DNA chip submicron precision large-scale copying method

Abstract

The invention provides a large-scale imprinting method for copying a template microchip.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application No. 63 / 388,326, filed on July 12, 2022, and is incorporated herein by reference in its entirety. Technical Field

[0002] The present application relates to an imprinting method for large-scale manufacturing of DNA chips. Background Art

[0003] In nucleic acid microchip (or DNA chip) technology, DNA of known sequence is deposited ("spotted") or synthesized in situ on a substrate to form a two-dimensional array chip, in which the DNA is covalently or non-covalently attached to the substrate. A 75×25 square millimeter glass slide can carry hundreds to billions of separated DNA chip spots (or feature spots), which can serve as probes to detect multiple target analytes in a complex mixture. Therefore, DNA chips provide a highly parallelized and miniaturized assay platform to maximize throughput and cost-effectiveness. DNA chips have been widely used for analysis of gene expression and transcription factor binding, genotyping, and DNA synthesis. Recently, some chips have been applied to spatial transcriptomics and proteomics.

[0004] Based on the manufacturing method, DNA chips can be divided into three major categories: spotting chips, in situ synthesis chips, and self-assembled (or random) chips. Microchip spotting is the most common method for depositing DNA, proteins, and other molecules on chip substrates. In 1995, Schena et al. developed a method to deposit complementary DNA (cDNA) on a glass slide and used this type of chip to monitor the expression of multiple genes in parallel (1). In 1996, DeRisi et al. used a robotic spotter to deposit more than 1,000 DNA probes on a poly-lysine-coated glass slide (2). Since then, contact and non-contact printing spotting devices have made significant progress, with significant improvements in throughput, quality, and reproducibility (3). However, the spotting method relies on the pre-synthesis and sequential transfer of multiple different DNAs to the substrate surface, which greatly limits the cost-effectiveness and throughput of chip manufacturing. For example, a non-covalently deposited DNA spot can diffuse to other chip locations, which necessitates the use of stringent washing to remove unbound DNA and separate adjacent feature spots with significant spacing to prevent them from merging with each other. Initial reports showed that the center spacing of feature points was 450 μm. With the development of advanced spotting equipment, the center spacing of feature points has been reduced to 60-150 μm (4, 5).

[0005] The second type, in situ synthesis chips, was first manufactured using photochemical synthesis. This method was first used by Fodor, the founder of Affymetrix, to synthesize peptide chips in 1991 (6), and by Pease et al. in 1994 for the synthesis of DNA chips (7). Photochemical synthesis uses photolithography to remove photosensitive protecting groups at specific chip locations, and then selectively adds four nucleotides to the deprotected DNA. Photochemical synthesis has high spatial resolution, which can achieve high feature point density of in situ synthesis chips. For example, 135,000 feature points of 35×35μm in size can be synthesized on each slide (8). Recently, the size of the feature points has been further reduced to 5μm (9). The main limitations of the in situ synthesis method are the need to use multiple photolithography masks to control multiple synthesis cycles, making the process slow and expensive; and the high synthesis error rate, which limits the maximum length of synthesized DNA to about 100 base pairs. Other in situ synthesis methods have also been developed: for example, inkjet printing is used to print nucleotide building blocks onto specific chip locations (10). Inkjet printing is compatible with classical DNA synthesis chemistry based on chemical removal of DNA protecting groups and has a lower error rate than photo-guided synthesis, so DNA lengths can be increased to 200 base pairs or more. A representative commercial example of this type of synthesis is Agilent's oligonucleotide microchip.

[0006] The characteristic points of self-assembled chips come in many forms, such as DNA-coated microbeads (11-14), DNA nanospheres (15), and DNA clone groups (hereinafter referred to as polony (16, 17) or DNA clusters (18)). Although these chips are manufactured using different methods, they have one thing in common: the characteristic points are randomly distributed on non-array (or random) or array chips, and the DNA sequence of each characteristic point needs to be determined through a decoding step after the chip is prepared. Walt et al. prepared wells slightly larger than a microbead at the end of the optical fiber array by photolithography, and flowed into the DNA-coated microbeads, thereby preparing a random microbead chip using an optical fiber chip. These microbeads are optically encoded by different combinations of fluorescent dyes. By decoding the optical fiber fluorescence imaging, the type of oligonucleotide at a certain position in the chip can be determined (11, 19-21). This technology has been licensed to Illumina, and after improvement, it can be used to prepare large-size chips with high microbead density. The optical decoding method of fluorescently labeled microbeads limits the number of overall microbeads. Therefore, a new decoding method similar to DNA sequencing technology was invented, which uses fluorescently labeled oligonucleotide probes to hybridize with DNA in glass wells and then perform sequencing decoding (12). In recent years, the application of second-generation sequencing platforms has produced more complex self-assembled chips. Complete Genomics uses rolling circle amplification (22) to amplify single-molecule templates into DNA nanoballs of approximately 200 nanometers in size in a capillary tube, and then fixes these nanoballs on a silicon chip for large-scale parallel DNA sequencing to determine the sequence of each feature point (15). Similarly, the most widely used Illumina second-generation sequencing uses an in-gel bridge amplification method to in situ amplify single-molecule templates randomly spotted on the linear polyacrylamide gel coating on the glass surface of the fluid tank into DNA clusters of approximately 1 μm in size for sequencing (18). Due to the small size of the feature points, this type of DNA chip has an ultra-high feature point density (for example, more than 1 million per square millimeter) and solves complex decoding problems through sequencing. However, this type of chip manufacturing method requires sequencing of each chip, which is an expensive and time-consuming process. In addition, the fluorescence imaging used in DNA nanospheres and DNA cluster sequencing often partially destroys the DNA on the chip. Therefore, these ultra-dense DNA chips have not yet been commercialized as standard DNA chip applications. Recently, spatial transcriptomics (23) for mapping biological tissues has begun to use high-resolution DNA chips to achieve cellular and subcellular resolution (13, 14, 24-28).

[0007] DNA chips in which each feature point carries a unique spatial code are called spatial position information encoding chips, which are increasingly used to capture and sequence RNA and proteins in situ to map the structure and function of heterogeneous tissues (13, 14, 23-27). To achieve single-cell resolution, the feature points of DNA chips need to be significantly smaller than cells, such as micrometer to submicrometer feature point diameters to depict different cell shapes. Traditional spotting (1, 2) or photosensitive synthesis (6) methods deposit or synthesize sequence-defined oligonucleotides at specific chip locations. The size of the generated feature points is usually larger than that of mammalian cells (>10μm) and there is a significant spacing between the feature points. Recent spatial transcriptomics uses random chips with smaller feature points, such as DNA-coated microbeads (13, 14), DNA nanospheres (26), and DNA clonal groups (polonies) (28) (or DNA clusters (25)), which requires the use of special equipment or fluid tanks to decode the individual spatial codes on each chip by sequencing (see Table 2). The ability to print high-resolution microarrays on a given substrate, such as a glass slide, without having to sequence each microarray would increase the ubiquity, scalability, and flexibility of microarray-based analytical methods. One possible approach to large-scale manufacturing is to use microcontact printing (29) to replicate the microarray molecules onto a substrate using an elastomeric stamp. However, an unresolved issue is how to construct an elastomeric stamp that encodes spatial position information in a way that does not progressively reduce the resolution and amount of DNA replicated during successive printing steps. Summarize

[0008] The embodiments of the present disclosure are based on the following discovery: when a DNA clone group (polonies) chip (hereinafter referred to as polony gel chip) formed on the surface of an elastic cross-linked polyacrylamide "stamp gel" is used as a template, the DNA array structure of the chip can be efficiently replicated to other "replication gel" surfaces by DNA polymerase-catalyzed chain extension. Because all primers and templates are covalently attached to the gel, the diffusion of DNA is prevented, and the gel-to-gel replication described herein reliably achieves submicron resolution accuracy. Unlike traditional stamping, which requires the stamp to be "re-inked" for continuous printing (see, for example, S. A. Lange et al., Anal. Chem. 76, 1641-1647 (2004)), the enzymatic DNA replication used in the present disclosure does not consume the template on the stamp. Imprint replication chips achieve high replication efficiency and strengthen amplification of weakly imprinted areas through bridge amplification on the gel surface (L. Gu et al., Nature 515, 554-557 (2014), and Bentley et al., Nature 456, 53-59 (2008)). The spatial encoding map of all imprinted replicated chips can be obtained by sequencing only one or a few replicated gel chips. The replicated polony gel chip can be used as a stamp for the next round of manufacturing. The polony gel chip exhibits a uniform and continuous distribution of feature points, which can limit the diffusion of templates in tissue sections and achieve high RNA capture efficiency (for example, 10×10μm mouse tissue). 2 The average number of unique molecular identifiers (UMIs) in the area is about 1000. The polony gel chip library sequencing method with molecular markers (abbreviated as Pixel-seq) has achieved spatial transcriptomics research on single cells in mouse brain tissue.

[0009] Spatial resolution of DNA chip-based spatial transcriptomic and proteomic assays is reduced by diffusion of analytes (e.g., RNA and DNA-tagged affinity molecules) within tissues and on the chip. Currently, these assays rely on the nondirectional migration of tissue analytes before capture by chip probes. Although tissue samples can be sliced ​​into thin sections (e.g., ≤10-μm thick) to facilitate capture of features that fit on the chip, lateral diffusion of analytes is a major limitation for assays that require single-cell or subcellular resolution. One solution to reduce diffusion is to apply an electric field perpendicular to the tissue slice for electrophoretic transfer to drive directional migration of tissue analytes onto the chip. However, DNA chips prepared using traditional methods or that require sequencing use solid support substrates such as glass, silicon, or polydimethylsiloxane, which are not compatible with electrophoretic transfer. Although surface modification of solid support substrates can make them conductive, such as tin oxide coating on glass surfaces (preprint doi.org / 10.1101 / 2022.01.12.476082), such modifications are costly and may affect the attachment of DNA to the solid substrate. To address this limitation, the DNA chip fabricated by DNA imprinting of the present disclosure is grown on the surface of a hydrogel (eg, polyacrylamide gel), allowing for the capture of tissue analytes by electrophoresis.

[0010] In some embodiments of the present disclosure, a method for replicating a template microchip is provided, which comprises the following steps: providing a template microchip comprising a plurality of nucleic acid sequence clusters; contacting the template microchip with a replicating microchip in the presence of a polymerase and nucleotides; removing the template microchip; and amplifying the nucleic acid sequences on the replicating microchip to form a plurality of nucleic acid clusters.

[0011] Some embodiments of the present disclosure provide a method for replicating a template microchip. The method includes the following steps: providing a template microchip including a plurality of nucleic acid feature points, each feature point including a group of single-stranded DNAs with the same sequence, and the distribution of the feature points on the chip can be random or arrayed; the template microchip contacts the replica microchip in the presence of a polymerase and nucleotides to synthesize a complementary DNA sequence on the replica microchip; removing the template microchip; amplifying the complementary DNA sequence synthesized on the replica microchip to form a plurality of nucleic acid feature points, and these replicated nucleic acid feature points have the same sequence and distribution as the corresponding feature points on the template microchip; repeating the above-mentioned replication process with the same template microchip and a plurality of replica microchips to produce a plurality of replica microchips.

[0012] In certain exemplary embodiments, the template microchip includes a solid support substrate having an upper surface with a plurality of nucleic acid feature points on the upper surface.

[0013] In certain exemplary embodiments, the solid support substrate is a non-porous substrate or a porous substrate, a rigid substrate or an elastomeric substrate, a single-layer substrate or a multi-layer substrate; the solid support substrate allows the nucleic acid sequence to be covalently or non-covalently attached to its surface, and the substrate may have a smooth surface, or a surface with a micro-well or nano-well structure generated by chemical or photoetching; the nucleic acid sequence is attached to the smooth surface or the inner surface of the micro-well or nano-well.

[0014] In certain exemplary embodiments, the nucleic acid feature spots in the template microchip are selected from clonal DNA clusters, DNA nanospheres, DNA-coated microbeads, DNA spots deposited or synthesized by spotting methods, and DNA spots synthesized by light-guided synthesis methods.

[0015] In certain exemplary embodiments, a plurality of nucleic acid feature points form a random chip or an array chip.

[0016] In certain exemplary embodiments, the synthesis of single-stranded DNA sequences in the template microchip can be performed by a method selected from chemical synthesis, enzymatic synthesis, template-dependent synthesis, template-independent synthesis, and synthesis of double-stranded DNA sequences followed by cleavage of one or both strands of the double-stranded DNA.

[0017] In certain exemplary embodiments, cleavage can be accomplished using one or more of a uracil-specific excision enzyme, an 8-oxoguanosine DNA glycosylase, and a restriction endonuclease, or a chemical reagent including an oxidizing agent and a reducing agent.

[0018] In certain exemplary embodiments, the oxidizing agent may be periodate and lead tetraacetic acid, and the reducing agent may be phosphine, dithiothreitol, dithioerythritol and L-glutathione.

[0019] In certain exemplary embodiments, the replicated microchip comprises an elastic solid substrate having an upper surface with a plurality of nucleic acid feature points on the upper surface.

[0020] In certain exemplary embodiments, the elastic solid substrate is a non-porous substrate or a porous substrate, a single-layer substrate or a multi-layer substrate; the elastic solid substrate allows nucleic acid primer sequences to be covalently attached to its surface, facilitating conformal contact between two microarrays, and solid-phase clonal DNA amplification on the substrate.

[0021] In certain exemplary embodiments, a plurality of nucleic acid primers are mixed and then uniformly or arrayed on the upper surface of the elastic solid substrate.

[0022] In certain exemplary embodiments, in the contacting step, part or all of the single-stranded DNA sequence in each feature point in the template microchip is hybridized with the primer sequence in the replica microchip to synthesize a complementary DNA sequence as a template.

[0023] In certain exemplary embodiments, before removing the template microchip, the complementary DNA sequence synthesized on the replication microchip is dissociated from the single-stranded DNA sequence on the template microchip by thermal and / or chemical denaturation.

[0024] In certain exemplary embodiments, the amplification step is solid phase clonal DNA amplification, optionally bridge amplification.

[0025] In certain exemplary embodiments, the same template microchip is sequentially contacted with multiple replica microchips to produce multiple replica microchips with almost identical nucleic acid sequences and distributions of characteristic points.

[0026] In certain exemplary embodiments, the replicated microchip can be used as a template microchip for additional microchip replication processes.

[0027] In certain exemplary embodiments, by sequentially contact-copying a plurality of different template microchips onto a replicated microchip, the goal of increasing the density of feature points and DNA coverage, as well as reducing the size of feature points, can be achieved on the replicated microchip.

[0028] In certain exemplary embodiments, the nucleic acid sequence includes a single or multiple probe sequences for hybridizing to complementary sequences in target nucleic acid molecules in a sample.

[0029] In certain exemplary embodiments, the target nucleic acid molecules are retained in the sliced ​​tissues with the original cells and tissues positions unchanged; or the target nucleic acid molecules are released from the homogenized cells or tissues after the cell membranes are broken.

[0030] In certain exemplary embodiments, the target nucleic acid molecule is an endogenous nucleic acid in a cell, or is an artificially synthesized nucleic acid tag covalently or non-covalently attached to an affinity molecule or polymerized to an endogenous nucleic acid sequence.

[0031] In certain exemplary embodiments, the affinity molecule can be selected from one or more of a binding protein, an antibody, an antigen-binding fragment of an antibody, a nanobody, a monomer, and a nucleic acid aptamer. In certain examples, the affinity molecule is used to detect one or more combinations of proteins, DNA, RNA, small molecule ligands, protein modifications, DNA modifications, RNA modifications, small molecule ligand modifications, and molecular complexes present in a sample.

[0032] In certain exemplary embodiments, different probe sequences are present in different feature points, whereby the probe sequences are associated with their positions in the microchip.

[0033] In certain exemplary embodiments, all feature points in the microchip carry the same probe or a plurality of different probes.

[0034] In certain exemplary embodiments, in addition to the probe sequence, the nucleic acid sequence of the feature point also includes a coding sequence associated with its position in the microchip.

[0035] In certain exemplary embodiments, the probe and / or spatial coding sequences are known or unknown during the construction of the microchip and can be subsequently determined by DNA sequencing or hybridization methods.

[0036] In certain exemplary embodiments, the built-in probe sequence in the DNA is exposed at the 3' end by a nucleic acid sequence-specific cleavage method including, but not limited to, restriction endonuclease digestion.

[0037] In certain exemplary embodiments, after the microchip is replicated, one or more probe sequences are added to the nucleic acid sequences without probe sequences by enzymatic methods (which can be selected from DNA ligation or primer extension).

[0038] In certain exemplary embodiments, the probes and / or spatial coding sequences corresponding to the characteristic points in the microchip replicas replicated from the same template microchip are substantially identical and can be determined by the sequences of the corresponding characteristic points in the template microchip or by sequencing one or a small number of replicated microchips.

[0039] In certain exemplary embodiments, the template microchip and the replica microchip are used to analyze gene expression and genetic variation of single nucleotide polymorphisms in a sample.

[0040] In certain exemplary embodiments, the template microchip and the replica microchip are used to analyze the binding of transcription factors to probe sequences.

[0041] In certain exemplary embodiments, the microchip is used to spatially encode nucleic acids, proteins, and small molecule ligands in tissue sections placed thereon by combining coding sequences with complementary DNA of endogenous nucleic acids synthesized in situ or artificially synthesized nucleic acid markers.

[0042] In certain exemplary embodiments, spatially encoded microchips analyze molecular interactions between proteins, nucleic acids, and / or small molecule ligands in tissue section samples or homogenous mixed samples.

[0043] In certain exemplary embodiments, microchip replicas are used to amplify natural or synthetic DNA and / or genes on the chip, thereby minimizing amplification distortion caused by the overall polymerase chain reaction (PCR).

[0044] In certain exemplary embodiments, nucleic acid templates and primers are covalently attached to the template microchip and the replication microchip, thereby limiting the diffusion of DNA to achieve feature point replication with sub-micron resolution accuracy.

[0045] In certain exemplary embodiments, there is minimal loss of nucleic acid templates covalently attached to the template microchip during each contacting step, and thus the template microchip can be reused for multiple copying cycles.

[0046] Some embodiments of the present disclosure provide a method for replicating a template microchip. The method comprises the following steps: providing a template microchip comprising a plurality of cloned DNA clusters, wherein each cluster comprises a plurality of identical single-stranded DNA sequences, and the distribution of the DNA clusters on the chip may be random or arrayed; contacting the template microchip with a replicating microchip in the presence of a polymerase and nucleotides to synthesize a complementary DNA sequence on the replicating microchip; removing the template microchip; amplifying the complementary DNA sequence synthesized on the replicating microchip to form a plurality of DNA clusters having substantially the same sequence and distribution as the cluster corresponding to the template microchip; repeating the above-mentioned replication process with the same template microchip and a plurality of replicating microchips to produce a plurality of replicating microchips.

[0047] In certain exemplary embodiments, the template microchip comprises a gel or a substrate with chemically or photoetched micro- or nano-well structures and the replica microchip comprises a gel.

[0048] In certain exemplary embodiments, the gel may be selected from polyacrylamide gel, hydrogel and polydimethylsiloxane gel.

[0049] In certain exemplary embodiments, the cloned DNA clusters in the template and replicate microchips are amplified by a bridge amplification method, and the DNA density in the clusters, the number of DNA copies, and the size of the clusters can be controlled by adjusting the number of amplification cycles.

[0050] Some embodiments of the present disclosure provide a method for replicating a template microchip. The method comprises the following steps: providing a template microchip having a polyacrylamide gel with multiple DNA clusters attached to the surface; contacting the template microchip with a replica microchip in the presence of a polymerase and nucleotides, the replica microchip comprising a polyacrylamide gel with multiple primer sequences attached to the surface; removing the template microchip; amplifying the nucleic acid sequence on the replica microchip to form multiple cloned DNA clusters having substantially the same sequence and distribution as the cluster corresponding to the template microchip; repeating the above-mentioned replication process with the same template microchip and multiple replica microchips to generate multiple replica microchips.

[0051] In certain exemplary embodiments, the polyacrylamide gel comprises a single layer or multiple layers of linear polyacrylamide and / or cross-linked polyacrylamide, and the cross-linking agent may be selected from one or more of N,N'-methylene-bisacrylamide, N,N'-cystathionamide-bisacrylamide and N,N'-diallyl-tartaric acid diamide.

[0052] Some embodiments of the present disclosure provide a method for generating a replica microchip on a gel suitable for electroblotting transfer analysis of tissue sections or homogenous mixed samples. The method comprises the following steps: replicating the template microchip information onto the replica microchip using the method described in claim 1, the replica microchip being attached to the upper surface of a porous gel, the upper and lower surfaces of the porous gel being exposed to a conductive medium to allow ions to enter and exit; attaching the tissue section or homogenous mixed sample to the microchip; applying an electric field perpendicular to the upper surface of the gel to drive the analyte in the sample to migrate to the upper surface of the gel and hybridize with the probe sequence in the microchip, thereby capturing the nucleic acid target on the gel surface.

[0053] In certain exemplary embodiments, the sample is a fresh snap-frozen or formalin-fixed paraffin-embedded (FFPE) tissue section or a homogenous mixture containing one or any combination of nucleic acids, proteins, small molecule ligands, and DNA-tagged affinity molecules.

[0054] In certain exemplary embodiments, transfer of the nucleic acid target is performed by a semi-dry or dry electroblotting device.

[0055] Some embodiments of the present disclosure provide a method for regional selective analysis of nucleic acid targets captured on one or more regions of a microchip, the method replicating a template microchip onto a replica microchip having a photodissociable probe sequence. The method comprises the following steps: providing a template microchip comprising a plurality of nucleic acid sequence clusters; contacting the template microchip with the replica microchip in the presence of a polymerase and nucleotides; removing the template microchip; amplifying the nucleic acid sequences on the replica microchip to form a plurality of nucleic acid clusters; attaching tissue sections or homogenized mixed samples to the replica microchip; capturing the nucleic acid targets on the gel surface by hybridization with the probe sequences on the microchip; synthesizing complementary DNA sequences on the microchip using the probe sequences as primers; cutting the probes with light in one or more selected local regions, thereby regionally selectively releasing the synthesized complementary DNA sequences; eluting the released complementary DNA, the eluted complementary DNA can be used for DNA amplification, sequencing and / or other analysis.

[0056] In certain exemplary embodiments, the probe sequences are linked to the microchip via one or more photodissociable spacers, including but not limited to 1-(2-nitrophenyl)ethyl ester.

[0057] Some embodiments of the present disclosure provide a method for regional selective analysis of nucleic acid targets captured on one or more regions of a microchip. The method replicates a template microchip to a replica microchip on a gel substrate by the following steps: providing a template microchip including multiple nucleic acid sequence clusters; contacting the template microchip with the replica microchip in the presence of a polymerase and nucleotides; removing the template microchip; amplifying the nucleic acid sequences on the replica microchip to form multiple nucleic acid clusters; attaching tissue sections or homogenized mixed samples to the replica microchip; capturing nucleic acid targets on the gel surface by hybridization with probe sequences on the microchip; using the probe sequences as primers to synthesize complementary DNA sequences on the microchip; mechanically cutting one or more local regions of the gel using a micro-cutting device and transferring them to a container, which can be a test tube; analyzing the complementary DNA in the cut gel slices by DNA amplification, sequencing and / or other assays.

[0058] In certain exemplary embodiments, the gel substrate is a flexible material suitable for mechanical cutting, including but not limited to polyacrylamide gel.

[0059] In certain exemplary embodiments, the micro-cutting device has a micro-cutting head for cutting small gel areas; a connecting tube that can apply vacuum and pressure to the end of the micro-cutting head to transfer the cut gel slices to a test tube; and an XYZ worktable for sampling different areas of the gel. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] The following detailed description of the specific embodiments and drawings can help to more fully understand the foregoing description of the present invention and its features and advantages. This patent or application document contains at least one color drawing. Upon request, the office will provide a color drawing copy of the patent or patent application text upon payment of the necessary fee.

[0061] Figure 1 Schematic diagram of the amplifiable DNA imprinting method. Linearized single-stranded polony DNA is copied from the stamp to multiple replicate gels, and the replicated DNA completes the entire gel replication process after bridge amplification. A small number of replicate gels are used for subsequent manufacturing rounds or for polony sequencing to create spatially encoded maps, and most replicate gels are used for other test experiments.

[0062] Figure 2A and Figure 2B Demonstrating gel-to-gel DNA replication ( Figure 2A ) and automated embossing and replication equipment ( Figure 2BThe device contains a tabletop robotic arm that places the stamp in four positions in sequence during each stamping cycle: A) formamide at 60°C; B) stamping buffer at 60°C; C) stamping mix containing Taq DNA polymerase and dNTPs at 4°C; and D) a specific position on the replica gel, where the temperature is reduced from 95 to 60°C. The stamping pressure is monitored by an electronic balance.

[0063] Figure 3A Images of SYBR Green-stained DNA in stamp and replica gels at the millimeter scale are shown. To compare the DNA patterns on the gels, the template was deposited on a 40 μm thick stamp gel with a mask and amplified into DNA clusters showing the “Pixel” pattern.

[0064] Figure 3B SYBR Green-stained images of DNA clusters at submicron resolution in replica gels generated from the 2nd, 10th, and 50th stamps are shown.

[0065] Figure 4 Depicts the continuous distribution of characteristic spots in the polony gel. (Left) 3D intensity distribution of discrete and continuous polony SYBR Green staining generated by 35 cycles of amplification with the same deposition density template, (Middle) a sequencing image merged with four colors, which is converted into a spatially encoded map by pixel-level base calling (right).

[0066] Figure 5A Bar graph showing the percentage of DNA clusters in replicate gels relative to the distribution consensus. Data are the average of six gel sampling positions where polony was found from 195 to 332; error bars are standard deviations.

[0067] Figure 5B 2D and 1D density plots showing the relative positions of polony centers from three different replicate gels. The inner and outer dashed circles mark the distances of 0.5 and 1 μm. n = 4,521.

[0068] Figure 5C The bar graph compares the coding error rates detected by Illumina sequencing of the coding DNA eluted from the two replicate gels.

[0069] Figure 5D Comparison of Polony amplification efficiency on different gel substrates. Linear polyacrylamide substrate was prepared according to the reported Illumina method. Four sets of data points on the left: cross-linked polyacrylamide; rightmost data point: linear polyacrylamide.

[0070] Figure 5E Violin plot showing the measured polony diameters at different densities. N≈0.6 to 1 million.

[0071] Figure 6 Demonstration of the principle of Pixel-seq. A polony gel captures RNA in a frozen tissue section and the contact surface of the gel. Spatially encoded complementary DNA is synthesized by template switching oligonucleotides, and a universal sequence at the 3′ end is added to facilitate amplification of the complementary DNA. After complementary DNA sequencing, RNA can be associated with gel positions to create a transcriptome map. A k-nearest neighbor network is constructed on the map, where each code represents a node. Edge weights are calculated as a function of the number of UMIs, the distance between two connected codes, and the transcript similarity. The weighted network is segmented by a graph algorithm to generate cell masks, and the transcript data are aggregated to facilitate single-cell data analysis.

[0072] Figure 7 Shows a gel that captures only RNA from a single cell layer on a tissue section. The analysis compares a confocal image of a mouse olfactory bulb section nucleus on a gel and a confocal image of a synthetic complementary DNA on the same gel. The nucleus image has two focus planes, 0μm and 6μm, which are superimposed on the complementary DNA image.

[0073] Fig. 8A A representative UMI density map of a coronal olfactory bulb section is shown. The UMI density is calculated based on the area of ​​the image pixel (0.325×0.325μm 2 ).

[0074] Figure 8B and Figure 8C The RNA capture efficiency of Pixel-seq and other spatial transcriptomics methods was compared. The latest reported olfactory bulb or other available datasets were used. The bin size for pixel-seq data calculations was 7 × 7 pixels (2 μm, Figure 8B ) and 33×33 pixels (10μm, Figure 8C ). This comparison does not take into account the spacing between feature points in other methods.

[0075] Fig. 9A Compared with about 1×10 4 , about 2×10 4 , and about 3×10 4 Polony distribution patterns at corresponding positions of three replicated gels stamped under three different Pascal pressures. Specifically, the applied stress is equivalent to a weight of about 50, about 100, and about 150 grams acting on a sheet of about 7×7 mm 2 On the stamp, the low-density polony was amplified to compare the distribution pattern of feature points.

[0076] Fig. 9BBar graph showing the percentage of polony matches in replicate gels. Data are the average of six gel sampling positions where polony was found ranging from 127 to 236; error bars are standard deviations.

[0077] Fig. 10A and Fig. 10B Demonstrated in cross-linking ( Fig. 10A ) and linear ( Fig. 10B ) Representative images of amplified polonies on polyacrylamide (PAA) gels. Linear PAA gels were prepared according to the reported Illumina protocol. Templates were deposited at the same density on the gels and amplified for the indicated number of cycles before DNA staining with SYBR green. All images were acquired with the same imaging settings. Background-subtracted fluorescence signals were Figure 5D A comparison was made in .

[0078] Fig.11 Four color sequencing images are shown, comparing the size of polony at increasing density. Templates were deposited at a specific density and then amplified for 35 cycles. Due to the repulsive nature of polony, even at higher than the commonly used feature point density (e.g., about 1.2 million / mm 2 In the case of 100 μm (average feature size of about 0.5 μm), most of the different colored polonies showed clear boundaries, indicating that they can be sequenced by the high-resolution imaging setting. Some dark areas in the image are also occupied by polonies without sequencing signals because some randomly generated codes have Taql cleavage sites.

[0079] Fig. 12A Cy5-labeled signals of complementary DNA synthesized from gel-captured RNA reveal the shape of cell bodies. 10-μm sections of mouse olfactory bulb-cortex were used. AON, anterior olfactory nucleus; AOB, accessory olfactory bulb; MOB, main olfactory bulb; CTX, cerebral cortex.

[0080] Fig. 12B Demonstrates a method for quantifying template lateral diffusion. A 10-μm mouse olfactory bulb cryosection was placed on a xerogel pre-impregnated with SYTOX Green and the stained nuclei were immediately photographed for reference and comparison with the Cy5-labeled complementary DNA signal. The images were taken with an epifluorescence microscope used for polony gel sequencing. The magnified images show significantly more nuclei than complementary DNA signals because the former are derived from multiple cell layers in the section, while the latter are derived from only a single cell in contact with the gel.

[0081] Fig. 12C Quantification of template lateral diffusion is shown. The scaling factor is given by Fig. 12BCalculated by image analysis in (B). Data are from ten areas sampled in (250 × 250 pixels, 1 pixel = 0.65 μm); error bars are standard error of the mean. The scaling factor of 1.028 ± 0.016 corresponds to a diffusion distance of 0.04 to 0.4 μm.

[0082] Fig.13 Schematic diagram of RNA capture from a cell monolayer at the moment the gel is wetted.

[0083] Fig.14 Flowchart showing the Pixel-seq method for constructing complementary DNA sequencing libraries.

[0084] Fig.15 The template sequence used for polony construction is given (SEQ ID NO: 1).

[0085] Fig.16 Demonstrates the replication of DNA clusters in an Illumina NovaSeq sequencing flow cell onto a replica gel. Matching concentric markers (no DNA clusters) are visible in the replica gel.

[0086] Fig.17 Depicts the capture of analytes from tissue sections onto the upper surface of a gel-based DNA microchip via electrophoresis. The tiny pores in the gel prevent the analytes from entering the interior of the gel.

[0087] Fig.18 Depicts the release of nucleic acid targets in selected areas of a DNA chip using a light-guided specific area release method.

[0088] Fig.19 Mechanical microdissection sampling of selected areas is demonstrated to allow analysis of specific regions of nucleic acid targets captured on a DNA chip. Detailed statement

[0089] Before describing the disclosure, it should be understood that the disclosure is not limited to the specific methods and experimental conditions described, because the methods and conditions may vary. It is also necessary to understand that the terminology used herein is only for the purpose of describing specific embodiments, and is not intended to be limiting, because the scope of the disclosure of the invention is limited only by the claims below.

[0090] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0091] When the term "about" is used herein to refer to a specific numerical value, it means that the value may vary from the listed value by no more than 1%. For example, the expression "about 100" herein includes 99 and 101 and all values ​​therebetween (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0092] In various embodiments, the methods disclosed herein include nucleic acid amplification, which includes, for example, polynucleotides, oligonucleotides and / or oligonucleotide fragments. The amplification method can include contacting the nucleic acid sequence with one or more primers (e.g., primers complementary to the coding sequence), which specifically hybridize to the nucleic acid under conditions suitable for hybridization and chain extension. Exemplary methods for amplifying nucleic acids include polymerase chain reaction (PCR) (see, e.g., Mullis et al. (1986) Cold Spring Harb. Symp. Quant. Biol. 51 Pt 1:263; Cleary et al. (2004) Nature Methods 1:241; and those described in U.S. Pat. Nos. 4,683,195 and 4,683,202), anchored PCR, RACE PCR, ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241: 1077-1080; and Nakazawa et al. (1994) Proc. Natl. Acad. Sci. USA 91: 360-364), self-sustaining sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874), transcription amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), Q-Beta replicase (Lizardi et al. (1988) BioTechnology 6:1197), recursive PCR (Jaffe et al. (2000) J. Biol. Chem. 275:2619; Williams et al. (2002) J. Biol. Chem. 277:7790), the application methods described in U.S. Pat. Nos. 6,391,544, 6,365,375, 6,294,323, 6,261,797, 6,124,090 and 5,612,199, isothermal amplification (e.g., isothermal bridge amplification (IBA)), rolling circle amplification (RCA), hyperbranched rolling circle amplification (HRCA), strand displacement amplification (SDA), helicase-dependent amplification (HAD), PWGA, or any other known nucleic acid amplification technique.

[0093] "Polymerase chain reaction" or "PCR" refers to an in vitro amplification reaction of a specific DNA sequence by simultaneous extension of primers of complementary DNA strands. In other words, PCR is a reaction in which multiple copies or replicas of a target nucleic acid are made through primer binding sites, the reaction comprising one or more repetitions of the following steps: (i) denaturation of the target nucleic acid, (ii) annealing of primers to primer binding sites, and (iii) extension of the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. The reaction is typically cycled in a thermal cycler at different temperatures optimized for each step. The specific temperature of each step, the duration, and the rate of change between steps depend on many factors well known to those of ordinary skill, such as exemplified in the references: PCR: A Practical Approach and PCR 2: A Practical Approach, edited by McPherson et al. (IRL Press, Oxford, 1991 and 1995). For example, in conventional PCR using Taq DNA polymerase, the double-stranded target nucleic acid can be denatured at a temperature greater than 90° C., the primers can be annealed at a temperature range of 50-75° C., and the primers can be extended at a temperature range of 72-78° C. In certain embodiments, the double-stranded target nucleic acid is denatured at a temperature greater than 90° C. in conventional PCR using Taq DNA polymerase, or formamide is added at 60° C. to complete the denaturation of the double-stranded target nucleic acid in isothermal bridge amplification using Bst polymerase.

[0094] The term "PCR" includes various derivative forms of the reaction, including but not limited to RT-PCR, real-time PCR, nested PCR, quantitative PCR, multiplex PCR, assembled PCR, etc. The reaction volume ranges from hundreds of nanoliters, such as 200 nanoliters, to hundreds of microliters, such as 200 microliters. "Reverse transcription PCR" or "RT-PCR" refers to PCR performed by a reverse transcription reaction, which first converts the target RNA into complementary single-stranded DNA and then amplifies the DNA, such as Tecott et al., U.S. Patent No. 5,168,038. "Real-time PCR" refers to PCR in which the number of reaction products (i.e., amplicons) is monitored in real time as the reaction proceeds. There are many forms of real-time PCR, which differ primarily in the detection chemistry used to monitor the reaction products, e.g., Gelfand et al., U.S. Pat. No. 5,210,015 ("Tagman"), Wittwer et al., U.S. Pat. Nos. 6,174,670 and 6,569,627 (intercalating dyes), Tyagi et al., U.S. Pat. No. 5,925,517 (molecular beacons). For a review of real-time PCR detection chemistries, see Mackay et al., Nucleic Acids Research, 30:1292-1305 (2002). "Nested PCR" refers to a two-stage PCR in which the amplicon from the first stage PCR is used in conjunction with a new set of primers to form the sample for the second stage PCR, with at least one primer in the second stage binding to a position within the first stage amplicon. As used herein in reference to a nested amplification reaction, "primary primers" refer to the primers used to generate the first stage amplicon, and "secondary primers" refer to the primer or primers used to generate the second stage or nested amplicon. "Multiplex PCR" refers to PCR of multiple target sequences (or a single target sequence and one or more reference sequences) performed simultaneously in the same reaction mixture, e.g., Bernard et al. (1999) Anal. Biochem., 273:221-228 (two-color real-time PCR). A different primer set is usually used for each sequence amplified. "Quantitative PCR" refers to PCR designed to measure the abundance of a specific target sequence in a sample or specimen.The technique of quantitative PCR is well known to ordinary technicians and is exemplified by the following literature: Freeman et al. (1999) Biotechniques, 26: 112-126, Becker-Andre et al. (1989) Nucleic Acids Research, 17: 9437-9447, Zimmerman et al. (1996) Biotechniques, 21: 268-279, Diviacco et al. (1992) Gene, 122: 3013-3020, Becker-Andre et al. (1989) Nucleic Acids Research, 17: 9437-9446, etc.

[0095] In some embodiments, a method for determining one or more target nucleic acid sequences, such as polynucleotides, oligonucleotides and / or oligonucleotide fragments, is provided. The sequencing of the target nucleic acid sequence can be performed by sequencing methods known in the literature, including but not limited to: hybridization sequencing (SBH), ligation sequencing (SBL), quantitative incremental fluorescent nucleotide addition sequencing (QIFNAS), step-by-step ligation and cleavage, fluorescence resonance energy transfer (FRET), molecular beacons, TaqMan probe digestion, pyrophosphate sequencing, in situ fluorescence sequencing (FISSEQ), FISSEQ beads (U.S. Patent No. 7,425,431), wobble sequencing (PCT / US05 / 27695), multiple sequencing (U.S. Patent Application No. 12 / 027,039, filed February 6, 2008; Porreca et al. (2007) Nat. Methods 4:931), polony sequencing (U.S. Patent Nos. 6,432,360, 6,485,944 and 6,511,803, and PCT / US05 / 06425), nanogrid rolling circle sequencing (rolony) (U.S. Patent Application No. 12 / 120,541, filed May 14, 2008), allele-specific oligo ligation (e.g., oligo ligation assay (OLA), single template molecule OLA using linear probes and rolling circle amplification (RCA) readouts, ligation of locked circle probes, and / or single template molecule OLA using circular locked circle probes and RCA readouts), etc. High-throughput sequencing methods can also be used, such as cycle sequencing using sequencing platforms such as Roche 454, Illumina Solexa, AB-SOLiD, Helicos, Polonator platforms, etc. High-throughput sequencing methods are described in U.S. Patent Application No. 61 / 162,913 (filed March 24, 2009). Various optical sequencing technologies have been reported in the literature (Landegren et al. (1998) Genome Res. 8: 769-76, Kwok (2000) Pharmocogenomics 1: 95-100, Shi (2001) Clin. Chem. 47: 164-172).

[0096] Embodiments of the present invention relate to polynucleotides, oligonucleotides, small molecules, matrices, test compounds, etc. connected to one or two or more tags (e.g., coding sequences). As used herein, the term "coding" refers to a unique oligonucleotide sequence that can be used to identify, retrieve and / or amplify a corresponding nucleic acid sequence (e.g., an oligonucleotide fragment). In certain embodiments, the length of the coding may range from 4 to 36 nucleotides, or from 6 to 30 nucleotides, or from 8 to 20 nucleotides. In certain exemplary embodiments, the length of the coding is 4 nucleotides. In certain embodiments, the melting temperatures in the coding set are within 10°C, within 5°C, or within 2°C of each other. In some embodiments, the coding belongs to a minimal cross-hybridization set. That is, the sequence of each member of this set is significantly different from the sequences of all other members of the set, so that under stringent hybridization conditions, any member cannot form a stable double strand with the complementary sequence of any other member. In some embodiments, the sequence of each member in the minimal cross-hybridization set is at least two different nucleotides from the other members. Encoding technology is widely reported in the literature, for details, see Winzeler et al. (1999) Science 285:901; Brenner (2000) Genome Biol. 1:1; Kumar et al. (2001) Nature Rev. 2:302; Giaever et al. (2004) Proc. Natl. Acad. Sci. USA 101:793; Eason et al. (2004) Proc. Natl. Acad. Sci. USA 101:11046; and Brenner (2004) Genome Biol. 5:240.

[0097] In certain embodiments, one or more labels are used to detect and / or obtain (i.e., purify) polynucleotides, oligonucleotides, small molecules, substrates, test compounds, and other targets described herein. Examples of detectable and / or obtainable labels include various radioactive groups, enzymes, precursor groups, fluorescent labels, luminescent labels, bioluminescent labels, metal particles, protein-protein binding pairs, protein-antibody binding pairs, etc. Detectable labels are available from a variety of sources on the market.

[0098] Certain embodiments of the present invention provide detectable and / or retrievable proteins and / or protein tags. Examples of detectable fluorescent proteins include, but are not limited to, yellow fluorescent protein (YFP), green fluorescent protein (GFP), cyan fluorescent protein (CFP), podophyllotoxin, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineamine fluorescein, dansyl chloride, phycoerythrin, etc. Examples of detectable bioluminescent proteins include, but are not limited to, luciferase (e.g., bacteria, fireflies, click beetles, etc.), luciferin, photoproteins, etc. Examples of detectable and / or retrievable enzyme systems include, but are not limited to, beta-galactosidase, glucuronidase, phosphatase, peroxidase, cholinesterase, etc.

[0099] Biotin or its derivatives can also be used as detectable and / or retrievable labels, which can then be combined with detectably labeled avidin / streptavidin derivatives (e.g., phycoerythrin-conjugated streptavidin) or labeled anti-biotin antibodies. Digoxin can be expressed for subsequent binding of labeled anti-digoxin antibodies (e.g., fluoresceinized anti-digoxin). Either member of the conjugate pair can generally be polymerized into a detection oligonucleotide, as long as a detectable labeled conjugated ligand can be combined to allow detection. The term "antibody" as used herein refers to any class of antibody molecules, or any subfragment thereof, such as an antigen binding fragment (Fab).

[0100] Other tags suitable for detection and / or retrieval include one or more protein tags.The term "protein tag" as used herein refers to a heterologous polypeptide sequence linked to a polymerase of the invention. Protein tags include, but are not limited to, Avi tag (GLNDIFEAQKIEWHE) (SEQ ID NO: 2), calmodulin tag (KRRWKKNFIAVSAANRFKKISSSGAL) (SEQ ID NO: 3), FLAG tag (DYKDDDDK) (SEQ ID NO: 4), HA tag (YPYDVPDYA) (SEQ ID NO: 5), His tag (HHHHHH) (SEQ ID NO: 6), Myc tag (EQKLISEEDL) (SEQ ID NO: 7), S tag (KETAAAKFERQHMDS) (SEQ ID NO: 8), SBP tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP) (SEQ ID NO: 9), Soffag 1 (SLAELLNAGLGGS) (SEQ ID NO: 10), Softag 3 (TQDPSRVG) (SEQ ID NO: 11), V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 12), NO: 12), Xpress tag (DLYDDDDK) (SEQ ID NO: 13), Isopep tag (TDKDMTITFTNKKDAE) (SEQ ID NO: 14), Spy tag (AHIVMVDAYKPTK) (SEQ ID NO: 15), Strep-tag II (Strep-tag II: WSHPQFEK) (SEQ ID NO: 16), etc.

[0101] The method for detecting and / or retrieving depends on the specific detectable label used in the microorganism. In certain exemplary embodiments, a microscope, a spectrophotometer, a tube luminometer or a plate luminometer, an X-ray film, a magnetic field, a scintillator, a fluorescence activated cell sorting (FACS) device, a chromatographic device, a microfluidic device, a microbead-based screening device or other similar equipment can be used to select, screen and / or retrieve microorganisms.

[0102] The terms and symbols of nucleic acid chemistry, biochemistry, genetics and molecular biology used in this article follow the standard monographs and textbooks in the field, such as Komberg and Baker, DNA Replication, 2nd edition (WH Freeman, New York, 1992); Lehninger, Biochemistry, 2nd edition (Worth Publishers, New York, 1975); Strachan and Read, Human Molecular Genetics, 2nd edition (Wiley-Liss, New York, 1999); Oligonucleotides and Analogs: A Practical Approach, edited by Eckstein (Oxford University Press, New York, 1991); Oligonucleotide Synthesis: A Practical Approach, edited by Gait (IRL Press, Oxford, 1984), etc.

[0103] "Complementary" or "substantially complementary" refers to hybridization or base pairing or formation of a double strand between nucleotides or nucleic acids, such as between the two strands of a double-stranded DNA molecule or between an oligonucleotide primer and a primer binding site on a single-stranded nucleic acid. Complementary nucleotides are typically A and T (or A and U), or C and G. Two single-stranded RNA or DNA molecules are said to be substantially complementary when the nucleotides of one strand are matched with at least about 80% (usually at least about 90% to 95%, or from about 98% to 100%) of the nucleotides of the other strand after optimal matching, comparison, and insertion or deletion of appropriate nucleotides, or substantial complementarity occurs when an RNA or DNA strand hybridizes to its complementary strand under selective hybridization conditions. Selective hybridization will usually occur when there is at least about 65% complementarity over a length of at least 14 to 25 nucleotides, preferably at least about 75% complementarity, and more preferably at least about 90% complementarity. See Kanehisa (1984) Nucl. Acids Res. 12:203.

[0104] "Substantially identical" means that two or more sequences (e.g., DNA sequences) or two or more microchips (e.g., between template microchips and / or replica microchips) are about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or about 100% identical to each other.

[0105] "Complex" refers to a combination or aggregate of direct or indirect contact between molecules. In some embodiments, reference to a molecular complex or specific or specific binding "contact", or more specifically "direct contact", refers to two or more molecules being close enough so that non-covalent interactions such as van der Waals forces, hydrogen bonds, ionic and hydrophobic interactions or similar dominate the interaction between the molecules. In some embodiments, the molecular complex is stable because the thermodynamic stability of the complex under experimental conditions is superior to the stability of its component molecules in a non-aggregated or non-complexed state. "Complex" as used herein refers to a duplex or triplex of a polynucleotide, or a stable aggregate of two or more proteins. In the case of the latter, the complex is formed via antibody specific binding to its corresponding antigen.

[0106] "Double-stranded" refers to at least two oligonucleotides and / or polynucleotides that are completely or partially complementary to each other and produce Watson-Crick type base pairing on most or all nucleotides, thereby forming a stable complex. The terms "annealing" and "hybridization" can be used interchangeably and refer to the formation of stable double strands. In some embodiments, stable double strands mean that the double-stranded structure will not be destroyed under stringent washing (such as conditions including about 5°C lower than the Tm of one strand of the double strand and a low monovalent salt concentration, such as less than 0.2M or less than 0.1M). In terms of double strands, "perfect match" means that when a polynucleotide or oligonucleotide chain forms a double strand, each nucleotide in one chain forms a Watson-Crick base pairing with the corresponding nucleotide in the other chain. The term "double-stranded" includes pairing of nucleoside analogs, such as deoxyxanthine, 2-aminopurine base nucleosides, PNA (peptide nucleic acid), etc. The "mismatch" of the double strand between two oligonucleotides or polynucleotides refers to a pair of nucleotides in the double strand that fail to form a Watson-Crick type pairing bond.

[0107] "Genetic locus" or "site" refers to a continuous sub-region or fragment of a genome. As used herein, a genetic locus or site can refer to the position of a nucleotide, gene or gene fragment in a genome, including mitochondrial DNA, or can refer to any continuous genomic sequence portion, whether or not it is within a gene or associated with a gene. In some embodiments, a genetic locus refers to any genomic sequence portion, including mitochondrial DNA, from a single nucleotide to a fragment of hundreds of nucleotides (e.g., 100-300) in length. A specific genetic locus can usually be identified by its nucleotide sequence or one or both of the nucleotide sequences in the adjacent or flanking regions on one or both sides. In other embodiments, a genetic locus refers to the expressed nucleic acid product of a gene, such as an RNA molecule or a complementary DNA copy.

[0108] "Hybridization" refers to the process of non-covalent binding of two single-stranded polynucleotides to form a stable double-stranded polynucleotide. The term "hybridization" can also refer to triple-stranded hybridization. The resulting (commonly) double-stranded polynucleotide is a "hybrid" or "double-stranded". "Hybridization conditions" generally include a salt concentration of no more than 1M, more often no more than 500mM, and more usually no more than 200mM. The hybridization temperature can be as low as 5°C, but is generally greater than 22°C, more generally greater than 30°C, and often greater than 37°C. Hybridization is generally performed under stringent conditions, i.e., the conditions under which the probe hybridizes with its target sequence. Stringent conditions are sequence-dependent and different in different situations. Longer fragments may require higher hybridization temperatures for specific hybridization. Because other factors may also affect the stringency of hybridization, including the base composition and length of the complementary chains, the presence of organic solvents, and the degree of base mismatching, the combination of parameters is more important than any single absolute condition. Typically stringent conditions are about 5°C lower than the Tm of a specific sequence at a specific ionic strength and pH. Exemplary stringent conditions include a sodium ion concentration of 0.01-1 M (or other salts) at a pH of 7.0 to 8.3 and a temperature of at least 25° C. For example, conditions of 5×SSPE (750 mM sodium chloride, 50 mM sodium phosphate, 5 mM ethylenediaminetetraacetic acid, pH 7.4) and a temperature of 25-30° C. are suitable for allele-specific probe hybridization. For stringent conditions, see, for example, Sambrook, Fritsche and Maniatis, Molecular Cloning A Laboratory Manual, 2nd edition, Cold Spring Harbor Press (1989) and Anderson, Nucleic Acid Hybridization, 1st edition, BIOS Scientific Publishers Limited (1999). "Specific hybridization" or "specific hybridization" or similar expressions refer to when one or more specific nucleic acid sequences are present in a complex mixture (such as total cell) DNA or RNA, a molecule completely or only partially binds, double-strands or hybridizes to this or those specific nucleic acid sequences under stringent conditions.

[0109] "Kit" refers to any delivery system for providing materials or reagents to perform a method or invention. In the article, such a delivery system includes a system that allows the storage, transportation or delivery of reaction reagents (e.g., substrates, enzymes, microchips, etc. in appropriate containers) and / or support materials (e.g., buffers, written instructions for performing the analysis, etc.) from one location to another. For example, the kit includes one or more closures (e.g., boxes) containing relevant reaction reagents and / or support materials for the analysis of the present invention. These contents can be delivered to the intended recipient together or separately. For example, a first container can contain an enzyme for analysis, while a second container contains primers.

[0110] "Ligation" refers to the formation of a covalent bond or connection between the ends of two or more nucleic acids (e.g., oligonucleotides and / or polynucleotides) by a template driven reaction. The nature of the bond or connection may vary widely, and the ligation may be performed enzymatically or chemically. As used herein, ligation is typically performed by an enzymatic reaction to form a phosphodiester linkage between the 5' carbon of the terminal nucleotide of one oligonucleotide and the 3' carbon of another oligonucleotide. The following references describe various template-driven ligation reactions: Whitely et al., U.S. Pat. No. 4,883,750; Letsinger et al., U.S. Pat. No. 5,476,930; Fung et al., U.S. Pat. No. 5,593,826; Kool, U.S. Pat. No. 5,426,180; Landegren et al., U.S. Pat. No. 5,871,921; Xu and Kool (1999) Nucl. Acids Res. 27:875; Higgins et al., Meth. in Enzymol. (1979) 68:50; Engler et al., The Enzymes, 15:3 (1982); and Namsaraev, U.S. Pat. No. 2004 / 0110213.

[0111] "Amplification" includes the process of generating copies of nucleic acid molecules on a chip or nucleic acid molecules bound to microbeads by multiple rounds of primer enzymatic synthesis. "In situ" amplification refers to amplification performed on template nucleic acid molecules placed on a substrate or microbeads, rather than in solution. In situ amplification methods are described in U.S. Patent No. 6,432,360.

[0112] "Support" refers to a solid substrate that is insoluble in water and is used to place the nucleic acid molecules of the nucleic acid chip. The support can be non-porous or porous, rigid or elastic, monolayer or multilayer. "Elastic solid support" refers to a soft support that can adjust its shape under pressure to achieve conformal contact between its surface and another solid surface, and then restore its original shape when the pressure is removed from the support. Typical non-porous supports include, but are not limited to glass, silicon and polydimethylsiloxane. Typical porous supports include, but are not limited to hydrogels, such as linear and cross-linked polyacrylamide gels. Examples of rigid supports include, but are not limited to glass and silicon. Examples of elastic supports include, but are not limited to polydimethylsiloxane, elastic gels and hydrogels. The support can be solid or semi-solid. "Semi-solid" refers to a compressible matrix containing solid and liquid components, wherein the liquid occupies the pores, spaces or other gaps between the solid matrix units. Semi-solid supports can be selected from polyacrylamide, cellulose, polyamide (nylon) and cross-linked agarose, dextran and polyethylene glycol.

[0113] "Random array pattern" or "random" refers to the non-ordered, non-Cartesian distribution of nucleic acid molecules on a support, in other words, there is no orderly arrangement at a predetermined point on the x-axis or y-axis of the grid or a "clock position", degree or radius defined from the center of the radial pattern, and this process is not achieved by purposeful design (or by a program that can achieve the design) or the placement of individual nucleic acid feature points. Such a "random array pattern" or "random" nucleic acid chip can be achieved by dripping, spraying, plating or extending a solution, emulsion, aerosol, steam or dry preparation containing nucleic acid molecules onto a support, allowing the nucleic acid molecules to settle freely on the support without any intervention to guide them to a specific position. The chip of the present invention can be a random array pattern or random.

[0114] "Heterogeneous" refers to a population or collection of nucleic acid molecules of multiple different sequences. According to some embodiments, a heterogeneous oligonucleotide sequence library is provided using a manufacturing method (eg, a microchip).

[0115] "Nucleosides" herein include natural nucleosides, including 2'-deoxy and 2'-hydroxy forms, as described in Komberg and Baker, DNA Replication, 2nd edition (Freeman, San Francisco, 1992). "Analogs" of nucleosides include synthetic nucleosides capable of specific hybridization, having modified base groups and / or modified sugar groups, see Scheit, Nucleotide Analogs (John Wiley, New York, 1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990) or similar literature. Such analogs include synthetic nucleosides designed to enhance binding properties, reduce complexity, increase specificity, etc. Polynucleotides containing analogs having enhanced hybridization or nuclease resistance properties are described in Uhlman and Peyman (supra); Crooke et al., Exp. Opin. Ther. Patents, 6: 855-870 (1996); Mesmaeker et al., Current Opinion in Structural Biology, 5: 343-355 (1995), or similar references. Exemplary types of polynucleotides that can increase double-stranded stability include oligonucleotide phosphoramidates (hereinafter referred to as "amides"), peptide nucleic acids (hereinafter referred to as "PNAs"), oligo-2'-O-alkyl ribonucleotides, polynucleotides containing C-5 allyl pyrimidine, locked nucleotides (LNAs), and the like. Such oligonucleotides can be synthesized by methods described in the literature or obtained from commercial sources.

[0116] As used herein, "nucleic acid molecule", "nucleic acid sequence", "nucleic acid fragment", "oligonucleotide", "oligonucleotide fragment" and "polynucleotide" are used interchangeably and are intended to include, but are not limited to, nucleotides of various lengths in polymeric form, which may be deoxynucleotides or ribonucleotides or their analogs. Nucleic acid molecules include single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), single-stranded RNA (ssRNA) and double-stranded RNA (dsRNA). Different nucleic acid molecules can have different three-dimensional structures and can perform various functions, whether known or not. Non-limiting examples of nucleic acid molecules include genes, gene fragments, genomic gaps, exons, introns, intergenic DNA (including but not limited to heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, small interfering RNA (siRNA), miRNA, small nucleolar RNA (snoRNA), complementary DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from a specific sequence, RNA isolated from a specific sequence, nucleic acid probes and primers. Nucleic acid molecules used in the methods described herein can comprise natural nucleic acid sequences and variants thereof, artificial nucleic acid sequences, or combinations of these sequences.

[0117] Oligonucleotide sequence refers to a linear polymer of natural or modified nucleotide monomers connected by phosphodiester bonds or their analogs. The term "oligonucleotide" generally refers to a shorter polymer, for example, comprising about 3 to about 100 monomers, and the term "polynucleotide" generally refers to a longer polymer, for example, comprising from 100 to tens of thousands of monomers, for example 10,000 monomers or more. "Oligonucleotide fragment" refers to an oligonucleotide sequence that has been split into two or more smaller oligonucleotide sequences. The length of the oligonucleotide containing a probe or primer is generally between 12 and 60 nucleotides, more commonly between 18 and 40 nucleotides. Oligonucleotides and polynucleotides can be natural or synthetic. Oligonucleotides and polynucleotides include deoxynucleotides, ribonucleotides and their non-natural analogs (such as benzoyl forms), peptide nucleic acids (PNAs), etc., provided that they can be specifically bound to the target genome by a monomer-to-monomer interaction mode, such as Watson-Crick type base pairing, base stacking, Hoostein or anti-Hoostein type base pairing or other similar means.

[0118] Nucleoside monomers are usually linked by phosphodiester bonds. Unless otherwise indicated, when an oligonucleotide is represented by a letter sequence (e.g., "ATGCCTG"), it is understood to be in 5' to 3' order from left to right, and "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents deoxythymidine, and "U" represents ribonucleoside / uridine. Typically, oligonucleotides contain four natural deoxynucleotides, but they may also contain ribonucleosides or non-natural nucleotide analogs. It is obvious to those skilled in the art that oligonucleotides containing natural or non-natural nucleotides can be used in this discussion. For example, when it comes to enzyme treatment, oligonucleotides composed entirely of natural nucleotides are generally required. Likewise, when an enzyme has a specific oligonucleotide or polynucleotide requirement for a substrate (e.g., single-stranded DNA, RNA / DNA duplex, or the like), the selection of an oligonucleotide or polynucleotide substrate of appropriate composition is within the knowledge of a practitioner with common sense, especially with the guidance of a monograph such as Sambrook et al., Molecula r Cloning, 2nd edition (Cold Spring Harbor Laboratory, New York, 1989) or other monographs. Oligonucleotides and polynucleotides can be single-stranded or double-stranded.

[0119] The nucleic acid molecule may comprise one or more non-standard nucleotides, nucleotide analogs or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, S2T, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylflocculin, inosine, N6-isopentenyladenine, 1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine, 2-methyladenine, 2-methylguanosine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanosine, 5-methylaminomethyl Uracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylfloccin, 5′-methoxycarboxymethyl uracil, 5-methoxyuracil, 2-methylthio-D46-isopentenyladenine, uracil-5-carboxylic acid (v), floccin oxygen, pseudouracil, floccin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-carboxylic acid methyl ester, uracil-5-carboxylic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, 2,6-diaminopurine, etc. Nucleic acid molecules can also be modified at the base groups (eg, at one or more atoms that can form hydrogen bonds with complementary nucleotides, and / or atoms that cannot form hydrogen bonds with complementary nucleotides), sugar groups, or the phosphate backbone.

[0120] In some embodiments, large polynucleotides are provided. In some embodiments, separation techniques that maximize the length of polynucleotides (e.g., DNA molecules) are used. For example, polynucleotides can be obtained by in situ cleavage or deproteinization (e.g., with EDTA, detergents, proteases, or any of their compositions or similar protocols) after agarose embedding (as routinely performed in pulsed field gel electrophoresis).

[0121] Nucleic acid molecules can be isolated from natural sources or purchased from commercial sources. Oligonucleotide sequences (e.g., coding) can also be prepared by any suitable method, such as the standard phosphoamino method (Beaucage and Carruthers ((1981) Tetrahedron Lett. 22: 1859); or according to the phosphite triester method of Matteucci et al. ((1981) J. Am. Chem. Soc. 103: 3185)); or by other chemical methods using commercial automated oligonucleotide synthesizers or known high-throughput high-density chip methods (see U.S. Pat. Nos. 5,602,244, 5,574,146, 5,554,744, 5,428,148, 5,264,566, 5,141,813, 5,959,463, 4,861,571 and 4,659,774, which are incorporated herein by reference in their entirety). Presynthesized oligonucleotides are also available commercially from a variety of suppliers.

[0122] Nucleic acid molecules can be obtained from one or more biological samples. "Biological samples" used herein can be single cells or multiple cells. Biological samples can contain a single cell type or a combination of two or more cell types. Biological samples also include cell collections that perform similar functions, such as cells found in tissues. Therefore, certain embodiments of the present invention are directed to biological samples containing one or more tissues. Tissues used herein include, but are not limited to, epithelial tissues (e.g., skin, inner layers of glands, intestines, skin, and organs such as liver, lungs, and kidneys), endothelium (e.g., inner layers of blood vessels and lymphatic vessels), mesothelium (e.g., inner layers of pleura, peritoneum, and pericardial cavity), interstitium (e.g., cells that fill the gaps between organs, including fat, muscle, bone, cartilage, and tendon cells), blood cells (e.g., red blood cells and white blood cells), neurons, germ cells (e.g., sperm, oocytes), amniotic fluid cells, placenta, stem cells, etc. Tissue samples include microscopic samples and macroscopic samples.

[0123] In certain embodiments, nucleic acid sequences derived or obtained from one or more organisms are provided. The term "organism" as used herein includes, but is not limited to, humans, non-human primates, cattle, horses, sheep, goats, pigs, dogs, cats, rabbits, mice, rats, gerbils, frogs, toads, fish (e.g., zebrafish), nematodes (e.g., Caenorhabditis elegans), and any transgenic species thereof. The term "organism" also includes, but is not limited to, yeast (e.g., Saccharomyces cerevisiae) cells, yeast tetrads, yeast colonies, bacteria, bacterial colonies, viral particles, virus-like particles, and / or cultures thereof, and the like.

[0124] Isolation, extraction or derivatization of nucleic acid sequences can be performed by any suitable method. Isolation of nucleic acid sequences from a biological sample generally involves treating the biological sample in a manner such that the nucleic acid sequences in the sample are extracted and available for analysis. Any separation method that results in the extraction of nucleic acid sequences can be used in the practice of the present invention. It is common knowledge to select a particular method for extracting nucleic acid sequences based on the characteristics of the source.

[0125] Methods for DNA extraction are well known in the art. The classical DNA isolation protocol is based on extraction with an organic solvent (e.g., a mixture of phenol and chloroform) followed by ethanol precipitation (J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 1989, 2nd ed., Cold Spring Harbor Laboratory Press: New York, NY). Other methods include: salting-out DNA extraction (Sunnucks et al., Genetics, 1996, 144: 747-756; SM Aljanabi and I. Martinez Nucl. Acids Res. 1997, 25: 4692-4693); trimethylammonium bromide DNA extraction (Gustincich et al., BioTechniques, 1991, 11: 298-302); and guanidine thiocyanate DNA extraction (Hammond et al., Biochemistry, 1996, 240: 298-300). A variety of kits are available for extracting DNA from biological samples, such as those available from BD Biosciences Clontech (Palo Alto, California), Epicentre Technologies (Madison, Wisconsin), Gentra Systems, Inc. (Minneapolis, Minnesota), MicroProbe Corp. (Bothell, Washington), Organon Teknika (Durham, North Carolina), and Qiagen Inc. (Valencia, California).

[0126] The method of RNA extraction is also well known in the art (see, for example, J. Sambrook et al., Molecula r Cloning: A Laboratory Manual, 1989, 2nd edition, Cold Spring Harbor Laboratory Press: New York, NY), and there are some kits for extracting RNA from body fluids on the market, such as the corresponding products of the following companies: Ambion, Inc. (Austin, Texas), Amersham Biosciences (Piscataway, New Jersey), BD Biosciences Clontech (Palo Alto, California), BioRad Laboratories (Hercules, California), Dynal Biotech Inc. (Lake Success, New York), Epicentre Technologies (Madison, Wisconsin), Gentra Systems, Inc. (Minneapolis, Minnesota), GIBCO BRL (Gaithersburg, Maryland), Invitrogen Life Technologies (Carlsbad, California), MicroProbe Corp. (Bothell, Washington), Organon Teknika (Durham, NC), Promega, Inc (Madison, WI), and Qiagen Inc (Valencia, CA).

[0127] "Polymorphism" or "genetic variation" refers to the presence of one or more nucleotide substitutions, inversions, insertions or deletions at a genetic locus, or the transfer of DNA from one genetic locus to another genetic locus. In some embodiments, polymorphism refers to one of a variety of alternative nucleotide sequences that may be present at an individual genetic locus, relative to other sequences at the locus of another individual in the same individual or a population, the sequence comprises substitutions, insertions or deletions of nucleotides. An individual may be homozygous or heterozygous at a genetic locus, i.e., an individual may have the same nucleotide sequence in two alleles, or may have different nucleotide sequences in each allele, respectively. In some embodiments, an insertion or deletion at a genetic locus refers to an increase or deletion of 1 to 10 nucleotides at this locus compared to another individual in a population (or another allele of the same individual). Usually an insertion or deletion is relative to the major allele of a locus in a population (e.g., an allele present in a population at a frequency of not less than 50 percent).

[0128] "Primer" includes a natural or synthetic oligonucleotide, which can serve as a starting point for nucleic acid synthesis by forming a double strand with a polynucleotide template, and can extend from its 3' end along the template to form an extended double strand. The nucleotide sequence added during the extension process is determined by the sequence of the template polynucleotide. Primers are usually extended by DNA polymerase. The length of the primer is usually between 3 and 36 nucleotides, and can also be between 5 and 24 nucleotides, or from 14 to 36 nucleotides. Primers in the present disclosure include orthogonal primers, amplification primers, construction primers, etc. Primer pairs can be flanked by a sequence or a group of sequences. The sequences of primers and probes can be degenerate. Primers within the scope of this article are bound to target sequences (e.g., oligonucleotide fragments, coding sequences, etc.).

[0129] "Specific" or "specificity" refers to when one molecule binds to another molecule, such as when an amplification primer or sequencing primer binds to a coding sequence, the two molecules recognize, contact and form a stable complex, and at the same time, the recognition, contact or complex formation of this molecule with other molecules is significantly less. In some embodiments, the "specificity" of the binding of a first molecule to a second molecule means that when the first molecule recognizes other molecules in a reaction or sample and forms a complex with them, the maximum number of complexes is formed with the second molecule. In some texts, the maximum number means at least fifty percent of the total. Molecules involved in specific binding events usually have regions on their surfaces or cavities that can lead to specific recognition between binding molecules. Examples of specific binding include antibody-antigen interactions, enzyme-substrate interactions, the formation of double or triple chains between polynucleotides and / or oligonucleotides, receptor-ligand interactions, etc. "Contact" in specific or specific binding herein refers to the close enough proximity between the two molecules so that weak non-covalent chemical interactions, such as van der Waals forces, hydrogen bonds, base stacking interactions, ionic and hydrophobic interactions, etc., dominate the intermolecular interactions.

[0130] "Spectrally resolvable" means that the fluorescence emission bands of multiple fluorescent labels are sufficiently different, i.e., sufficiently non-overlapping, that the molecular tags bound by these labels can be distinguished by the fluorescence signals generated by the corresponding labels through a standard photodetection system (e.g., a system containing bandwidth filters and photomultiplier tubes or similar systems, such as the system described in U.S. Patent No. 4,230,558 or similar patents, or see Wheeless et al., Flow Cytometry: Instrumentation and Data Analysis (Academic Press, New York, 1985) pp. 21-76). In some embodiments, spectrally resolvable organic dyes (such as fluorescein, rhodamine, etc.) refer to wavelength emission maxima that are at least 20 nm apart, and in other texts at least 40 nm apart. In other embodiments, spectrally resolvable chelated lanthanide compounds, quantum dots, etc. refer to wavelength emission maxima that are at least 10 nm apart, or further at least 15 nm apart.

[0131] "Tm" refers to "melting temperature". Melting temperature refers to the temperature at which half of a population of double-stranded nucleic acid molecules dissociate into single strands. Several equations for calculating the Tm of nucleic acids are well known in the art. As discussed in standard references, a simple estimate of the Tm of nucleic acids in 1M sodium chloride aqueous solution can be calculated by the equation Tm = 81.5 + 0.41 (% G + C) (see, for example, Anderson and Young in "Nucleic Acid Hybridization" on "Quantitative Filter Hybridization", 1985). Other references (e.g., Allawi, HT and Santa Lucia, J., Jr. Biochemistry 36, 10581-94 (1997)) provide alternative calculation methods that take into account structural, environmental and sequence characteristics when calculating Tm.

[0132] The present disclosure is further illustrated by the following examples, which should not be construed as additional limitations. The figures, tables and all references, patents and published patent applications cited herein are expressly incorporated herein by reference for all purposes.

[0133] In addition, the present disclosure may employ conventional molecular biology, microbiology, and recombinant DNA techniques, which are within the skill of the skilled person. These techniques are explained in detail in the literature. See, e.g., Green and Sambrook, Molecula r Cloning: A Laboratory Manual, 4th edition (2012), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York; DN Glover, ed., DNA Cloning: A Practical Approach, Volumes I and II (1985); MJ Gait, ed., Oligonucleotide Synthesis (1984); BD Hames and SJ Higgins, eds., Nucleic Acid Hybridization (1985); BD Hames and SJ Higgins, eds., Transcription And Translation (1984); RI Freshney, ed., Animal Cell Culture (1986); IRL Press, Immobilized Cells And Enzymes (1986); B. Perbal, A Practical Guide To Molecula rCloning (1984); FM Ausubel et al., ed., Current Protocols in Molecula rBiology, John Wiley & Sons, Inc. (1994).

[0134] The contents of all articles, patents, patent applications, and other documents and electronically available information mentioned or cited herein are hereby incorporated by reference to the same extent as if each individual publication was individually and specifically indicated to be cited. Applicants reserve the right to physically incorporate into this application all material and information from such articles, patents, patent applications, or other documents, both physical and electronic, into this application.

[0135] Although the present invention has been described by specific embodiments, it will be appreciated by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention. It will be readily apparent to those skilled in the art that appropriate modifications and adjustments to the methods described herein may be made with appropriate equivalents without departing from the scope of the disclosed embodiments. In addition, many modifications may be made to accommodate specific circumstances, materials, combinations of substances, processes, process steps, to achieve the goals, spirit and scope of the present invention. All of these modifications are intended to be included within the scope of the appended claims. Certain embodiments have now been described in detail, and these embodiments will be more clearly understood with reference to the following examples, which are intended to be illustrative only and not restrictive. Examples

[0136] The following examples are intended to provide a person of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions described in the claims, and are not intended to limit the scope of what the inventors consider to be their invention. Although every effort has been made to ensure the accuracy of the numbers used (e.g., amounts, temperatures, etc.), experimental errors and deviations should be considered. Unless otherwise indicated, parts are by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Example 1. DNA chip replication with submicron resolution and amplification

[0137] This example describes a stamping method for large-scale fabrication of polony gel microarrays, where each ~1-micron clonal DNA cluster carries a unique sequence. By combining enzymatic DNA replication between hydrogel surfaces and bridge amplification of the replicated DNA, this method enables reproducible replication of spatially arrayed sequences on gels with submicron resolution. Gel stamping is achieved with a simple robotic arm and readily available reagents. Compared to methods for fabricating similar DNA cluster microarrays that require sequencing of each chip, our cost and time are reduced by at least 35-fold and 7-fold, respectively. This amplifiable replication method can be widely used to replicate and amplify DNA from many types of DNA microarrays onto hydrogel surfaces. Polony gels provide high-resolution and high-sensitivity tissue mapping assays, particularly for chip-based spatial transcriptomics and spatial proteomics applications. In addition, they can reduce the cost and increase the throughput and resolution of other DNA microarray-based applications, such as gene expression analysis, transcription factor binding analysis, and genotyping. Experimental setup

[0138] Materials and methods

[0139] Polony template construction

[0140] DNA templates with 24 random sequences and a total length of 370 base pairs (bp) were synthesized by Integrated DNA Technologies (IDT). They were PCR amplified for 15 cycles (Taq2× master mix; New England Biolabs (NEB), M0270) with bridge PCR primers (BA(+) and BA(-), Table 1) and size-selected by 2% agarose gel. The purified DNA was quantified by QUBIT 4 fluorometer (Thermo Fisher Scientific), diluted to 1 nM, and aliquoted for stamp gel preparation or stored at -20°C.

[0141] Template sequence - see Fig.15 .

[0142] Table 1. Oligonucleotides used in this study "r" before the base indicates a RNA base; “+” indicates locked nucleic acid (LNA); "5'PS" indicates a 5' phosphorothioate modification. Gel Casting

[0143] Polony gel was prepared by tape casting on a 75×25 mm 2 The slides were cleaned by ultrasonic cleaning with 5% CONTRAD 70, 0.5 M sodium hydroxide solution, 0.1 N hydrochloric acid solution and Milli-Q water, then air dried in an AirClean PCR hood and coated with Bind-silane (Sigma M6514) as described previously (Reference 17). 2A tape with a center hole (Grainer Carton Sealing Tape, 1.6 mil) was pasted on each glass slide to form a gel flow channel. The casting of cross-linked polyacrylamide was carried out in an anaerobic box (Coy Lab) using the same method as described in reference 17. Briefly, the gel runner solution contained 8% acrylamide / bisacrylamide (w / v, 19:2; Sigma-Aldrich, A9099 and M7279), 16 mg / mL N-(5-bromoacetamidopentyl) acrylamide (Combi-blocks, HD-8626), 0.015% (w / v) ammonium thiopersulfate (freshly prepared; Sigma-Aldrich, A3678) and 0.025% (v / v) N, N, N', N'tetramethylethylenediamine (Thermo Fisher Scientific, 15524-010), which was mixed and immediately pipetted into the gel runner trough covered with a 60×24 mm cover glass (Corning, 2940-246). About 40 μL of runner solution was usually used to cast a 40-μm thick gel. The gel was polymerized at room temperature (RT) for 2 hours in an anaerobic chamber and then transferred to a PCR hood. After removing the cover glass, the slide coated with gel was assembled into a modified fluidic tank (BioSurface Technologies, FC 81-PC) with a central fluidic channel measuring 55 × 9 × 0.3 mm 3 The volume is about 150 μL. The gel prepared on the round cover glass is used to sequence the code on the gel. A cut 8×40 mm 2 The glass slide was placed on the cover glass to form a uniform liquid layer between the cover glass and the top glass slide, and then polymerized in an oxygen-free chamber at room temperature for 90 minutes, and then condensed into a gel less than 5-μm thick. After removing the top glass, the cover glass coated with gel was assembled into an FCS2 fluid cell (Bioptechs) and a 0.2-mm thick gasket was used to form an 8×35 mm 2 , a channel volume of approximately 60 μL. Primer grafting

[0144] The fluid reservoir was rinsed with 1 mL of MILLI-Q water and 500 μL of grafting buffer (10 mM potassium phosphate buffer, pH 7), and then 25 μM of 5′ phosphorothioate-modified primers (PS-BA(+) and PS-BA(-); Table 1) in the grafting buffer were injected and incubated at 50° C. for 1 hour for grafting. The fluid reservoir was then washed with 500 μL of hybridization buffer (5×SSC, 0.05% TWEEN-20; Invitrogen, AM9763 and Sigma-Aldrich, P9416) to remove unhybridized primers and stored at 4° C. or used in the next step. Stamp gel preparation

[0145] When seeding the DNA template on the grafted primer gel, 1 nM of the template was denatured in freshly diluted 0.2 N sodium hydroxide (Sigma-Aldrich, 72068), neutralized with 200 mM Tris-HCl solution, pH 7, and then diluted to a working concentration of 8-12 pM with ice-cold hybridization buffer, resulting in a density of approximately 600,000 to 800,000 features per square millimeter. 500 μL of the diluted template was injected into the grafted fluid reservoir, incubated at 75°C for 2 minutes on a heating / cooling block (CPACHT 2-TEC, Inheco), air-cooled to 40°C to complete the hybridization of the template to the gel, and then washed with 500 μL of amplification buffer (2 M betaine, 20 mM Tris-HCl (pH 8.8), 10 mM ammonium sulfate (Sigma-Aldrich A4418), 2 mM magnesium sulfate (Sigma-Aldrich, M2773), 0.1% (v / v) Triton-X-100 (Sigma-Aldrich T8787), and 1.3% (v / v) DMSO (Sigma-Aldrich, D8418)) to remove unhybridized template. The method for synthesizing the first-strand DNA anchored on the gel is as follows: 150 μL of Taq DNA polymerase mixture (50 U / mL Taq DNA polymerase (NEB M0267) and 200 μM of each dNTPs (GenScript C01582) mixed in amplification buffer) was injected into the fluid tank, incubated at 74°C for 5 minutes, then cooled to 60°C, and then bridge amplification was performed. Polony amplification was performed using an automated fluidic device with a P625 pump set (Instech Laboratories) on a HT 2-TEC heating / cooling block set at 60°C for 22 amplification cycles, each cycle including: i) denaturation: pumping in 500 μL of deionized formamide (Emd millipore, 4670) and staying for 1 minute; ii) annealing: pumping in 500 μL of amplification buffer and staying for 2 seconds; iii) extension: pumping in 500 μL of Bst DNA polymerase mix (80 U / mL Bst DNA polymerase (NEB, M0275 or laboratory purified Bst of equivalent performance) and each 200 MM dNTPs mixed in amplification buffer) and staying for 1 minute. The flow rate was 3 mL / min. After amplification, the fluid cell was rinsed with 500 μL 1× CUTSMART buffer (NEB), and then 150 μL of a mixture of 100 U / mL USER (NEB M5505) in 1× CUTSMART buffer was injected and incubated at 37° C. for 1 hour to complete the linearization of the double-stranded DNA.The gel with linearized DNA was stored in 100% formamide and washed with 500 μL elution buffer (1× SSC and 70% formamide (v / v)) before printing. Gel Imprinting

[0146] The slide with the grafted primers is used as a replica gel. The slide with the stamp gel is cut into stamps of specific sizes (e.g., 7 × 7 mm) using a glass cutter. 2 The stamp was fixed to the φ8 mm flat surface of the stamp fixture on a desktop 4-axis robot arm (DOBOT, MG400) by double-sided tape (3M, 468MP), and the positioning repeatability of the robot arm was 0.05 mm. Each stamping cycle consisted of seven steps, involving placing the stamp at four locations (AD) ( Figure 2A and Figure 2B ): 1) Immerse the stamp in formamide at 60°C at position A; 2) Move the stamp from position A to position B and wash the stamp with 60°C imprinting buffer (2M betaine, 20mM Tris-HCl (pH 8.8), 10mM ammonium sulfate, 2mM magnesium sulfate, 0.1% (v / v) Triton-X-100, and 1.3% (v / v) DMSO) for 10 seconds; 3) Move the stamp from position B to position C and immerse the stamp in 4°C imprinting mixture (100U / mL Taq DNA polymerase and 200μM dNTPs each mixed in imprinting buffer) for 30 seconds; 4) Move the stamp from position C to position D and place the stamp on the designated replica gel position at 95°C and apply an imprinting pressure of 10 to 30kPa for 1 minute. The replica gel was pre-incubated in the imprinting mixture at 95°C before imprinting. The pressure is monitored by an electronic scale (e.g., a weight of about 50 to 150 grams is applied to a surface of about 7 x 7 mm. 2 5) Lower the temperature of the replica gel to 60°C at position D and keep it for 1 minute; 6) Increase the temperature of the replica gel to 95°C at position D and add 2 mL of formamide to soak the stamp and replica gel for 3 minutes to dissociate double-stranded DNA; 7) Move the stamp from position D to position A to prepare for the next cycle. At the same time, wash the replica gel with 3×1 mL of MILLI-Q water to prepare for the next position on it. A 55×9mm 2 Six 7×7 mm replica gels can be printed on 2 The chip is then placed in position D for imprinting. Post-processing of the imprinted gel

[0147] After stamping, the replica gel was assembled into the fluid cell and 22 cycles of bridge amplification were performed as described in the preparation of stamp gels. To expose the 3′ poly(T) probe for RNA capture, the fluid cell was first washed with 500 μL 1× CUTSMART buffer (NEB), then injected with 160 μL of 2,000 U / mL Taql (NEB, R0149) CUTSMART mix and incubated at 60°C for 1.5 hours. The Taql-treated polony gel was stored in 100% formamide and washed with 500 μL elution buffer (1× SSC and 70% formamide (v / v)) before Pixel-seq analysis or polony sequencing. Pixel-seq experiments Tissue preparation

[0148] Mice were anesthetized with pentobarbital (0.2 mL, Merck) and then decapitated. The brains were rapidly dissected out, frozen on crushed dry ice, and stored at -80°C until cryosectioning. Transcription capture and complementary DNA synthesis

[0149] The polony gel slide was removed from the fluid tank, washed with MILLI-Q water and 3×200 μL wash buffer 1 (0.1×SSC and 0.4×MAXIMA H Minus RT buffer (Thermo Fisher, EP0753)) and dried in a PCR hood for use. For frozen section preparation, the frozen tissue block was placed in a Cryostat NX70 (Thermo Scientific), equilibrated at -20°C for 15 minutes, mounted on a holder with OCT (Fisher Healthcare, 4585) and cut into 10 μm thick sections. The tissue section was immediately placed on the imprinted chip position on the dried coal gel surface, 50 μL tissue hybridization buffer (6×SSC and 2U / μL RNAseOUT (Thermo Fisher, 10777019)) was gently dripped, and then incubated at room temperature for 15 minutes. After hybridization, the buffer was removed with a pipette, and the polony gel slide was assembled into a multi-well reaction chamber (PROPLATE; GraceBio-Labs, 246868). Complementary DNA synthesis was completed by adding 100 μL of reverse transcription (RT) mixture (5 μL MAXIMA H-reverse transcriptase (Thermo Fisher, EP0753), 20 μL 5× MAXIMA RT buffer, 20 μL 20% Ficoll PM-400 (Sigma-Aldrich, F4375), 10 μL of each dNTP 10 mM, 5 μL 50 μM template switching oligonucleotide (Qiagen, 339414YCO0076714), 2.5 μL RNAseOUT (40 U / μL) and 37.5 μL water) to each well and incubating at 42°C for 1 hour. The conditions for the Cy5-dCTP-labeled complementary DNA experiment were the same, except that the dNTPs were replaced by 500 μM each of dATP / dGTP / dTTP, 12.5 μM dCTP, and 25 μM Cy5-dCTP (PerkinElmer, NEL577001EA). Tissue cleaning

[0150] After complementary DNA synthesis, the reaction buffer was removed and the tissue was washed with 3×200 μL 0.1×SSC. 100 μL proteinase K digestion solution (10 μL proteinase K (Qiagen, 19131) and 90 μL PKD buffer (Qiagen, 1034963)) was added to each reaction chamber and incubated at 55°C for 30 minutes. In order to remove digested proteins, genomic DNA, etc., each reaction chamber was washed with 3×200 μL elution buffer 2 (2×SSC and 0.1% SDS) and 3×200 μL washing solution 2 (0.1×SSC). Sequencing library construction

[0151] Complementary DNA with spatial encoding is recovered from the gel by double-strand synthesis and primer extension, and unique molecular identifiers (UMIs) are introduced. For example, 70 μL of double-strand mixture (7 μL of 10× isothermal amplification buffer (NEB, B0357), 7 μL of each dNTP 10mM mixture, 3.5 μL of 10 μM TSO primer, 0.5 μL of 20 mg / mL BSA (NEB, B9000), 3 μL of BST2.0WARMSTART DNA polymerase (NEB, M0538) and 49 μL of water) are added to each reaction chamber, and the reaction chamber is sealed with a sealing film. After incubation at 65°C for 15 minutes, the reagents are removed and the gel is washed with 3×200 μL of 0.1×SSC. To elute the DNA, 35 μL of 0.05 M potassium hydroxide denaturation and elution mixture was added to each reaction chamber, incubated at room temperature for 10 minutes, and neutralized with 5 μL Tris (1 M, pH 7.0). Approximately 35 μL of sample was transferred from each reaction chamber to a test tube, and 65 μL of UMI introduction mixture (50 μL 2×Q5 Ultra II master mix (NEB, M0544), 2.5 μL 10 μM UMI primer (Table 1), and 12.5 μL water) was added. UMI introduction was completed by denaturation at 95°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 5 minutes in a PCR machine. Unbound primers were removed by incubating 1 μL of thermosensitive exonuclease I (20 U; NEB, M0568) at 37°C for 4 minutes, and the exonuclease was inactivated by incubation at 80°C for 1 minute. To amplify the complementary DNA library, 2 μL of 10 μM TSO, 2 μL TruSeq LibP5 sequencing primer (Table 1) and 1 μL Q5 HotStart polymerase (NEB M0493) were added to the tube for PCR amplification, and 5-10 ng DNA was obtained for each sample. The PCR reaction was as follows: annealing at 95°C for 3 minutes; 4 amplification cycles, each including 98°C for 20 seconds, 65°C for 45 seconds, and 72°C for 3 minutes; 8 amplification cycles, each including 98°C for 20 seconds, 67°C for 20 seconds, and 72°C for 3 minutes; and a final incubation at 72°C for 5 minutes. Complementary DNA was amplified from a mouse olfactory bulb section using 12 PCR cycles. After amplification, approximately 1 ng of DNA was usually used for sequencing library construction, which was performed using the NexteraXT kit (Illumina FC-131-1024) and TruSeq LibP5 primers and Nextera index primers (Table 1) according to the manufacturer's protocol. Polony sequencing

[0152] To determine the coding sequence and distribution, gel stamping was performed between replicate gels on a stamp and a circular coverslip, similar to the method described previously. The coverslip was assembled into an FCS2 fluid cell and polony sequencing was performed using the HiSeq SBS kit v4 (Illumina, FC-401-4002). Images were captured using a Nikon Ti-E automated inverted fluorescence microscope (equipped with a perfect focus system, Nikon CFl60 Plan Fluor 40× / 1.3-NA oil immersion objective, linear encoding motorized stage (Nikon Ti-S-ER), and Andor iXon Ultra 888EMCCD camera (16-bit dynamic range, 1024×1024 array, 13 μm pixels)). The four-channel imaging system included two laser lines (Laser Quantum GEM 532 nm (500 mW) and Melles Griot 85-RCA-400 660 nm (400 mWS)) and two filters with emission filters (610 / 60-730 / 60 or 555 / 40-685 / 20; Chroma Technology) and 532 / 660 polarizer (Chroma Technology). Sequencing reagents were added to the fluid reservoir via a fluidic system including multi-position micro-motorized valves (Valco Instruments EMH2CA) and multi-channel syringe pumps (Kloehn V6 12K). An application was built in Java 1.6 using jSerialComm (website: fadecast.github.io / jSerialComm / ) to control the fluidic system and control Micro-Manager v1.4.22 (website: micro-manager.org) to acquire images from selected positions to automate the sequencing process. Sequencing was performed using the reagents provided by the HiSeq SBS kit according to the standard HiSeq sequencing protocol. Each sequencing cycle included: i) pre-cutting wash with cutting buffer; ii) removal of dye and protecting group with cutting mixture; iii) post-cutting wash with high salt buffer; iv) pre-polymerization wash with polymerization buffer; v) matching polymerization with polymerization mixture; vi) image acquisition in scanning mixture. Template diffusion analysis

[0153] To evaluate template diffusion during RNA capture, a mouse olfactory bulb (OB) slice was placed on a dry coal gel pre-soaked with a nuclear staining cocktail (0.1× SSC, 2.5× SYTOX Green, 0.4× RT buffer), and the SYTOX-stained nuclei were immediately imaged using the FITC channel (Ex488 nm / Em520 nm) of a polony sequencing fluorescence microscope. Cy5-labeled complementary DNA was synthesized on the same tissue slice following the above experimental procedures and then imaged in the Cy5 channel (Ex640 nm / Em665 nm). The nuclear image was used as a reference for analyzing template diffusion in the Cy5 image. Ten regions (250×250 pixels, 1 pixel = 0.65 μm) were randomly selected from both images using the MATLAB built-in function imregister to determine the transformation matrix and scaling factor. Data analysis Polony Image Analysis

[0154] To compare polony imprints on different replicate gels, all polony images were registered to the image with the highest signal-to-noise ratio using the im regcorr function in MATLAB. Polony was detected by local thresholding and its intensity, size, and centroid were measured using the regionprops function. A consensus map was constructed using polony with normalized intensity > 0.1 and detected in at least two of the three replicate gel images. The polony center offset was calculated as the Euclidean distance between the detected polony and the consensus. Base calling in Polony sequencing

[0155] The intensities of the raw sequencing tiff images were processed and extracted using a custom-built Dlight tool in MATLAB. All images were registered to the merged image of the first sequencing cycle Cy3 and Cy5 channels using im regcorr. Next, images from the first eight sequencing cycles (referred to as template cycles) were used to generate a polony reference map. Polony sites were identified by searching for a regional signal threshold (> median + 2 × standard deviation) in all template cycle images, and then the polony centroid with the best purity value was found. Dlight parameters were optimized using the PhiX control library (Illumina FC-110-3001). The intensity values ​​of polony centroids and image pixels were analyzed by the 3Dec base calling program (36) and allowed correction of signal crosstalk between adjacent polony sites. Unassigned image pixels were compared to polony centroids with a distance of less than 5 pixels to match the spatial code to find polony boundaries. These pixels were assigned to adjacent identified matching spatial codes with a signal correlation coefficient higher than 0.7. All sequencing gel positions were merged into a single image to construct the final spatial code map. Transcriptional mapping

[0156] After complementary DNA library sequencing, FASTQ files were processed to map transcripts onto the spatial encoding map. The spatial encoding and UMI sequences were first extracted by Flexbar (37). The indexed sequences were mapped back to the spatial encoding map using Bowtie (38) with up to 2 mismatches allowed. Paired-end reads of the mapped indexes were aligned to the mouse transcriptome (GRCm38) using STARv2.7.0 (39) with default settings. Sequencing reads with the same transcriptome mapping site, UMI, and spatial encoding were collapsed into unique records for subsequent analysis. result

[0157] Polony gels enable amplifiable DNA imprints and demonstrate continuous feature point distribution

[0158] The choice of cross-linked polyacrylamide as the stamp gel and the replica gel allows for conformal contact between the template and the replica DNA under low pressure, and bridge amplification of the template and the replica DNA. Unlike previous methods of generating polonies embedded in gels (16, 17), the polonies in this paper are amplified on the surface of the gel, which can promote DNA replication between gels. To automate the stamping process, a benchtop device with a robotic arm to position the stamp, a thermocycler to control the gel temperature, a digital balance to monitor the stamping pressure, and a fluidic system to amplify the DNA ( Figure 2A and Figure 2B ). Gels of different thicknesses attached to glass coverslips and slides of different sizes were compared: with increasing gel thickness (e.g., 40 to 100 μm; Figure 3A), the DNA replication efficiency between large-area gels is improved. To test the repeatability and stability, 50 cycles of imprinting were performed continuously. The distribution pattern of characteristic points on the replicated gels showed consistency ( Figure 3B ) and is stable under different imprint pressures ( Fig. 9A -9C).

[0159] High density polonies (≥600,000 / mm 2 ) typically form a continuous DNA distribution with very small gaps between feature points, which is different from the discrete, peak-like distribution of dots produced by amplification in Illumina non-arrayed fluidic tanks ( Figure 4 , left). One explanation for this difference is that the polonys on the gel surface may be less constrained by the gel during bridge amplification, resulting in faster size expansion. Such polonys are obviously susceptible to restriction digestion, and 93.6% of the double-stranded DNA was digested by Taql to expose the 3' poly-T probe. For spatial transcriptomics analysis, continuously distributed poly-T probes can minimize uneven tissue RNA capture on the chip. Although the polonys are continuous, they rarely penetrate each other due to the repulsive effect between polony (31), and their boundaries can be resolved by sequencing gel ( Figure 4 Because polonies vary in size and shape, a base calling pipeline was developed to determine the exact location of each pixel (0.325 × 0.325 μm) in the gel image in order to maximize feature resolution. 2 ) to construct a spatial coding map ( Figure 4 (right side).

[0160] Efficient replication of polony gels requires bridge amplification of the replicated DNA after stamping, which increases DNA density and compensates for inefficient replication of certain gel regions. However, excessive amplification may cause polony size expansion and introduce errors into spatial encoding, which affect resolution and accuracy, respectively. To evaluate this issue, replicate gels made in consecutive stamping experiments were quantitatively compared by analyzing the pattern of feature point arrays in multiple gel regions. Single gels were compared to a consensus feature map constructed using aligned images of three replicate gels. Repeated stamping was stable, with only <15% of feature points lost after 50 stamps, which is likely caused by slow loss of template from the stamp ( Figure 5A ). The matching polonys on the gel were extracted and their centers were measured for offset from the consensus center. The offset showed a normal distribution, with 93.1% and 65.7% of the center distances being less than 1 and 0.5 μm, respectively ( Figure 5B). By sequencing a 24-base pair spatial code and allowing a maximum of two mismatched bases, 93.43±0.04% of the matching polonys in the two gels had the same spatial code ( Figure 5C ). The amplified polony contained ultra-dense capture probes, with an average of 20,337 template copies per polony after 35 amplification cycles ( Figure 5D and Figures 10A-10B ), which is about 9 times more than the clusters prepared by the Illumina method (18). Limited by the aforementioned sequencing imaging equipment, the gels that can be reliably produced so far have 600,000 to 800,000 feature points passing through the filter per square millimeter, with an average diameter of 1.07 to 0.906 μm ( Figure 5E ). It is possible to create gels with smaller and denser features at higher resolution, because even more crowded polonies still show clear boundaries ( Fig.11 ), but sequencing them requires improved imaging resolution. Polony gel-based spatial transcriptomics shows high resolution and sensitivity

[0161] Pixel-seq was developed to convert the 1-μm feature resolution of gels to single-cell resolution for mapping complex tissues such as the brain. Figure 6 ). To test analytical conditions and compare performance, we analyzed the mouse olfactory bulb (OB). The OB is a layered structure of morphologically diverse cells that is often used to validate spatial transcriptomic analyses (13, 14, 23, 24, 26). We investigated two issues that commonly limit single-cell resolution in DNA chip-based analytical approaches: lateral diffusion of RNA between cells and intercellular mixing of RNA in multiple layers found even in thin tissue sections. Even without tissue fixation, polony gel-based RNA capture produced strong complementary DNA signals, clearly delineated the boundaries of neuronal cell bodies, and had minimal template drift ( Figures 12A-12C ), indicating that the gel substrate can greatly restrict the lateral diffusion of RNA on its surface. In addition, when frozen sections were placed on the dry coal gel, the gel apparently captured tissue RNA only from a single layer of cells. The Cy5-labeled complementary DNA signal produced by the captured mRNA only coincided with the position of the cell nuclei in the gel contact layer, but not in the deep cell nuclei ( Figure 7 The selective RNA capture can be explained by the rapid occupation of the gel surface by the contacting RNA as soon as the tissue sections wet the gel ( Fig.13 ). Gel-based capture not only improves resolution but also facilitates rapid preparation of complementary DNA sequencing libraries (about 6 hours; Fig.14 ).

[0162] To evaluate performance, 10-μm coronal sections of the olfactory bulb were analyzed to obtain a spatially resolved transcriptome. 2 In the olfactory bulb sections of the 2017 study, approximately 83% of the raw reads were mapped to the ENCODE map, resulting in approximately 82.5 million UMIs, with a density range of 1 to 678 UMIs per ENCODE. The UMI density map shows the continuous, pixel-resolution, multi-layer organization of the cell bodies ( Fig. 8A ). Approximately 23,000 unique genes were detected with more than 10 UMIs in at least one of the three replicates, and the data showed high correlation (R ≥ 0.968). Although the above methods only captured RNA from the cell layer in contact with the gel rather than the entire 10-μm slice, Pixel-seq provided capture efficiency similar to other top-performing methods ( Figure 8B and Table 2); for example, at 80% sequencing depth, Pixel-seq at 2×2 and 10×10 μm in the whole olfactory bulb 2 The median UMIs detected were 47 and 977 in the basal regions, and 83 and 1,695 in densely packed cell body regions such as the mitotic cell layer (MCL). Replicate DNA cluster chips from Illumina NovaSeq fluidics cells onto polony gels

[0163] Polony gel imprinting can replicate DNA chips not only between two gel surfaces, but also between a rigid surface and an elastic gel. The rigid surface can even have a chemically or photolithographically etched micro-well or nano-well structure. Because the elasticity of the gel allows it to conformally contact the inner surface of the micro-well or nano-well structure to replicate DNA. The DNA template in the Illumina NovaSeq fluid tank is efficiently replicated on the polony gel, and the concentric markers match perfectly ( Fig.16 ). Replication of arrayed DNA clusters to polony gels not only improves the feature point resolution of polony gels (from 1 μm to 0.65 μm or less), but also provides RNA capture efficiency far higher than that of nanowell sequencing fluidics. DNA probes in nanowells have lower molecular density and low tissue RNA accessibility.

[0164] Table 2. Comparison of chip-based spatial transcriptomics (ST) methods Table 2 continued a: Percentages for Visium, DBiT-Seq, Slide-seqV2, HDST, and Stereo-seq were calculated based on reported feature size, density, and spatial array distribution. Percentages for Seq-scope were measured using cluster image analysis of the flow cell hybridized at the highest reported template concentration (100 mM). b :PAA, polyacrylamide c : Different methods use different RNA capture conditions, and some may capture RNA from multiple cell layers throughout the tissue section. d :TSO (Template Switching Oligonucleotide), MDA (Multiple Site Displacement Amplification), T7 aRNA (T7 RNA polymerase-based amplification). Different amplification methods have different yields. MDA amplifies complementary DNA fragments, which usually has a higher yield than PCR amplification of full-length complementary DNA. e : UMI counts are obtained from the original publication. Feature point gaps are usually not considered in the calculation. discuss

[0165] This disclosure demonstrates the scalable, rapid, and low-cost fabrication of 1-micron resolution clonal DNA cluster chips. 2 The consumables cost for chips with an area of ​​≥30 million unique feature points is reduced to about US$3 (Table 3), and the time is shortened to about 6 hours.

[0166] Table 3. List of consumables for making polony gels.

[0167] The amplifiable imprinting method is suitable for replicating other chips. Some spot deposition, in situ synthesis or self-assembled chips (such as DNA-coated microbeads, DNA nanospheres and non-arrayed and arrayed DNA clusters in Illumina sequencing fluid tanks) can be used as stamps, and the templates of these chips can be replicated on the hydrogel surface, and the replicated DNA increases the DNA density through bridge amplification. The gel with replicated DNA can be used as a stamp to make more copies.

[0168] The sequential stamping method can increase the coverage ratio and density of chip feature points. Use multiple different stamps to stamp in sequence at the same replicate gel position to achieve a higher chip coverage ratio and feature point density, which cannot be analyzed by second-generation sequencing based on fluorescence imaging due to the limitation of optical resolution. In fact, all imaging-based sequencing methods, such as the Illumina second-generation sequencing platform, have a maximum feature point density limit (for example, 1 million clusters per square millimeter). Sequential stamping can exceed this limit.

[0169] In addition to being compatible with electroblotting transfer, hydrogel substrates are superior to other solid surface substrates (e.g., glass, silica gel, and polydimethylsiloxane) not only because of their ability to limit template diffusion and enhance capture efficiency, but also because of their compatibility with electrophoresis.

[0170] The hydrogel substrate can be combined with electrophoresis-assisted capture (or electrophoretic transfer similar to protein blotting) to improve spatial resolution and capture efficiency ( Fig.17 ). In situ capture of tissue analytes based on DNA chips usually relies on the non-directional migration of tissue analytes to the chip probes. Non-directional migration leads to lateral diffusion, which prevents the analytical method from achieving high spatial resolution (such as single cell or subcellular resolution) and increases the capture loss of analytes in deep tissues. The DNA imprinting method in this article can generate a replica chip on a polyacrylamide gel, and the upper and lower surfaces of the gel are exposed to a conductive medium, allowing ions to migrate in and out. Therefore, an electric field perpendicular to the upper surface of the gel can be applied to drive the analytes in the sample to migrate to the upper surface of the gel. The small pore size of the gel prevents the analytes from migrating further into the gel. The target analyte (such as a nucleic acid target) is hybridized and captured by the probe sequence in the chip on the gel surface.

[0171] DNA chips made by polony gel imprinting are more suitable for local area selective analysis of DNA captured on the chip. A major cost component of DNA chip-based spatial encoding combined with next-generation sequencing of encoded complementary DNA is DNA sequencing. The sequencing cost is linearly related to tissue size and RNA detection sensitivity. Many users are only interested in specific tissue regions and cell types, and specifically expect to sequence only the encoded complementary DNA of the selected tissue region.

[0172] One approach is to photocleave a photodissociable spacer that is incorporated into the bridge primer at a selected gel region, thereby releasing the complementary DNA encoding that region ( Fig.18 ). Multiple rounds of fluorescence imaging sequencing of DNA clusters in gels may cause DNA loss. The gel imprinting method avoids light exposure, thereby protecting the photosensitive primers in the gel.

[0173] Another approach is to mechanically cut the gel region of interest, since gel is an ideal soft material for mechanical cutting ( Fig.19 ). A microcutter device can be designed with a cutting head for cutting small gel regions, a tube that can apply vacuum and pressure to transfer the cut gel fragments to a container, and a computer-controlled XYZ stage for sampling multiple gel regions.

[0174] In addition to polyacrylamide gels, other elastic materials can also be used as substrates for imprinting, such as other hydrogels and polydimethylsiloxane with surfaces modified to allow bridge amplification.

[0175] Polony gels can be used for spatial omics analysis to detect RNA, protein, genomic DNA, and small molecule analytes. In this work, polony gels were used only for spatial transcriptomics, but they can be applied to detect proteins and epigenetic groups modified by DNA-labeled antibodies (32, 33) or other affibodies (e.g., nanobodies, monomers, and computationally designed binding proteins), enzyme (e.g., Tn5)-fragmented genomic DNA, and possibly small molecule analytes detected by innovative affinity reagents (34).

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Claims

1. A method for replicating a template microchip, comprising: o providing a template microchip composed of a plurality of nucleic acid feature points randomly or in an array, each feature point comprising a plurality of identical single-stranded DNA sequences; o the template microchip contacts the replica microchip in the presence of a polymerase and nucleotides to synthesize a complementary DNA sequence on the replica microchip; ο removing the template microchip; o amplifying the complementary DNA sequence synthesized on the replica microchip to form a plurality of nucleic acid feature points, the nucleic acid feature points on the replica microchip and the corresponding feature points on the template microchip having substantially the same sequence and distribution; o Repeat the above replication process using the same template microchip and multiple replicated microchips to manufacture multiple replicated microchips.

2. According to claim 1, the template microchip comprises a solid phase support having an upper surface with a plurality of nucleic acid feature points on the upper surface.

3. According to claim 2, wherein the solid support is: ο non-porous substrate or porous substrate; ο Rigid substrate or elastic substrate; o a single-layer substrate or a multi-layer substrate, a solid support allowing nucleic acid sequences to be covalently or non-covalently attached to its surface; o The substrate has a smooth surface, or the substrate surface has a micro-well or nano-well structure generated by chemical or photoetching; the nucleic acid sequence is attached to the smooth surface or the inner surface of the micro-well or nano-well.

4. According to claim 2, the nucleic acid feature spots in the template microchip are selected from cloned DNA clusters, DNA nanospheres, DNA coated microbeads, DNA spots deposited or synthesized by spotting methods, and DNA spots synthesized by photoconductive synthesis methods.

5. according to claim 2, wherein a plurality of nucleic acid feature points constitute a random chip or an array chip.

6. According to claim 2, the synthesis of single-stranded DNA sequences in the template microchip can be performed by one of the following methods: chemical synthesis, enzymatic synthesis, template-dependent synthesis, template-independent synthesis, synthesis of double-stranded DNA sequences and then cleavage of one or both strands of the double-stranded DNA.

7. According to claim 6, wherein the cutting is performed using: o one or more enzyme reagents selected from uracil-specific excision enzyme, 8-oxoguanine DNA glycosidase and restriction enzyme; o One or more chemical reagents selected from oxidizing agents and reducing agents.

8. According to claim 7, wherein the oxidizing agent is selected from periodate and lead tetraacetate, and the reducing agent is selected from phosphine, dithiothreitol, dithioerythritol and L-glutathione.

9. According to claim 1, wherein the replica microchip comprises an elastic solid phase support having an upper surface with a plurality of nucleic acid feature points on the upper surface.

10. According to claim 9, wherein the elastic solid support is: ο non-porous substrate or porous substrate; o Single-layer substrate or multi-layer substrate; wherein the flexible solid phase support allows nucleic acid primer sequences to be covalently attached to its surface, facilitating conformal contact between two microarrays and solid phase clonal DNA amplification on the support.

11. According to claim 9, a plurality of nucleic acid primers are mixed and coated on the upper surface of the elastic solid support uniformly or in an array distribution manner.

12. According to claim 1, wherein in the contacting step, part or all of the single-stranded DNA sequence in each characteristic point in the template microchip is hybridized with the primer sequence in the replica microchip to synthesize a complementary DNA sequence as a template.

13. According to claim 1, wherein before removing the template microchip, the complementary DNA sequence synthesized on the replication microchip is dissociated from the single-stranded DNA sequence on the template microchip by thermal and / or chemical denaturation.

14. According to claim 1, wherein the amplification step is solid phase clonal DNA amplification, optionally bridge amplification.

15. According to claim 1, wherein the same template microchip is sequentially contacted with a plurality of replica microchips to produce a plurality of replica microchips having substantially the same nucleic acid sequence and distribution of characteristic points.

16. According to claim 1, wherein the replicated microchip can be used as a template microchip for another microchip replication process.

17. According to claim 1, wherein a plurality of contacting steps are performed sequentially to replicate a plurality of different template microchips onto a replicated microchip, thereby increasing the feature point density and DNA coverage, and reducing the feature point size on the replicated microchip.

18. According to claim 1, wherein the nucleic acid sequence comprises a single or multiple probe sequences for hybridizing with complementary sequences in target nucleic acid molecules in a sample.

19. According to claim 18, the target nucleic acid molecules are retained in the sliced ​​tissues with the original cells and tissues positions unchanged; or the target nucleic acid molecules are released from the homogenized cells or tissues when the cell membrane is broken.

20. The method according to claim 18, wherein the target nucleic acid molecule is an endogenous nucleic acid in a cell, or an artificially synthesized nucleic acid marker covalently or non-covalently attached to an affinity molecule or polymerized to an endogenous nucleic acid sequence.

21. according to claim 20, wherein affinity molecule is selected from binding protein, antibody, antigen-binding fragment of antibody, nanobody, monomer and nucleic acid aptamer.

22. According to claim 21, wherein the affinity molecule is used to detect one or any combination of proteins, DNA, RNA, small molecule ligands, protein modifications, DNA modifications, RNA modifications, small molecule ligand modifications and molecular complexes present in a sample.

23. According to claim 18, wherein different probe sequences are present in different characteristic spots, whereby the probe sequences are associated with their positions in the microchip.

24. According to claim 18, all feature points in the microchip are provided with the same probe or a plurality of different probes.

25. According to claim 24, in addition to the probe sequence, the nucleic acid sequence of the feature point also includes a coding sequence associated with its position in the microchip.

26. According to claim 25, the probe and / or spatial coding sequence is known or unknown during the construction of the microchip and can be subsequently determined by DNA sequencing or hybridization.

27. According to claim 18, wherein the built-in probe sequence in the DNA is exposed at the 3' end by a nucleic acid sequence-specific cleavage method including but not limited to restriction endonuclease digestion.

28. According to claim 1, after the microchip is replicated, one or more probe sequences are added to the nucleic acid sequence not containing the probe sequence by enzymatic methods (DNA ligation or primer extension can be selected).

29. According to claim 1, the probes and / or spatial coding sequences corresponding to the characteristic points in the microchip copies replicated from the same template microchip are basically the same, and can be determined by the sequences of the corresponding characteristic points in the template microchip, or by sequencing one or a small number of replicated microchips.

30. According to claim 1, wherein the template microchip and the replica microchip are used to analyze gene expression and genetic variation of single nucleotide polymorphisms in a sample.

31. According to claim 1, wherein the template microchip and the replica microchip are used to analyze the binding of transcription factors to the probe sequences.

32. According to claim 1, the microchip is used to spatially encode nucleic acids, proteins and / or small molecule ligands in tissue sections placed thereon by combining the coding sequence with complementary DNA labeled with endogenous nucleic acids synthesized in situ or artificially synthesized nucleic acids.

33. According to claim 1, wherein the spatially encoded microchip is used to analyze molecular interactions between proteins, nucleic acids and / or small molecule ligands in tissue section samples or homogenous mixed samples.

34. According to claim 1, wherein the microchip replica is used to amplify natural or synthetic DNA and / or genes on the chip, thereby minimizing distortion caused by the overall polymerase chain reaction (PCR).

35. According to claim 1, wherein the nucleic acid template and primers are covalently attached to the template microchip and the replication microchip to restrict the diffusion of DNA to achieve sub-micron resolution accuracy of feature point replication.

36. According to claim 1, the nucleic acid template covalently attached to the template microchip is minimally lost in each contacting step, so that the template microchip can be reused for multiple replication cycles.

37. A method for replicating a template microchip, comprising: Providing a template microchip including a plurality of cloned DNA clusters, each cluster containing a plurality of identical single-stranded DNA sequences, wherein the DNA clusters are randomly or array-distributed; The template microchip contacts the replica microchip in the presence of a polymerase and nucleotides to synthesize a complementary DNA sequence on the replica microchip; Remove the template microchip; amplifying the complementary DNA sequence synthesized on the replica microchip to form a plurality of DNA clusters having substantially the same sequence and distribution as the corresponding cluster on the template microchip; Repeat the above replication process using the same template microchip and multiple replicate microchips to produce multiple replicate microchips.

38. According to claim 37, wherein the template microchip comprises a gel or a substrate with a chemically or photoetched micro- or nano-well structure, and the replica microchip comprises a gel.

39. According to claim 38, wherein the gel is selected from polyacrylamide gel, hydrogel and polydimethylsiloxane gel.

40. According to claim 39, the cloned DNA clusters in the template microchip and the replica microchip are amplified by a bridge amplification method, and the DNA density in the cluster, the number of DNA copies and the cluster size can be controlled by adjusting the number of amplification cycles.

41. A method for replicating a template microchip, comprising: Provide a template microchip with a polyacrylamide gel with multiple cloned DNA clusters attached to the surface; The template microchip is contacted with a replica microchip in the presence of a polymerase and nucleotides, wherein the replica microchip comprises a polyacrylamide gel with a plurality of primer sequences attached to the surface; Remove the template microchip; amplifying the nucleic acid sequences on the replicate microchip to form a plurality of clonal DNA clusters having substantially the same sequence and distribution as the corresponding clusters on the template microchip; Repeat the above replication process using the same template microchip and multiple replicate microchips to produce multiple replicate microchips.

42. According to claim 41, the polyacrylamide gel comprises a single layer or multiple layers of linear polyacrylamide and / or cross-linked polyacrylamide, and the cross-linking agent can be selected from one or more of N,N'-methylene-bisacrylamide, N,N'-cystathionamide-bisacrylamide and N,N'-diallyl-tartaric acid diamide.

43. A method for producing replicate microchips on gels suitable for electroblotting analysis of tissue sections or homogenized mixed samples, comprising: The method of claim 1 is used to replicate the information of the template microchip onto a replica microchip, wherein the replica microchip is attached to the upper surface of a porous gel, and the upper and lower surfaces of the gel are exposed to a conductive medium, allowing ions to enter and exit; Attach tissue sections or homogenized mixed samples to microchips; Applying an electric field perpendicular to the upper surface of the gel to drive the analytes in the sample to migrate to the upper surface of the gel; Capture of nucleic acid targets on the gel surface by hybridization with probe sequences in the microchip.

44. According to claim 43, wherein the sample is a fresh quick-frozen or formalin-fixed paraffin-embedded (FFPE) tissue section or a homogenous mixture containing one or any combination of nucleic acids, proteins, small molecule ligands and DNA-labeled affinity molecules.

45. The method of claim 43, wherein the transfer of the nucleic acid target is performed by a semi-dry or dry electroblotting device.

46. ​​A method for performing region-selective analysis of nucleic acid targets captured on one or more regions of a microchip, comprising: Replicating the template microchip onto a replica microchip having a photodissociable probe sequence by the method of claim 1; Attach tissue sections or homogenized mixture samples to replicate microchips; Capture nucleic acid targets on the gel surface by hybridizing to probe sequences on the microchip; Use the probe sequence as a primer to synthesize complementary DNA sequences on a microchip; Using light to dissociate the probes in one or more localized areas on the microchip, thereby regioselectively releasing the synthesized complementary DNA sequence; The released complementary DNA is eluted and can be used for DNA amplification, sequencing and / or other analysis.

47. According to claim 46, wherein the probe sequences are attached to the microchip via one or more photodissociable spacers, including but not limited to 1-(2-nitrophenyl)ethyl ester.

48. A method for performing region-selective analysis of nucleic acid targets captured on one or more regions of a microchip, comprising: Replicating the template microchip onto a replica microchip on a gel substrate by the method of claim 1; Attach tissue sections or homogenized mixture samples to replicate microchips; Capture nucleic acid targets on the gel surface by hybridizing to probe sequences on the microchip; Use the probe sequence as a primer to synthesize complementary DNA sequences on a microchip; Mechanically cutting one or more localized gel regions using a micro-cutting device and transferring them to a container, such as a test tube; Analyze the complementary DNA in the excised gel slices by DNA amplification, sequencing, and / or other analytical methods.

49. According to claim 48, wherein the gel substrate is a non-rigid material suitable for mechanical cutting, including but not limited to polyacrylamide gel.

50. According to claim 48, wherein the micro-cutting device has a micro-cutting head for cutting small gel areas; A connection line that can apply vacuum and pressure to the end of the microdissection tip to transfer the cut gel slices to a container (optionally a test tube); and an XYZ stage for sampling different areas of the gel.

Images