Array and method for detecting spatial information of nucleic acids

Abstract

The present application provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, a method for detecting spatial information of nucleic acids in a sample on the basis of the array, and a method for producing the nucleic acid array.

Description

Array and detection method for detecting nucleic acid spatial information Technical Field

[0001] The present application relates to the field of biomolecule spatial detection. Specifically, the present application provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, a method for detecting spatial information of nucleic acids in a sample based on the array, and a method for generating the nucleic acid array. Background Art

[0002] Spatial omics technology is a technology that obtains omics information such as transcriptome, genome, epigenome and proteome in cells in situ in tissues. Spatial omics technology was named the technical method of the year by Nature Methods in 2020, and was listed as one of Nature's seven most noteworthy technologies of the year in 2022. Spatial omics technology can be divided into four categories based on different principles, namely microdissection, in situ hybridization, in situ sequencing, chip in situ capture and high-throughput sequencing. Spatial omics technology based on chip in situ capture and high-throughput sequencing occupies a dominant position due to its advantages such as simple operation and high throughput, among which the capture chip is the key to this technology.

[0003] Chip-based spatial transcriptomics uses probes with spatial information on a chip to capture nucleic acids and other molecules in situ from tissue sections. High-throughput sequencing is then used to obtain spatial and intracellular nucleic acid information on the chip. Algorithmic analysis then restores the molecular information within the tissue. Since Joakim Lundeberg of the Royal Institute of Technology in Sweden first published spatial transcriptomics in Science in 2016, this field has rapidly developed due to its advantages, such as high-throughput whole-transcriptome analysis. Other similar spatial transcriptomics technologies include HDST (high-definition spatial transcriptomics) based on silica magnetic beads, slide-seq (sequencing by oligonucleotide ligation and detection) based on barcoded microbeads, DBiT-seq (deterministic barcoding in tissue) based on microfluidics, Seq-Scope based on the 11μMina sequencing platform, and stereo-seq (spatial enhanced resolution omics sequencing) based on the DNB sequencing platform. Among them, Joakim's spatial transcriptome technology was acquired and commercialized by 10x Genomics at the end of 2018, and the VisiμM product was released in 2019. Based on this technology, 10x Genomics has formed another spatial omics product line in addition to the single-cell production line, including instruments and equipment, analysis software and reagents and consumables. At the same time, Biomark Biotech also released a spatial transcriptome product based on this technology in 2022.

[0004] The core of existing spatial omics technology based on in situ capture on chips lies in the resolution and capture efficiency of the capture chip. The resolution of the chip is determined by the spatial information density on the chip, which is related to whether the cells on the tissue section can be finely analyzed with spatial single-cell or subcellular resolution, while the capture efficiency is determined by the capture information on the chip, which is related to whether all molecular information in the cell can be fully captured without bias. In the existing technology that uses sequencing chips as capture chips, the capture function area and the spatial information area of ​​the chip are on the same molecule of the nucleic acid array. However, due to the influence of the resolution of the sequencing chip, the nucleic acid molecules containing the spatial information area in the nucleic acid array need to be separated by a certain distance before they can be recognized by the sequencer. If the separation distance is too small, each nucleic acid molecule cannot be accurately identified. When the capture function area and the spatial information area are on the same nucleic acid molecule, the resolution is limited, and the capture efficiency is also limited.

[0005] In addition, since existing capture chips are all based on poly T probes to capture mRNA, in order to simultaneously obtain the expression map and spatial information of the transcripts, they can only capture the 3' end transcripts of mRNA, while losing the information of its 5' end transcripts. For the immune repertoire, important molecular information is concentrated at the 5' end, so the existing technology is lacking in capturing the 5' end transcripts.

[0006] Summary of the Invention

[0007] In order to simultaneously achieve high-resolution and high-capture efficiency spatial omics technology, this application splits the capture area and spatial information on the capture chip probe in the existing technology into two independent molecules, namely the positioning probe and the capture probe. The positioning probe is used to provide spatial position information of spatial omics, while the capture probe is used to capture molecules in the cell, thereby getting rid of the limitation that the number of capture probes is affected by spatial density, so as to achieve the goal of improving the capture efficiency of molecules while ensuring high resolution.

[0008] In addition, the nucleic acid array (eg, capture chip) provided in this application can also achieve the acquisition of 5' transcript information.

[0009] Nucleic acid array

[0010] Therefore, in one aspect, the present application provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, comprising a solid support to which one or more capture probes and at least two localization probes are attached;

[0011] The capture probe and the localization probe are each independently connected to the solid support;

[0012] Each localization probe occupies a different position on the solid support;

[0013] The positioning probe comprises a first universal sequence and a positioning sequence, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the positioning probe on the solid support;

[0014] The capture probe contains a capture sequence that is capable of annealing to the nucleic acid molecule to be captured and initiating an extension reaction.

[0015] In certain embodiments, each of said localization probes consists of one or more localization probe molecules.

[0016] In certain embodiments, the localization sequences contained in localization probe molecules belonging to the same type of localization probe are identical to each other, and the localization sequences contained in localization probe molecules belonging to different types of localization probes are different from each other.

[0017] Those skilled in the art will readily understand that one or more localization probe molecules of the same localization probe have the same localization sequence. However, it is not necessary for each localization probe molecule of each localization probe to have the same complete sequence.

[0018] In certain embodiments, the localization probe molecules and the capture probes of the nucleic acid array are separated from each other.

[0019] In certain embodiments, the first universal sequence is located 3' to the localization sequence.

[0020] In certain embodiments, the localization probe further comprises a second universal sequence.

[0021] In certain embodiments, the second universal sequence is located 5' to the localization sequence.

[0022] In certain embodiments, the second universal sequence comprised by different types of localization probes attached to the solid support is the same or different. In certain embodiments, the second universal sequence comprised by different types of localization probes attached to the solid support is the same. In certain embodiments, the second universal sequence comprised by the same type of localization probe attached to the solid support is the same or different.

[0023] In certain embodiments, the first universal sequence comprised by the same type of localization probes attached to the solid support is the same or different.

[0024] In certain embodiments, the localization probe does not comprise or further comprises a molecular identifier (MID) sequence.

[0025] In certain embodiments, the localization probe further comprises a MID sequence.

[0026] In certain embodiments, the MID sequence is located 5' to the first universal sequence, and / or the MID sequence is located 3' to the second universal sequence.

[0027] In certain embodiments, cognate localization probes comprise MID sequences that differ from each other.

[0028] In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

[0029] In certain embodiments, the 3' end of the capture sequence has a free hydroxyl group (-OH).

[0030] In certain embodiments, the capture probe further comprises an immobilization sequence.

[0031] In certain embodiments, the fixed sequences comprised by different capture probes attached to the solid support are the same or different. In certain embodiments, the fixed sequences comprised by different capture probes attached to the solid support are the same.

[0032] In certain embodiments, the immobilization sequence is located 5' to the capture sequence.

[0033] In certain embodiments, the capture probe contains a single-stranded region comprising the capture sequence.

[0034] In certain embodiments, the capture probe exists in single-stranded form or in duplex form containing the single-stranded region.

[0035] In certain embodiments, the capture probe exists in a single-stranded form, and the capture sequence is located at the 3' end of the capture probe; or

[0036] The capture probe exists in the form of a duplex containing a single-stranded region, which includes a first chain directly connected to the solid support and a second chain hybridized with the first chain; and the 3' end of the second chain includes a single-stranded region, and the capture sequence is located at the 3' end of the single-stranded region.

[0037] In certain embodiments, the second strand comprises the capture sequence and the immobilization sequence.

[0038] In certain embodiments, the nucleic acid array has one or more features selected from the group consisting of:

[0039] (1) the positioning sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) random nucleotides, and in certain embodiments, each random nucleotide is independently any one of the deoxyribonucleotides A, C, G, and T;

[0040] (2) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; in certain embodiments, the solid support is a chip; in certain embodiments, the solid support can be used in a sequencing platform; in certain embodiments, the solid support is a sequencing chip (e.g., a high-throughput sequencing chip), such as a sequencing chip (e.g., a high-throughput sequencing chip) used in an Illumina, MGI, or Thermo Fisher sequencing platform;

[0041] (3) The same type of positioning probes (i.e., positioning probes containing the same positioning sequence) occupy the same area on the surface of the solid support, and different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm);

[0042] (4) the first universal sequence is capable of annealing to (i) a nucleic acid molecule captured by the capture sequence, or (ii) a nucleic acid molecule derived from (i);

[0043] (5) the first universal sequences contained in the different types of localization probes connected to the solid support are the same or different;

[0044] (6) The solid support is capable of releasing the localization probe and / or capture probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.).

[0045] In certain embodiments, the localization probe and / or the capture probe is covalently or non-covalently linked to the solid support.

[0046] In certain embodiments, the localization probe and / or the capture probe is covalently linked to the solid support.

[0047] In certain embodiments, the localization probe and the capture probe are each independently linked to the solid support via the same or different click chemistry reactions.

[0048] In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is selected from: alkynyl / azido, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol. In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is azide / alkynyl.

[0049] In certain embodiments, the surface of the solid support is modified with a molecule or group X, the positioning probe is modified with a molecule or group Y, the molecule or group X is capable of undergoing a click chemistry reaction with the molecule or group Y, and the positioning probe is connected to the solid support through the click chemistry reaction between the molecule or group X and the molecule or group Y; and / or, the surface of the solid support is modified with the molecule or group X', the capture probe is modified with a molecule or group Y', the molecule or group X' is capable of undergoing a click chemistry reaction with the molecule or group Y', and the capture probe is connected to the solid support through the click chemistry reaction between the molecule or group X' and the molecule or group Y'.

[0050] In certain embodiments, the pair of molecules or groups XY and X'-Y' is each independently selected from the group consisting of: alkynyl / azide, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, alkene / amine, alkene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone.

[0051] In certain embodiments, the molecule or group X and / or X' is an azide group, and the molecule or group Y and / or Y' is

[0052] In certain embodiments, the surface of the solid support is modified with an azide group, and the localization probe and / or the capture probe is modified with The positioning probe and / or the capture probe is connected to the solid support through a click chemistry reaction between the azide group and the DBCO.

[0053] It is easy for those skilled in the art to understand that any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the positioning probe in performing its function (for example, the function of annealing with the target sequence and / or initiating an extension reaction at the 3' end). In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is capable of initiating an extension reaction. In this case, any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the positioning probe to the target sequence and / or the 3' end initiating an extension reaction. In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is closed. In this case, any part of the positioning probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the positioning probe to the target sequence. In certain embodiments, the positioning sequence of the positioning probe is not directly connected to the solid support. In certain embodiments, the first universal sequence of the positioning probe is not directly connected to the solid support. In certain embodiments, neither the first universal sequence nor the localization sequence of the localization probe is directly attached to the solid support. In certain embodiments, the localization probe is attached to the solid support via the second universal sequence. In certain embodiments, the localization probe is attached to the solid support via the 5' end (e.g., the 5' terminus).

[0054] It is easy for those skilled in the art to understand that any part of the capture probe can be used to connect to the solid support, as long as the connection does not affect the function of the capture probe (for example, annealing with the target sequence and the function of the 3' end initiation extension reaction). In certain embodiments, the capture probe exists in a single-stranded form. In this case, any part of the capture probe can be used to connect to the solid support, as long as the connection does not affect the annealing of the capture probe to the target sequence and the function of the 3' end initiation extension reaction. In certain embodiments, the capture sequence of the capture probe is not directly connected to the solid support. In certain embodiments, the capture probe is connected to the solid support via its 5' end (for example, the 5' end).

[0055] In certain embodiments, the capture probe exists in the form of a duplex containing a single-stranded region, and the duplex comprises a first chain directly connected to the solid support and a second chain hybridized to the first chain. In this case, any portion of the first chain can be used to connect to the solid support, as long as the connection does not affect the function of the first chain (e.g., the function of annealing to the second chain). In certain embodiments, the first chain is connected to the solid support by a portion that is not annealed to the second chain. In certain embodiments, the first chain is connected to the solid support by its end (e.g., 5' end or 3' end). In certain embodiments, the second chain is not directly connected to the solid support.

[0056] In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, the nucleic acid molecule annealed to the capture probe, or the nucleic acid molecule obtained by extending the capture probe through nucleic acid polymerization reaction) can contact the positioning probe adjacent to it.

[0057] In certain embodiments, there are at least 100 capture probes adjacent to each of the positioning probes, for example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, or at least 10000 capture probes.

[0058] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each localization probe.

[0059] In certain embodiments, the expression "adjacent to each of the positioning probes" refers to an area on the surface of a solid support near the positioning probe, wherein nucleic acid molecules connected to capture probes distributed in the area (e.g., nucleic acid molecules annealed to the capture probes, or nucleic acid molecules obtained by extending the capture probes by nucleic acid polymerization) are able to contact the positioning probes.

[0060] In certain embodiments, at least 100 capture probes are distributed within a radius of 200 nm to 300 nm around the center of the position occupied by each positioning probe on the solid support (e.g., the center of the area occupied by each positioning probe on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 of the capture probes.

[0061] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed within a radius of 200 nm-300 nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

[0062] As used herein, the expression "the area occupied by each of the positioning probes on the surface of the solid support" or similar expressions has the same meaning as "the position occupied by each of the positioning probes on the solid support" and can be used interchangeably.

[0063] In certain embodiments, the nucleic acid array comprises one or more of the capture probes.

[0064] In certain embodiments, different species of capture probes contain different capture sequences.

[0065] In certain embodiments, the nucleic acid molecule to be captured is RNA (e.g., mRNA), and the capture sequence of the capture probe contains a poly (dT) sequence or a random oligonucleotide sequence;

[0066] The nucleic acid molecule to be captured is a target nucleic acid (e.g., a target DNA and / or RNA) or a nucleic acid molecule derived from the target nucleic acid, the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid, and the capture sequence of the capture probe comprises a sequence that can specifically anneal to the target nucleotide sequence; or

[0067] The nucleic acid molecules to be captured are RNA (e.g., mRNA) and target nucleic acids (e.g., target target DNA and / or RNA), and the nucleic acid array comprises a first capture probe for capturing RNA (e.g., mRNA) and a second capture probe for capturing the target nucleic acid or a nucleic acid molecule derived from the target nucleic acid, wherein the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has a target target nucleotide sequence contained in the target nucleic acid; wherein the capture sequence of the first capture probe contains a poly (dT) sequence or a random oligonucleotide sequence, and the capture sequence of the second capture probe contains a sequence that can specifically anneal to the target target nucleotide sequence.

[0068] It is readily understood by those skilled in the art that, unless otherwise specified herein or clearly contradicted by the context, the above definition / description of the capture probe also applies to the first capture probe and the second capture probe.

[0069] In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and / or abundance of the nucleic acid.

[0070] In certain embodiments, the first universal sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

[0071] In certain embodiments, the second universal sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

[0072] In certain embodiments, the fixed sequence is a nucleotide sequence consisting of 5-80 (e.g., 5-20, 5-30, 5-40, 5-50, 5-70, 10-30, 10-50, 10-70, 20-30, 20-50, 20-70, 25-40, 25-50, 25-70) nucleotides.

[0073] In certain embodiments, the capture sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) nucleotides.

[0074] In certain embodiments, the random oligonucleotide sequence is a nucleotide sequence consisting of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) random nucleotides. In certain embodiments, each random nucleotide is independently any one of deoxyribonucleotides A, C, G, and T.

[0075] In certain embodiments, the poly (dT) sequence consists of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) thymidine deoxyribonucleotide residues.

[0076] In certain embodiments, the first universal sequence, the positioning sequence, the second universal sequence, the immobilization sequence, and the capture sequence each independently comprise or do not comprise non-natural nucleotide residues (eg, modified nucleotide residues).

[0077] Nucleic acid array preparation method

[0078] In another aspect, the present application provides a method for preparing the nucleic acid array as described above, comprising the following steps:

[0079] (A) attaching and / or synthesizing a localization probe on a solid support, said localization probe being as defined above; and,

[0080] (B) attaching and / or synthesizing capture probes on a solid support, wherein the capture probes are as defined above;

[0081] The (A) and (B) can be carried out in any order or simultaneously (for example, in the same reaction system).

[0082] In certain embodiments, (A) is performed before (B), and the solid support in (B) is a solid support to which the localization probe has been attached.

[0083] In certain embodiments, (B) is performed before (A), and the solid support in (A) is a solid support to which the capture probe has been attached.

[0084] In certain embodiments, (A) and (B) are performed simultaneously, and the solid phase support in (A) and (B) is the same solid phase support.

[0085] In certain embodiments, step (A) comprises:

[0086] (1) providing: (a) the free positioning probe, wherein the positioning probe is modified with a molecule or group A; and (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B, wherein the molecule or group A is capable of forming a connection (e.g., covalent or non-covalent connection) with the molecule or group B; and

[0087] (2) contacting the positioning probe with the solid support under conditions suitable for forming a connection between the molecule or group A and the molecule or group B, thereby obtaining a solid support to which the positioning probe is connected.

[0088] In certain embodiments, step (A) further comprises step (3): performing bridge amplification on the positioning probes on a solid support to which the positioning probes are attached, thereby obtaining multi-copy clusters of each positioning probe.

[0089] It is readily understood by those skilled in the art that any portion of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the function of the positioning probe (e.g., annealing with the target sequence and / or the function of initiating an extension reaction at the 3' end). In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is capable of initiating an extension reaction. In this case, any portion of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the annealing of the positioning probe to the target sequence and / or the function of initiating an extension reaction at the 3' end. In certain embodiments, the positioning probe is capable of annealing to the target sequence and the 3' end of the positioning probe is blocked. In this case, any portion of the positioning probe can be used to modify the molecule or group A, as long as it does not affect the annealing of the positioning probe to the target sequence. In certain embodiments, the positioning sequence of the positioning probe does not modify the molecule or group A. In certain embodiments, the first universal sequence of the positioning probe does not modify the molecule or group A. In certain embodiments, neither the first universal sequence nor the positioning sequence of the positioning probe modifies the molecule or group A. In certain embodiments, the second universal sequence of the localization probe is modified with the molecule or group A. In certain embodiments, the 5' end (eg, the 5' terminus) of the localization probe is modified with the molecule or group A.

[0090] In certain embodiments, the molecule or group A is capable of undergoing a click chemistry reaction with the molecule or group B.

[0091] In certain embodiments, the molecules or groups A / B are selected from the group consisting of: alkynyl / azide, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, alkene / amine, alkene / thiol, alkyne / thiol, 1,3 / diol-aldehyde, 1,3 / diol-ketone.

[0092] In certain embodiments, the molecules or groups A / B are azide / alkynyl or alkynyl / azide.

[0093] In certain embodiments, the molecule or group A is DBCO and the molecule or group B is azide.

[0094] In certain embodiments, the localization probe is modified with The surface of the solid support is modified with an azide group, and the positioning probe and the solid support are connected through a click chemical reaction between DBCO and the azide group.

[0095] In certain embodiments, step (A) comprises:

[0096] (1) providing at least two vectors, each vector comprising at least one copy of a vector sequence, wherein the vector sequence comprises: a complementary sequence of a positioning sequence and a complementary sequence of a first universal sequence; the first universal sequence and the positioning sequence are as defined above; wherein the complementary sequence of the positioning sequence of each vector sequence is different from each other;

[0097] (2) placing at least two of the carriers on the surface of the solid support;

[0098] (3) providing a fixed primer and performing a nucleic acid polymerization reaction using the vector sequence as a template to generate an extension product, wherein the extension product is a positioning probe; wherein the fixed primer can anneal to the vector sequence and initiate an extension reaction; and,

[0099] (4) connecting the immobilized primer to the surface of the solid support;

[0100] Wherein, steps (3) and (4) are performed in any order (for example, step (3) is performed before or after step (4), or step (3) and step (4) are performed simultaneously).

[0101] In certain embodiments, in step (2), the carrier is placed on the surface of the solid support by forming a bond (eg, a non-covalent bond or a covalent bond) with the surface of the solid support.

[0102] In certain embodiments, the vector sequence comprises, in order from the 5' end to the 3' end: a complementary sequence to the universal sequence, and a complementary sequence to the positioning sequence.

[0103] In certain embodiments, the extension product comprises, in order from 5' to 3', the positioning sequence and the first universal sequence.

[0104] In certain embodiments, each vector is a DNB formed from concatemers of multiple copies of the vector sequence.

[0105] In certain embodiments, the method optionally comprises step (5): digesting the vector sequence, and / or separating (eg, melting) the extension product of step (3) from the vector sequence to which it annealed.

[0106] In certain embodiments, the vector sequence further comprises a cleavage site. In certain embodiments, the cleavage is selected from nicking enzyme cleavage, USER cleavage, photoexcision, chemical excision, or CRISPR excision. In certain embodiments, in step (5), the cleavage site comprised by the vector sequence is cleaved, so that the vector sequence and the extension product of step (3) annealed thereto are separated.

[0107] In certain embodiments, the vector is provided in step (1) by:

[0108] (i) providing a vector template sequence, wherein the vector template sequence comprises a complementary sequence to the vector sequence;

[0109] (ii) performing a nucleic acid amplification reaction using the vector template sequence as a template to obtain an amplification product of the vector template sequence, wherein the amplification product comprises at least one copy of the vector sequence; in certain embodiments, performing rolling circle replication to obtain a DNB formed by concatemers of the vector sequence.

[0110] As used herein, a "DNB" (DNA nanoball) is a typical RCA (rolling circle amplification) product, sharing the characteristics of an RCA product. The RCA product is a multi-copy single-stranded DNA sequence that forms a "spherical" structure due to the interactions between the bases within the DNA sequence. Typically, library molecules are circularized to form single-stranded circular DNA, which is then amplified by multiple orders of magnitude using rolling circle amplification (RCA). The resulting amplification product is called a DNB.

[0111] In certain embodiments, the vector sequence further comprises a complementary sequence to a second universal sequence or a partial sequence thereof (eg, the vector sequence further comprises a complementary sequence to the second universal sequence or a 3' partial sequence thereof), wherein the second universal sequence is as defined above.

[0112] In certain embodiments, the complementary sequence of the second universal sequence or a partial sequence thereof is located 3' to the complementary sequence of the positioning sequence.

[0113] In certain embodiments, the fixed primer comprises the second universal sequence or a partial sequence thereof (eg, the fixed primer comprises the second universal sequence or a partial sequence at the 5' end thereof).

[0114] In certain embodiments, the extension product comprises, in order from 5' to 3' direction: the second universal sequence, the positioning sequence, and the first universal sequence.

[0115] In certain embodiments, the vector sequence does not comprise or further comprises a template sequence for a MID sequence.

[0116] In certain embodiments, the vector sequence further comprises a template sequence for a MID sequence. In certain embodiments, the template sequence for the MID sequence is located at the 3' end of the complementary sequence of the first universal sequence, and / or the template sequence for the MID sequence is located at the 5' end of the complementary sequence of the second universal sequence or a partial sequence thereof. In certain embodiments, the MID sequence consists of 5-50 (e.g., 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35, or 20-45) degenerate deoxyribonucleotide residues.

[0117] In certain embodiments, the immobilized primer is covalently or non-covalently attached to the solid support.

[0118] In certain embodiments, the immobilized primer is covalently linked to the solid support.

[0119] In certain embodiments, the immobilized primer is linked to the solid support via a click chemistry reaction.

[0120] In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is selected from: alkynyl / azido, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol. In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is azide / alkynyl.

[0121] In certain embodiments, the fixed primer is modified with The surface of the solid support is modified with an azide group, and the fixed primer and the solid support are connected through a click chemical reaction between DBCO and the azide group.

[0122] In certain embodiments, the capture probe exists in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

[0123] (1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and

[0124] (2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

[0125] It is readily understood by those skilled in the art that any portion of the capture probe can be used to modify the molecule or group A', as long as it does not affect the function of the capture probe (e.g., annealing with the target sequence and the function of the 3' end initiation extension reaction). In certain embodiments, the capture sequence of the capture probe does not modify the molecule or group A'. In certain embodiments, the 5' end (e.g., the 5' end) of the capture probe is modified with the molecule or group A'.

[0126] In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

[0127] In certain embodiments, the capture probe exists in a duplex form containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

[0128] (I)(1) providing: (a) a free first strand of the capture probe, wherein the first strand is modified with a molecule or group A'; and, (b) the solid support, wherein the surface of the solid support is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent and / or non-covalent connection) with the molecule or group B'; and, (c) the free second strand of the capture probe, wherein the second strand comprises the capture sequence and is capable of annealing to the first strand to form a duplex;

[0129] (2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and,

[0130] (3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe;

[0131] or,

[0132] (II)(1) providing: (a) a duplex formed by annealing a first strand and a second strand of the capture probe, wherein the first strand is linked to a molecule or group A', and the second strand contains the capture sequence; and, (b) a solid support, wherein the surface of the solid support is modified with a molecule or group B', and the molecule or group A' is capable of forming a connection (e.g., covalent and / or non-covalent connection) with the molecule or group B'; and,

[0133] (2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

[0134] In certain embodiments, any portion of the first strand may be used to connect to the solid support, as long as the connection does not affect the first strand's ability to perform its function (e.g., annealing to the second strand). In certain embodiments, the portion of the first strand that is not annealed to the second strand is modified with the molecule or group A'. In certain embodiments, the end (e.g., 5' end or 3' end) of the first strand is modified with the molecule or group A'.

[0135] In certain embodiments, the second strand is not directly attached to the solid support.

[0136] In certain embodiments, in the duplex formed by annealing the first and second strands of the capture probe, the 3' end of the second strand comprises the capture sequence, and the capture sequence does not anneal to the first strand.

[0137] In certain embodiments, the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'.

[0138] In certain embodiments, the molecules or groups A' / B' are selected from the group consisting of: alkynyl / azide, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, alkene / amine, alkene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone.

[0139] In certain embodiments, the molecules or groups A' / B' are azide / alkynyl or alkynyl / azide.

[0140] In certain embodiments, the molecule or group A' is DBCO and the molecule or group B' is azide.

[0141] In certain embodiments, the first strand of the capture probe is modified with The surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemistry reaction between DBCO and the azide group.

[0142] In certain embodiments, the solid support has one or more characteristics selected from the group consisting of:

[0143] (1) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; in certain embodiments, the solid support is a chip;

[0144] (2) The solid support is planar, spherical or porous;

[0145] (3) The solid support can be used in a sequencing platform; in certain embodiments, the solid support is a sequencing chip for an 111 μMina, MGI, or Thermo Fisher sequencing platform; and

[0146] (4) The solid support is capable of releasing the localization probe and / or capture probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.).

[0147] In certain embodiments, the same type of positioning probes (i.e., positioning probes comprising the same positioning sequence) occupy the same area on the surface of the solid support, different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm).

[0148] In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, the nucleic acid molecule annealed to the capture probe, or the nucleic acid molecule obtained by extending the capture probe through nucleic acid polymerization reaction) can contact the positioning probe adjacent to it.

[0149] In certain embodiments, there are at least 100 capture probes adjacent to each of the positioning probes, for example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, or at least 10000 capture probes.

[0150] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each localization probe.

[0151] In certain embodiments, the expression "adjacent to each of the positioning probes" refers to an area on the surface of a solid support near the positioning probe, wherein nucleic acid molecules connected to capture probes distributed in the area (e.g., nucleic acid molecules annealed to the capture probes, or nucleic acid molecules obtained by extending the capture probes by nucleic acid polymerization) are able to contact the positioning probes.

[0152] In certain embodiments, at least 100 capture probes are distributed within a radius of 200 nm to 300 nm around the center of the position occupied by each positioning probe on the solid support (e.g., the center of the area occupied by each positioning probe on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 of the capture probes.

[0153] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed within a radius of 200 nm-300 nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

[0154] Nucleic acid array preparation method

[0155] In another aspect, the present application provides a method for preparing the nucleic acid array as described above, comprising the following steps:

[0156] (A) providing a nucleic acid array to be processed, wherein the nucleic acid array to be processed comprises a solid support to which at least two oligonucleotide molecules are linked;

[0157] Each oligonucleotide molecule occupies a different position on the solid support;

[0158] The oligonucleotide molecule contains a positioning sequence and a first universal sequence from the 5' end to the 3' end, wherein the positioning sequence has a nucleotide sequence corresponding to the position of the oligonucleotide molecule on the solid support; the first universal sequence is as defined above;

[0159] and,

[0160] (B) attaching and / or synthesizing capture probes on the solid support contained in the nucleic acid array to be processed, wherein the capture probes are as defined above.

[0161] In certain embodiments, each oligonucleotide molecule consists of one or more oligonucleotide molecules.

[0162] In certain embodiments, the positioning sequences contained in the same oligonucleotide molecules are identical to each other, and the positioning sequences contained in different oligonucleotide molecules are different from each other.

[0163] It is readily understood by those skilled in the art that one or more oligonucleotide molecules of the same oligonucleotide molecule may have the same positioning sequence, however, it is not necessary for each oligonucleotide molecule to have the same complete sequence.

[0164] In certain embodiments, the oligonucleotide molecule does not comprise a capture sequence at the 3' end of the first universal sequence, the capture sequence being as defined above.

[0165] In certain embodiments, the oligonucleotide molecule comprises a capture sequence at the 3′ end of the first universal sequence, the capture sequence being as defined above, and the method further comprises step (B′): removing the capture sequence contained in the oligonucleotide molecule;

[0166] The steps (B) and (B') can be performed in any order or simultaneously (for example, in the same reaction system).

[0167] In certain embodiments, step (B') removes the capture sequence contained in the oligonucleotide molecule by the steps comprising:

[0168] (i) providing a blocking probe capable of annealing to (a) the first universal sequence or a partial sequence thereof (e.g., a partial sequence at the 3' end of the first universal sequence) of the oligonucleotide molecule, or (b) a sequence located at the 5' end of the capture sequence and at the 3' end of the first universal sequence in the oligonucleotide molecule, or (c) a combination of (a) and (b);

[0169] (ii) contacting the blocking probe with the nucleic acid array to be processed containing the oligonucleotide molecules under conditions suitable for annealing the blocking probe to the oligonucleotide molecules;

[0170] (iii) contacting the product of step (ii) with an exonuclease under conditions that allow the exonuclease to exert its cleavage activity; wherein the exonuclease has an exonuclease activity that exocleaves the 3' to 5' end of a single-stranded nucleic acid.

[0171] In certain embodiments, the exonuclease is Exonuclease I.

[0172] In certain embodiments, the step (B') further comprises the step of removing the blocking probe (eg, removing the blocking probe by unzipping the blocking probe from the oligonucleotide molecule).

[0173] In certain embodiments, in the method, step (B) is performed after step (B').

[0174] In certain embodiments, the oligonucleotide molecule further comprises a second universal sequence, the second universal sequence being as defined above.

[0175] In certain embodiments, the second universal sequence is located 5' to the localization sequence.

[0176] In certain embodiments, the oligonucleotide molecule does not comprise or further comprises a MID sequence.

[0177] In certain embodiments, the oligonucleotide molecule further comprises a MID sequence. In certain embodiments, the MID sequence is located 5' to the first universal sequence, and / or the MID sequence is located 3' to the second universal sequence. In certain embodiments, the MID sequences comprised by the same oligonucleotide molecule are different from each other.

[0178] In certain embodiments, in the nucleic acid array to be processed, the oligonucleotide molecules are covalently or non-covalently linked to the solid support.

[0179] In certain embodiments, the oligonucleotide molecule is covalently linked to the solid support.

[0180] In certain embodiments, the oligonucleotide molecule is linked to the solid support via a click chemistry reaction.

[0181] In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is selected from: alkynyl / azido, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol. In certain embodiments, the molecule or group pair capable of undergoing the click chemistry reaction is azide / alkynyl.

[0182] In certain embodiments, the surface of the solid support is modified with an azide group, and the oligonucleotide molecule is modified with The oligonucleotide molecule is connected to the solid support through a click chemistry reaction between the azide group and the DBCO.

[0183] In certain embodiments, the capture probe exists in a single-stranded form comprising a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

[0184] (1) providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and (b) the nucleic acid array to be treated or the nucleic acid array after treatment in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' is capable of forming a connection (e.g., covalent or non-covalent connection) with the molecule or group B'; and

[0185] (2) contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

[0186] It is readily understood by those skilled in the art that any portion of the capture probe can be used to modify the molecule or group A', as long as it does not affect the function of the capture probe (e.g., annealing with the target sequence and the function of the 3' end initiation extension reaction). In certain embodiments, the capture sequence of the capture probe does not modify the molecule or group A'. In certain embodiments, the 5' end (e.g., the 5' end) of the capture probe is modified with the molecule or group A'.

[0187] In certain embodiments, the capture sequence is located at the 3' end of the capture probe.

[0188] In certain embodiments, the capture probe exists in a duplex form containing a single-stranded region comprising a capture sequence, wherein the capture sequence is as defined above, and step (B) comprises:

[0189] (I)(1) providing: (a) a free first chain of the capture probe, wherein the first chain is modified with a molecule or group A'; and, (b) the nucleic acid array to be treated or the nucleic acid array after treatment in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', wherein the molecule or group A' is capable of forming a connection (e.g., covalent and / or non-covalent connection) with the molecule or group B'; and, (c) the free second chain of the capture probe, wherein the second chain comprises the capture sequence and is capable of annealing with the first chain to form a duplex;

[0190] (2) contacting the first chain with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the first chain is connected; and,

[0191] (3) contacting the free second strand with the solid support connected to the first strand formed in step (2) under conditions allowing annealing, thereby obtaining a solid support connected to the capture probe;

[0192] or,

[0193] (II)(1) providing: (a) a duplex formed by annealing the first and second chains of the capture probe, wherein the first chain is connected to a molecule or group A', and the second chain contains the capture sequence; and, (b) the nucleic acid array to be treated or the nucleic acid array after treatment in step (B'), wherein the solid support surface of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' is capable of forming a connection (e.g., covalent and / or non-covalent connection) with the molecule or group B'; and,

[0194] (2) contacting the duplex with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B', thereby obtaining a solid support to which the capture probe is connected.

[0195] In certain embodiments, any portion of the first strand may be used to connect to the solid support, as long as the connection does not affect the first strand's ability to perform its function (e.g., annealing to the second strand). In certain embodiments, the portion of the first strand that is not annealed to the second strand is modified with the molecule or group A'. In certain embodiments, the end (e.g., 5' end or 3' end) of the first strand is modified with the molecule or group A'.

[0196] In certain embodiments, the second strand is not directly attached to the solid support.

[0197] In certain embodiments, in the duplex formed by annealing the first and second strands of the capture probe, the 3' end of the second strand comprises the capture sequence, and the capture sequence does not anneal to the first strand.

[0198] In certain embodiments, the molecule or group A' is capable of undergoing a click chemistry reaction with the molecule or group B'.

[0199] In certain embodiments, the molecules or groups A' / B' are selected from the group consisting of: alkynyl / azide, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, alkene / amine, alkene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone.

[0200] In certain embodiments, the molecules or groups A' / B' are azide / alkynyl or alkynyl / azide.

[0201] In certain embodiments, the molecule or group A' is DBCO and the molecule or group B' is azide.

[0202] In certain embodiments, the first strand of the capture probe is modified with The surface of the solid support is modified with an azide group, and the first chain of the capture probe and the solid support are connected through a click chemistry reaction between DBCO and the azide group.

[0203] In certain embodiments, the solid support has one or more characteristics selected from the group consisting of:

[0204] (1) The solid support is selected from the group consisting of latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; in certain embodiments, the solid support is a chip;

[0205] (2) The solid support is planar, spherical or porous;

[0206] (3) The solid support can be used in a sequencing platform; in certain embodiments, the solid support is a sequencing chip for an 111 μMina, MGI, or Thermo Fisher sequencing platform; and

[0207] (4) The solid support is capable of releasing the localization probe and / or capture probe spontaneously or upon exposure to one or more stimuli (e.g., temperature change, pH change, exposure to specific chemicals or phases, exposure to light, reducing agents, etc.).

[0208] In certain embodiments, the same type of positioning probes (i.e., positioning probes comprising the same positioning sequence) occupy the same area on the surface of the solid support, different types of positioning probes occupy different areas on the surface of the support, and the center distance between any two adjacent areas is less than 1 μm (e.g., less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm).

[0209] In certain embodiments, the distribution of the capture probe and the positioning probe on the solid support is configured as follows: the nucleic acid molecule connected to the capture probe (for example, the nucleic acid molecule annealed to the capture probe, or the nucleic acid molecule obtained by extending the capture probe through nucleic acid polymerization reaction) can contact the positioning probe adjacent to it.

[0210] In certain embodiments, there are at least 100 capture probes adjacent to each of the positioning probes, for example, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500, or at least 10000 capture probes.

[0211] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed adjacent to each localization probe.

[0212] In certain embodiments, the expression "adjacent to each of the positioning probes" refers to an area on the surface of a solid support near the positioning probe, wherein nucleic acid molecules connected to capture probes distributed in the area (e.g., nucleic acid molecules annealed to the capture probes, or nucleic acid molecules obtained by extending the capture probes by nucleic acid polymerization) are able to contact the positioning probes.

[0213] In certain embodiments, at least 100 capture probes are distributed within a radius of 200 nm to 300 nm around the center of the position occupied by each positioning probe on the solid support (e.g., the center of the area occupied by each positioning probe on the surface of the solid support). For example, at least 100, at least 200, at least 300, 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 3500, at least 4000, at least 4500, at least 5000, at least 5500, at least 6000, at least 6500, at least 7000, at least 7500, at least 8000, at least 8500, at least 9000, at least 9500 or at least 10000 of the capture probes.

[0214] In certain embodiments, there are 500-10,000 (e.g., 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-4,000, 1,000-5,000, 1,000-8,000, 1,000-10,000) capture probes distributed within a radius of 200 nm-300 nm around the center of the position occupied by each of the positioning probes on the solid support (e.g., the center of the area occupied by each of the positioning probes on the surface of the solid support).

[0215] Nucleic acid spatial information detection method

[0216] In another aspect, the present application provides a method for detecting spatial information of nucleic acids in a sample, comprising the following steps:

[0217] (1) providing a nucleic acid array as described above;

[0218] (2) contacting the nucleic acid array with a sample to be tested under conditions that allow annealing, so that the nucleic acid derived from the sample to be tested anneals with the capture sequence in the capture probe of the nucleic acid array;

[0219] (3) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a first extension product, wherein the first extension product comprises a capture sequence and a complementary sequence of a nucleic acid molecule or a partial sequence thereof that anneals to the capture sequence, and the first extension product is connected to the solid support via the capture sequence contained therein;

[0220] (4) annealing the first extension product connected to the solid support with the adjacent positioning probe of the nucleic acid array under conditions allowing annealing;

[0221] wherein the first universal sequence of the positioning probe is capable of annealing to the first extension product; and the first universal sequence is located at the 3' end of the positioning sequence of the positioning probe;

[0222] (5) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization to produce a second extension product and / or its complementary chain, wherein the second extension product comprises: (a) the positioning sequence of the positioning probe and the complementary sequence of the first extension product or its partial sequence, or (b) the first extension product sequence or its partial sequence and the complementary sequence of the positioning sequence of the positioning probe; the positioning sequence or its complementary sequence contained in the second extension product serves as its spatial information marker, and the complementary sequence of the positioning sequence or the positioning sequence contained in the complementary chain of the second extension product serves as its spatial information marker, so that the position of the nucleic acid derived from the sample to be tested in the sample to be tested is mapped to the position of the positioning probe corresponding to the positioning sequence; thereby obtaining a nucleic acid molecule containing the spatial information marker; and

[0223] (6) Analyzing: (i) the nucleic acid molecule containing the spatial information marker obtained in step (5), and / or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i).

[0224] It is easy for those skilled in the art to understand that in steps (4) to (5), the first extension product anneals with the adjacent positioning probe and undergoes nucleic acid polymerization reaction to produce the second extension product. As a result, the second extension product contains the positioning sequence or its complementary sequence in the positioning probe, so that the position of the nucleic acid derived from the sample to be tested captured by the capture probe in the sample to be tested is corresponded to the position of the positioning probe on the nucleic acid array that anneals to the first extension product.

[0225] In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and / or abundance of the nucleic acid.

[0226] In certain embodiments, in step (3), the nucleic acid polymerization reaction uses the nucleic acid molecule annealed to the capture sequence as a template to extend the capture sequence.

[0227] In certain embodiments, the capture sequence is located at the 3' end of the capture probe, and / or the 3' end of the capture sequence has a free hydroxyl group (-OH).

[0228] In certain embodiments, in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe; and / or uses the positioning probe as a template to extend the first extension product.

[0229] In certain embodiments, in step (5), the polymerization reaction uses the first extension product as a template to extend the positioning probe to produce a second extension product and / or its complementary strand, wherein the second extension product comprises the positioning sequence of the positioning probe and a complementary sequence to the first extension product or a partial sequence thereof. In certain embodiments, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and / or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH).

[0230] In certain embodiments, in step (5), the polymerization reaction uses the positioning probe as a template to extend the first extension product to produce a second extension product and / or its complementary strand, wherein the second extension product comprises the first extension product sequence or a partial sequence thereof and a complementary sequence to the positioning sequence of the positioning probe. In certain embodiments, the first universal sequence of the positioning probe is located or not located at the 3' end of the positioning probe, and / or the 3' end of the first universal sequence of the positioning probe is blocked or unblocked.

[0231] In certain embodiments, in step (5), in the polymerization reaction, the first extension product and the positioning probe serve as templates for each other, and the first extension product and the positioning probe are extended to produce a second extension product and / or its complementary chain, wherein the second extension product comprises the positioning sequence of the positioning probe and the complementary sequence of the first extension product or a partial sequence thereof, and the second extension product comprises the first extension product sequence or a partial sequence thereof and the complementary sequence of the positioning sequence of the positioning probe. In certain embodiments, the first universal sequence of the positioning probe is located at the 3' end of the positioning probe, and / or the 3' end of the first universal sequence of the positioning probe has a free hydroxyl group (-OH).

[0232] In certain embodiments, in step (6), the sequence of the nucleic acid molecule containing the spatial information label attached to the solid support of the nucleic acid array is analyzed.

[0233] In certain embodiments, before step (6) and after step (5), the method further comprises a step pre-(6): releasing at least a portion of the nucleic acid molecules containing spatial information labels from the surface of the nucleic acid array.

[0234] In certain embodiments, in step (6), (i) the nucleic acid molecule containing the spatial information marker released in step pre-(6), and / or, (ii) the sequence of the nucleic acid molecule containing the spatial information marker derived from (i) is analyzed.

[0235] In certain embodiments, in step pre-(6), the nucleic acid molecule is released from the solid support surface by: (i) nucleic acid shearing; and / or, (ii) denaturation.

[0236] In certain embodiments, after step pre-(6) and before step (6), the method further comprises a step of amplifying the released nucleic acid molecules.

[0237] In certain embodiments, before performing step (6), the method further comprises a step of purifying the released nucleic acid molecules.

[0238] In certain embodiments, the method has one or more features selected from the group consisting of:

[0239] (i) In step (1), the nucleic acid array is provided by the method for preparing a nucleic acid array as described above;

[0240] (ii) in step (2), the sample to be tested is treated (e.g., permeabilized or cell lysed) to release nucleic acid from the sample to be tested, so that the nucleic acid anneals with the capture sequence;

[0241] (iii) after step (3) and before step (4), the method further comprises the step of removing the template strand bound to the first extension product (for example, removing the template strand bound to the first extension product by enzyme cleavage or denaturing and melting);

[0242] (iv) after step (3) and before step (4), the method further comprises a step of washing the nucleic acid array to remove residual sample (e.g., tissue or cells);

[0243] (v) After step (5) and before step (6) (e.g., after step (5) and before step pre-(6)), the method further comprises a step of amplifying (e.g., in situ amplification) the nucleic acid molecule containing the spatial information marker. In certain embodiments, the nucleic acid molecule containing the spatial information marker is attached to a solid support of the nucleic acid array.

[0244] In certain embodiments, the sample is a tissue sample (eg, a tissue section) or a single cell sample (eg, a single cell suspension).

[0245] In certain embodiments, the tissue sample (eg, tissue section) is prepared from fixed tissue, eg, formalin-fixed paraffin-embedded (FFPE) tissue, deep-frozen tissue, or fresh tissue.

[0246] In certain embodiments, the cell is an immune cell, such as a B cell or a T cell. In certain embodiments, the nucleic acid derived from the sample to be tested comprises a T cell receptor gene or its nucleic acid product (e.g., mRNA), or a B cell receptor gene or its nucleic acid product (e.g., mRNA).

[0247] In certain embodiments, the nucleic acid derived from the sample to be tested is selected from: RNA molecules (e.g., mRNA) molecules, target nucleic acid molecules (e.g., target DNA and / or RNA), genomic nucleic acid fragments in open chromatin regions, and any combination thereof.

[0248] In certain embodiments, in step (6), the analysis comprises sequencing and / or sequence-specific PCR reaction.

[0249] In certain embodiments, before sequencing, the method further comprises the step of constructing a sequencing library for the nucleic acid molecule containing the spatial information marker or its amplified product obtained in step (5).

[0250] In certain embodiments, the methods are used to detect spatial information of RNA (eg, mRNA) in cells in a sample.

[0251] In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested is RNA (eg, mRNA) in cells of the sample to be tested, and the capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence.

[0252] In certain embodiments, step (3) comprises:

[0253] (a) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization, using a nucleic acid molecule annealed to the capture sequence as a template, extending the capture sequence to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the capture sequence as a reverse transcription primer, and a 3' end overhang;

[0254] (b) annealing the template switching sequence to the cDNA chain generated in (a) under conditions that allow nucleic acid polymerization, and continuing nucleic acid polymerization using the template switching sequence as a template to generate the first extension product;

[0255] Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; in certain embodiments, the MID sequences comprised by each template switching sequence are different from each other;

[0256] The first extension product comprises: a capture sequence, the cDNA chain sequence or a partial sequence thereof, and a complementary sequence of the common sequence.

[0257] In certain embodiments, the 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0258] In certain embodiments, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product (eg, removing the template switching sequence bound to the first extension product by enzyme cleavage or denaturing and melting).

[0259] In certain embodiments, in step (4) of the method, the first universal sequence of the localization probe is capable of annealing to the complement of the consensus sequence.

[0260] In certain embodiments, the positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; alternatively, the positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence; alternatively, both the positioning probe and the template switch sequence contain a MID sequence.

[0261] In certain embodiments, the method is used to detect the spatial information of a target nucleic acid (eg, target DNA and / or RNA) of a cell in a sample, wherein the target nucleic acid comprises a target nucleotide sequence.

[0262] In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested comprises the target nucleotide sequence. In certain embodiments, the nucleic acid derived from the sample to be tested comprises the target nucleic acid and / or a nucleic acid molecule derived from the target nucleic acid, and the nucleic acid molecule derived from the target nucleic acid comprises the target nucleotide sequence.

[0263] In certain embodiments, in step (2), the capture sequence comprises a sequence that specifically recognizes the target nucleotide sequence.

[0264] In certain embodiments, in step (2), the capture sequence comprises a sequence that specifically recognizes the first segment of the target target nucleotide sequence; in step (4), the first universal sequence of the positioning probe comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target target nucleotide sequence, or, the first universal sequence comprises a random oligonucleotide sequence; in certain embodiments, in the target target nucleotide sequence, the first segment is located at the 3' end of the second segment.

[0265] In certain embodiments, in step (2), the first segment is present in a single-stranded region of the nucleic acid molecule derived from the sample to be tested that anneals to the capture sequence.

[0266] In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested that anneals to the capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region comprising the first segment.

[0267] In certain embodiments, the method is used to detect spatial information of RNA (e.g., mRNA) and target nucleic acid (e.g., target DNA and / or RNA) of cells in a sample, wherein the target nucleic acid comprises a target nucleotide sequence.

[0268] In certain embodiments, the nucleic acid array contains a first capture probe capable of capturing the RNA (e.g., mRNA) and a second capture probe capable of capturing a nucleic acid molecule containing the target nucleotide sequence; the first capture probe contains a first capture sequence, which comprises a poly (dT) sequence or a random oligonucleotide sequence; and the second capture probe contains a second capture sequence, which comprises a sequence that specifically recognizes the target nucleotide sequence.

[0269] It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradicted by the context, the definition / description of the capture probe above also applies to the first capture probe and the second capture probe, and the definition / description of the capture sequence above also applies to the first capture sequence and the second capture sequence.

[0270] In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested comprises RNA (e.g., mRNA) derived from the sample to be tested and a nucleic acid molecule comprising a target nucleotide sequence; in certain embodiments, the nucleic acid molecule comprising a target nucleotide sequence comprises the target target nucleic acid and / or a nucleic acid molecule derived from the target target nucleic acid.

[0271] In certain embodiments, step (3) comprises:

[0272] (A)(a) performing a nucleic acid polymerization reaction under conditions that allow nucleic acid polymerization, using a nucleic acid molecule annealed to the first capture sequence as a template, extending the first capture sequence to generate a cDNA chain, wherein the cDNA chain comprises a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the first capture sequence as a reverse transcription primer, and a 3' end overhang;

[0273] (b) annealing the template switching sequence to the cDNA chain generated in (a) under conditions that allow nucleic acid polymerization, and continuing nucleic acid polymerization using the template switching sequence as a template to generate a first extension product I;

[0274] Wherein, the template switching sequence comprises a consensus sequence, a 3'-terminal overhang complementary sequence, and an optional MID sequence; in certain embodiments, the MID sequences comprised by each template switching sequence are different from each other;

[0275] as well as,

[0276] (B) performing a nucleic acid polymerization reaction using the nucleic acid molecule annealed to the second capture sequence as a template under conditions that allow nucleic acid polymerization, extending the second capture sequence to produce a first extension product II, wherein the first extension product II comprises the second capture sequence and a complementary sequence to the nucleic acid molecule annealed to the second capture sequence or a partial sequence thereof;

[0277] Wherein, step (B) and step (A) are performed in any order; for example, step (B) is performed before or after step (A), or step (B) and step (A) are performed simultaneously (for example, in the same reaction system).

[0278] In certain embodiments, the 3' terminal overhang is at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides in length. In certain embodiments, the 3' terminal overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0279] In certain embodiments, in step (4), the positioning probe contains a first universal sequence I that can anneal to the first extension product I, and a first universal sequence II that can anneal to the first extension product II; in certain embodiments, the first universal sequence I and the first universal sequence II are present in the same positioning probe, or, respectively, in different positioning probes.

[0280] In certain embodiments, the nucleic acid array comprises a first positioning probe comprising the first universal sequence I and a second positioning probe comprising the first universal sequence II.

[0281] It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradicted by the context, the definition / description of the positioning probe above also applies to the first positioning probe and the second positioning probe, and the definition / description of the first universal sequence above also applies to the first universal sequence I and the first universal sequence II.

[0282] In certain embodiments, in step (4) of the method, the first universal sequence I is capable of annealing to the complementary sequence of the consensus sequence.

[0283] In certain embodiments, in step (2) of the method, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence; in step (4), the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, in the target nucleotide sequence, the first segment is located at the 3' end of the second segment.

[0284] In certain embodiments, the method further comprises, before step (4), a step of removing the template switching sequence bound to the first extension product I (e.g., removing the template switching sequence bound to the first extension product I by enzyme cleavage or denaturing and melting).

[0285] In certain embodiments, in step (2), the first segment is present in a single-stranded region of the nucleic acid molecule derived from the test sample that anneals to the second capture sequence.

[0286] In certain embodiments, in step (2), the nucleic acid derived from the sample to be tested that anneals to the second capture sequence is a single-stranded nucleic acid or a double-stranded nucleic acid containing a single-stranded region comprising the first segment.

[0287] In certain embodiments, the first positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; alternatively, the first positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence; alternatively, both the first positioning probe and the template switch sequence contain a MID sequence.

[0288] Reagent test kit

[0289] In another aspect, the present application provides a kit comprising the nucleic acid array as described above.

[0290] In certain embodiments, the kit further comprises instructions for use. In certain embodiments, the instructions for use describe the method for detecting nucleic acid spatial information as described above.

[0291] In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and / or abundance of the nucleic acid.

[0292] For mRNA capture

[0293] In certain embodiments, the capture sequence of the nucleic acid array comprises a poly(dT) sequence or a random oligonucleotide sequence.

[0294] In certain embodiments, the kit further comprises a template switch sequence comprising a consensus sequence, a sequence complementary to the cDNA 3' overhang, and an optional MID sequence; in certain embodiments, the MID sequence comprised by each template switch sequence is different from one another. In certain embodiments, the cDNA 3' overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides. In certain embodiments, the cDNA 3' overhang is an overhang of 2-5 cytosine nucleotides (e.g., a CCC overhang).

[0295] In certain embodiments, the complement of the consensus sequence of the template switch sequence is capable of annealing to the first universal sequence of the positioning probe of the nucleic acid array.

[0296] In certain embodiments, the positioning probe does not contain a MID sequence and the template switch sequence contains a MID sequence; alternatively, the positioning probe contains a MID sequence and the template switch sequence does not contain a MID sequence; alternatively, both the positioning probe and the template switch sequence contain a MID sequence.

[0297] For target nucleic acid capture

[0298] In certain embodiments, the capture sequence of the nucleic acid array comprises a sequence that specifically recognizes a target nucleotide sequence comprised by a target nucleic acid (eg, a specific target DNA and / or RNA).

[0299] In certain embodiments, the first universal sequence comprises a sequence that specifically recognizes the complementary sequence of the target nucleotide sequence of interest.

[0300] In certain embodiments, the capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence, the first universal sequence of the positioning probe of the nucleic acid array comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence.

[0301] In certain embodiments, in the target nucleotide sequence of interest, the first segment is located 3' to the second segment.

[0302] Dual capture of mRNA and target nucleic acid

[0303] In certain embodiments, the nucleic acid array contains a first capture probe and a second capture probe; the first capture probe contains a first capture sequence, and the first capture sequence comprises a poly (dT) sequence or a random oligonucleotide sequence; the second capture probe contains a second capture sequence, and the second capture sequence comprises a sequence that specifically recognizes a target target nucleotide sequence contained in a target target nucleic acid (e.g., a specific target DNA and / or RNA).

[0304] It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradicted by the context, the definition / description of the capture probe above also applies to the first capture probe and the second capture probe, and the definition / description of the capture sequence above also applies to the first capture sequence and the second capture sequence.

[0305] In certain embodiments, the kit further comprises a template switch sequence comprising a consensus sequence, a cDNA 3' end overhang complementary sequence, and an optional MID sequence. In certain embodiments, the MID sequences comprised by each template switch sequence are different from each other. In certain embodiments, the cDNA 3' end overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5, or 2-10 nucleotides. In certain embodiments, the cDNA 3' end overhang is an overhang of 2-5 cytosine nucleotides (e.g., CCC overhang).

[0306] In certain embodiments, the positioning probe of the nucleic acid array comprises a first universal sequence I and a first universal sequence II; wherein the first universal sequence I is capable of annealing to a complementary sequence of the consensus sequence, and the first universal sequence II comprises a sequence that specifically recognizes a complementary sequence of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, the first universal sequence I and the first universal sequence II are present in the same positioning probe, or in different positioning probes.

[0307] In certain embodiments, the nucleic acid array comprises a first positioning probe comprising the first universal sequence I and a second positioning probe comprising the first universal sequence II. In certain embodiments, the first positioning probe does not comprise a MID sequence, and the template switch sequence comprises a MID sequence; alternatively, the first positioning probe comprises a MID sequence, and the template switch sequence does not comprise a MID sequence; or alternatively, both the first positioning probe and the template switch sequence comprise a MID sequence.

[0308] It is easy for those skilled in the art to understand that, unless otherwise specified herein or clearly contradicted by the context, the definition / description of the positioning probe above also applies to the first positioning probe and the second positioning probe, and the definition / description of the first universal sequence above also applies to the first universal sequence I and the first universal sequence II.

[0309] In certain embodiments, the second capture sequence comprises a sequence that specifically recognizes the first segment of the target nucleotide sequence, the first universal sequence II comprises a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence. In certain embodiments, in the target nucleotide sequence, the first segment is located at the 3' end of the second segment.

[0310] In certain embodiments, the first universal sequence comprised by the positioning probe of the nucleic acid array is located at or not located at the 3' end of the positioning probe, and / or the 3' end of the first universal sequence of the positioning probe of the nucleic acid array is blocked or unblocked.

[0311] In certain embodiments, the kit further comprises: reagents for nucleic acid hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, reagents for recovering or purifying nucleic acids, reagents for constructing a transcriptome sequencing library, reagents for sequencing (e.g., second-generation sequencing or third-generation sequencing), or any combination thereof.

[0312] In another aspect, the present application provides the use of the nucleic acid array or kit as described above for constructing a nucleic acid molecule library, for performing nucleic acid sequencing, or for detecting spatial information of nucleic acids in a sample.

[0313] In certain embodiments, the spatial information of the nucleic acid includes the location, distribution and / or abundance of the nucleic acid.

[0314] As used herein, "target nucleotide sequence" and "target nucleotide sequence" have the same meaning and can be used interchangeably.

[0315] Advantageous Effects of the Invention

[0316] The capture area and spatial information of the nucleic acid array (e.g., capture chip) provided by the present application exist in two independent molecules, for example, the capture area and spatial information exist in the capture probe and the positioning probe, respectively, wherein the positioning probe is used to provide spatial position information of spatial omics, and the capture probe is used to capture molecules in the cell, thereby getting rid of the limitation that the number of capture probes is affected by spatial density, and being able to make full use of all areas on the chip to graft capture probes, thereby improving the capture efficiency of intracellular molecules while ensuring high resolution.

[0317] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are intended only to illustrate the present invention and are not intended to limit the scope of the invention. Various objects and advantages of the present invention will become apparent to those skilled in the art based on the following detailed description of the accompanying drawings and preferred embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0318] Figure 1: Schematic diagram of an exemplary capture chip of the present application, wherein the capture probe (including the capture domain) and the localization probe (including the spatial barcode) on the chip are located on two independent molecules.

[0319] Figure 2: Exemplary implementation process of spatial transcriptomics in this application.

[0320] Figure 3: Chip probe detection results.

[0321] Figure 4: cDNA Bioanalyzer 2100 detection results.

[0322] Figure 5: Spatial expression map of mouse brain sections.

[0323] Figure 6: Cell types in mouse brain slices.

[0324] Figure 7: Gene coverage analysis results of the captured transcriptome.

[0325] Sequence information

[0326] A description of the sequences involved in this application is provided in the table below.

[0327] Table 1: Sequence information

[0328] Note: V=A, C, or G; N=A, T, C, or G; “r” indicates that the nucleotide at the 3' adjacent position is a ribonucleotide; “iXNA” indicates an LNA locked nucleotide. DETAILED DESCRIPTION

[0329] The present invention will now be described with reference to the following examples which are intended to illustrate the invention rather than to limit the scope of the invention as claimed.

[0330] An exemplary implementation process for obtaining sample spatial transcriptomic information based on the chip provided in this application is shown in Figure 2:

[0331] (1) Prepare a capture chip as shown in FIG1 ;

[0332] (2) mRNA capture: Tissue sections are attached to a capture chip, and the tissue is processed to release mRNA molecules;

[0333] (3) The released mRNA molecules are captured by the capture probes on the chip and synthesized into cDNA molecules in situ under the action of reverse transcriptase with terminal transferase activity; because the reverse transcriptase with terminal transferase activity will add some additional nucleotides C to the 3' end of the newly synthesized cDNA molecules, forming base complementary pairing with the 3' end of TSO (template switching oligonucleotide), the reverse transcriptase "converts" the template and continues to synthesize cDNA molecules using TSO as a template until the 5' end of TSO;

[0334] (4) The 3' end of the cDNA molecule can be captured by the adjacent positioning probe containing positional information and further extended, thereby adding spatial information to the cDNA molecule and synthesizing a cDNA molecule with spatial information;

[0335] (5) collecting cDNA molecules from the chip and performing cDNA amplification, or performing PCR directly on the chip to amplify cDNA;

[0336] (6) Library construction and sequencing;

[0337] (7) Data analysis: restore spatial information and RNA expression to chip locations.

[0338] In addition, the applicant wishes to emphasize that based on the different designs of the capture zone sequence, it can be designed to target a variety of target sequences, not limited to mRNA. Therefore, the chip provided in this application can not only be used to obtain spatial transcriptomics information, but it can also be used to obtain genomic, epigenomic, multi-omics and other information, and can also be used to obtain information on specific target molecules.

[0339] 1. Capture Chip Preparation

[0340] An exemplary capture chip of the present application comprises a positioning probe and a capture probe, wherein the capture probe comprises a linker sequence and a capture sequence in sequence from the 5' end to the 3' end, and the positioning probe comprises a first linker sequence, a spatial position sequence (i.e., a positioning sequence) and a second linker sequence in sequence from the 5' end to the 3' end.

[0341] This embodiment shows an exemplary method for preparing the capture chip, which includes:

[0342] (a) providing a conventional chip commonly used in the art that includes a positioning sequence and a capture sequence in the same probe (for example, the conventional chip can be purchased or prepared by the user); the probe of the conventional chip includes, from the 5' end to the 3' end, a first linker sequence, a spatial position sequence (i.e., a positioning sequence), a second linker sequence, and a capture sequence;

[0343] (b) blocking the second linker, for example, hybridizing an oligonucleotide molecule capable of annealing to the second linker sequence with the probe, thereby forming a double-stranded structure at the position of the second linker of the probe;

[0344] (c) incubating the product of (b) with a nuclease having 3' to 5' end single-stranded nuclease activity; thereby, the capture sequence of the probe of the conventional chip is cleaved by the nuclease, thereby forming a positioning probe that does not contain a capture sequence but contains a positioning sequence;

[0345] (d) Connecting capture probes to the chip containing the positioning probes obtained in (c) through a click chemistry reaction, thereby obtaining an exemplary capture chip comprising capture probes and positioning probes of the present application.

[0346] Specifically, the capture chip preparation steps are as follows:

[0347] (1) Block and remove the conventional chip capture area to form a positioning probe: synthesize an oligo sequence: GTCTTAGGAAGACAA (SEQ ID NO: 1) from Bio-Technology, and use 5×SSC (saline sodium citrate) to prepare it into a 1μM oligo solution; take out the chip in the stereo-seq transcriptomics T kit (BGI, catalog number: 111KT114) (conventional chip, the structure of each nucleic acid molecule from the 5' end to the 3' end is: linker 1-spatial position sequence-linker 2-capture sequence poly T), draw 100μL of 1μM oligo solution onto the chip, and hybridize at room temperature for 30 minutes to block the linker 2 of the nucleic acid molecule on the conventional chip; draw off the above hybridization solution, and wash the chip with 0.1×SSC, add 50μL Exonuclease I (ThermoFisher: EN0581) reaction solution (5μL Exonuclease I, 5μL 10x Reaction buffer, 40μL The chip was reacted with ddH2O) at 37°C for 20 min to remove the capture sequence poly T of the nucleic acid molecule on the conventional chip; the reaction solution was discarded and the chip was washed with 0.1×SSC; the chip only contained "linker 1-spatial position sequence-linker 2".

[0348] (2) Chip surface modification: Prepare 0.1% poly-lysine solution (PLL, Sigma P8920) and add it to the treated chip. Incubate at 30°C for 3 h to modify the chip surface with amino groups. Discard the reaction solution, rinse the chip with ddH2O, and dry it for 1 h. Prepare NHS-PEG-N3 reaction solution (Sigma JKA5088) according to the instructions and add it to the chip. Incubate at 37°C for 5 h to modify the chip surface with azide groups.

[0349] (3) Modification of oligo dT on chip capture probe: Bioengineering synthesized a capture probe with a linker and oligo dT: 5'-CCTCCGACTGTGTGACTTAGACTCCTGCCACCTCCTGATGTGCTTTTTTTTTTTTTTTTTTTTTTV-3' (SEQ ID NO: 2). The 5' end of the probe was modified with DBCO (dibenzocyclooctyne). The probe was diluted to 1 μM with PBS and added to the chip for reaction at 37°C overnight. This allowed the azide groups on the chip surface to undergo a click reaction with the DBCO-modified probe, and then the oligo dT probe was connected to the chip to obtain a chip containing the capture probe.

[0350] (4) Chip probe detection: A detection probe AAAAAAAAAAAAAAAAGCACA (SEQ ID NO: 3) with a 3'-end modified cy5 fluorescence was used for hybridization photography to detect the synthesis of the capture probe on the chip and the excision of the capture region on the chip in step 1. The results are shown in Figure 3: Figure ① is a photograph of the PLL-modified chip obtained in step (2) after adding the detection probe, and Figure ② is the background of the positioning probe chip obtained in step (1) (no detection probe added); Figure ③ is a photograph of the positioning probe chip obtained in step (1) after adding the detection probe; Figure ④ is the fluorescence of the chip after oligo dT connection obtained in step (3) after adding the detection probe. The results show that Figures ② and ③ show that the original capture region poly T on the commercial chip was successfully excised, and Figure ④ shows that the newly synthesized capture probe oligo dT was successfully modified on the chip.

[0351] 2. mRNA molecule capture

[0352] Refer to the instructions of the Stereo-seq Transcriptomics T Kit and attach tissue slices, such as mouse brain slices, to the chip. After permeabilization, the slices are treated to release mRNA, allowing the mRNA in the tissue to be captured by the capture probes on the chip.

[0353] 3. In situ cDNA synthesis

[0354] Refer to the instructions of the Stereo-seq Transcriptomics T Kit to configure the following reverse transcription reaction system and incubate at 42°C for 5 hours.

[0355] 4. Add spatial information

[0356] After cDNA synthesis is complete, discard the reaction solution and add TR buffer (1000028507) according to the manufacturer's instructions. Incubate for 30 minutes before removing the tissue. Prepare a formamide solution and add it to the chip surface. Incubate at 80°C for 3 hours. Aspirate the reaction solution, remove the mRNA, and rinse the chip surface with ddH2O. Add 5×SSC reaction solution to the chip surface and incubate at 37°C for 30 minutes. Remove the reaction solution, rinse the chip surface with 0.1×SSC, and then add the following reaction solution. Incubate at 37°C for 3 hours to extend spatial information.

[0357] 5. cDNA Release and cDNA Amplification

[0358] Prepare 100 μL of 100 mM KOH solution, add it to the chip and react at 55°C for 3 hours. Collect the reaction supernatant into a PCR tube.

[0359] The chip was then washed with 50 μL of ddH2O. The wash solution and supernatant were combined into a PCR tube. The reaction solution was neutralized to pH 8.5 with 0.1 M HCl and mixed thoroughly before being divided into three tubes for PCR reaction. The reaction solution was as follows. PCR reactions were performed according to the instructions. The obtained cDNA was detected using a Bioanalyzer 2100. The results are shown in Figure 4.

[0360] 6. Library Construction and Sequencing

[0361] Refer to the instructions of the stereo-seq transcriptomics T kit to shear the cDNA library and prepare DNBs.

[0362] Following the MGISEQ 2000 sequencer operating instructions, DNBs were loaded onto the MGISEQ 2000 sequencing chip for sequencing. Sequencing was performed using a 25bp sequencer for spatial information decoding, followed by a 22bp dark reaction, a 5bp sequencer to obtain the molecular identifier (MID), and a 50bp-100bp sequencer to obtain information on the 5' end of the cDNA transcript.

[0363] 7. Data Analysis

[0364] (1) Log in to the website http: / / stereomap.cngb.org / Stereo-Draftsman / report / index and perform data analysis according to the website's operating instructions. Compare the first 25bp of the read1 sequence (from the first-strand sequencing) obtained in step 6 with the 25bp position information of the capture chip in step 1. Keep the reads that can be matched to the position information on the chip and map them to the corresponding chip positions. Find the 46bp starting sequence corresponding to the reads corresponding to the chip position, which is the cDNA sequence. After reverse complementation of the sequence, compare it with the mouse brain genome. Remove duplicate reads based on the MID information obtained from 41bp-45bp to obtain the number of each gene expressed in the mouse brain.

[0365] (2) The expression number of each gene was further mapped to obtain the spatial expression map of the mouse brain slice as shown in Figure 5. The expression level was subjected to spatial cluster analysis to obtain the cell type of the mouse brain slice as shown in Figure 6.

[0366] (3) 5'-end transcript capture analysis

[0367] The captured transcriptome was subjected to gene coverage analysis, and it can be seen that most of the captured transcriptome is concentrated in the transcription start region (TSS), proving that this scheme can capture 5'-end transcripts, as shown in Figure 7.

[0368] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all the teachings published, and these changes are all within the scope of protection of the present invention. The entire invention is given by the appended claims and any equivalents thereof.

Claims

1. A nucleic acid array for detecting the spatial information of nucleic acids in a sample, comprising a solid support, wherein the solid support is linked to one or more capture probes and at least two positioning probes; The capture probe and the positioning probe are each independently linked to the solid support; Each positioning probe occupies a different position on the solid support; The positioning probe contains a first universal sequence and a positioning sequence, wherein, The positioning sequence has a nucleotide sequence corresponding to the position of the positioning probe on the solid support; The capture probe contains a capture sequence that can anneal to the nucleic acid molecule to be captured and initiate an extension reaction.

2. The nucleic acid array of claim 1, wherein, The first universal sequence is located at the 3'-end of the positioning sequence; Preferably, the positioning probe further comprises a second universal sequence; Preferably, the second universal sequence is located at the 5'-end of the positioning sequence; Preferably, the second universal sequences contained in different positioning probes linked to the solid support are the same or different; preferably, the second universal sequences contained in different positioning probes linked to the solid support are the same; Preferably, the positioning probe does not contain or further contains a molecular tag sequence (MID, molecular identifier); Preferably, the positioning probe further comprises an MID sequence; Preferably, the MID sequence is located at the 5'-end of the first universal sequence, and / or, the MID sequence is located at the 3'-end of the second universal sequence; Preferably, the MID sequences contained in the same kind of positioning probes are different from each other.

3. The nucleic acid array of claim 1 or 2, wherein, The capture sequence is located at the 3'-end of the capture probe; Preferably, the 3'-end of the capture sequence has a free hydroxyl group (-OH); Preferably, the capture probe further comprises a fixation sequence; Preferably, the fixation sequences contained in different capture probes linked to the solid support are the same or different; preferably, the fixation sequences contained in different capture probes linked to the solid support are the same; Preferably, the fixation sequence is located at the 5'-end of the capture sequence.

4. The nucleic acid array according to any one of claims 1-3, wherein, The capture probe contains a single-stranded region containing the capture sequence; Preferably, the capture probe exists in a single-stranded form or a duplex form containing the single-stranded region; Preferably, the capture probe exists in a single-stranded form, and the capture sequence is located at the 3'-end of the capture probe; or, The capture probe exists in a duplex form containing a single-stranded region, which comprises a first strand directly linked to the solid support and a second strand hybridized to the first strand; and, the 3'-end of the second strand contains a single-stranded region, and the capture sequence is located at the 3'-end of the single-stranded region.

5. The nucleic acid array according to any one of claims 1-4, having one or more of the following features: (1) The positioning sequence is a nucleotide sequence consisting of 5 to 50 (such as 5 to 25, 5 to 35, 5 to 45, 10 to 25, 10 to 35, 10 to 45, 15 to 25, 15 to 35, 15 to 45, 20 to 25, 20 to 35, or 20 to 45) random nucleotides. Preferably, each random nucleotide is independently any one of deoxyribonucleotides A, C, G, and T; (2) The solid support is selected from latex beads, dextran beads, polystyrene surfaces, polypropylene surfaces, polyacrylamide gels, gold surfaces, glass surfaces, chips, sensors, electrodes, and silicon wafers; preferably, the solid support is a chip; preferably, the solid support can be used in a sequencing platform; preferably, the solid support is a sequencing chip, such as a sequencing chip for the Illumina, MGI, or Thermo Fisher sequencing platform; (3) The same type of positioning probe occupies the same region on the surface of the solid support, and different types of positioning probes occupy different regions on the surface of the support. Moreover, the center distance between any two adjacent regions is less than 1 μm (for example, less than 900 nm, less than 800 nm, less than 700 nm, less than 600 nm, less than 500 nm, less than 400 nm, less than 300 nm, less than 200 nm); (4) The first universal sequence can anneal with (i) the nucleic acid molecule captured by the capture sequence, or (ii) a nucleic acid molecule derived from (i); (5) The first universal sequences contained in different types of positioning probes connected to the solid support are the same or different; (6) The solid support can release the positioning probe and / or the capture probe spontaneously or when exposed to one or more stimuli (such as temperature change, pH change, exposure to a specific chemical substance or phase, exposure to light, reducing agent, etc.).

6. The nucleic acid array according to any one of claims 1-5, wherein, The positioning probe and / or the capture probe is covalently or non-covalently connected to the solid support; Preferably, the positioning probe and / or the capture probe is covalently connected to the solid support; Preferably, the positioning probe and the capture probe independently form a connection with the solid support through the same or different click chemical reactions; Preferably, the molecule or group pairs capable of undergoing the click chemical reaction are selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol; preferably, the molecule or group pair capable of undergoing the click chemical reaction is azide / alkynyl; For example, the surface of the solid support is modified with a molecule or group X, the positioning probe is modified with a molecule or group Y, the molecule or group X can undergo a click chemical reaction with the molecule or group Y, and the positioning probe forms a connection with the solid support through the click chemical reaction between the molecule or group X and the molecule or group Y; and / or, the surface of the solid support is modified with a molecule or group X', the capture probe is modified with a molecule or group Y', the molecule or group X' can undergo a click chemical reaction with the molecule or group Y', and the capture probe forms a connection with the solid support through the click chemical reaction between the molecule or group X' and the molecule or group Y'; Preferably, the molecule or group pairs X / Y and X' / Y' are each independently selected from: alkynyl / azide, azide / cyano, amine / alkene, thiol / alkene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, alkene / amine, alkene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone; Preferably, the surface of the solid support is modified with azide groups, and the positioning probe and / or the capture probe is modified with (DBCO), the positioning probe and / or the capture probe forms a connection with the solid support through the click chemical reaction between the azide group and the DBCO.

7. The nucleic acid array according to any one of claims 1-6, wherein, There are at least 100 capture probes distributed adjacent to each positioning probe, preferably 500 - 10,000 capture probes; Preferably, at least 100 capture probes, preferably 500 - 10,000 capture probes, are distributed within a range of a radius of 200 nm - 300 nm around the center of the position occupied by each positioning probe on the solid support (for example, the center of the area occupied by each positioning probe on the surface of the solid support).

8. The nucleic acid array according to any one of claims 1-7, wherein, The nucleic acid array contains one or more of the capture probes; Preferably, different capture probes contain different capture sequences; Preferably, the nucleic acid molecule to be captured is RNA (for example, mRNA), and the capture sequence of the capture probe contains a poly(dT) sequence or a random oligonucleotide sequence; or, The nucleic acid molecule to be captured is a target nucleic acid (for example, target DNA and / or RNA) or a nucleic acid molecule derived from the target nucleic acid, the target nucleic acid or the nucleic acid molecule derived from the target nucleic acid has the target nucleotide sequence contained in the target nucleic acid, and the capture sequence of the capture probe contains a sequence capable of specifically annealing to the target nucleotide sequence; or, The nucleic acid molecules to be captured are RNA (e.g., mRNA) and target nucleic acids (e.g., target DNA and / or RNA). The nucleic acid array comprises a first capture probe capable of capturing RNA (e.g., mRNA) and a second capture probe capable of capturing the target nucleic acid or a nucleic acid molecule derived from the target nucleic acid, and the target nucleic acid or a nucleic acid molecule derived from the target nucleic acid has a target nucleotide sequence contained in the target nucleic acid; wherein, the capture sequence of the first capture probe contains a poly(dT) sequence or a random oligonucleotide sequence, and the capture sequence of the second capture probe contains a sequence capable of specifically annealing to the target nucleotide sequence.

9. A method for preparing the nucleic acid array according to any one of claims 1-8, which comprises the following steps: (A) Connecting and / or synthesizing a positioning probe on a solid support, wherein the positioning probe is defined as in claim 1, 2 or 5; and, (B) Connecting and / or synthesizing a capture probe on a solid support, wherein the capture probe is defined as in any one of claims 1, 3-5, 8; wherein, (A) and (B) can be carried out in any order or simultaneously (e.g., simultaneously in the same reaction system); for example, (A) is carried out before (B), and the solid support in (B) is a solid support already connected with the positioning probe; for example, (B) is carried out before (A), and the solid support in (A) is a solid support already connected with the capture probe; for example, (A) and (B) are carried out simultaneously, and the solid supports in (A) and (B) are the same solid support.

10. The method of claim 9, wherein, Step (A) comprises: (1) Providing: (a) the free positioning probe, wherein the positioning probe is modified with a molecule or group A; and, (b) the solid support, the surface of the solid support is modified with a molecule or group B, and the molecule or group A can form a connection with the molecule or group B (e.g., covalent or non-covalent connection); and, (2) Contacting the positioning probe with the solid support under conditions suitable for forming a connection between the molecule or group A and the molecule or group B, so as to obtain a solid support connected with the positioning probe; Preferably, the molecule or group A can undergo a click chemical reaction with the molecule or group B; Preferably, the molecule or group A / B is selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, ene / amine, ene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone; Preferably, the molecule or group A / B is azide / alkynyl or alkynyl / azide; (DBCO), the surface of the solid support is modified with an azide group, and the positioning probe and the solid support form a connection through the click chemical reaction between DBCO and the azide group. Preferably, the positioning probe is modified with Step (A) comprises:

11. The method of claim 9, wherein, ​ (1) Provide at least two vectors, each vector containing at least one copy of a vector sequence, the vector sequence comprising: a complementary sequence of a localization sequence and a complementary sequence of a first universal sequence; the first universal sequence and the localization sequence are defined as in any one of claims 1, 2, and 5; wherein, the complementary sequences of the localization sequences of each vector sequence are different from each other; (2) Place at least two of the vectors on the surface of the solid support; (3) Provide a fixed primer and perform a nucleic acid polymerization reaction using the vector sequence as a template to generate an extension product, which is the localization probe; wherein, the fixed primer can anneal to the vector sequence and initiate the extension reaction; and, (4) Connect the fixed primer to the surface of the solid support; Wherein, steps (3) and (4) are carried out in any order (for example, step (3) is carried out before or after step (4), or, step (3) and step (4) are carried out simultaneously); Preferably, the vector sequence sequentially comprises, in the direction from the 5'-end to the 3'-end: a complementary sequence of the universal sequence, and, a complementary sequence of the localization sequence; Preferably, the extension product sequentially comprises, in the direction from the 5' to the 3': the localization sequence, and the first universal sequence; Preferably, each vector is a DNB formed by a concatemer of multiple copies of the vector sequence; Preferably, the method optionally includes step (5): digesting the vector sequence, and / or, separating the extension product in step (3) from the vector sequence annealed thereto; Preferably, in step (1), the vector is provided by the following steps: (i) Provide a vector template sequence, the vector template sequence containing a complementary sequence of the vector sequence; (ii) Perform a nucleic acid amplification reaction using the vector template sequence as a template to obtain an amplification product of the vector template sequence, the amplification product containing at least one copy of the vector sequence; preferably, perform rolling circle replication to obtain a DNB formed by a concatemer of the vector sequence.

12. The method of claim 11, wherein, The vector sequence further comprises a complementary sequence of a second universal sequence or a partial sequence thereof (for example, the vector sequence further comprises a complementary sequence of the second universal sequence or its 3'-end partial sequence), the second universal sequence being defined as in claim 2; Preferably, the complementary sequence of the second universal sequence or its partial sequence is located at the 3'-end of the complementary sequence of the localization sequence; Preferably, the fixed primer contains the second universal sequence or a partial sequence thereof (for example, the fixed primer contains the second universal sequence or its 5'-end partial sequence); Preferably, the extension product sequentially comprises, in the direction from the 5' to the 3': the second universal sequence, the localization sequence, and the first universal sequence; Preferably, the vector sequence does not contain or further contains a template sequence of the MID sequence; Preferably, the carrier sequence further comprises a template sequence of the MID sequence; preferably, the template sequence of the MID sequence is located at the 3' end of the complementary sequence of the first universal sequence, and / or, the template sequence of the MID sequence is located at the 5' end of the complementary sequence of the second universal sequence or a partial sequence thereof; preferably, the MID sequence is composed of 5-50 (such as 5-25, 5-35, 5-45, 10-25, 10-35, 10-45, 15-25, 15-35, 15-45, 20-25, 20-35 or 20-45) degenerate deoxyribonucleotide residues.

13. The method of claim 11 or 12, wherein, The immobilized primer is covalently or non-covalently linked to the solid support; Preferably, the immobilized primer is covalently linked to the solid support; Preferably, the immobilized primer forms a connection with the solid support through a click chemical reaction; Preferably, the pair of molecules or groups capable of undergoing the click chemical reaction is selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol; preferably, the pair of molecules or groups capable of undergoing the click chemical reaction is azide / alkynyl; Preferably, the fixed primer is modified with (DBCO), the surface of the solid support is modified with azide, and the immobilized primer and the solid support form a connection through the click chemical reaction of DBCO with azide to form a connection.

14. The method according to any one of claims 9 - 13, wherein, The capture probe exists in a single-stranded form containing a single-stranded region containing a capture sequence, wherein the capture sequence is defined in any one of claims 1, 3-5, 8, and the step (B) includes: (1) Providing: (a) the free capture probe, wherein the capture probe is modified with a molecule or group A'; and, (b) the solid support, the surface of the solid support is modified with a molecule or group B', and the molecule or group A' can form a connection (such as a covalent or non-covalent connection) with the molecule or group B'; and, (2) Contacting the capture probe with the solid support under conditions suitable for forming a connection between the molecule or group A' and the molecule or group B' to obtain a solid support linked with the capture probe; Preferably, the capture sequence is located at the 3' end of the capture probe.

15. The method according to any one of claims 9-13, wherein, The capture probe exists in a double-stranded form containing a single-stranded region containing a capture sequence, wherein the capture sequence is defined in any one of claims 1, 3-5, 8, and the step (B) includes: (I)(1) Providing: (a) the first strand of the free capture probe, the first strand is modified with a molecule or group A'; and, (b) the solid support, the surface of the solid support is modified with a molecule or group B', and a connection can be formed between the molecule or group A' and the molecule or group B' (such as a covalent and / or non-covalent connection); and, (c) the second strand of the free capture probe, the second strand contains the capture sequence and can anneal with the first strand to form a double-stranded body; (2) Under conditions suitable for forming a linkage between the molecule or group A' and the molecule or group B', contact the first strand with the solid support, thereby obtaining a solid support linked with the first strand; and, (3) Under conditions allowing annealing, contact the free second strand with the solid support linked with the first strand formed in step (2), thereby obtaining a solid support linked with the capture probe; Or, (II) (1) Provide: (a) a duplex formed by annealing the first strand and the second strand of the capture probe, the first strand being linked with a molecule or group A', and the second strand containing the capture sequence; and, (b) a solid support, the surface of which is modified with a molecule or group B', and a linkage (e.g., covalent and / or non-covalent linkage) can be formed between the molecule or group A' and the molecule or group B'; and, (2) Under conditions suitable for forming a linkage between the molecule or group A' and the molecule or group B', contact the duplex with the solid support, thereby obtaining a solid support linked with the capture probe; Preferably, in the duplex formed by annealing the first strand and the second strand of the capture probe, the 3'-end of the second strand contains the capture sequence, and the capture sequence does not anneal with the first strand.

16. The method of claim 14 or 15, wherein, The molecule or group A' can undergo a click chemical reaction with the molecule or group B'; Preferably, the molecule or group A' / B' is selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, ene / amine, ene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone; Preferably, the molecule or group A' / B' is azide / alkynyl or alkynyl / azide; Preferably, the first strand of the capture probe is modified with (DBCO), the surface of the solid support is modified with an azide group, and the first strand of the capture probe and the solid support form a linkage through the click chemical reaction between DBCO and the azide group.

17. A method for preparing a nucleic acid array according to any one of claims 1-8, which comprises the following steps: (A) Provide a nucleic acid array to be processed, the nucleic acid array to be processed comprising a solid support, and at least two oligonucleotide molecules are linked to the solid support; Each oligonucleotide molecule occupies a different position on the solid support; The oligonucleotide molecule sequentially contains a positioning sequence and a first universal sequence from the 5'-end to the 3'-end, wherein the positioning sequence has a nucleotide sequence corresponding to the position of this oligonucleotide molecule on the solid support; the first universal sequence is as defined in any one of claims 1, 2, 5; and, (B) Link and / or synthesize a capture probe on the solid support comprised in the nucleic acid array to be processed, the capture probe being as defined in any one of claims 1, 3-5, 8.

18. The method of claim 17, wherein, The oligonucleotide molecule does not contain a capture sequence at the 3'-end of the first universal sequence, and the capture sequence is as defined in any one of claims 1, 3-5, 8.

19. The method of claim 17, wherein, The oligonucleotide molecule comprises a capture sequence at the 3'-end of the first universal sequence, the capture sequence being defined in any one of claims 1, 3-5, 8, and the method further comprises step (B'): removing the capture sequence contained in the oligonucleotide molecule; Wherein, steps (B) and (B') can be carried out in any order or simultaneously (for example, simultaneously in the same reaction system). The method of claim 19, wherein, In step (B'), the capture sequence contained in the oligonucleotide molecule is removed by steps including the following: (i) providing a blocking probe capable of annealing to (a) the first universal sequence of the oligonucleotide molecule or a partial sequence thereof (for example, the 3'-terminal partial sequence of the first universal sequence), or (b) the sequence in the oligonucleotide molecule located at the 5'-end of the capture sequence and at the 3'-end of the first universal sequence, or (c) a combination of (a) and (b); (ii) contacting the blocking probe with the nucleic acid array to be processed containing the oligonucleotide molecule under conditions suitable for annealing the blocking probe to the oligonucleotide molecule; (iii) contacting an exonuclease with the product of step (ii) under conditions allowing the exonuclease to exert its cleavage activity; wherein the exonuclease has 3'- to 5'-exonuclease activity for single-stranded nucleic acids; Preferably, the exonuclease is Exonuclease I; Preferably, step (B') further comprises the step of removing the blocking probe (for example, by denaturing the blocking probe from the oligonucleotide molecule to remove the blocking probe); Preferably, in the method, step (B) is carried out after step (B').

21. The method according to any one of claims 17 - 20, wherein The oligonucleotide molecule further comprises a second universal sequence, the second universal sequence being defined in claim 2 or 5; Preferably, the second universal sequence is located at the 5'-end of the positioning sequence; Preferably, the oligonucleotide molecule does not contain or further contains a MID sequence; Preferably, the oligonucleotide molecule further contains a MID sequence; preferably, the MID sequence is located at the 5'-end of the first universal sequence, and / or the MID sequence is located at the 3'-end of the second universal sequence; preferably, the MID sequences contained in the same kind of oligonucleotide molecules are different from each other.

22. The method according to any one of claims 17 - 21, wherein, In the nucleic acid array to be processed, the oligonucleotide molecule is covalently or non-covalently linked to the solid support; Preferably, the oligonucleotide molecule is covalently linked to the solid support; Preferably, the oligonucleotide molecule forms a linkage with the solid support through click chemistry; Preferably, the molecule or group pairs capable of undergoing the click chemistry are selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol; preferably, the molecule or group pair capable of undergoing the click chemistry is azide / alkynyl; Preferably, the surface of the solid support is modified with azide groups, and the oligonucleotide molecule is modified with (DBCO), and the oligonucleotide molecule forms a linkage with the solid support through a click chemical reaction between the azide group and the DBCO.

23. The method according to any one of claims 17-22, wherein, The capture probe exists in a single-stranded form containing a single-stranded region with a capture sequence, where the capture sequence is defined in any one of claims 1, 3-5, and 8, and step (B) includes: (1) Providing: (a) the free capture probe, where the capture probe is modified with a molecule or group A'; and (b) the nucleic acid array to be processed or the nucleic acid array processed by step (B'), where the surface of the solid support of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a linkage (e.g., covalent or non-covalent linkage) with the molecule or group B'; and, (2) Contacting the capture probe with the solid support under conditions suitable for forming a linkage between the molecule or group A' and the molecule or group B' to obtain a solid support linked with the capture probe; Preferably, the capture sequence is located at the 3'-end of the capture probe.

24. The method according to any one of claims 17-23, wherein, The capture probe exists in a double-stranded form containing a single-stranded region with a capture sequence, where the capture sequence is defined in any one of claims 1, 3-5, and 8, and step (B) includes: (I)(1) Providing: (a) the first strand of the free capture probe, which is modified with a molecule or group A'; (b) the nucleic acid array to be processed or the nucleic acid array processed by step (B'), where the surface of the solid support of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a linkage (e.g., covalent and / or non-covalent linkage) with the molecule or group B'; and (c) the second strand of the free capture probe, which contains the capture sequence and can anneal with the first strand to form a double-stranded body; (2) Contacting the first strand with the solid support under conditions suitable for forming a linkage between the molecule or group A' and the molecule or group B' to obtain a solid support linked with the first strand; and, (3) Contacting the free second strand with the solid support linked with the first strand formed in step (2) under annealing-permitting conditions to obtain a solid support linked with the capture probe; Or, (II)(1) Providing: (a) a double-stranded body formed by annealing the first strand and the second strand of the capture probe, where the first strand is linked with a molecule or group A' and the second strand contains the capture sequence; and (b) the nucleic acid array to be processed or the nucleic acid array processed by step (B'), where the surface of the solid support of the nucleic acid array is modified with a molecule or group B', and the molecule or group A' can form a linkage (e.g., covalent and / or non-covalent linkage) with the molecule or group B'; and, (2) Under conditions suitable for forming a linkage between the molecule or group A' and the molecule or group B', contact the double-stranded body with the solid support to obtain a solid support linked with the capture probe; Preferably, in the double-stranded body formed by annealing the first strand and the second strand of the capture probe, the 3'-end of the second strand contains the capture sequence, and the capture sequence does not anneal with the first strand.

25. The method of claim 23 or 24, wherein, The molecule or group A' is capable of undergoing a click chemical reaction with the molecule or group B'; Preferably, the molecule or group A' / B' is selected from: alkynyl / azide, azide / cyano, amine / ene, thiol / ene, thiol / alkyne, aldehyde / 1,3-diol, ketone / 1,3-diol, azide / alkynyl, cyano / azide, ene / amine, ene / thiol, alkyne / thiol, 1,3-diol / aldehyde, 1,3-diol / ketone; Preferably, the molecule or group A' / B' is azide / alkynyl or alkynyl / azide; Preferably, the first strand of the capture probe is modified with (DBCO), the surface of the solid support is modified with azide groups, and the first strand of the capture probe and the solid support form a linkage through the click chemical reaction between DBCO and azide groups.

26. A method for detecting the spatial information of nucleic acids in a sample, which comprises the following steps: (1) Provide a nucleic acid array according to any one of claims 1-8; (2) Under conditions allowing annealing, contact the nucleic acid array with the sample to be tested, so that the nucleic acids derived from the sample to be tested anneal with the capture sequences in the capture probes of the nucleic acid array; (3) Under conditions allowing nucleic acid polymerization, carry out a nucleic acid polymerization reaction to produce a first extension product, the first extension product containing the capture sequence and the complementary sequence of the nucleic acid molecule or its partial sequence that anneals with the capture sequence, and the first extension product is linked to the solid support through the capture sequence it contains; (4) Under conditions allowing annealing, the first extension product linked to the solid support anneals with the positioning probe of the adjacent nucleic acid array; Wherein, the first universal sequence of the positioning probe can anneal with the first extension product; and, the first universal sequence is located at the 3'-end of the positioning sequence of the positioning probe; (5) Under conditions allowing nucleic acid polymerization, carry out a nucleic acid polymerization reaction to produce a second extension product and / or its complementary strand, the second extension product containing: (a) the positioning sequence of the positioning probe and the complementary sequence of the first extension product or its partial sequence, or, (b) the sequence of the first extension product or its partial sequence, and the positioning probe The positioning sequence or its complementary sequence contained in the second extension product is used as its spatial information marker, and the complementary sequence of the positioning sequence or the positioning sequence contained in the complementary strand of the second extension product is used as its spatial information marker, so that the position of the nucleic acids derived from the sample to be tested in the sample to be tested is corresponding to the position of the positioning probe corresponding to the positioning sequence; thereby obtaining a nucleic acid molecule containing the spatial information marker; and (6) Analysis: (i) the nucleic acid molecule containing the spatial information label obtained in step (5), and / or (ii) the sequence of the nucleic acid molecule containing the spatial information label derived from (i); Preferably, the spatial information of the nucleic acid includes the location, distribution and / or abundance of the nucleic acid.

27. The method of claim 26, wherein, In step (3), the nucleic acid polymerization reaction uses the nucleic acid molecule annealed to the capture sequence as a template to extend the capture sequence; Preferably, the capture sequence is located at the 3'-end of the capture probe, and / or the 3'-end of the capture sequence has a free hydroxyl group (-OH).

28. The method of claim 26 or 27, wherein, In step (5), the polymerization reaction uses the first extension product as a template to extend the localization probe; and / or uses the localization probe as a template to extend the first extension product; For example, in step (5), the polymerization reaction uses the first extension product as a template to extend the localization probe to generate a second extension product and / or its complementary strand, and the second extension product contains the localization sequence of the localization probe and the complementary sequence of the first extension product or a part of its sequence; preferably, the first universal sequence of the localization probe is located at the 3'-end of the localization probe, and / or the 3'-end of the first universal sequence of the localization probe has a free hydroxyl group (-OH); For example, in step (5), the polymerization reaction uses the localization probe as a template to extend the first extension product to generate a second extension product and / or its complementary strand, and the second extension product contains the sequence of the first extension product or a part of its sequence and the complementary sequence of the localization sequence of the localization probe; preferably, the first universal sequence of the localization probe is located or not located at the 3'-end of the localization probe, and / or the 3'-end of the first universal sequence of the localization probe is blocked or unblocked; For example, in step (5), in the polymerization reaction, the first extension product and the localization probe are used as templates for each other to extend the first extension product and the localization probe to generate a second extension product and / or its complementary strand, and the second extension product contains the localization sequence of the localization probe and the complementary sequence of the first extension product or a part of its sequence, and the second extension product contains the sequence of the first extension product or a part of its sequence and the complementary sequence of the localization sequence of the localization probe; preferably, the first universal sequence of the localization probe is located at the 3'-end of the localization probe, and / or the 3'-end of the first universal sequence of the localization probe has a free hydroxyl group (-OH). The method according to any one of claims 26 - 28, wherein, In step (6), analyze the sequence of the nucleic acid molecule containing the spatial information label connected to the solid support of the nucleic acid array. The method according to any one of claims 26-28, wherein, Before step (6) and after step (5), the method further includes step pre-(6): releasing at least a part of the nucleic acid molecule containing the spatial information label from the surface of the nucleic acid array; Preferably, in step (6), analyze (i) the nucleic acid molecules containing the spatial information marker released in step pre-(6), and / or, (ii) the sequences of the nucleic acid molecules containing the spatial information marker derived from (i); Preferably, in step pre-(6), the nucleic acid molecules are released from the surface of the solid support by the following methods: (i) nucleic acid shearing; and / or, (ii) denaturation; Preferably, after step pre-(6) and before step (6), the method further includes a step of amplifying the released nucleic acid molecules; Preferably, before performing step (6), the method further includes a step of purifying the released nucleic acid molecules.

31. The method according to any one of claims 26-30, having one or more of the following features selected from: (i) In step (1), the nucleic acid array is provided by the method according to any one of claims 9-25; (ii) In step (2), the test sample is processed (e.g., permeabilization treatment or cell lysis treatment) to release the nucleic acids derived from the test sample and anneal them to the capture sequence; (iii) After step (3) and before step (4), the method further includes a step of removing the template strand bound to the first extension product (e.g., removing the template strand bound to the first extension product by enzymatic digestion or denaturation and strand separation); (iv) After step (3) and before step (4), the method further includes a step of washing the nucleic acid array to remove residual samples (e.g., tissues or cells); (v) After step (5) and before step (6) (e.g., after step (5) and before step pre-(6)), the method further includes a step of amplifying the nucleic acid molecules containing the spatial information marker (e.g., in situ amplification); preferably, the nucleic acid molecules containing the spatial information marker are linked to the solid support of the nucleic acid array. The method according to any one of claims 26-31, wherein, The sample is a tissue sample (e.g., tissue section) or a single-cell sample (e.g., single-cell suspension); Preferably, the tissue sample (e.g., tissue section) is prepared from fixed tissue, e.g., formalin-fixed paraffin-embedded (FFPE) tissue, cryopreserved tissue, or fresh tissue.

33. The method according to any one of claims 26 - 32, wherein, The nucleic acids derived from the test sample are selected from: RNA molecules (e.g., mRNA) molecules, target nucleic acid molecules (e.g., target DNA and / or RNA), genomic nucleic acid fragments in chromatin open regions, and any combination thereof.

34. The method according to any one of claims 26 - 33, wherein, In step (6), the analysis includes sequencing and / or sequence-specific PCR reactions; Preferably, before performing sequencing, the method further includes a step of constructing a sequencing library for the nucleic acid molecules containing the spatial information marker obtained in step (5) or their amplification products. The method according to any one of claims 26 to 34, wherein, The method is used to detect the spatial information of RNA (e.g., mRNA) in cells of a sample.

36. The method of claim 35, wherein, In step (2), the nucleic acids derived from the test sample are RNA (e.g., mRNA) in the cells of the test sample, and the capture sequence contains a poly(dT) sequence or a random oligonucleotide sequence; Preferably, step (3) includes: (a) Under conditions allowing nucleic acid polymerization, using a nucleic acid molecule annealed to the capture sequence as a template, performing a nucleic acid polymerization reaction to extend the capture sequence, generating a cDNA strand. The cDNA strand contains a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the capture sequence as a reverse transcription primer, and a 3' end overhang; (b) Under conditions allowing nucleic acid polymerization, annealing the template switching sequence to the cDNA strand generated in (a), and continuing the nucleic acid polymerization reaction using the template switching sequence as a template to generate the first extension product; Wherein, the template switching sequence contains a consensus sequence, a 3' end overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; The first extension product contains: the capture sequence, the cDNA strand sequence or a partial sequence thereof, and a complementary sequence of the consensus sequence; Preferably, before step (4), the method further includes a step of removing the template switching sequence bound to the first extension product (e.g., removing the template switching sequence bound to the first extension product by enzymatic digestion or denaturation and strand separation); Preferably, in step (4) of the method, the first universal sequence of the localization probe can anneal to the complementary sequence of the consensus sequence; Preferably, the localization probe does not contain an MID sequence, and the template switching sequence contains an MID sequence; or, the localization probe contains an MID sequence, the template switching sequence does not contain an MID sequence, or both the localization probe and the template switching sequence contain MID sequences. The method according to any one of claims 26 to 34, wherein, The method is used to detect the spatial information of the target nucleic acid (e.g., target DNA and / or RNA) of cells in a sample, and the target nucleic acid contains a target nucleotide sequence; Preferably, in step (2), the nucleic acid derived from the test sample contains the target nucleotide sequence; preferably, the nucleic acid derived from the test sample contains the target nucleic acid and / or a nucleic acid molecule derived from the target nucleic acid, and the nucleic acid molecule derived from the target nucleic acid contains the target nucleotide sequence; Preferably, in step (2), the capture sequence contains a sequence that specifically recognizes the target nucleotide sequence; Preferably, in step (2), the capture sequence contains a sequence that specifically recognizes the first segment of the target nucleotide sequence; in step (4), the first universal sequence of the localization probe contains a sequence that specifically recognizes the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence contains a random oligonucleotide sequence; preferably, in the target nucleotide sequence, the first segment is located at the 3' end of the second segment; Preferably, in step (2), the first segment is present in the single-stranded region of the nucleic acid molecule derived from the test sample that anneals to the capture sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested that anneals to the capture sequence is a single-stranded nucleic acid or double-stranded nucleic acid containing a single-stranded region comprising the first segment. The method according to any one of claims 26 to 34, wherein, The method is used to detect the spatial information of RNA (e.g., mRNA) and target nucleic acid (e.g., target DNA and / or RNA) in a sample, wherein the target nucleic acid comprises a target nucleotide sequence; Preferably, the nucleic acid array contains a first capture probe capable of capturing the RNA (e.g., mRNA), and a second capture probe capable of capturing a nucleic acid molecule containing the target nucleotide sequence; the first capture probe contains a first capture sequence, and the first capture sequence comprises a poly(dT) sequence or a random oligonucleotide sequence; the second capture probe contains a second capture sequence, and the second capture sequence comprises a sequence that specifically recognizes the target nucleotide sequence; Preferably, in step (2), the nucleic acid derived from the sample to be tested comprises RNA (e.g., mRNA) derived from the sample to be tested and a nucleic acid molecule containing the target nucleotide sequence; preferably, the nucleic acid molecule containing the target nucleotide sequence comprises the target nucleic acid and / or a nucleic acid molecule derived from the target nucleic acid; Preferably, step (3) includes: (A) (a) Under conditions allowing nucleic acid polymerization, using the nucleic acid molecule annealed to the first capture sequence as a template, performing a nucleic acid polymerization reaction to extend the first capture sequence, generating a cDNA strand, the cDNA strand comprising a cDNA sequence complementary to the RNA (e.g., mRNA) formed using the first capture sequence as a reverse transcription primer, and a 3'-end overhang; (b) Under conditions allowing nucleic acid polymerization, annealing a template switching sequence to the cDNA strand generated in (a), and continuing the nucleic acid polymerization reaction using the template switching sequence as a template to generate a first extension product I; Wherein, the template switching sequence comprises a consensus sequence, a 3'-end overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; And, (B) Under conditions allowing nucleic acid polymerization, using the nucleic acid molecule annealed to the second capture sequence as a template, performing a nucleic acid polymerization reaction to extend the second capture sequence, generating a first extension product II, the first extension product II comprising the second capture sequence and a complementary sequence of the nucleic acid molecule annealed to the second capture sequence or a partial sequence thereof; Wherein, step (B) and step (A) are carried out in any order; for example, step (B) is carried out before or after step (A), or step (B) and step (A) are carried out simultaneously (e.g., in the same reaction system); Preferably, in step (4), the positioning probe contains a first universal sequence I capable of annealing with the first extension product I and a first universal sequence II capable of annealing with the first extension product II; preferably, the first universal sequence I and the first universal sequence II coexist in the same positioning probe, or exist in different positioning probes respectively; Preferably, the nucleic acid array includes a first positioning probe containing the first universal sequence I and a second positioning probe containing the first universal sequence II; Preferably, in step (4) of the method, the first universal sequence I can anneal with the complementary sequence of the common sequence; Preferably, in step (2) of the method, the second capture sequence includes a sequence specifically recognizing the first segment of the target nucleotide sequence; in step (4), the first universal sequence II includes a sequence specifically recognizing the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II includes a random oligonucleotide sequence; preferably, in the target nucleotide sequence, the first segment is located at the 3'-end of the second segment; Preferably, before step (4) of the method, there is also a step of removing the template conversion sequence bound to the first extension product I (for example, removing the template conversion sequence bound to the first extension product I by enzymatic digestion or denaturation and strand separation); Preferably, in step (2), the first segment exists in the single-stranded region of the nucleic acid molecule derived from the test sample that anneals with the second capture sequence; Preferably, in step (2), the nucleic acid derived from the test sample that anneals with the second capture sequence is single-stranded nucleic acid or double-stranded nucleic acid containing a single-stranded region including the first segment; Preferably, the first positioning probe does not contain a MID sequence, and the template conversion sequence contains a MID sequence; or the first positioning probe contains a MID sequence, and the template conversion sequence does not contain a MID sequence, or both the first positioning probe and the template conversion sequence contain a MID sequence.

39. A kit, which includes the nucleic acid array according to any one of claims 1-8.

40. The kit of claim 39, wherein, The capture sequence of the nucleic acid array includes a poly(dT) sequence or a random oligonucleotide sequence; Preferably, the kit further includes a template conversion sequence, which includes a common sequence, a cDNA 3'-end overhang complementary sequence, and an optional MID sequence; preferably, the MID sequences included in each template conversion sequence are different from each other; preferably, the cDNA 3'-end overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides; preferably, the cDNA 3'-end overhang is an overhang of 2-5 cytosine nucleotides (for example, a CCC overhang); Preferably, the complementary sequence of the common sequence of the template conversion sequence can anneal with the first universal sequence of the positioning probe of the nucleic acid array; Preferably, the positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; alternatively, the positioning probe contains a MID sequence, and the template switching sequence does not contain a MID sequence; alternatively, both the positioning probe and the template switching sequence contain a MID sequence.

41. The kit of claim 39, wherein, The capture sequence of the nucleic acid array comprises a sequence that specifically recognizes a target nucleotide sequence contained in a target nucleic acid (e.g., a specific target DNA and / or RNA); Preferably, the first universal sequence comprises a sequence that specifically recognizes a complementary sequence of the target nucleotide sequence; Preferably, the capture sequence comprises a sequence that specifically recognizes a first segment of the target nucleotide sequence, and the first universal sequence of the positioning probe of the nucleic acid array comprises a sequence that specifically recognizes a complementary sequence of a second segment of the target nucleotide sequence, or the first universal sequence comprises a random oligonucleotide sequence; Preferably, in the target nucleotide sequence, the first segment is located at the 3'-end of the second segment.

42. The kit of claim 39, wherein, The nucleic acid array contains a first capture probe and a second capture probe; the first capture probe contains a first capture sequence, and the first capture sequence comprises a poly(dT) sequence or a random oligonucleotide sequence; the second capture probe contains a second capture sequence, and the second capture sequence comprises a sequence that specifically recognizes a target nucleotide sequence contained in a target nucleic acid (e.g., a specific target DNA and / or RNA); Preferably, the kit further comprises a template switching sequence, and the template switching sequence comprises a common sequence, a cDNA 3'-end overhang complementary sequence, and optionally a MID sequence; preferably, the MID sequences contained in each template switching sequence are different from each other; preferably, the cDNA 3'-end overhang has a length of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, 1-10, 1-5 or 2-10 nucleotides; preferably, the cDNA 3'-end overhang is a 2-5 cytosine nucleotide overhang (e.g., CCC overhang); Preferably, the positioning probe of the nucleic acid array comprises a first universal sequence I and a first universal sequence II; wherein, the first universal sequence I can anneal with a complementary sequence of the common sequence, and the first universal sequence II comprises a sequence that specifically recognizes a complementary sequence of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence; preferably, the first universal sequence I and the first universal sequence II coexist in the same positioning probe, or are present in different positioning probes respectively; Preferably, the nucleic acid array comprises a first positioning probe containing the first universal sequence I and a second positioning probe containing the first universal sequence II; preferably, the first positioning probe does not contain a MID sequence, and the template switching sequence contains a MID sequence; alternatively, the first positioning probe contains a MID sequence, the template switching sequence does not contain a MID sequence, or both the first positioning probe and the template switching sequence contain a MID sequence; Preferably, the second capture sequence comprises a sequence specifically recognizing the first segment of the target nucleotide sequence, and the first universal sequence II comprises a sequence specifically recognizing the complementary sequence of the second segment of the target nucleotide sequence, or the first universal sequence II comprises a random oligonucleotide sequence; preferably, in the target nucleotide sequence, the first segment is located at the 3'-end of the second segment. The kit according to any one of claims 39 to 42, wherein, The first universal sequence contained in the positioning probe of the nucleic acid array is located or not located at the 3'-end of the positioning probe, and / or the 3'-end of the first universal sequence of the positioning probe of the nucleic acid array is blocked or unblocked; Preferably, the kit further comprises: reagents for nucleic acid hybridization, reagents for nucleic acid extension, reagents for nucleic acid amplification, reagents for nucleic acid recovery or purification, reagents for constructing a transcriptome sequencing library, reagents for sequencing (such as next-generation sequencing or third-generation sequencing), or any combination thereof.

44. Use of the nucleic acid array according to any one of claims 1-8 or the kit according to any one of claims 39-43 for constructing a nucleic acid molecule library, for nucleic acid sequencing, or for detecting the nucleic acid spatial information in a sample.

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