety of analytes that must be detected, characterized, modified, synthesized, or the like can be present in or on the gel material of a substrate disclosed here. Exemplary analytes include, but are not limited to, nucleic acids (for example, DNA, RNA or analogs thereof), proteins, polysaccharides, cells, antibodies, epitopes, receptors, ligands, enzymes (for example, kinase, phosphatase or polymerase), small molecule drug candidates, or the like. A structured substrate can include multiple different species from an analyte library. For example, the species may be antibodies different from an antibody library, nucleic acids that have different sequences from a nucleic acid library, proteins that have a different structure and / or function from a protein library, candidates for the drug of a small molecule combination library, etc.
[063] In some embodiments, the analytes can be distributed on a structured substrate so that they can be individually resolved. For example, a single molecule from each analyte can be present in each well containing a structured substrate gel. Alternatively, analytes can be present as colonies or populations so that individual molecules are not necessarily resolved. Colonies or populations can be homogeneous with respect to containing only a single species of analyte (albeit in multiple copies). Taking nucleic acids as an example, each well on a structured substrate can include a colony or population of nucleic acids and each nucleic acid in the colony or population can have the same nucleotide sequence (either single or double standard). Such colonies can be created by cluster amplification or bridge amplification as revealed in greater detail elsewhere here. Multiple repetitions of a target sequence can be present in a single nucleic acid molecule, such as a catalyst created using a rolling circle amplification procedure. Therefore, the gel material in each well on a structured substrate can contain multiple copies of a single species of an analyte. Alternatively, a colony or population of analytes that are in a well may include two or more different species. For example, one or more wells on a structured substrate may contain a mixed colony that has two or more different nucleic acid species (that is, nucleic acid molecules with different sequences). The two or more different nucleic acid species in a mixed colony can be present in insignificant amounts, for example, allowing more than one nucleic acid to be detected in the mixed colony.
[064] The analytes can be attached to a gel material. The fixation can be covalent or non-covalent. Exemplary methods and reagents for attaching nucleic acids to gels are described, for example, US Patent Application Publication 2011/0059865 A1 or US Provisional Patent Application 61 / 753,833, the descriptions of which are incorporated herein by reference. The analytes can be nucleic acids and the nucleic acids can be attached to the gel by means of 3 'oxygen, 5' hydrogen or in other places along its length such as by means of a base half of the 3 'nucleotide terminal, a base half of the 5 'nucleotide, and / or one or more halves elsewhere in the molecule. Non-covalent modes of attachment include, for example, ionic interactions between nucleic acid and gel, entrapment of nucleic acid within pores of a gel, protein-protein interactions, binding between receptors and ligands in the gel and / or nucleic acid, and other known methods.
[065] In some embodiments, a gel coating that is applied to a surface contains one or more analytes before removing gel material from the interstitial regions. Therefore, the gel material can be present in the interstitial regions and the gel material in the interstitial regions can be attached to one or more different analytes. Alternatively, the analytes are added to a gel material in concave resources after removing the interstitial regions.
[066] A structured substrate of the present disclosure can occur in a flow cell. Exemplary flow cells, methods for their manufacture and methods for their use are described in US Patent Application Publication 2010/0111768 A1 or 2012-0270305 A1; or WO 05/065814, the descriptions of which are incorporated herein by reference; Flow cells provide a convenient format for housing an array that is produced by the methods of the present disclosure for a synthesis sequence (SBS) or other technique that involves repeated distributions of reagents in the cycles (for example, synthesis techniques or detection techniques that have repetitive or cyclical steps). Exemplary detection methods are revealed in more detail below.
[067] In some embodiments, a flow cell or other container having multiple surfaces is used. Containers that have multiple surfaces can be used in such a way that only a single surface has concave features containing gel (for example, wells). Alternatively, two or more surfaces present in the container may have concave features containing gel. One or more surfaces of a flow cell can be selectively detected. For example, opposite surfaces within a flow cell can be selectively treated with focused radiation using methods known in the art such as confocal techniques. Useful confocal techniques and devices for selectively targeting radiation to multiple surfaces of a container (for example, a flow cell) are described, for example, in US Patent Application Publication 2009/0272914 A1 or US Patent 8,039,817, whose descriptions are hereby incorporated by reference.
[068] The present disclosure provides a method of making a substrate. The method may include the steps of (a) providing a solid support that has a flat surface, where the flat surface is interrupted by one or more concave features and where the one or more concave features are bounded by one or more interstitial regions on the flat surface; (b) coating a part of the solid support with gel material, where the part includes at least one of the concave features and at least one of the interstitial regions; and (c) polishing the flat surface to remove the gel material from at least one of the interstitial regions and maintaining the gel material in at least one concave feature.
[069] A substrate can be manufactured to be provided with concave features using any of a variety of techniques known in the art. In many embodiments, the concave resources will be small, in the order of nanomedical or micro-measured dimensions. In such cases, nanofabrication or microfabrication techniques can be used. Examples of such techniques are disclosed elsewhere in this application as in Example II below. Additional nanofabrication or microfabrication techniques are described in Document US 13 / 661,524 and US Patent Application Publication 2012/0316086 A1, the descriptions of which are incorporated herein by reference.
[070] One or more concave features, such as wells, can be coated with preformed gel material or with a liquid that subsequently forms a gel material. An example of the previous approach is the coating of a substrate with preformed PAZAM using spin coating, dip, gel flow under positive or negative pressure or techniques disclosed in Provisional Patent Application US 61 / 753,833, the disclosure of which is incorporated present as a reference. The coating of a well arrangement with preformed PAZAM is shown below in Example III. An example of applying a liquid that subsequently forms a gel material is to coat a well arrangement with silane without acrylamide and N- [5- (2-bromoacetyl) aminopentyl] acrylide (BRAPA) in liquid form and allowing the reagents form a gel by polymerizing on the surface. The coating of such an arrangement is shown in Example I below and may use chemical reagents and procedures as disclosed in US Patent Application Publication 2011/0059865 A1, the disclosure of which is hereby incorporated by reference. In some embodiments, for example, when a well-containing substrate is dipped in a preformed gel material, the gel material can selectively fill the wells and polishing may not be necessary.
[071] Analytes can be added to a gel material before contacting with a solid support or later. In addition, the analytes can be added to a gel (that is, after the gel is formed from its precursor reagents) or the analytes can be added to a gel-forming reagent solution (i.e., before gel formation ). In some embodiments, several analytes can be added before gel formation and others can be added after gel formation. In one example, the initiator nucleic acids are added to a gel-forming solution and the solution is then allowed to form in a gel (for example, by polymerization as occurs for SFA and PAZAM). Gel formation can occur on a solid support or the gel can be preformed and then coated on a solid support. In any case, the initiators will be fixed to the gel that is present in the concave resources such as wells. Target nucleic acids that are complementary to the primers can then be added to the gel containing primer so that the target nucleic acids become attached to the gel (via hybridization) after the gel material has been coated on the solid support. Hybridization of target nucleic acids can optionally occur after a polishing step has been performed (polishing is described in more detail below). The preceding example describes several cases where nucleic acids (acting as primers or targets) are added to the gel at different stages in the manufacture of a structured substrate.
[072] In various embodiments, initiator nucleic acids that are attached to a gel (or otherwise present in or on a gel) can be used to capture and / or amplify standard nucleic acids. Primers can be universal primers that hybridize to a universal adapter sequence that is fixed on different target nucleic acids in a library (that is, each target nucleic acid includes a target region that differs from other target nucleic acids in the library and several target nucleic acids in the library have the same universal adapter sequence). In some embodiments, a target nucleic acid can be attached to the gel material, and the primers (whether in solution or also attached to the gel) can be used to amplify the fixed target nucleic acid (for example, the target nucleic acid can serve as standard for amplification).
[073] A method revealed here can use any of a variety of amplification techniques. Exemplary techniques that can be used include, but are not limited to, polymerase chain reaction (PCR), rolling circle amplification (RCA), multiple displacement amplification (MDA), or random start amplification (RPA). In specific embodiments, one or more primers used for amplification can be attached to a gel material. In PCR embodiments, one or both of the primers used for amplification can be attached to a gel material. Formats using two types of fixed primer are often referred to as bridge amplification because a double stranded amplicon forms a bridge-like structure between the two fixed primers that form the standard sequence that has been copied. Exemplary reagents and conditions that can be used for bridge amplification are described, for example, in US Patent 5,641,658; in US Patent Publication 2002/0055100; US Patent 7,115,400; US Patent Publication 2004/0096853; US Patent Publication No. 2004/0002090; US Patent Publication No. 2007/0128624; and US Patent Publication No. 2008/0009420, the descriptions of which are incorporated herein by reference. PCR amplification can also be performed with one of the amplification primers attached to the gel material and the second primer in solution. An exemplary format that uses a combination of a fixed solid phase primer and a solution phase primer is PCR emulsion as described, for example, in Dressman etal., Proc. Natl. Acad. Sci. USA 100: 8817-8822 (2003), WO 05/010145, or US Patent Publication 2005/0130173 or 2005/0064460, the descriptions of which are incorporated herein by reference. The PCR emulsion is illustrative of the format and it will be understood that for the purposes of the methods disclosed here the use of an emulsion is optional and, in fact, for various embodiments an emulsion is not used. In addition, the primers do not need to be attached directly to the solid supports as revealed in the references and PCR and can instead be attached to the gel material as disclosed here. In some formats of solid phase PCR or bridge amplification, a target nucleic acid can be attached to a gel material and used as a standard for amplification.
[074] RCA techniques can be modified for use in a method of the present disclosure. Exemplary components that can be used in an RCA reaction and principles by which RCA produces amplicons are described, for example, in Lizardi et al., Nat. Genet. 19: 225-232 (1998) and Publication of US Patent Application 2007/0099208 A1, the descriptions of which are incorporated herein by reference. The primers used for RCA may be in solution or attached to the gel material.
[075] MDA techniques can be modified to be used in a method of the present disclosure. Some basic principles and conditions useful for MDA are described, for example, in Dean et al., Proc Natl. Acad. Sci. USA 99: 5261-66 (2202); 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 Patents 5,455,166; 5,130,238 and 6,214,587, the descriptions of which are incorporated herein by reference. The primers used for MDA can be in solution or attached to a gel material.
[076] In specific embodiments, a combination of the amplification techniques exemplified above can be used. For example, RCA and MDA can be used in a combination where RCA is used to generate a catammeric amplicon in solution (for example, using solution phase initiators). The amplicon can then be used as a standard for MDA using primers that are attached to the gel material. In this example, the amplicons produced after the combined RCA and MDA steps will be attached to the gel material. Amplicons generally contain concatameric repeats of a target nucleotide sequence.
[077] Amplification techniques, such as those explained above, can be used to produce resources that contain gels that have multiple copies of target nucleic acids. An individual resource, such as a well, can have a clonal population of nucleotide sequences in the form of a single molecule concaamer, such as those produced by RCA, or in the form of many nucleic acid molecules that have the same sequence such as those produced by PCR bridge. Usually the nucleic acid (s) that have multiple copies of the amplified target will be attached to the gel material.
[078] For some applications, an individual well containing gel (or other concave resource) may be predominantly populated with amplicons from a first target nucleic acid and may also have a low level of contaminating amplicons from a second target nucleic acid or a spontaneous mutation that occurs during amplification. An arrangement may have one or more amplification sites that have a sufficiently low level of contamination amplicons to have an unacceptable impact on a subsequent use of the arrangement. For example, when an arrangement is to be used in a detection application, an acceptable level of contamination could be a level that does not impact signal to noise or resolution of the detection technique in an unacceptable manner. Therefore, the apparent clonality will generally be relevant to a specific use or application of an arrangement made by the methods disclosed here. Exemplary levels of contamination that may be acceptable in an individual well or other resource for specific applications include, but are not limited to, at most 0.1%, 0.5%, 1%, 5%, 10% or 25% of contaminating amplicons. An arrangement may include one or more wells or other resources that have these exemplary levels of contaminating amplicons. For example, up to 5%, 10%, 25% 50%, 75% or even 100% of the resources in an arrangement can have some contaminating amplicons.
[079] A gel material that has been coated on the surface of a solid support can be covalently attached to the support. As disclosed above, the step of fixing an analyte, such as a nucleic acid, to the gel material can be carried out at several different stages in the manufacture of a structured substrate. Therefore, a gel material can be attached to the solid support before or after attaching an analyte to the gel material. The fixation of gel material to a solid support can be performed using any useful chemistry including without limitation those disclosed in Provisional Patent Application US 753,833, the disclosure of which is hereby incorporated by reference, or demonstrated in Example III below. It will be understood that covalent attachment of gel material to a solid support is not necessarily in all embodiments. Therefore, subsequent steps of polishing a gel-coated support or using a polished substrate can be performed for a substrate that has gel material that is optionally, but not necessarily, covalently attached to concave resources, such as wells.
[080] A method disclosed here may include a step of removing gel material from the surface of a solid support. Gel material that is coated on a solid support can be selectively removed from the interstitial regions using any of a variety of techniques. For example, the gel material can be removed from a solid support that contains concave features and interstitial regions by a mechanical polishing technique. Mechanical polishing can be carried out by applying abrasive forces to the surface of the solid support. Exemplary methods include abrasion with granule paste, rubbing with a blade or cloth, fragmenting or the like. An example of polishing includes the use of lint-free (cleanroom grade) towels coated with 3 pm silica granule paste (10% by weight / volume in water) to remove interstitial gel. A polishing wheel / grinder can also be used with this slurry. Mechanical polishing can also be achieved using a fluid jet or air jet to remove gel from the interstitial regions.
[081] Polishing may involve chemical polishing such as hydrolysis or degradation based on acrylamide radical (for example, through exposure to benzene peroxide or diluted hydrogen peroxide as described in Kuren- kov, et al., Russian Journal of Chemistry applied, 75: 1039-1050 (2002); Caulfield et al., Polym. 44: 1331-1337 (2003); and Caulfield et al., Chem. Rev. 102: 3067-3038 (2002)).
[082] Polishing can also involve a combination of chemical and mechanical polishing methods where a chemical paste containing a colloidal suspension of particles is used to mechanically exfoliate and then chemically dissolve part of the gel material from the interstitial regions. Other methods for polishing or cleaning interstitial regions include adhesive-based techniques, for example, techniques in which a rigid planar adhesive film with an affinity for gel material is coated on the surface, thereby making an intimate count (for example, by bonding chemistry) with the gel material in the interstitial regions. The mechanical removal / flaking of this adhesive film will result in the mechanical removal of the gel material from the interstitial regions, while leaving gel material in the concave resources.
[083] In another example, SFA grafted thiophosphate can be removed from the interstitial regions on a surface as follows. A Whatman wipe moistened with water can be rubbed in Aluminum oxide (~ 100mg, 0.3pm) or steel granules. Then, the formed paste can be rubbed onto the surface of a solid support, in small concentric circles, using the same pressure. A Whatman wipe moistened with clean water can then be used to remove the paste on the surface. The mechanical and chemical polishing methods exemplified here to remove gel material from interstitial regions can also be used to inactivate gel material in interstitial regions, whether or not the gel material is removed. For example, the gel material can be inactivated with respect to the ability to fix analytes such as nucleic acids or with respect to the ability to withstand nucleic acid amplification.
[084] A method for producing an arrangement can include the steps of (a) providing a solid support having a surface with a plurality of wells, the wells containing a gel material, the wells being separated from each other by interstitial regions on the surface , the interstitial regions segregating the gel material in each of the wells of the gel material in other wells among the plurality; (b) deliver a library of target nucleic acids to the wells of the solid support to produce an array of wells that have a single species of nucleic acid attached to the gel material in each well, where different wells in the array have different species of nucleic acid target of the library; and (c) amplifying the target nucleic acids trapped in the gel material in the wells of the array to produce a clonal population of an individual target nucleic acid in each of the wells of the array.
[085] In several embodiments, the structured substrates disclosed here provide the advantage of convenient delivery of multiple different analytes from a mixture of individualized locations on the substrate, thereby forming an arrangement. Structured substrates facilitate selective capture of a single analyte in each well (or other concave resource) containing individual gel from a mixture of analytes in contact with the substrate. The pattern of wells (or other concave resources) containing gel in the structured substrate and the loading efficiency can be adjusted to obtain arrangements having desired characteristics such as analyte density and purity of each resource with respect to having a single species of analyte. For example, a higher density of wells can be used to obtain a higher density of analytes in the array, and conversely, a lower density of wells can be used to obtain a lower density of analytes in the array. Alternatively or additionally, the concentration of the amount of analyte in the solution can be increased to obtain a higher density of analytes in the array or decreased to obtain a lower density of analytes in the array. The average analyte purity in each well (or other concave resource) containing gel can be adjusted by changing the substrate properties or conditions for analyte delivery as revealed in more detail below and demonstrated in the Examples section.
[086] In particular embodiments, the size or volume of the walls (or other concave features) can be adjusted to influence the purity of captured analytes. For example, a well may have an area or volume of gel material that accommodates only a single analyte of a particular type so that steric exclusion prevents more than one analyte molecule from being captured in or seed the well. Steric exclusion can be particularly useful for large analytes such as nucleic acids. More specifically, wells (or other concave resources) may have a gel surface having an area that is equivalent to or less than the diameter of the volume excluded from the target nucleic acids that must be seeded on the substrate. The excluded volume for a target nucleic acid and its diameter can be determined, for example, from the length of the target nucleic acid. Methods for determining the excluded volume of target nucleic acids and their diameter are described, for example, in US patent 7,785,790; Rybenkov et al., Proc. Natl. Acad. Know. US A. 90: 5307-5311 (1993); Zimmerman and others; J. Mol. Biol. 222: 599-620; or Sobel et al., Bioploymers 31: 1559-1564 (1991), each of which is incorporated herein by reference. Conditions for steric exclusion are disclosed in US 13 / 661,254 and US patent 7,785,790, each of which is incorporated herein by reference, and can be readily used for structured substrates of the present disclosure.
[087] It will be understood that in some embodiments, wells (or other concave resources) may have a gel surface having an area that is substantially larger than the diameter of the excluded volume of the target nucleic acids that are transported to the amplification sites. Thus, the area for resources may be large enough that steric exclusion does not occur.
[088] In some embodiments, such as the exclusion embodiments disclosed above, a library of target nucleic acids can be sent to wells (or other concave resources) containing gel from a solid support prior to initiating an amplification process. For example, target nucleic acids can be sent to a structured substrate under conditions to seed the gel material on the substrate with the target nucleic acids. The substrate can optionally be washed to remove target nucleic acids that do not sow the gel and also any other materials that are undesired for subsequent processing or use of the substrate. The amplification can include one or more of the techniques previously revealed here.
[089] In alternative embodiments, a library of target nucleic acids can be sent to the wells (or other concave resources) of a solid support and an amplification process can occur simultaneously with the seeding event. For example, seeding can occur under a regime that exploits kinetic exclusion as described, for example, in US 67 / 715.478, which is incorporated herein by reference. Kinetic exclusion can occur when a process occurs at a rate fast enough to effectively exclude the occurrence of another event or process. In the case of a gel array of wells, the wells can be seeded randomly with target nucleic acids from a solution and copies of the target nucleic acids can be generated in an amplification process to fill each of the entire seeded sites. The sowing and amplification processes can proceed simultaneously under conditions where the amplification rate exceeds the sowing rate. Therefore, the relatively fast rate at which copies are made at a site that was seeded by a first target nucleic acid will not effectively exclude seeding the site for amplification by a second nucleic acid. Similarly, kinetic exclusion can exploit a relatively low rate for making a first copy of a target nucleic acid as opposed to a relatively quick rate for making subsequent copies of the target nucleic acid or the first copy. For example, kinetic exclusion can occur due to a delay in the formation of a first copy of a target nucleic acid that has seeded a well containing gel (for example, delayed or slow activation) as opposed to the relatively fast rate at which subsequent copies are made to fill the place. In this example, a well containing an individual gel may have been seeded with several different target nucleic acids (for example, several target nucleic acids may be present at each site prior to amplification). However, the first copy formation for any given target nucleic acid can be activated at random so that the average rate of the first copy formation is relatively slow compared to the rate at which subsequent copies are generated. In that case, although a well containing an individual gel may have been seeded with several different target nucleic acids, the kinetic exclusion will allow only one of these target nucleic acids to be amplified. Generally, a gel-containing well (or other concave resource) can serve as a location for amplification and arrangement formation in a method disclosed in US 61 / 715.478, which is incorporated here by reference.
[090] As an alternative to stuffing different analytes from a mixture of individual concave resources containing gel, analytes can be sent separately to individual resources from pure stocks. Similarly, analytes can be synthesized in individual resources by sending separate synthetic building blocks (for example, nucleotide precursors can be sent in sequence to synthesize nucleic acids). Exemplary methods for sending pure analytes or building blocks to synthesize analytes in their natural place include, but are not limited to, inkjet array identification and photolithographic array synthesis. Useful lithographic methods include those used commercially by Affymetrix (Santa Clara, CA) to manufacture GeneChip® microarrays or described in US patents 5,324,633; 5,744,305; 5,624,711; 6,022,963; 6,291,183; and 6,416,949, each of which is incorporated herein by reference. Also useful are inkjet identification techniques such as those marketed by Agilent (Santa Clara, CA) for printing SurePrint ™ arrangements or described in US patents 6,337,393; 6,419,883; 6,240,180 or 6,689,319, each of which is incorporated herein by reference. These methods can be readily modified to direct the delivery of the gel-containing resources of this disclosure.
[091] The gel material in a particular concave resource need not contain just a single species of analyte. On the contrary, in some embodiments, a concave resource may contain several different species of analyte in the gel in it. An example is shown by the center fiducial markers in Figure 5. The fiducial markers include two “clear” ring-shaped channels, each of the two channels contains gel material, and the gel material in each clear channel is attached to a plurality of different nucleic acid colonies. Colonies of nucleic acid in the clear channels were formed by sowing each ring with several different species of target nucleic acid that served as a model in an amplification procedure. The fiducial marker also includes two “dark” ring-shaped regions. The dark rings are formed by interstitial surface patterns. The exemplary aiming center is formed by alternating dark and light rings in a concentric pattern. In the example in Figure 5 the structured substrate also includes gel-containing wells that each contain a clonal population derived from a single target nucleic acid. Wells occur and a ring-shaped ribbon between the light and dark rings. Thus, fiducials have an alternating pattern of interstitial ring, tape containing well and channel ring. The same amplification procedure was used to simultaneously develop the nucleic acid colonies in the wells and the mixed population on the fiducial marker (see Example III, below). Other examples of fiducials having alternative ring patterns are shown in Figure 3B and Figure 3C.
[092] This disclosure also provides a method of detecting analytes. The method may include the steps of (a) providing a solid support having a flat surface, where the flat surface is interrupted by one or more concave features, where the concave features contain gel material, where the one or more features concave are bounded by one or more interstitial regions on the flat surface, the interstitial regions being substantially devoid of gel material, and where the gel material is attached to or contains target analytes; (b) contacting the solid support with probes under conditions where the target analytes specifically interact with the probes; and (c) detecting the solid support to distinguish at least a subset of the target analytes that interact with one or more of the probes.
[093] In particular embodiments nucleic acids are the analytes that are detected and the concave resources are wells. For example, a method of detecting nucleic acids may include the steps of (a) providing a solid support having a surface and a library of nucleic acids, the surface having a plurality of wells, wells containing gel material, wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells of the gel material in other wells of the plurality, a single species of the target nucleic acids of the library being attached to the gel material in each of the wells; (b) contacting the solid support with at least one probe that binds to the target nucleic acids; and (c) detecting the solid support to distinguish the wells having a kind of nucleic acid that binds to at least one probe.
[094] Structured substrates of the present disclosure that contain arrays of nucleic acids can be used for any of a variety of purposes. A particularly desirable use for nucleic acid is to serve as capture probes that hybridize to target nucleic acids having complementary sequences. Target nucleic acids once hybridized to the capture probes can be detected, for example, by means of a tag recruited to the capture probe. Methods for detecting target nucleic acids by hybridization to capture probes are known in the art and include, for example, those described in US patents 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US patent 2005/00553980 A1; 2009/0186349 A1 or 2005/0181440 A1, each of which is incorporated herein by reference. For example, a tag can be recruited for a capture probe by virtue of hybridizing the capture probe to a target probe carrying the tag. In another example, a tag can be recruited to a capture probe by hybridizing a target probe to the capture probe so that the capture probe can be extended by binding to a labeled oligonucleotide (for example, through an activity ligase) or by adding a labeled nucleotide (for example, via polymerase activity).
[095] A nucleic acid array can also be used in a sequencing procedure, such as a sequencing-by-synthesis (SBS) technique. In summary, SBS can be initiated by contacting the target nucleic acids with one or more labeled nucleotides, DNA polymerase, etc. Those features where a primer is extended using the target nucleic acid as a template will incorporate a labeled nucleotide that can be detected. Optionally, the labeled nucleotides may further include a reversible termination property that terminates the extension of the additional primer once a nucleotide has been added to a primer. For example, an analog nucleotide having a reversible terminator half can be added to a primer so that subsequent extension cannot occur until an unlocking agent is delivered to remove the half. Thus, for embodiments using reversible termination, an unlocking agent can be sent to the flow cell (before or after detection occurs). Washes can be performed between the various stages of delivery. The cycle can then be repeated n times to extend a primer by n nucleotides, thereby detecting a sequence of length n. Exemplary SBS procedures, fluid systems and detection platforms that can be readily adapted for use with an array produced by the methods of the present disclosure are described, for example, in Bentley et al., Nature 456: 53-59 (2008), WO 04 / 018497; WO 91/06678; WO 07/123744, US patents 7,057,026; 7,329,492; 7,211,414; 7,315,019 or 7,405,281, and US patent application 2008/0108082 A1, each of which is incorporated herein by reference.
[096] Other sequencing procedures that use cyclic reactions can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are embedded in a nascent nucleic acid strand (Ronaghi, Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3 -11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); US Patent 6,210,891; 6,258,568 and 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected by means of photons produced by luciferase. Thus, the sequencing reaction can be monitored using a luminescence detection system. Excitation radiation sources used for fluorescence-based detection systems are not required for pyosequencing procedures. Useful fluid systems, detectors and procedures that can be used to apply pyrosequencing in arrangements of the present disclosure are described, for example, in patent application publication WIPO PCT / US11 / 5711, published patent application US 2005 / 0191698A1, US patent 7,595,883 and US patent 7,244,559, each of which is incorporated herein by reference.
[097] Link sequencing reactions are also useful including, for example, those described in Shendure and others Science 309: 1728-17732 (2005); US patent 5,599,675; and US patent 5,750,341, each of which is incorporated herein by reference. Some embodiments may include sequencing hybridization procedures as described, for example, in Bains et al., Journal of Theoretical Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology 16, 54-58 (1998); Fodor et al., Science 251 (4995), 767-773 (1995); and WO 1989/10977, each of which is incorporated herein by reference. In both sequencing by ligation and sequencing by hybridization procedures, nucleic acids that are present in wells containing gel (or other concave resources) undergo repeated cycles of nucleotide delivery and detection. Fluid systems for SBS methods as disclosed herein, or in references cited here, can be readily adapted for delivery of reagents for ligation sequencing or hybridization sequencing procedures. Typically, oligonucleotides are fluorescently labeled and can be detected using fluorescence detectors similar to those described with respect to the SBS procedures described here or in references cited here.
[098] Some embodiments may use methods involving real-time monitoring of NA polymerase activity. For example, nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fluorophore-bearing polymerase and / -phosphate-labeled nucleotides, or with zero mode waveguides. Techniques and reagents for FRET-based sequencing are described, for example, in Levene and others Science 299, 682-686 (2003); Lundquist and others Opt. Lett. 33, 1026-1028 (2008); Korlach and others Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference.
[099] Some embodiments of SBS include detecting a proton released by incorporating a nucleotide into an extension product. For example, sequencing based on the detection of released protons may use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a subsidiary of Life Technologies) or sequencing methods and systems described in the published patent applications 2009 / 0026082 A1; 2009/0127589 A1; 2010/0137143 A1; or 2010/0282617 A1, each of which is incorporated herein by reference. In particular embodiments, electrical detectors that are used to detect released protons can be modified to include wells and the wells can contain gel material as disclosed here.
[0100] Another useful application for an arrangement of the present disclosure is analysis of gene expression. Gene expression can be detected or quantified using RNA sequencing techniques, such as those called digital RNA sequencing. Digital RNA sequencing techniques can be performed using sequencing methodologies known in the art such as those noted above. Gene expression can also be detected or quantified using hybridization techniques performed by direct hybridization to an array or using a multiplex assay, whose products are detected in an array. An arrangement of the present disclosure can also be used to determine genotypes for a genomic DNA sample from one or more individuals. Exemplary methods for expression analysis and genotyping based on an arrangement that can be performed on an arrangement of the present invention are described in US patents 7,582,420; 6,890,741; 9,913,884 or 6,335,431 or published patent applications US 2005/0053980 A1; 2009/0186349 A1 or 2005/0181440 A1, each of which is incorporated herein by reference.
[0101] Several applications for the arrangements of the present disclosure have been exemplified above here for pool detection, in which multiple copies of a target nucleic acid are present in each resource and are detected together. In alternative embodiments, a single nucleic acid, be it a target nucleic acid or amplicon thereof, can be detected in each resource. For example, a gel-containing well (or other concave resource) can be configured to contain a single nucleic acid molecule having a target nucleotide sequence that must be detected. Any of a variety of single molecule detection techniques can be used including, for example, modifications of the joint detection techniques revealed above to detect the sites in increased resolution or using more sensitive tags. Other examples of single molecule detection methods that can be used are disclosed in published patent application US 2011/0312529 A1; US 61 / 578,684; and US 61 / 540,714 each of which is incorporated herein by reference.
[0102] It will be understood that a gel-containing substrate of the present disclosure, for example, having been produced by a method disclosed herein, need not be used for a detection method. In contrast, the structured substrate can be used to store a nucleic acid library. Consequently, the structured substrate can be stored in a state that preserves the nucleic acids in it. For example, a substrate having wells containing gel that are attached to nucleic acids can be stored in a desiccated, frozen state (for example, in liquid nitrogen, or in a solution that is protective of nucleic acids. Alternatively or additionally, the substrate structured can be used to replicate a nucleic acid library, for example, a substrate having wells containing gel that are attached to nucleic acids can be used to create replicated amplicons from one or more of the wells in the array.
[0103] The following examples are intended to illustrate, but not to limit the present invention. EXAMPLE I
[0104] Multipoint substrates coated with silane-free acrylamide
[0105] This example demonstrates the coating of nanowell substrates with silane-free acrylamide (SFA), followed by grafting with thiophosphate primers into complementary fluorescent oligonucleotide to confirm the success of the functionalization approach.
[0106] Chip substrates commonly used for making Bead-Chips were obtained from Illumina (San Diego, CA). The chips were made from silicon or Zeonor (Zeon Corp., Tokyo, Japan) having 0.5 pm wells positioned in a hexagonal pattern having a 1.5 pm pitch, but the wells did not contain granules. The chips were standardized with gel pads as shown below and diagrammed in Figure 1.
[0107] The chips were enclosed in a gasket-sealed chamber and oxygen was removed by displacement with fluid reagents to form SFA. SFA was polymerized on the chips in the chamber. The reagents for SFA formation and conditions for polymerization were in other respects as described in published patent application 2011/0059865 A1, which is incorporated herein by reference. The sealed chamber was used to bring the polymerization mixture into direct contact with the chip and ensure complete elimination of air since the polymerization of free radical SFA is an air sensitive process. After polymerization, primers were grafted onto the SFA polymer in the sealed chamber as disclosed in published patent application US 2011/0059865 A1 as follows. A solution containing the primers was extracted through the polymer-coated surface of the BeadChip and the mixture was then incubated for 1.25h at 65 ° C (the entire sealed set was placed in a large oven).
[0108] This approach generated uniformly coated substrates. A sample image is shown in Figure 2, Panel A. In order to create separate polymer regions, the excess polymer located between the wells in the substrate was removed using a mechanical polishing technique using a nanoparticle paste. aluminum oxide (300 mm in diameter) in Dl water. A 10% thick paste of 3 micron silica particles (Kisker Biotech GmbH, Stein-furt, Germany) can also be used. The surface was manually rubbed with the nanoparticle paste using an optical fabric without cotton. After washing to remove the paste and polymer debris, a labeled probe solution was hybridized on a chip. Images captured using a fluorescence microscope showed that this approach was able to provide polymer resources with clean interstitial regions (Figure 2, Panels B to C).
[0109] These results indicated that the fluorescent intensities in the gel-filled wells were spatially separated in contrast to the lack of signal from the interstitial regions. The results also demonstrated that gel standardization can be achieved using gel material attached to a substrate having nanofabricated wells. EXAMPLE II
[0110] Manufacture of a Substrate Having Nanowells Containing Gel
[0111] Multiple techniques can be used to manufacture structured arrangements that can subsequently be loaded with gel material.
[0112] The process can start with a blank substrate / tablet and a pattern is introduced into the substrate using micro or non-manufacturing techniques. The substrate / tablet material can be silicon, glass, conventional plastic, COC or any of a variety of materials that can be structured. Exemplary techniques for introducing standardization into the substrate include photolithography, nanoprint lithography, engraving the structures on a plastic / COC based material and injection molding of a plastic or COC into a main mold that has the standardized structures themselves. Approaches based on photolithography will typically involve the use of a photoresist material that is standardized with a step recording or a mask aligner, exposed with radiation that transfers the pattern present in a reticule / photomembrane into the interior of the photoresist material, and then the coating material is developed to provide a structured film (photoresist material) on top of the substrate. The structured coating material is potentially the final substrate that can be used for subsequent gel coating or the pattern in the coating material can be transferred into the substrate by further processing. The continuation process steps will typically include reactive ion carving (plasma based carving) or a wet carving process (chemical based). If the pattern is transferred into the substrate, the photoresist material is subsequently removed to provide the patterned substrate for subsequent gel coating. It may be desirable to sacrifice a film of a material such as Chromium or Titanium (a metal) under the photoresist material, and first transfer the pattern in the photoresist material to the metal film and then use that film as a rigid mask so the pattern is trans-wound into the substrate and, therefore, considered sacrificable for the manufacturing process. If nanoprint lithography is used, the printed photoresist material can be a expendable material and similarly be used as an intermediate tool to transfer the coating material into the substrate or a variation of the coating material can be used so that the material printed coating layer serves as the entrance to a subsequent coating step. An example of a coating material that would remain following standardization would be a material based on Sol-Gel.
[0113] A diagrammatic representation of how a structured substrate can be manufactured is shown in Figure 3 and described below. Standardized substrate images are shown at varying levels of magnification in Figure 3B and Figure 3C.
[0114] The creation of chemically specific gel pads on a substrate / sequencing flow cell may involve one or more of the nanofabrication techniques previously disclosed in this Example. The process can optionally then include one or more steps, such as silanization to allow subsequent bonding of a polymer from a gel polymer to the substrate through the silane. Then chemical / mechanical polishing (CMP) is used to remove all interstitial polymer on the substrate surface. The polishing process will remove material in a top-down manner and since the features structured on the substrate are effectively decentralized to the plane of the interstitial regions of the arrangement, the polishing will remove the polymer from the interstitial before removing the structured features . If the polishing process is interrupted after the ideal time, the structures will retain the polymer coating and the interstitial regions will be empty of the polymer. The gel pad substrate is then grafted with primers, target nucleic acids are seeded on the gel pads, and the target nucleic acids are used as templates for creating clusters of genes on the gel pads. EXAMPLE III
[0115] Multi-well substrates coated with PAZAM
[0116] This example shows how the fabrication of a well arrangement containing gel, the amplification of nucleic acid clusters in the wells and the sequencing of nucleic acids in the clusters.
[0117] Substrates were manufactured as follows. A nanowell substrate (400nm diameter well, 1.5 pm pitch, 300nm depth) was manufactured using nanoprint lithography. An aminosilane monolayer / multilayer (APTES or APTMS) was deposited on the entire surface of the substrate using chemical vapor deposition. Then, a saline solution (pH 7.4) buffered with acrylic N-hydroxysuccinimate ester phosphate (Aldrich PN 8060) at a concentration of 10mM was reacted with the aminosilane surface by adding 1 ml of the NHS acrylate solution on the surface, covering it with a glass cover with thin glass and allowing the reaction to proceed for one hour at room temperature. A polymer (PAZAM) is then applied to the surface by spinning 500 pl of a 2% by weight solution of PAZAM in water on the newly formed functionalized acrylamide surface. PAZAM was synthesized as described in provisional patent application US 61 / 753,833, which is incorporated herein by reference. Subsequent heating of the PAZAM-coated substrate at 60 ° C for one hour resulted in a bond between the polymer and the surface. The covalently bonded interstitial polymer was removed by polishing the surface with a 3 pm 10 wt% SiO2 microparticle paste. A Janeway surface (acryloyl chloride with DIPEA in MeCN) can be used in place of the aminosilane-coated surface in the above procedure.
[0118] The polymer substrate was then grafted with primers as described in provisional patent application US 61 / 753,833, which is incorporated herein by reference. Then, reverse complements labeled with dye (Cy5) from the grafted primers were exposed to the surface in a 1xPBS buffer solution at a complement concentration of 20 pM, and then the surface was washed with 50 ml of 1PBS buffer applied with a bottle of splash. The complements labeled on the substrate had their image represented on a Typhoon Imager FLA 9500 adjusted on the Cy5 scanning channel and using a PMT configuration of 450. The complements labeled on the substrate also had their image represented by a high resolution microscope, showing the standardization or polymer / initiators without polymer / interstitial initiators remaining. (Figure 4). The substrate was then seeded with phiX DNA, and clusters developed as described in US 61 / 715.478, which is incorporated herein by reference.
[0119] A flow cell containing the substrate containing the cluster was sequenced on a HiSeq 2000 (Illumina, Inc. San Diego, CA). An algorithm to extract the locations of the groups in standardized sequencing (rigid registration) was employed, successfully generating high quality sequencing measures (Figure 5 and Figure 6). The sequencing results showed that the occupancy versus clonality ratio was surprisingly higher than expected for a standard Poisson distribution. Specifically, the measure of the occupancy versus clonality relationship for the sequencing assay, as indicated by the “x” in Figure 7, is above the limits of the Poisson curve and is very close to the line for the ideal clonal fraction.
[0120] Throughout this report, references were made to various publications, patents or patent applications. The disclosures of these publications are incorporated herein entirely as a reference to more fully describe the state of the art to which this invention pertains.
[0121] The term “comprising” is intended here and to be open, including not only the elements listed, but also covering any additional elements.
[0122] Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the invention. Consequently, the invention is limited only by the claims.

权利要求:
Claims (15)
[0001]
1. Arrangement, FEATURED for understanding: a solid support comprising a surface, the surface comprising a plurality of wells, the wells coated with a gel material that conforms to the shape of the well where it resides, the wells being separated from each other by interstitial regions on the surface, the interstitial regions segregating the gel material in each of the wells of the gel material in other wells of the plurality, where the volume of each of the wells is a maximum of 1,000 pm3, and in which the volume the gel in each of the wells is at most 1,000 pm3; and a library of target nucleic acids in the gel material, wherein the gel material in each well comprises a single species of the target nucleic acids in the library.
[0002]
2. Arrangement, according to claim 1, CHARACTERIZED by the fact that the plurality of wells form an arrangement having a repetition pattern and, optionally, in which the wells in the pattern have a step of no more than 5 micrometers.
[0003]
Arrangement according to either of claims 1 or 2, CHARACTERIZED by the fact that the gel comprises a hydrogel, and, optionally, the gel comprises silane-free acrylamide (SFA) or poly (N- (5-azidoacetamidylpentyl) ) acrylamide-co-acrylamide (PAZAM).
[0004]
4. Arrangement according to any one of claims 1 to 3, CHARACTERIZED by the fact that the gel material in each well comprises a single species of the target nucleic acids in the library, and, optionally, in which multiple copies of the single species form a clonal population of nucleic acid molecules in each well.
[0005]
5. Arrangement according to any one of claims 1 to 4, CHARACTERIZED by the fact that the surface comprises a density of at least 1,000 of the wells per mm2.
[0006]
6. Arrangement according to any one of claims 1 to 5, CHARACTERIZED by the fact that the gel material is covalently bonded to the surface of the wells.
[0007]
7. Method for producing a substrate, CHARACTERIZED by comprising (a) providing a solid support comprising a flat surface, in which the flat surface is interrupted by one or more wells and in which the one or more wells are bounded by one or more regions interstitials on the flat surface; (b) coating at least a portion of the solid support with a gel material that conforms to the shape of the well where it resides, wherein the portion comprises at least one of the wells and at least one of the interstitial regions; and (c) polishing the flat surface to remove the gel material from at least one of the interstitial regions and to maintain the gel material in at least one well.
[0008]
8. Method, according to claim 7, CHARACTERIZED by the fact that the volume of each of the wells is a maximum of 1,000 pm3, or that the volume of the gel in each of the wells is a maximum of 1,000 pm3.
[0009]
9. Method according to claim 7, CHARACTERIZED by the fact that the polishing comprises mechanical abrasion with a granule paste, or in which the polishing comprises mechanical abrasion by cleaning or scraping the surface of the solid support, or in which the polishing comprises chemical polishing.
[0010]
10. Method for producing an arrangement, CHARACTERIZED by the fact that it comprises: (a) providing a solid support comprising a surface, the surface comprising a plurality of wells, the wells containing a gel material that conforms to the shape of the well where it resides, the wells being separated from each other by interstitial regions on the surface, the surface being polished to remove the gel material from the interstitial regions, the interstitial regions segregating the gel material in each of the wells of the gel material in other wells plurality, in which the volume of each well is a maximum of 1,000 pm3, and in which the volume of gel in each of the wells is a maximum of 1,000 pm3; (b) delivering a library of target nucleic acids to the wells of the solid support to produce an array of wells that have a single species of target nucleic acid attached to the gel material in each well, where different wells in the array comprise different species of acid target nucleic acid from the library; and (c) amplifying the target nucleic acids trapped in the gel material in the wells of the array to produce a clonal population of an individual target nucleic acid in each of the wells of the array.
[0011]
11. Method according to claim 10, CHARACTERIZED by the fact that delivering in step (b) comprises providing the library as a mixture of the target nucleic acids in a fluid and contacting the fluid with the solid support, through which the acids target nucleics have fluidic access to wells and interstitial regions.
[0012]
12. Method according to claim 10 or 11, CHARACTERIZED by the fact that the amplification comprises extension by polymerase of at least one type of initiator that is attached to the gel material in the wells, and, optionally, in which the amplification comprises bridge amplification using at least two species of initiator that are attached to the gel material in the wells.
[0013]
13. Method of detecting nucleic acids, CHARACTERIZED in that it comprises (a) providing the arrangement as defined in any of claims 1 to 6; (b) contact the array with at least one probe that binds to the target nucleic acids; and (c) detecting the array to distinguish the wells having a kind of target nucleic acid that binds to at least one probe.
[0014]
14. Method according to claim 13, CHARACTERIZED by the fact that the at least one probe comprises (i) at least one nucleic acid that is complementary to at least a portion of at least one of the target nucleic acids, and optionally , wherein the at least one probe comprises a fluorecent tag that is detected in step (c), or (ii) a polymerase and a nucleotide, and, optionally, where the detection in step (c) comprises detecting the incorporation of the nucleotide into the probe or into the target nucleic acid. (c) detecting the array to distinguish the wells having a kind of target nucleic acid that binds to at least one probe.
[0015]
15. Method according to claim 14, CHARACTERIZED by the fact that the at least one probe (i) comprises at least one nucleic acid that is complementary to at least a portion of at least one of the target nucleic acids, and optionally by at least one probe comprises a fluorecent tag that is detected in step (c), or (ii) a polymerase and a nucleotide, and optionally detection in step (c) comprises detecting the incorporation of the nucleotide into the probe or within the target nucleic acid .

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法律状态:
2018-11-13| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-06-30| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-10-27| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 21/02/2014, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201361769289P| true| 2013-02-26|2013-02-26|

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US61/769.289|2013-02-26|

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US13/787.396|2013-03-06|

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