WO2020228788A1 - 用于检测核酸空间信息的阵列及检测方法 - Google Patents

用于检测核酸空间信息的阵列及检测方法 Download PDF

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WO2020228788A1
WO2020228788A1 PCT/CN2020/090340 CN2020090340W WO2020228788A1 WO 2020228788 A1 WO2020228788 A1 WO 2020228788A1 CN 2020090340 W CN2020090340 W CN 2020090340W WO 2020228788 A1 WO2020228788 A1 WO 2020228788A1
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sequence
nucleic acid
acid molecule
complementary
vector
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English (en)
French (fr)
Inventor
陈奥
徐讯
杨晋
刘龙奇
王欧
黎宇翔
廖莎
唐国鑫
江媛
徐崇钧
倪鸣
章文蔚
德马纳克•拉多杰
德马纳克•斯内扎娜
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Shenzhen BGI Life Science Research Institute
MGI Tech Co Ltd
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Shenzhen BGI Life Science Research Institute
MGI Tech Co Ltd
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Priority to CN202411741947.8A priority Critical patent/CN119530347A/zh
Priority to US17/611,099 priority patent/US12516446B2/en
Priority to CN202411735916.1A priority patent/CN119530344A/zh
Priority to CA3140512A priority patent/CA3140512A1/en
Priority to CN202411741948.2A priority patent/CN119753087A/zh
Priority to CN202411740879.3A priority patent/CN119530346A/zh
Priority to BR112021022865-9A priority patent/BR112021022865B1/pt
Priority to SG11202112703QA priority patent/SG11202112703QA/en
Priority to JP2021568140A priority patent/JP2022540744A/ja
Priority to CN202080036185.5A priority patent/CN114207105B/zh
Priority to KR1020217041221A priority patent/KR20220009445A/ko
Priority to EP20806227.3A priority patent/EP3971282A4/en
Priority to CN202411741950.XA priority patent/CN119685448A/zh
Priority to CN202411741603.7A priority patent/CN119592665A/zh
Priority to MX2021013907A priority patent/MX2021013907A/es
Priority to AU2020274774A priority patent/AU2020274774C1/en
Application filed by Shenzhen BGI Life Science Research Institute, MGI Tech Co Ltd filed Critical Shenzhen BGI Life Science Research Institute
Priority to CN202411737361.4A priority patent/CN119530345A/zh
Priority to CN202411732967.9A priority patent/CN119592664A/zh
Publication of WO2020228788A1 publication Critical patent/WO2020228788A1/zh
Anticipated expiration legal-status Critical
Priority to JP2024126026A priority patent/JP2024156848A/ja
Priority to US19/397,772 priority patent/US20260193814A1/en
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q2533/00—Reactions characterised by the enzymatic reaction principle used
    • C12Q2533/10—Reactions characterised by the enzymatic reaction principle used the purpose being to increase the length of an oligonucleotide strand
    • C12Q2533/101—Primer extension

Definitions

  • the invention relates to the field of biomolecule spatial detection. Specifically, the present invention provides a method for detecting spatial information of nucleic acids in a sample, a nucleic acid array used in the method, and a method for producing the nucleic acid array.
  • the spatial location of cells in a tissue significantly affects their functions. In order to explore this spatial heterogeneity, it is necessary to quantify and analyze the cell's genome or transcriptome when the spatial coordinates are known. However, collecting small tissue regions or even single cells for genome or transcriptome analysis is very laborious, costly, and low precision. Therefore, it is very necessary to develop a method that can achieve single-cell or even subcellular level and high-throughput detection of spatial information (for example, nucleic acid location, distribution and/or expression) of biomolecules (for example nucleic acids).
  • spatial information for example, nucleic acid location, distribution and/or expression
  • biomolecules for example nucleic acids
  • the prior art combines array technology with high-throughput DNA sequencing technology to capture and locate nucleic acids in labeled tissue samples, and sequence and analyze them.
  • a probe capable of capturing nucleic acid is immobilized on the chip by spotting or bead-mediated.
  • the active area size of the chip obtained by using the micro-volume spotting system for the droplet plane spotting method is as high as 200 microns, and the cell observation accuracy is only 20 cells; use the beads with positioning markers for plane spreading beads
  • the size of the active area of the chip obtained in this way is up to 10 microns, and the cell observation accuracy can only reach a single cell, and the positioning of sub-cells cannot be achieved.
  • the present invention provides a novel nucleic acid array for nucleic acid spatial detection, a preparation method thereof, and a nucleic acid spatial detection method based on the array, which can simultaneously realize high-precision subcellular positioning and high-throughput tissue positioning, and has important applications value.
  • the present invention provides a method for generating a nucleic acid array for detecting spatial information of nucleic acids in a biological sample, the method comprising the following steps:
  • each vector sequence contains multiple copies of the vector sequence, and the vector sequence from 5'to 3'includes a positioning sequence and a first fixed sequence,
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction
  • the first primer includes a first fixed sequence complement at its 3'end
  • the complementary region of the first fixed sequence includes the complementary sequence of the first fixed sequence or a fragment thereof, and has a 3'free end.
  • the vector sequence and the first nucleic acid molecule are single-stranded nucleic acid sequences. In some embodiments, the vector sequence and the first nucleic acid molecule are single-stranded DNA sequences.
  • step (3) while performing the extension reaction, the vector sequence is sequenced to obtain the sequence information of the positioning sequence contained in the vector sequence.
  • step (1) multiple vector sequences are provided through the following steps:
  • each vector template sequence as a template, perform a nucleic acid amplification reaction to obtain an amplified product of each vector template sequence, and the amplified product contains multiple copies of the vector sequence.
  • the amplification is selected from rolling circle replication (RCA), bridge PCR amplification, multiple strand displacement amplification (MDA), or emulsion PCR amplification.
  • rolling circle replication is performed to obtain DNB formed by concatemers of the vector sequence.
  • a circular template sequence is provided in step (i).
  • the method of preparing circular nucleic acid molecules is a conventional method in the art, and those skilled in the art can choose according to needs.
  • a linear nucleic acid template can be obtained first, and then circularization of the linear nucleic acid template can be realized by a ligase (such as DNA ligase).
  • bridge PCR amplification, emulsion PCR amplification, or multiple strand displacement amplification is performed to obtain DNA clusters in the form of clonal populations of the vector sequence.
  • the method further includes the following steps:
  • the capture sequence can hybridize with all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or , (C) a specific sequence for a specific target nucleic acid; and, the capture sequence has a 3'free end so that the second nucleic acid molecule can serve as an extension primer,
  • the second nucleic acid molecule is a single-stranded nucleic acid sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded DNA sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded RNA sequence.
  • the second nucleic acid molecule comprises a fixed region and a capture sequence in the 5'to 3'direction, and the fixed region comprises a double-stranded nucleic acid sequence, such as a double-stranded DNA sequence.
  • the capture sequence contained in the second nucleic acid molecule is a single-stranded nucleic acid sequence, such as a single-stranded DNA sequence or a single-stranded RNA sequence. It is easy to understand that in such embodiments, the second nucleic acid molecule has a partially double-stranded structure, that is, its fixed region has a double-stranded structure, and the capture sequence has a single-stranded structure.
  • the length of the double-stranded nucleic acid sequence is 1 bp-50 bp, for example, 10 bp-50 bp, 10 bp-40 bp, or 10 bp-30 bp.
  • the method further includes the following steps:
  • the second nucleic acid molecule comprising a second fixed sequence complementary sequence and a capture sequence from 5'to 3';
  • the complementary sequence of the second fixed sequence allows hybridization to its complementary nucleotide sequence
  • the capture sequence can hybridize with all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or , (C) a specific sequence for a specific target nucleic acid; and, the capture sequence has a 3'free end so that the second nucleic acid molecule can be used as an extension primer;
  • connect the first nucleic acid molecule and the second nucleic acid molecule respectively hybridized to the carrier sequence for example, use a ligase to connect the second nucleic acid molecule to the first nucleic acid molecule.
  • each vector sequence further comprises a second fixed sequence at its 5' end, which allows annealing of the nucleotide sequence complementary to it.
  • the second immobilized sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction (for example, it can serve as a binding site for bridge PCR primers).
  • the second nucleic acid molecule is a single-stranded nucleic acid sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded DNA sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded RNA sequence.
  • the second fixed sequence is adjacent to the positioning sequence.
  • the length of the second fixed sequence is 1bp-50bp, for example, 10bp-50bp, 10bp-40bp, 10bp-30bp, or 10bp-20bp.
  • the first primer further includes a unique molecular identifier (UMI) sequence at the 5'end of the complementary region of the first fixed sequence, so that the first nucleic acid molecule
  • UMI unique molecular identifier
  • the second nucleic acid molecule further includes the UMI sequence, and the UMI sequence is located at the 5'end of the capture sequence;
  • the UMI sequence is composed of at least 1 (for example, at least 2, at least 3, at least 4, or at least 5; for example, 5-100, 5-50, 5-20, such as 10) N
  • Each N is independently any one of A, C, G, and T.
  • the first primer when the first primer includes a unique molecular identifier (UMI) sequence at the 5'end of the complementary region of the first fixed sequence, the first primer may be at the 5'end of the UMI sequence.
  • UMI unique molecular identifier
  • the oligonucleotide sequence capable of capturing mRNA includes a sequence capable of hybridizing to the poly-A tail of the mRNA. In certain embodiments, the oligonucleotide sequence capable of capturing mRNA includes a poly-T oligonucleotide sequence. In certain embodiments, the poly-T oligonucleotide sequence includes at least 10 (e.g., at least 20) deoxythymidine residues.
  • the solid support is a chip. In some embodiments, the solid support can be used as a sequencing platform, such as a sequencing chip. In certain embodiments, the solid support is a high-throughput sequencing chip, such as a high-throughput sequencing chip used in Illumina, MGI, or Thermo Fisher sequencing platforms.
  • the present invention provides a method for generating a nucleic acid array for detecting spatial information of nucleic acids in a biological sample, the method comprising the following steps:
  • each vector sequence contains multiple copies of the vector sequence
  • the vector sequence from 5'to 3'in includes: capture template sequence, positioning sequence and first fixed sequence
  • the capture template sequence includes the complementary sequence of the capture sequence, which can hybridize to all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) Random or degenerate oligonucleotide sequence; or, (c) specific sequence for specific target nucleic acid;
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence is allowed to anneal to its complementary nucleotide sequence and initiate an extension reaction; and, the first fixed sequence also includes a cleavage site, and the cleavage may be selected from a nicking enzyme.
  • Resection USER restriction, photoresection, chemical resection or CRISPR resection;
  • the first primer includes a binding region, a resection region and a first fixed sequence complementary region in the 5'to 3'direction, and the first fixed sequence is complementary
  • the region includes the complementary sequence of the first fixed sequence or a fragment thereof, and has a 3'free end;
  • the binding region includes a linker that can be connected to the surface of the solid support;
  • the excision region includes a cleavage site;
  • the first primer as a primer and the vector sequence as a template
  • a primer extension reaction is performed, so that the first fixed sequence, positioning sequence, and capture template sequence region of the vector sequence form a double-stranded strand.
  • the hybridized strand is the first nucleic acid molecule, and the first nucleic acid molecule includes a first fixed sequence complementary sequence, a positioning sequence complementary sequence, and a capture sequence from 5'to 3'; the first nucleic acid molecule can also be called Capture probe
  • step (3) Optionally cut the cleavage site contained in the first fixed sequence of the vector sequence to digest the vector sequence, so that the extension product in step (3) and the template for forming the extension product (ie, the vector The sequence) is separated, so that the first nucleic acid molecule (capture probe) is attached to the surface of a solid support (such as a chip).
  • a solid support such as a chip
  • the capture probe that is, the first nucleic acid molecule
  • the capture probe includes the same type of capture probe (the carrier sequence) on the array
  • the position of the capture probe on the array can be determined by analyzing the complementary sequence of the positioning sequence.
  • each vector sequence refers to a vector sequence containing the same positioning sequence.
  • step (1) multiple vector sequences are provided through the following steps:
  • each vector template sequence as a template, perform a nucleic acid amplification reaction to obtain an amplified product of each vector template sequence, and the amplified product contains multiple copies of the vector sequence.
  • the amplification is selected from rolling circle replication (RCA), bridge PCR amplification, multiple strand displacement amplification (MDA), or emulsion PCR amplification.
  • rolling circle replication is performed to obtain DNB formed by concatemers of the vector sequence.
  • a circular template sequence is provided in step (i).
  • the method of preparing circular nucleic acid molecules is a conventional method in the art, and those skilled in the art can choose according to needs.
  • a linear nucleic acid template can be obtained first, and then circularization of the linear nucleic acid template can be realized by a ligase (such as DNA ligase).
  • each vector sequence is a DNB formed by a concatemer of multiple copies of the vector sequence.
  • step (1) includes the following steps:
  • the circular nucleic acid template includes a vector template sequence
  • the vector template sequence includes the complementary sequence of the vector sequence, that is, the vector template sequence is in the 5'to 3'direction
  • the above includes the first fixed sequence complementary sequence, the positioning sequence complementary sequence and the capture sequence
  • Rolling circle amplification is performed using the circular nucleic acid template as a template to obtain nanospheres (DNB) formed by concatemers of the carrier sequence.
  • bridge PCR amplification, emulsion PCR amplification, or multiple strand displacement amplification is performed to obtain DNA clusters in the form of clonal populations of the vector sequence.
  • the cleavage site included in the first fixed sequence is a nicking site.
  • the nicking enzyme is selected from USER, BamHI, BmtI and the like.
  • the restriction site is as shown in SEQ ID NO: 14.
  • the first fixed sequence further includes a sequencing primer hybridization region and/or an amplification primer hybridization region; wherein the sequencing primer hybridization region allows the sequencing primer to anneal and initiate a sequencing reaction, and the amplification The primer hybridization region allows the amplification primer to anneal and initiate extension and amplification reactions.
  • the length of the first fixed sequence is greater than 1 bp, such as greater than 10 bp, or greater than 20 bp. In some embodiments, the length of the first fixed sequence is 20-100 bp, such as 20-80 bp.
  • the length of the positioning sequence is greater than 1 bp, such as greater than 10 bp. In certain embodiments, the length of the positioning sequence is 10-100 bp, such as 10-50 bp, such as 10-30 bp, such as 20 bp.
  • the oligonucleotide sequence capable of capturing mRNA includes a sequence capable of hybridizing to the poly-A tail of the mRNA. In certain embodiments, the oligonucleotide sequence capable of capturing mRNA includes a poly-T oligonucleotide sequence. In certain embodiments, the poly-T oligonucleotide sequence includes at least 10 (e.g., at least 20) deoxythymidine residues.
  • the capture sequence is greater than 1 bp in length. In some embodiments, the capture sequence is 1-100 bp in length, such as 10-50 bp, such as 10-30 bp.
  • the vector sequence further includes a UMI complementary sequence (also referred to as a probe tag region) located downstream of the capture template sequence and upstream of the first fixed sequence, which is similar to UMI
  • the sequence is complementary, and the UMI sequence is composed of at least 1 (for example, at least 2, at least 3, at least 4, or at least 5; for example, 5-100, 5-50, 5-20, such as 10).
  • a nucleotide sequence composed of N, each N is independently any one of A, C, G and T.
  • the UMI complementary sequence is located between the positioning sequence and the capture template sequence.
  • the UMI complementary sequence is located between the first fixed sequence and the positioning sequence.
  • step (3) when the primer extension reaction is performed using the carrier sequence as a template, the first nucleic acid molecule/capture probe that hybridizes to the carrier sequence will correspondingly contain the UMI Sequence (can also be called probe tag).
  • the template sequence of the vector sequence includes a UMI template sequence at a corresponding position, and the UMI template sequence is composed of modified bases Sequence, the modified base can form a hydrogen bond complementary pairing with a variety of main bases (for example, C, G, A, T, U), such as Inosine, which can be complementary paired with bases A, C, and U.
  • main bases for example, C, G, A, T, U
  • Inosine which can be complementary paired with bases A, C, and U.
  • the UMI template sequence includes a plurality of Inosines (e.g., at least 10, e.g., 10-100).
  • the length of the UMI template sequence is greater than 1 bp.
  • the length of the UMI template sequence is greater than 5 bp.
  • the length of the UMI template sequence is 5-100 bp, such as 5-50 bp, such as 5-20 bp, such as 5-15 bp, such as 10 bp.
  • the solid support is a chip. In some embodiments, the solid support can be used as a sequencing platform. In certain embodiments, the solid phase support is a sequencing chip (made by MGI), such as a sequencing chip of the BGISEQ-500 platform. In some embodiments, the solid support is a high-density array chip, which can be obtained, for example, as described in the patent CN103180496B.
  • the carrier sequence (eg, DNB) can be attached to the surface of the solid support by any suitable method known in the art.
  • non-limiting examples of the methods include nucleic acid hybridization, biotin-streptavidin binding, sulfhydryl binding, photoactivated binding, covalent binding, antibody-antigen, via hydrogel or other porous Physical limitations of the polymer, etc., or any combination thereof.
  • the solid support is selected from the following materials: glass, silicon, polylysine coating materials, nitrocellulose, polystyrene, cyclic olefin copolymers (COCs), cyclic olefins Polymers (COPs), polypropylene, polyethylene or polycarbonate, etc.
  • step (3) while performing the primer extension reaction, the vector sequence (for example, the positioning sequence contained therein) is sequenced to obtain the positioning sequence contained in the vector sequence The sequence information.
  • the vector sequence for example, the positioning sequence contained therein
  • step (3) before step (3), it further includes a step of sequencing the vector sequence (for example, the positioning sequence contained therein). In some embodiments, after sequencing is completed, washing is performed to remove dNTPs added to the synthetic strand due to sequencing.
  • the linker is a linking group capable of coupling with an activating group (e.g., NH 2 ).
  • the surface of the solid support is modified with an activating group (e.g., NH 2 ).
  • the linker includes -SH, -DBCO, -NHS, and the like.
  • the linker is DBCO, and the surface of the solid support has 8-NHS ester.
  • the cleavage site contained in the excision region of the first primer is a site where controlled cleavage can be performed by chemical, enzymatic or photochemical methods.
  • the cleavage site is a restriction site.
  • the cleavage site is a USER enzyme cleavage site (UUU).
  • the excision region of the first primer is different from the cleavage site included in the first fixed sequence of the vector sequence.
  • the amplification comprises PCR.
  • the present invention provides a nucleic acid array for detecting spatial information of nucleic acids in a sample, which includes a solid support (such as a chip) with multiple carrier sequences attached to the surface, and each carrier sequence is in the array.
  • a solid support such as a chip
  • Each vector sequence contains multiple copies of the vector sequence, and the vector sequence from 5'to 3'includes a positioning sequence and a first fixed sequence,
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction.
  • the diameter of each carrier sequence (that is, the carrier sequence containing the same positioning sequence) on the surface of the solid support (that is, the active area) is less than 1 micron, For example, about 900 nanometers, about 800 nanometers, about 700 nanometers, about 600 nanometers, or about 500 nanometers.
  • the nucleic acid array further comprises a first nucleic acid molecule, and the first nucleic acid molecule in a direction from 5'to 3'comprises: a first fixed sequence complementary sequence and a positioning sequence complementary sequence, and the The first fixed sequence of the vector sequence and the positioning sequence hybridize to form a double strand. It is easy to understand that only the first fixed sequence complementary sequence and the positioning sequence complementary sequence in the first nucleic acid molecule are complementary to the corresponding sequence of the carrier sequence to form a double strand, so the double strand formed by the first fixed sequence and the carrier sequence It is an incomplete double-stranded structure, that is, a partial double-stranded structure.
  • each copy of the vector sequence in each vector sequence includes a first nucleic acid molecule that hybridizes to it.
  • the vector sequence and the first nucleic acid molecule are single-stranded nucleic acid sequences. In some embodiments, the vector sequence and the first nucleic acid molecule are single-stranded DNA sequences.
  • the nucleic acid array further includes a second nucleic acid molecule that is linked to the first nucleic acid molecule to be immobilized to the nucleic acid array, and the second nucleic acid molecule includes a capture sequence;
  • the capture sequence can hybridize with all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or , (C) a specific sequence for a specific target nucleic acid; and, the capture sequence has a 3'free end so that the second nucleic acid molecule can be used as an extension primer.
  • each first nucleic acid molecule is linked to the second nucleic acid molecule.
  • the 5'end of the second nucleic acid molecule is connected to the 3'end of the first nucleic acid molecule.
  • the nucleic acid array further includes a second nucleic acid molecule that hybridizes with the carrier sequence to be immobilized to the nucleic acid array.
  • each vector sequence further comprises a second fixed sequence at its 5'end, which allows annealing of the nucleotide sequence complementary to it;
  • the second nucleic acid molecule includes a second fixed sequence complementary sequence and a capture sequence from 5'to 3'direction; the second fixed sequence complementary sequence hybridizes with the second fixed sequence of the carrier sequence to form a double strand;
  • the capture sequence can hybridize with all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or , (C) a specific sequence for a specific target nucleic acid; and, the capture sequence has a 3'free end so that the second nucleic acid molecule can be used as an extension primer.
  • each copy of the vector sequence in each vector sequence includes a second nucleic acid molecule that hybridizes to it.
  • the second immobilized sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction (for example, it can serve as a binding site for bridge PCR primers).
  • the second fixed sequence is adjacent to the positioning sequence.
  • the second nucleic acid molecule has a 5' end modification.
  • the modification is phosphorylation or biotin modification.
  • the second nucleic acid molecule is a single-stranded nucleic acid sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded DNA sequence. In certain embodiments, the second nucleic acid molecule is a single-stranded RNA sequence.
  • the vector sequence of the multiple copies is an amplification product formed by amplification of the complementary sequence of the vector sequence as a template, and the amplification is selected from rolling circle replication (RCA), bridge PCR amplification, multiple strand displacement amplification (MDA) or emulsion PCR amplification.
  • RCA rolling circle replication
  • MDA multiple strand displacement amplification
  • emulsion PCR amplification emulsion PCR amplification
  • the multiple copies of the vector sequence are DNBs formed by concatemers of the vector sequence. In some embodiments, the multiple copies of the vector sequence are DNBs formed by rolling circle replication using the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by clonal populations of the vector sequence.
  • the multiple copies of the vector sequence are DNA clusters formed by bridge PCR amplification using the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by emulsion PCR amplification of the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by multiple strand displacement amplification using the complementary sequence of the vector sequence as a template.
  • the first nucleic acid molecule further comprises a unique molecular identifier (UMI) sequence, the UMI sequence is located at the 5'end of the complementary sequence of the first fixed sequence; or, the second nucleic acid molecule further comprises UMI sequence, the UMI sequence is located at the 5'end of the capture sequence;
  • UMI unique molecular identifier
  • the UMI sequence is composed of at least 1 (for example, at least 2, at least 3, at least 4, or at least 5; for example, 5-100, 5-50, 5-20, such as 10) N
  • Each N is independently any one of A, C, G, and T.
  • the solid support is a chip. In some embodiments, the solid support can be used as a sequencing platform, such as a sequencing chip. In certain embodiments, the solid support is a high-throughput sequencing chip, such as a high-throughput sequencing chip used in Illumina, MGI, or Thermo Fisher sequencing platforms.
  • the oligonucleotide sequence capable of capturing mRNA includes a sequence capable of hybridizing to the poly-A tail of the mRNA. In certain embodiments, the oligonucleotide sequence capable of capturing mRNA includes a poly-T oligonucleotide sequence. In certain embodiments, the poly-T oligonucleotide sequence includes at least 10 (e.g., at least 20) deoxythymidine residues.
  • the length of the positioning sequence is greater than 1 nt, such as greater than 5 nt. In some embodiments, the length of the positioning sequence is 5-50 nt, such as 10-50 nt, 10-30 nt, or 20-30 nt. In some embodiments, the length of the positioning sequence contained in the different vector sequences may be the same or different.
  • the length of the capture sequence is greater than 1 nt. In certain embodiments, the capture sequence is 1-100 nt in length, such as 1-50 nt, such as 10-30 nt.
  • the length of the first fixed sequence is greater than 1 nt, such as greater than 10 nt. In some embodiments, the length of the first fixed sequence is 10-200 nt. In some embodiments, the length of the first fixed sequence is 20-100 nt, such as 20-50 nt.
  • the length of the second fixed sequence is greater than 1 nt, such as greater than 10 nt. In certain embodiments, the length of the second fixed sequence is 10-200 nt, such as 10-100 nt, 10-50 nt, 10-30 nt, or 10-20 nt.
  • the present invention provides a kit for detecting spatial information of nucleic acids in a sample, comprising: (i) the nucleic acid array according to the third aspect, wherein the nucleic acid array does not include a second nucleic acid molecule And, (ii) a second nucleic acid molecule, the second nucleic acid molecule comprising a fixed region and a capture sequence from 5'to 3'direction;
  • the capture sequence can hybridize with all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) a random or degenerate oligonucleotide sequence; or , (C) a specific sequence for a specific target nucleic acid; and, the capture sequence has a 3'free end so that the second nucleic acid molecule can be used as an extension primer.
  • the kit includes: (i) a nucleic acid array, which includes a solid support (such as a chip) with multiple carrier sequences attached to the surface, each carrier sequence occupies a different position in the array, Each vector sequence contains multiple copies of the vector sequence, and the vector sequence from 5'to 3'includes a positioning sequence and a first fixed sequence,
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction
  • the nucleic acid array further includes a first nucleic acid molecule, the first nucleic acid molecule from 5'to 3'direction includes: a first fixed sequence complementary sequence and a positioning sequence complementary sequence, and is first fixed with the carrier sequence The sequence and the positioning sequence hybridize to form a double strand;
  • a second nucleic acid molecule the fixed region of the second nucleic acid molecule comprises a double-stranded DNA sequence.
  • the kit further comprises a ligase.
  • the kit includes: (i) a nucleic acid array, which includes a solid support (such as a chip) with multiple carrier sequences attached to the surface, each carrier sequence occupies a different position in the array
  • a nucleic acid array which includes a solid support (such as a chip) with multiple carrier sequences attached to the surface, each carrier sequence occupies a different position in the array
  • Each vector sequence contains multiple copies of the vector sequence, and the vector sequence from 5'to 3'includes: a second fixed sequence, a positioning sequence and a first fixed sequence,
  • the second fixed sequence allows annealing of its complementary nucleotide sequence
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction
  • the nucleic acid array further includes a first nucleic acid molecule, the first nucleic acid molecule from 5'to 3'direction includes: a first fixed sequence complementary sequence and a positioning sequence complementary sequence, and is first fixed with the carrier sequence The sequence and the positioning sequence hybridize to form a double strand;
  • a second nucleic acid molecule the fixed region of the second nucleic acid molecule includes a second fixed sequence complementary sequence.
  • the second fixed sequence is adjacent to the positioning sequence.
  • the second nucleic acid molecule described in (ii) can hybridize with the complementary region of the carrier sequence contained in the nucleic acid array described in (i), and the second nucleic acid molecule can then be combined with a ligase.
  • the nucleic acid molecule is linked to the first nucleic acid molecule. Therefore, in certain embodiments, the kit further comprises a ligase.
  • the present invention also relates to the use of the nucleic acid array according to the third aspect or the kit according to the fourth aspect for detecting spatial information of nucleic acids in a sample, or in preparing spatial information for detecting nucleic acids in a sample.
  • detection reagents used to detect spatial information of nucleic acids in a sample.
  • the spatial information of the nucleic acid includes the location, distribution and/or expression of the nucleic acid.
  • the sample is a tissue sample, such as a tissue sample containing cells.
  • the sample is a tissue section.
  • the tissue section is prepared from fixed tissue, for example, formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.
  • FFPE formalin-fixed paraffin-embedded
  • the present invention also relates to a nucleic acid array for detecting spatial information of nucleic acids in a sample, which includes a solid support (such as a chip) with multiple carrier sequences attached to the surface, and each carrier sequence is Each carrier sequence occupies different positions in the array, and each vector sequence contains multiple copies of the vector sequence, and the vector sequence from 5'to 3'includes: a capture template sequence, a positioning sequence and a first fixed sequence, wherein,
  • the capture template sequence includes the complementary sequence of the capture sequence, which can hybridize to all or part of the nucleic acid to be captured, and includes: (a) an oligonucleotide sequence capable of capturing mRNA; and/or, (b) Random or degenerate oligonucleotide sequence; or, (c) specific sequence for specific target nucleic acid;
  • the positioning sequence has a unique nucleotide sequence corresponding to the position of the vector sequence on the array
  • the first fixed sequence allows annealing of its complementary nucleotide sequence and initiates an extension reaction, and the first fixed sequence also includes a cleavage site, and the cleavage may be selected from nicking enzyme enzymes.
  • Resection USER restriction, photoresection, chemical resection or CRISPR resection;
  • the nucleic acid array also includes a first nucleic acid molecule (also referred to as a capture probe), and the first nucleic acid molecule in a direction from 5'to 3'includes a binding region, a resection region, and a carrier sequence complementary region,
  • the binding region includes a linker that can be connected to the surface of the solid support
  • the resection zone includes a cutting site
  • the carrier sequence complementary region includes a sequence that can be complementary to the carrier sequence, and from 5'to 3', it includes: a first fixed sequence complementary sequence, a positioning sequence complementary sequence, and a capture sequence; and, the capture sequence Having a 3'free end so that the first nucleic acid molecule can be used as an extension primer;
  • the complementary region of the vector sequence of the first nucleic acid molecule hybridizes with the vector sequence to form a double strand.
  • the vector sequence and the first nucleic acid molecule are single-stranded nucleic acid sequences. In some embodiments, the vector sequence and the first nucleic acid molecule are single-stranded DNA sequences.
  • each copy of the vector sequence in each vector sequence includes the first nucleic acid molecule described above that hybridizes to it.
  • the linker of the first nucleic acid molecule is a linking group capable of coupling with an activation group (such as NH 2 ), and the solid support has an activation group (such as NH 2 ) on the surface Retouch.
  • the linker includes -SH, -DBCO, or -NHS.
  • the linker is (DBCO), the surface of the solid support is connected with ( 8-NHS ester).
  • the cleavage site included in the first fixed sequence is a nicking site.
  • the nicking enzyme is selected from USER, BamHI, BmtI and the like.
  • the restriction site is as shown in SEQ ID NO: 14.
  • the cleavage site contained in the excision region of the first nucleic acid molecule is a site where controlled cleavage can be performed by chemical, enzymatic or photochemical methods.
  • the cleavage site is a restriction site.
  • the cleavage site is a USER enzyme cleavage site (UUU).
  • the excision region of the first nucleic acid molecule is different from the cleavage site contained in the first fixed sequence of the vector sequence.
  • the nucleic acid array is prepared by the method described in the second aspect.
  • the vector sequence of the multiple copies is an amplification product formed by amplification of the complementary sequence of the vector sequence as a template, and the amplification is selected from rolling circle replication (RCA), bridge PCR amplification, multiple strand displacement amplification (MDA) or emulsion PCR amplification.
  • RCA rolling circle replication
  • MDA multiple strand displacement amplification
  • emulsion PCR amplification emulsion PCR amplification
  • the multiple copies of the vector sequence are DNBs formed by concatemers of the vector sequence. In some embodiments, the multiple copies of the vector sequence are DNBs formed by rolling circle replication using the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by clonal populations of the vector sequence.
  • the multiple copies of the vector sequence are DNA clusters formed by bridge PCR amplification using the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by emulsion PCR amplification of the complementary sequence of the vector sequence as a template.
  • the multiple copies of the vector sequence are DNA clusters formed by multiple strand displacement amplification using the complementary sequence of the vector sequence as a template.
  • the vector sequence further comprises a UMI complementary sequence located downstream of the capture template sequence and upstream of the first fixed sequence, which is complementary to the UMI sequence, and the UMI sequence is composed of at least 1 (for example, at least 2, at least 3, at least 4, or at least 5; for example, 5-100, 5-50, 5-20, such as 10) N nucleotide sequence, each Each N is independently any one of A, C, G and T;
  • the carrier sequence complementary region of the first nucleic acid molecule further includes a UMI sequence located upstream of the capture sequence and downstream of the first fixed sequence complementary sequence.
  • the UMI complementary sequence is located between the positioning sequence and the capture template sequence, or between the first fixed sequence and the positioning sequence.
  • the vector sequence of each copy of each vector sequence (that is, a vector sequence containing the same positioning sequence) has UMI complementary sequences that are different from each other.
  • the first nucleic acid molecule (capture probe) that hybridizes to each copy of the vector sequence also has UMI sequences different from each other.
  • the vector sequence is removed from the nucleic acid array through the cleavage site contained in the first fixed sequence of the vector sequence.
  • the nucleic acid array includes a solid support (such as a chip) to which a plurality of capture probes (first nucleic acid molecules) are attached to the surface, and each capture probe (first nucleic acid molecule) is The array occupies different positions and is oriented to have 3'free ends so that the capture probe (first nucleic acid molecule) can be used as an extension primer, wherein each capture probe (first nucleic acid molecule) ranges from 5'to 3'
  • the direction includes: binding region, excision region, positioning sequence complementary sequence and capture sequence, where,
  • the binding region includes a linker that can be connected to the surface of the solid support
  • the resection zone includes a cutting site
  • the positioning sequence corresponds to the position of the capture probe on the array
  • the capture sequence can hybridize to all or part of the nucleic acid to be captured, and includes: (1a) an oligonucleotide sequence capable of capturing mRNA; and/or, (1b) a random or degenerate oligonucleotide sequence; or , (C) Specific sequence for specific target nucleic acid.
  • each of the capture probes ie, capture probes containing the same positioning sequence/positioning sequence complementary sequence
  • each of the capture probes has UMI sequences that are different from each other, and the UMI sequence Located upstream of the capture sequence and downstream of the excision zone.
  • the UMI sequence is located at the 5' end of the capture sequence, for example between the capture sequence and the complementary sequence of the positioning sequence.
  • the UMI sequence is located at the 5'end of the complementary sequence of the positioning sequence, for example, between the excision region and the complementary sequence of the positioning sequence.
  • each carrier sequence that is, a carrier sequence containing the same positioning sequence
  • each capture probe that is, a capture probe containing the same positioning sequence/sequence complementary to the positioning sequence
  • the diameter of the area occupied by the surface of the solid support is less than 1 micrometer, for example, about 900 nanometers, about 800 nanometers, about 700 nanometers, about 600 nanometers, or about 500 nanometers. In certain embodiments, the diameter of the active region of each carrier sequence or each capture probe is about 500 nanometers.
  • the solid support is a chip. In some embodiments, the solid support can be used as a sequencing platform. In some embodiments, the solid phase support is a sequencing chip (made by MGI), such as the BGISEQ-500 platform. In some embodiments, the solid support is a high-density array chip, which can be obtained, for example, as described in the patent CN103180496B.
  • the length of the first fixed sequence is greater than 1 bp, such as greater than 10 bp, or greater than 20 bp. In some embodiments, the length of the first fixed sequence is 20-100 bp, such as 20-80 bp.
  • the length of the positioning sequence is greater than 1 bp, such as greater than 10 bp. In certain embodiments, the length of the positioning sequence is 10-100 bp, such as 10-50 bp, such as 10-30 bp, such as 20 bp.
  • the oligonucleotide sequence capable of capturing mRNA includes a sequence capable of hybridizing to the poly-A tail of the mRNA. In certain embodiments, the oligonucleotide sequence capable of capturing mRNA includes a poly-T oligonucleotide sequence. In certain embodiments, the poly-T oligonucleotide sequence includes at least 10 (e.g., at least 20) deoxythymidine residues.
  • the capture sequence is greater than 1 bp in length. In some embodiments, the capture sequence is 1-100 bp in length, such as 10-50 bp, such as 10-30 bp.
  • the present invention provides a method for detecting spatial information of nucleic acids in a sample, which includes the following steps:
  • the nucleic acid array includes multiple carrier sequences attached to the surface of a solid support (such as a chip), each carrier sequence occupies a different position in the array, and each carrier sequence includes multiple copies of the carrier sequence;
  • Each vector sequence includes a first nucleic acid molecule and a second nucleic acid molecule hybridized therewith, and the first nucleic acid molecule and the second nucleic acid molecule are not connected to each other;
  • the first nucleic acid molecule includes a positioning sequence complementary sequence corresponding to the position of the vector sequence on the array,
  • the second nucleic acid molecule includes a capture sequence capable of capturing nucleic acid in the sample
  • the first and second nucleic acid molecules connected are used as primers, and the captured nucleic acid molecule is used as a template to perform a primer extension reaction to produce an extension product, which hybridizes with the captured nucleic acid molecule.
  • the complementary sequence of the positioning sequence contained in the first nucleic acid molecule as a spatial information marker; and/or,
  • the captured nucleic acid molecule is used as a primer, and the first and second connected nucleic acid molecules are used as templates to perform a primer extension reaction to produce an extended captured nucleic acid molecule, which has all
  • the positioning sequence is used as a spatial information mark;
  • the second nucleic acid molecule as a primer and using the captured nucleic acid molecule as a template to perform a primer extension reaction under conditions that allow primer extension to produce an extended second nucleic acid molecule.
  • the extended second nucleic acid molecule contains the complementary sequence of the captured nucleic acid; the first nucleic acid molecule hybridized to each vector sequence and the extended second nucleic acid molecule (for example, using a ligase) are connected to the first nucleic acid molecule
  • the linked extended second nucleic acid molecule has the complementary sequence of the positioning sequence contained in the first nucleic acid molecule as a spatial information marker;
  • the first nucleic acid molecule and the second nucleic acid molecule on the nucleic acid array are not connected.
  • the present invention provides a method for detecting spatial information of nucleic acids in a sample, which includes the following steps:
  • the nucleic acid array includes multiple carrier sequences attached to the surface of a solid support (such as a chip), each carrier sequence occupies a different position in the array, and each carrier sequence includes multiple copies of the carrier sequence;
  • Each vector sequence contains a first nucleic acid molecule hybridized to it, and the first nucleic acid molecule is linked to a second nucleic acid molecule;
  • the first nucleic acid molecule includes a positioning sequence complementary sequence corresponding to the position of the vector sequence on the array,
  • the second nucleic acid molecule includes a capture sequence capable of capturing nucleic acid in the sample
  • the captured nucleic acid molecule is used as a primer, and the first and second connected nucleic acid molecules are used as templates to perform a primer extension reaction to produce an extended captured nucleic acid molecule, which has all
  • the positioning sequence is used as a spatial information mark;
  • the first nucleic acid molecule and the second nucleic acid molecule on the nucleic acid array have been connected.
  • the multiple copies of the vector sequence are DNBs formed by the concatemers of the vector sequences, or the multiple copies of the vector sequence It is a DNA cluster formed by a clonal population of the vector sequence.
  • the vector sequence and the first nucleic acid molecule are single-stranded DNA.
  • the second nucleic acid molecule is single-stranded DNA or single-stranded RNA.
  • the present invention provides a method for detecting spatial information of nucleic acids in a sample, which includes the following steps:
  • nucleic acid array (1) Provide the nucleic acid array according to the fifth aspect, or obtain the nucleic acid array by the method according to the second aspect; wherein the nucleic acid array comprises a plurality of carrier sequences attached to the surface of a solid support (such as a chip) , Each vector sequence occupies a different position in the array, and each vector sequence contains multiple copies of the vector sequence;
  • Each vector sequence includes a first nucleic acid molecule hybridized therewith, and the first nucleic acid molecule includes a positioning sequence complementary sequence corresponding to the position of the vector sequence on the array and a capture sequence capable of capturing nucleic acid in the sample;
  • the method before step (2), further includes cleaving the cleavage site contained in the first fixed sequence of the vector sequence to digest the vector sequence and simultaneously A nucleic acid molecule (capture probe) is attached to the surface of a solid support (such as a chip).
  • the nucleic acid array includes a plurality of capture probes attached to the surface of a solid support (e.g., a chip), each capture probe occupies a different position in the array, and the capture probe
  • the needle includes a positioning sequence complementary sequence corresponding to the position of the capture probe on the array and a capture sequence capable of capturing nucleic acid in the sample;
  • the capture probe as a primer and the captured nucleic acid molecule as a template to perform a primer extension reaction
  • the resulting extension product contains the complementary sequence of the positioning sequence as a spatial information marker and The complementary sequence of the captured nucleic acid molecule, thereby generating the DNA molecule of the spatial information marker; optionally, generating the complementary strand of the DNA molecule of the spatial information marker, and/or optionally, amplifying the DNA of the spatial information marker molecular;
  • the first nucleic acid molecule and capture probe are DNA molecules, such as single-stranded DNA.
  • the spatial information of the nucleic acid includes the location, distribution and/or expression of the nucleic acid.
  • the sample is a tissue sample, such as a tissue section.
  • the tissue section is prepared from fixed tissue, for example, formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.
  • FFPE formalin-fixed paraffin-embedded
  • the method is for non-diagnostic use.
  • any nucleic acid analysis method can be used in step (5).
  • this step may include sequencing.
  • sequence-specific analysis methods can be used. For example, a sequence-specific amplification reaction can be performed, such as by using a primer, and the primer is specific to the localization domain and/or a specific target sequence (for example, a specific target DNA to be tested). Therefore, in certain embodiments, this step may include a sequence-specific PCR reaction.
  • the sequence analysis information obtained in step (5) can be used to obtain spatial information (ie, position information) of the nucleic acid in the sample.
  • the spatial information can be inferred from the nature of the sequence analysis information measured, for example, it can reveal the existence of a specific nucleic acid, and the nucleic acid may have space in the environment of the sample being tested.
  • Information meaning, and/or spatial information can be inferred by combining the position of the sample on the array and the sequencing information. Therefore, the method may only include the step of associating the sequence analysis information with a certain position of the sample, for example, using a positioning mark and its association with a certain position on the sample.
  • the method of any one of the sixth to eighth aspects further includes step (6): Associating the sequence analysis information obtained in step (5) with the image of the sample, wherein the sample is Image before or after step (3).
  • the imaging of the sample uses light, bright field, dark field, phase difference, fluorescence, mirror image, interference or confocal microscope, or a combination thereof.
  • the method is used to detect the transcriptome in a sample.
  • step (3)(i) using the connected first and second nucleic acid molecules as reverse transcription primers, cDNA molecules are synthesized from the captured RNA molecules, and the cDNA molecules are provided with the first The complementary sequence of the positioning sequence contained in the nucleic acid molecule is used as a spatial information marker, and optionally, the cDNA molecule is amplified; or, in step (3)(ii), the second nucleic acid molecule is used as a reverse transcription primer, Synthesize a cDNA molecule from the captured RNA molecule, ligate the first nucleic acid molecule hybridized to each vector sequence and the cDNA molecule (for example, using a ligase) to generate a complementary sequence with the positioning sequence contained in the first nucleic acid molecule As a spatial information marker, and optionally, amplify the cDNA molecule; and, in step (4), release at least a part
  • the method is used to detect the transcriptome in a sample.
  • step (3)(iii) using the connected first and second nucleic acid molecules as reverse transcription primers, cDNA molecules are synthesized from the captured RNA molecules, and the cDNA molecules are provided with the first
  • the complementary sequence of the positioning sequence contained in the nucleic acid molecule is used as a spatial information marker, and optionally, the cDNA molecule is amplified; and, in step (4), at least a part of the cDNA molecule and/or it is released from the array surface An amplicon, wherein the released nucleic acid molecule may be the first and/or second strand of a cDNA molecule or an amplicon thereof, and wherein the part comprises a spatial information sequence or a complementary strand thereof.
  • the capture sequence includes an oligonucleotide sequence capable of capturing mRNA.
  • the method is used to detect the transcriptome in a sample.
  • step (3) using the capture probe as an RT primer, cDNA molecules are synthesized from the captured RNA molecules, and the cDNA molecules are provided with spatial information markers, and optionally, amplified The cDNA molecule; in step (4), at least a portion of the cDNA molecule and/or amplicon thereof is released from the surface of the array, wherein the released nucleic acid molecule may be the first and/or second of the cDNA molecule A strand or an amplicon thereof, and wherein the portion includes a spatial information marker sequence or a complementary strand thereof.
  • the capture sequence includes an oligonucleotide sequence capable of capturing mRNA.
  • the spatial information-labeled nucleic acid molecule such as a DNA molecule
  • the spatial information-labeled cDNA molecule is released from the array surface, The complementary strand or the second cDNA strand is generated.
  • the second-strand DNA (for example, cDNA) synthesis step can be performed in situ on the array, can be a separate second-strand synthesis step, or can be used as the initial step of the amplification reaction.
  • the first strand of DNA e.g. cDNA
  • the second strand synthesis can be performed, for example, reaction in a solution , Can be used as a separate step or as a step in an amplification reaction.
  • the method may include an optional step of removing the captured nucleic acid molecules (such as RNA) before the second-strand synthesis, such as RNA nitration Enzyme (RNase), such as RNase H.
  • RNase RNA nitration Enzyme
  • the operation process of this step is well known and documented in the art. However, this step is generally unnecessary, and in most cases, RNA will degrade naturally.
  • the step of removing the sample from the array generally also removes the RNA from the array.
  • the second strand of DNA (e.g., cDNA) is produced in a single reaction, and second strand synthesis can be achieved by any suitable method known in the art.
  • the first strand of cDNA released from the array is then used to perform a DNA synthesis reaction using random primers (such as hexamer primers) and DNA polymerase (such as strand displacement polymerase) using the first strand as a template. Therefore, in certain embodiments, the synthesis of the complementary strand or the second strand uses random primers and strand displacement polymerase.
  • the step of amplifying the nucleic acid molecule (such as a DNA molecule) or cDNA molecule labeled with the spatial information is further included before the sequence analysis.
  • the amplification step is performed after the spatial information-labeled nucleic acid molecules (such as DNA molecules) or cDNA molecules are released from the array, or the amplification step is performed in situ on the array (ie , When the first nucleic acid molecule and/or carrier sequence and/or capture probe are still attached to the surface of the solid support).
  • the amplification step includes PCR.
  • step (4) the molecule is released from the surface of the array by the following methods: (i) nucleic acid shearing; (ii) denaturation; and /Or (iii) physical methods.
  • the molecule is released by applying hot water or a buffer to the solid support.
  • the step of purifying the released molecules is further included before sequencing.
  • a step of replenishing the sample is further included.
  • the method before step (4), further includes a step of washing the array to remove residual samples (e.g., tissue).
  • residual samples e.g., tissue
  • the array includes at least one orientation mark to orient the samples on the array.
  • the sequence analysis step includes a sequencing step.
  • the sequencing step includes a sequencing reaction based on a reversible stain terminator.
  • the method for detecting spatial information of nucleic acid can be used for RNA detection, transcriptome analysis, DNA detection, and genome analysis. Spatial information is of great significance to transcriptomics and genomics related research, especially useful in the study of transcriptome or genomic variation in different cells or regions of tissues, such as comparing normal and diseased cells or tissues, or studying disease processes Transcriptome or genome changes, etc.
  • the pathophysiological analysis of Alzheimer’s disease shows that the pathological process involves the interaction of neurons and glial cells, and related transcriptome and epigenome studies have also found that the brain of Alzheimer’s patients Neuronal function is severely damaged, and the innate immune response is abnormal.
  • population-level research cannot reveal the complexity of changes between cells and within cell populations, especially for those rare cell types.
  • Research at the ordinary single-cell level cannot distinguish the characteristics of specific cell types in different tissue regions at the same period from the changes in cell composition during neurodegeneration. Therefore, in order to further reveal the pathogenic mechanism and development mode of diseases, it is urgent to obtain single-cell transcriptome information with spatial dimensions.
  • the method for detecting spatial information of nucleic acid can fix the nucleic acid molecules in different regions of the brain tissue sample to the chip through the capture sequence with position markers connected to the chip, and perform sequencing, In this way, transcriptome results containing accurate location information can be obtained to detect changes in specific cell types in different regions during Alzheimer's disease.
  • the active area of DNB or DNA clusters on the chip of the present invention is as low as nanometers, and the cell diameter is about 12um, the chip of the present invention can realize spatial positioning information with subcellular resolution.
  • Item 1 A method for generating a nucleic acid array for detecting spatial information of biomolecules (such as nucleic acids) in a sample, the method comprising the following steps:
  • a circular nucleic acid template comprising a template sequence of a capture probe, the template sequence comprising a linker region, a spatial tag region and a capture region in the 5'to 3'direction;
  • the linker region includes a cleavage site, and the cleavage can be selected from nicking enzyme cleavage, USER cleavage, photoresection, chemical resection, or CRISPR resection;
  • the spatial tag region includes a spatial tag sequence, and the spatial tag sequence corresponds to the position of the capture probe on the array;
  • the capture area includes a capture sequence capable of capturing biological molecules (such as nucleic acid) in a sample; wherein the capture sequence includes: (1a) an oligonucleotide sequence capable of capturing mRNA; and/or, (1b) random or simple The combined oligonucleotide sequence; or, (c) a specific sequence for a specific target molecule (such as a target nucleic acid);
  • the probe primer includes a binding region, a resection region and a primer adapter region in the 5'to 3'direction;
  • the binding region includes a linker capable of connecting with the surface of the solid support
  • the resection zone includes a cutting site
  • the primer linker region is complementary to all or part of the linker region sequence of the template complementary sequence contained in the DNB (ie, the complementary sequence of the template sequence linker region), and has a 3'free end, so that the The probe primer can be used as a primer to initiate an extension reaction; preferably, the primer linker region contains the sequence of the linker region of the template sequence or a fragment thereof;
  • step (4) Cut the cleavage site contained in the linker region to digest the DNB, so that the extension product in step (4) is separated from the template DNB that forms the extension product, thereby connecting the capture probe to the solid phase carrier (such as chip) surface;
  • the circular nucleic acid template, DNB and capture probe are DNA;
  • each circular nucleic acid template contains different template sequences of capture probes, so as to obtain a solid-phase carrier with multiple capture probes attached to the surface ( For example chip).
  • Item 2 The method of item 1, wherein the cleavage site contained in the linker region is a nicking enzyme cleavage site;
  • the nicking enzyme is selected from USER, BamHI and BmtI.
  • Item 3 The method of item 1 or 2, wherein the adapter region further includes a sequencing primer hybridization region and/or an amplification primer hybridization region; wherein the sequencing primer hybridization region allows the sequencing primer to anneal and initiate a sequencing reaction, so The hybridization region of the amplification primer allows the amplification primer to anneal and initiate extension and amplification reactions.
  • Item 4 The method of any one of items 1 to 3, wherein the oligonucleotide sequence capable of capturing mRNA includes a sequence capable of hybridizing with the poly-A tail of the mRNA;
  • the oligonucleotide sequence capable of capturing mRNA includes a poly-T oligonucleotide sequence
  • the poly-T oligonucleotide sequence includes at least 10 (for example, at least 20) deoxythymidine residues.
  • Item 5 The method of any one of items 1 to 4, wherein the template sequence further comprises a probe tag region located upstream of the capture region and downstream of the linker region, and the probe tag region includes modified
  • the probe tag complementary sequence composed of bases, the modified bases can form hydrogen bond complementary pairings with a variety of main bases (for example, C, G, A, T, U);
  • the probe tag area is located between the space tag area and the capture area, or between the linker area and the space tag area;
  • the probe tag complementary sequence includes multiple (for example, at least 10) Inosines.
  • Item 6 The method of any one of items 1-5, which has one or more of the following characteristics:
  • the length of the linker region is greater than 1 bp, for example, greater than 10 bp, or greater than 20 bp; preferably, the length of the linker region is 20-100 bp;
  • the length of the space tag region is greater than 1 bp, for example, greater than 10 bp; preferably, the length of the space tag region is 10-100 bp;
  • the length of the capture area is greater than 1 bp; preferably, the length of the capture area is 1-100 bp;
  • the length of the probe tag region is greater than 1 bp, for example, greater than 5 bp; preferably, the length of the probe tag region is 5-100 bp.
  • Item 7 The method of any one of items 1-6, wherein the solid phase carrier is a chip;
  • the solid phase carrier can be used as a sequencing platform, such as a sequencing chip.
  • Item 8 The method of any one of items 1-7, wherein in step (4), the complementary sequence of the spatial tag sequence is sequenced while the primer extension reaction is performed, so as to obtain the corresponding capture probe Contains the sequence information of the spatial tag sequence.
  • Item 9 The method of any one of items 1-7, wherein, before step (4), further comprising the step of sequencing the complementary sequence of the spatial tag sequence contained in the DNB;
  • washing is performed to remove dNTPs added to the synthetic strand due to sequencing.
  • Item 10 The method of any one of items 1-9, wherein the linker is a linking group capable of coupling with an activating group (such as NH 2 ), and the surface of the solid support has an activating group (such as NH 2 ) Modification;
  • the linker includes -SH, -DBCO or -NHS;
  • Item 11 The method of any one of items 1-10, wherein the cleavage site contained in the excision zone is a site that can be controlled cleavage by chemical, enzymatic or photochemical methods;
  • the cutting site is a restriction site
  • the cutting sites included in the resection zone and the linker zone are different.
  • Item 12 The method of any one of items 1-11, wherein the amplification includes PCR.
  • Item 13 A nucleic acid array prepared by the method of any one of items 1-12.
  • a nucleic acid array for detecting spatial information of biomolecules (such as nucleic acids) in a sample which includes a solid-phase carrier (such as a chip) with multiple capture probes attached to the surface, and each capture probe is in the The array occupies different positions and is oriented to have 3'free ends so that the capture probes can be used as extension primers, wherein each capture probe in the direction from 5'to 3'includes: binding zone, excision zone, space Tag sequence and capture sequence, where
  • the binding region includes a linker capable of connecting with the surface of the solid support
  • the resection zone includes a cutting site
  • the spatial tag sequence corresponds to the position of the capture probe on the array
  • the capture sequence can hybridize with all or part of a biological molecule (such as a nucleic acid) to be captured, and includes: (1a) an oligonucleotide sequence capable of capturing mRNA; and/or, (1b) a random or degenerate oligonucleotide A nucleotide sequence; or (c) a specific sequence for a specific target molecule (for example, a target nucleic acid).
  • a biological molecule such as a nucleic acid
  • Item 15 The nucleic acid array of Item 14, wherein each of the capture probes (ie, capture probes containing the same spatial tag sequence) in each of the capture probes has different probe tag sequences from each other, and The probe tag sequence is located upstream of the capture sequence and downstream of the cleavage region;
  • the probe tag sequence is located between the capture sequence and the spatial tag sequence, or between the cleavage region and the spatial tag sequence.
  • Item 16 The nucleic acid array of Item 14 or 15, wherein the area occupied by each of the capture probes (ie, capture probes containing the same spatial tag sequence) on the surface of the solid support (ie, the activity The diameter of the area) is less than 1 micron;
  • the diameter of the active region of each capture probe is about 500 nanometers.
  • Item 17 The nucleic acid array of any one of Items 14-16, wherein the solid phase carrier is a chip;
  • the solid phase carrier can be used as a sequencing platform, such as a sequencing chip.
  • Item 18 The nucleic acid array of any one of items 14-17, wherein the nucleic acid array is prepared by the method of any one of items 1-12.
  • Item 19 A method for detecting spatial information of biomolecules in a sample, which includes the following steps:
  • nucleic acid array of any one of items 13-18, or obtain the nucleic acid array by the method of any one of items 1-12;
  • the nucleic acid array includes multiple captures attached to the surface of a solid phase carrier (such as a chip) Probes, each capture probe occupies a different position in the array, and the capture probe contains a spatial tag sequence corresponding to the position of the capture probe on the array and can capture organisms in the sample Molecular capture sequence;
  • Item 20 A method for detecting the spatial information of nucleic acids in a sample, which includes the following steps:
  • the nucleic acid array comprises a nucleic acid array connected to the surface of a solid phase carrier (such as a chip)
  • a capture probe each capture probe occupies a different position in the array, and the capture probe contains a spatial tag sequence corresponding to the position of the capture probe on the array and can capture the sample The capture sequence of the nucleic acid;
  • the spatial information of the nucleic acid includes the location, distribution and/or expression of the nucleic acid
  • the capture probe is a DNA molecule
  • the sample is a tissue sample, such as a tissue section;
  • the tissue section is prepared from fixed tissue, for example, formalin-fixed paraffin-embedded (FFPE) tissue or deep-frozen tissue.
  • FFPE formalin-fixed paraffin-embedded
  • Item 21 The method of item 20, in step (5), the sequence analysis includes sequencing or sequence-specific PCR reaction.
  • Item 22 The method of Item 20 or 21, the method further comprising step (6): Associating the sequence analysis information obtained in step (5) with the image of the sample, wherein the sample is before or after step (3) Imaging.
  • Item 23 The method of any one of Items 20-22, which is used to detect the transcriptome in a sample, wherein:
  • step (3) using the capture probe as an RT primer, synthesize a cDNA molecule from the captured RNA molecule, the cDNA molecule is provided with a space tag label, and optionally, the cDNA molecule is amplified;
  • step (4) at least a part of the cDNA molecules and/or amplicons thereof are released from the surface of the array, wherein the released nucleic acid molecules may be the first and/or second strands of the cDNA molecules or the amplification thereof ⁇ , and wherein said part comprises a spatial tag sequence or its complement;
  • the capture sequence includes an oligonucleotide sequence capable of capturing mRNA.
  • Item 24 The method of any one of items 20-23, wherein the complementary strand or the cDNA is generated before or after the space tag-labeled DNA molecule or the space tag-labeled cDNA molecule is released from the array surface Second chain
  • the synthesis of the complementary strand or the second strand uses random primers and strand displacement polymerase.
  • Item 25 The method of any one of items 20-24, wherein before the sequence analysis, it further comprises the step of amplifying the DNA molecules or cDNA molecules marked by the spatial tags;
  • the amplification step is performed after the DNA or cDNA molecules labeled by the space tag are released from the array, or the amplification step is performed in situ on the array;
  • the amplification step includes PCR.
  • Item 26 The method of any one of items 20-25, wherein the sequence analysis further includes a step of purifying the released molecules.
  • Item 27 The method of any one of items 20-26, before step (4), the method further comprises a step of washing the array to remove residual samples (for example, tissue).
  • residual samples for example, tissue
  • Item 28 The method of any one of items 20-27, in step (4), the molecule is released from the surface of the array by the following methods: (i) nucleic acid shearing; (ii) denaturation; and/or (iii) physical method;
  • the molecule is released from the excision zone of the capture probe by restriction enzyme cleavage.
  • Item 29 The method of any one of items 20-28, in step (6), the sample is imaged using light, bright field, dark field, phase difference, fluorescence, mirror image, interference or confocal microscope or Its combination.
  • the present invention provides a novel array for detecting spatial information of nucleic acid and a preparation method thereof.
  • the nucleic acid array is applied to the detection of spatial information of nucleic acid, high-precision subcellular positioning and high-throughput tissue positioning can be realized at the same time.
  • the array of the present invention and the detection method based on the array have great application value in cell positioning, subcellular positioning, organelle positioning, cell interaction, organelle interaction, molecular pathway research, disease diagnosis and the like.
  • Figure 1 shows a schematic diagram of the structure of cDNA synthesis after capturing mRNA in Example 2.
  • Figure 2 shows a schematic diagram of the structure of the molecule released from the chip in Example 2.
  • Figure 3 shows the results of 2100 detection of cDNA fragment distribution in Example 2.
  • Figure 4 shows the matching result of the 25bp sequence of one strand obtained by cDNA sequencing in Example 3 and the fq of the positioning sequence on the capture chip.
  • Figure 5 shows a graph of mRNA expression in tissue sections in Example 3.
  • FIG. 6 shows a schematic diagram of the probe primer and the vector sequence structure contained in the DNB of an exemplary embodiment in Example 4.
  • FIG. 7 shows a schematic diagram of the probe structure connected on the chip in Embodiment 4.
  • Figure 8 shows a schematic diagram of the structure of the captured nucleic acid molecule in Example 5 after cDNA synthesis.
  • Figure 9 shows a schematic diagram of the structure of the molecule released from the chip in Example 5.
  • Figure 10 shows the results of 2100 detection of cDNA fragment distribution in Example 5.
  • Figure 11 shows the matching result of the one-stranded 20bp sequence obtained by cDNA sequencing in Example 6 with the fq of the positioning sequence on the capture chip.
  • Figure 12 shows a graph of the expression of mRNA in the tissue section in Example 6.
  • linker A SEQ ID NO: 1
  • N location sequence complementary sequence
  • N represents any base, such as C, G, A or T
  • CTGATAAGGTCGCCA second fixed sequence complementary sequence, SEQ ID NO :2
  • CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT connector B, SEQ ID NO: 3'.
  • the linker A includes a partial sequence of the complementary sequence of the first fixed sequence and a circularization site
  • the linker B includes another part of the complementary sequence of the first fixed sequence, a restriction site, and a circularization site.
  • DNA nanosphere (DNB) preparation configure the following reaction system 40ul, put in 80fmol of the above-mentioned DNA library, DNB primer sequence is GGCCTCCGACTTAAGTCGGATCGT (SEQ ID NO: 4), synthesized by BGI Liuhe.
  • the reaction conditions are as follows: 95°C for 3 minutes, 40°C for 3 minutes; after the reaction, place on ice, and add 40ul DNBSEQ sequencing kit of DNB preparation required mixed enzyme I, 2ul mixed enzyme II, and 1ul ATP (mother liquor 100mM, Thermo Fisher), 0.1ul T4 ligase (BGI self-produced), after mixing, put the above reaction system in a PCR machine at 30°C for 20 minutes to form DNB. Load the DNB on the BGISEQ500 sequencing chip according to the method described in the BGISEQ500SE50 kit.
  • Sequencing and decoding of the positioning sequence According to the BGISEQ500 SE50 sequencing kit instructions, the positioning sequence is decoded and sequenced, and the sequencing length is set to 25bp. The fq file formed by sequencing is stored for later use.
  • Chip dicing Cut the prepared chip into several small pieces. The size of the slice is adjusted according to the needs of the experiment. The chip is soaked in a 50mM pH8.0 tris buffer and prepared at 4°C for later use.
  • mRNA capture According to the size of the sliced tissue, take a suitable size of the chip prepared in Example 1 and place it at room temperature. After the liquid on the chip has evaporated, use the temperature difference between the sliced tissue and the chip in the microtome to attach the sliced tissue to the capture chip . Place the pasted tissue section at room temperature, add 5 ⁇ SSC reaction solution (full coverage of the tissue-attached area) on the chip, and react at 30°C for 30 minutes to fully hybridize the mRNA in the tissue with the capture area on the chip.
  • 5 ⁇ SSC reaction solution full coverage of the tissue-attached area
  • the spatial positioning area is connected with the capture area.
  • wash the chip twice with 5 ⁇ SSC configure the following reaction system 1ml, pump an appropriate volume into the chip, ensure that the chip is filled with the following ligation reaction solutions, and fill in the nick shown in Figure 1. React at room temperature for 30 minutes. After the reaction, the chip was cleaned with 5 ⁇ SSC at a temperature of 55° C., and cleaned 3 times, 5 min each time.
  • cDNA release After the cDNA strand is synthesized on the chip, add an appropriate amount of formamide solution to the chip and react at 55°C for 10 minutes to release the cDNA strand from the chip.
  • the released molecules have the structure shown in Figure 2. Collect the reaction solution released from the chip, use 2 ⁇ XP magnetic beads to purify the cDNA strand, and finally use 45ul TE buffer (Thermo fisher) to recover the product.
  • the qubit ssDNA detection kit was used to quantitatively detect cDNA single strands.
  • reaction system on the PCR machine and set the following reaction program: 95°C for 3min, 11 cycles (98°C for 20s, 58°C for 20s, 72°C for 3min), 72°C for 5min, 4°C ⁇ .
  • reaction program 95°C for 3min, 11 cycles (98°C for 20s, 58°C for 20s, 72°C for 3min), 72°C for 5min, 4°C ⁇ .
  • XP beads for purification and recovery of magnetic beads.
  • the dsDNA concentration was quantified using the qubit kit, and the cDNA fragment distribution was detected by 2100.
  • the 2100 test results are shown in Figure 3.
  • the cDNA length is normal.
  • Tn5 interrupted According to the cDNA concentration, take 20ng cDNA, add 0.5uM Tn5 interrupting enzyme and corresponding buffer (Tn5 interrupting enzyme coating method according to stLFR library building kit operation), mix well to form a 20ul reaction system, react at 55°C for 10min , Add 5ul 0.1% SDS and mix at room temperature for 5 minutes to end the Tn5 interruption step.
  • the reaction conditions are as follows: 95°C for 3 minutes, 40°C for 3 minutes; after the reaction, place on ice, and add 40ul DNBSEQ sequencing kit of DNB preparation required mixed enzyme I, 2ul mixed enzyme II, and 1ul ATP (mother liquor 100mM, Thermo Fisher), 0.1ul T4 ligase (BGI self-produced), after mixing, put the above reaction system in a PCR machine at 30°C for 20 minutes to form DNB.
  • Select the PE50 sequencing model in which the one-strand sequencing is divided into two segments and sequenced 25bp and then 15cycle dark After the reaction, 10bp UMI sequence is measured again, and the second chain is set to 50bp.
  • the fixed sequence 1 includes a partial sequence of the complementary sequence of the first fixed sequence and a circularization site
  • the fixed sequence 2 includes a partial sequence of the complementary sequence of the first fixed sequence and a circularization site.
  • DNA nanosphere (DNB) preparation configure the following reaction system 40ul, put into 80fmol of the above-mentioned DNA library, the DNB primer sequence is GACATGGCTACGTGTGAGCCAAGG (SEQ ID NO: 16), synthesized by BGI Liuhe.
  • the reaction conditions are as follows: 95°C for 3 minutes, 40°C for 3 minutes; after the reaction, place on ice, and add 40ul DNBSEQ sequencing kit of DNB preparation required mixed enzyme I, 2ul mixed enzyme II, and 1ul ATP (mother liquor 100mM, Thermo Fisher), 0.1ul T4 ligase (BGI self-produced), after mixing, put the above reaction system in a PCR machine at 30°C for 20 minutes to form DNB. Load the DNB on the BGISEQ500 sequencing chip according to the method described in the BGISEQ500SE50 kit.
  • the chip surface modification was carried out according to the following method: NHS-PEG8-Azido (564.58g/mol) concentration was 45 ⁇ M, and 100ml method was prepared:
  • DBCO-primer concentration is 1uM, diluted with PBS
  • DBCO connecting group-UUU (USER restriction site)TTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG (SEQ ID NO: 17, the sequence includes the first fixed sequence complementary sequence, PCR amplification site sequence, intermediate sequence). Dilute 1uM of the above primer probe sequence with PBS and pass it on the modified azido chip, and react at room temperature for 1h or overnight.
  • NNNNNNNNNNNNNNNNNNNNN positioning sequence complementary sequence, which is the same as the positioning sequence template sequence in the DNA library sequence in step 1
  • NNNNNNNNNN UI sequence, which It is the complementary sequence of the random base sequence obtained from the UMI template sequence in step 1 as a template
  • TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT (capture sequence, SEQ ID NO: 20).
  • mRNA capture According to the size of the sliced tissue, take a suitable size of the chip prepared in Example 4 and place it at room temperature. After the liquid on the chip has evaporated, use the temperature difference between the sliced tissue and the chip in the microtome to attach the sliced tissue to the capture chip on. Place the pasted tissue section at room temperature, add 5 ⁇ SSC reaction solution (full coverage of the tissue-attached area) on the chip, and react at 30°C for 30 minutes to fully hybridize the mRNA in the tissue with the capture area on the chip.
  • 5 ⁇ SSC reaction solution full coverage of the tissue-attached area
  • Tn5 interrupted According to the cDNA concentration, take 20ng cDNA, add 0.5uM Tn5 interrupting enzyme and corresponding buffer (Tn5 interrupting enzyme coating method according to stLFR library building kit operation), mix well to form a 20ul reaction system, react at 55°C for 10min , Add 5ul 0.1% SDS and mix at room temperature for 5 minutes to end the Tn5 interruption step.
  • the reaction conditions are as follows: 95°C for 3 minutes, 40°C for 3 minutes; after the reaction, place on ice, and add 40ul DNBSEQ sequencing kit of DNB preparation required mixed enzyme I, 2ul mixed enzyme II, and 1ul ATP (mother liquor 100mM, Thermo Fisher), 0.1ul T4 ligase (BGI self-produced), after mixing, put the above reaction system in a PCR machine at 30°C for 20 minutes to form DNB.

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Abstract

提供了检测样品中核酸的空间信息的方法，以及用于所述方法的核酸阵列及产生所述核酸阵列的方法。

Description

用于检测核酸空间信息的阵列及检测方法 技术领域
本发明涉及生物分子空间检测领域。具体地，本发明提供了检测样品中核酸的空间信息的方法，以及用于所述方法的核酸阵列及产生所述核酸阵列的方法。
背景技术
组织中细胞的空间位置显著影响其功能，为探究这种空间异质性，需要在获知空间坐标的情况下，对细胞的基因组或转录组进行量化及分析。然而要收集较小的组织区域甚至单个细胞用于基因组或转录组分析，非常费力、费钱且精确度低。因此，开发一种能够实现单细胞甚至亚细胞级别、且高通量的检测生物分子(例如核酸)的空间信息(例如，核酸的定位、分布和/或表达)的方法是十分必要的。
发明内容
为实现核酸的空间检测，现有技术通过将阵列技术与高通量DNA测序技术联用，捕获并定位标记组织样本中的核酸，并对其进行测序和分析。为获得能够实现所述目的的芯片，现有技术通过点样或微珠介导的方式将能够捕获核酸的探针固定于芯片。然而，使用微量体积点样系统进行液滴平面点样的点样方式获得的芯片的活性区域大小高达200微米，细胞观测精度仅为20细胞；使用带定位标记的微珠进行平面铺展的微珠方式获得的芯片的活性区域大小达10微米，细胞观测精度仅能达到单细胞，无法实现对亚细胞的定位。本发明提供了一种全新的用于核酸空间检测的核酸阵列及其制备方法、以及基于该阵列的核酸空间检测方法，其能够同时实现高精度亚细胞定位和高通量组织定位，具备重大应用价值。
核酸阵列的制备
在第一方面，本发明提供了一种产生用于检测生物样品中核酸的空间信息的核酸阵列的方法，所述方法包括以下步骤：
(1)提供多种载体序列，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：定位序列和第一固定序列，
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应；
(2)将所述多种载体序列连接于固相支持物(例如芯片)表面；
(3)提供第一引物，并以所述载体序列为模板，进行引物延伸反应，使得所述载体序列的第一固定序列和定位序列区域形成双链，其中与所述载体序列杂交的链即为第一核酸分子，所述第一核酸分子从5’至3’方向上包含第一固定序列和定位序列的互补序列；其中，所述第一引物在其3’端包含第一固定序列互补区，所述第一固定序列互补区包含所述第一固定序列的互补序列或其片段，且具有3’自由端。
在某些实施方案中，所述载体序列、第一核酸分子是单链核酸序列。在某些实施方案中，所述载体序列、第一核酸分子是单链DNA序列。
在某些实施方案中，在步骤(3)中，在进行延伸反应的同时，对所述载体序列进行测序，从而获得所述载体序列所包含的定位序列的序列信息。
在某些实施方案中，在步骤(1)中通过以下步骤提供多种载体序列：
(i)提供多种载体模板序列，所述载体模板序列包含所述载体序列的互补序列；
(ii)以每种载体模板序列为模板，进行核酸扩增反应，以获得每种载体模板序列的扩增产物，所述扩增产物包含多个拷贝的载体序列。
在某些实施方案中，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增。
在某些实施方案中，进行滚环复制，以获得由所述载体序列的多联体所形成的DNB。在此类实施方案中，在步骤(i)中提供环状模板序列。制备环状核酸分子的方法是本领域的常规方法，本领域技术人员可以根据需要进行选择，例如可以首先获得线性核酸模板，然后通过连接酶(例如DNA连接酶)实现线性核酸模板的环化。
在某些实施方案中，进行桥式PCR扩增、乳液PCR扩增或多重链置换扩增，以获得由所述载体序列的克隆群形的DNA簇。
在一些实施方案中，所述方法进一步包括以下步骤：
(4)提供第二核酸分子，所述第二核酸分子包含捕获序列；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物，
(5)将所述第二核酸分子与所述第一核酸分子连接(例如，使用连接酶将所述第二核 酸分子与第一核酸分子连接)。
在某些实施方案中，所述第二核酸分子是单链核酸序列。在某些实施方案中，所述第二核酸分子是单链DNA序列。在某些实施方案中，所述第二核酸分子是单链RNA序列。
在某些实施方案中，所述第二核酸分子从5’至3’方向上包含固定区和捕获序列，所述固定区包含双链核酸序列，例如双链DNA序列。在某些实施方案中，所述第二核酸分子所包含的捕获序列是单链核酸序列，例如单链DNA序列或单链RNA序列。易于理解的是，在此类实施方案中，所述第二核酸分子具有部分双链结构，也即，其固定区具有双链结构，捕获序列为单链结构。
在某些实施方案中，所述双链核酸序列的长度为1bp-50bp，例如10bp-50bp，10bp-40bp或10bp-30bp。
在另一些实施方案中，所述方法进一步包括以下步骤：
(4)提供第二核酸分子，所述第二核酸分子从5’至3’方向上包含第二固定序列互补序列和捕获序列；
所述第二固定序列互补序列允许与其互补的核苷酸序列发生杂交；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物；
(5)在允许退火的条件下使所述第二固定序列互补序列与第二固定序列发生杂交，从而将所述第二核酸分子连接至所述载体序列；
(6)任选地，将分别杂交在载体序列上的第一核酸分子和第二核酸分子连接(例如，使用连接酶将所述第二核酸分子与第一核酸分子连接)。
在此类实施方案中，每个载体序列在其5’端进一步包含第二固定序列，所述第二固定序列允许与其互补的核苷酸序列发生退火。在某些实施方案中，所述第二固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应(例如可作为桥式PCR引物的结合位点)。
在某些实施方案中，所述第二核酸分子是单链核酸序列。在某些实施方案中，所述第二核酸分子是单链DNA序列。在某些实施方案中，所述第二核酸分子是单链RNA序列。
在某些实施方案中，所述第二固定序列与定位序列邻接。
在某些实施方案中，所述第二固定序列的长度为1bp-50bp，例如10bp-50bp，10bp-40bp，10bp-30bp，或10bp-20bp。
在某些实施方案中，在步骤(3)中，所述第一引物在其第一固定序列互补区的5’端还包含唯一分子标识符(UMI)序列，从而使得所述第一核酸分子在其第一固定序列互补序列的5’端包含该UMI序列；或者，在步骤(4)中，所述第二核酸分子还包含UMI序列，所述UMI序列位于捕获序列的5’端；
所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个，5-50个，5-20个，例如10个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种。
在某些实施方案中，当所述第一引物在其第一固定序列互补区的5’端包含唯一分子标识符(UMI)序列时，所述第一引物可以在所述UMI序列的5’端进一步包含额外的序列。
在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列。在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列。在某些实施方案中，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
在某些实施方案中，所述固相支持物是芯片。在某些实施方案中，所述固相支持物能够用作测序平台，例如测序芯片。在某些实施方案中，所述固相支持物是高通量测序芯片，例如用于Illumina、MGI或Thermo Fisher测序平台的高通量测序芯片。
在第二方面，本发明提供了一种产生用于检测生物样品中核酸的空间信息的核酸阵列的方法，所述方法包括以下步骤：
(1)提供多种载体序列，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：捕获模板序列、定位序列和第一固定序列，
所述捕获模板序列包含捕获序列的互补序列，所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应；并且，所述第一固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；
(2)将所述多种载体序列连接于固相支持物(例如芯片)表面；
(3)提供第一引物(或称为探针引物)，所述第一引物在5’至3’方向上包含结合区、切除区和第一固定序列互补区，所述第一固定序列互补区包含所述第一固定序列的互补序列或其片段，且具有3’自由端；所述结合区包含能够与所述固相支持物表面连接的连接子；所述切除区包含切割位点；
以所述第一引物为引物，以所述载体序列为模板，进行引物延伸反应，使得所述载体序列的第一固定序列、定位序列以及捕获模板序列区域形成双链，其中与所述载体序列杂交的链即为第一核酸分子，所述第一核酸分子从5’至3’方向上包含第一固定序列互补序列、定位序列互补序列和捕获序列；所述第一核酸分子又可称为捕获探针；
(4)将所述第一引物与所述固相支持物表面连接；其中，步骤(3)与(4)以任意顺序进行；
(5)任选地对所述载体序列的第一固定序列所包含的切割位点进行切割，以消化所述载体序列，使得步骤(3)中的延伸产物与形成延伸产物的模板(即载体序列)分离，从而将所述第一核酸分子(捕获探针)连接于固相支持物(例如芯片)表面。
由于捕获探针(即第一核酸分子)是以所述载体序列为模板并通过引物延伸获得，因此，所述捕获探针包含与所述阵列上该种捕获探针(该种载体序列)的位置相对应的独一无二的定位序列互补序列，以及能够与待捕获核酸分子的全部或部分杂交的捕获序列。通过分析该定位序列互补序列能够确定该种捕获探针在阵列上的位置。
在本文中，表述“每种载体序列”是指包含相同定位序列的载体序列。
在某些实施方案中，在步骤(1)中通过以下步骤提供多种载体序列：
(i)提供多种载体模板序列，所述载体模板序列包含所述载体序列的互补序列；
(ii)以每种载体模板序列为模板，进行核酸扩增反应，以获得每种载体模板序列的扩增产物，所述扩增产物包含多个拷贝的载体序列。
在某些实施方案中，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增。
在某些实施方案中，进行滚环复制，以获得由所述载体序列的多联体所形成的DNB。在此类实施方案中，在步骤(i)中提供环状模板序列。制备环状核酸分子的方法是本领域的常规方法，本领域技术人员可以根据需要进行选择，例如可以首先获得线性核酸模板，然后通过连接酶(例如DNA连接酶)实现线性核酸模板的环化。
在某些实施方案中，每种载体序列是由多个拷贝的载体序列的多联体所形成的DNB。
在某些实施方案中，步骤(1)包括以下步骤：
(1a)提供环状核酸模板，所述环状核酸模板包含一种载体模板序列，所述载体模板序 列包含所述载体序列的互补序列，也即所述载体模板序列在5’至3’方向上包含第一固定序列互补序列、定位序列互补序列和捕获序列；
(1b)以所述环状核酸模板为模板进行滚环扩增(RCA)，以获得由所述载体序列的多联体所形成的纳米球(DNB)。
在某些实施方案中，进行桥式PCR扩增、乳液PCR扩增或多重链置换扩增，以获得由所述载体序列的克隆群形的DNA簇。
在某些实施方案中，所述第一固定序列包含的切割位点是切刻酶酶切位点。在某些实施方案中，所述切刻酶选自USER、BamHI、BmtI等。在某些示例性实施方案中，所述酶切位点如SEQ ID NO:14所示。
在某些实施方案中，所述第一固定序列还包含测序引物杂交区和/或扩增引物杂交区；其中，所述测序引物杂交区允许测序引物退火并起始测序反应，所述扩增引物杂交区允许扩增引物退火并起始延伸及扩增反应。
在某些实施方案中，所述第一固定序列的长度大于1bp，例如大于10bp，或大于20bp。在某些实施方案中，所述第一固定序列的长度为20-100bp，例如20-80bp。
在某些实施方案中，所述定位序列的长度大于1bp，例如大于10bp。在某些实施方案中，所述定位序列的长度为10-100bp，例如10-50bp，例如10-30bp，例如20bp。
在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列。在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列。在某些实施方案中，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
在某些实施方案中，所述捕获序列的长度大于1bp。在某些实施方案中，所述捕获序列的长度为1-100bp，例如10-50bp，例如10-30bp。
在某些实施方案中，所述载体序列还包含位于所述捕获模板序列的下游、且位于所述第一固定序列的上游的UMI互补序列(亦可称为探针标签区)，其与UMI序列互补，所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个，5-50个，5-20个，例如10个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种。在一些实施方案中，所述UMI互补序列位于所述定位序列和捕获模板序列之间。在另一些实施方案中，所述UMI互补序列位于所述第一固定序列与定位序列之间。在此类实施方案中，在步骤(3)中，当以所述载体序列为模板进行引物延伸反应时， 与所述载体序列杂交的第一核酸分子/捕获探针会相应地包含所述UMI序列(也可称为探针标签)。
在某些实施方案中，为获得上述UMI序列或其互补序列，所述载体序列的模板序列(即载体模板序列)在对应位置包含UMI模板序列，所述UMI模板序列是由修饰碱基组成的序列，所述修饰碱基能够与多种主要碱基(例如，C、G、A、T、U)形成氢键互补配对，如Inosine，可以与碱基A、C、U互补配对。不受理论约束，认为当载体模板序列包含所述UMI模板序列时，在滚环复制过程中，每进行一次复制，与所述UMI模板序列能够互补配对的碱基随机结合，从而使得每一次的复制产物带有独特的随机形成的UMI序列，进而将每一次的复制产物区分开来，如可以捕获不同的核酸分子，进行拷贝数定量。在某些实施方案中，所述UMI模板序列包含多个Inosine(例如至少10个，例如10-100个)。在某些实施方案中，所述UMI模板序列的长度大于1bp。在某些实施方案中，所述UMI模板序列的长度大于5bp。在某些实施方案中，所述UMI模板序列的长度为5-100bp，例如5-50bp，例如5-20bp，例如5-15bp，例如10bp。
在某些实施方案中，所述固相支持物是芯片。在某些实施方案中，所述固相支持物能够用作测序平台。在某些实施方案中，所述固相支持物是测序芯片(华大智造)，如BGISEQ-500平台的测序芯片。在某些实施方案中，所述固相支持物是高密度阵列芯片，例如可以如专利CN103180496B所描述的方法获得。
可以通过本领域已知的任何合适方法将载体序列(例如DNB)连接至固相支持物表面。在某些实施方案中，所述方法的非限制性示例包括核酸杂交、生物素链霉亲和素结合、巯基结合、光活化结合、共价结合、抗体-抗原、经由水凝胶或其他多孔聚合物的物理限制等，或其任意组合。
在某些实施方案中，所述固相支持物选自下列材料：玻璃、硅、多聚赖氨酸涂层材料、硝化纤维、聚苯乙烯、环状烯烃共聚物(COCs)、环状烯烃高分子(COPs)、聚丙烯、聚乙烯或聚碳酸酯等。
在某些实施方案中，在步骤(3)中，在进行引物延伸反应的同时，对所述载体序列(例如其所包含的定位序列)进行测序，从而获得所述载体序列所包含的定位序列的序列信息。
在某些实施方案中，在步骤(3)之前，还包括对所述载体序列(例如其所包含的定位序列)进行测序的步骤。在某些实施方案中，在完成测序之后，通过洗涤以去除因测序而被添加至合成链中的dNTP。
在某些实施方案中，所述连接子是能够与活化基团(例如NH 2)偶联的连接基团。在 此类实施方案中，所述固相载体表面具有活化基团(例如NH 2)修饰。在某些实施方案中，所述连接子包括-SH、-DBCO或-NHS等。在某些实例性实施方案中，所述连接子是DBCO，所述固相载体表面连接有
Figure PCTCN2020090340-appb-000001
8-NHS ester。
Figure PCTCN2020090340-appb-000002
在某些实施方案中，所述第一引物的切除区所包含的切割位点是可以通过化学、酶或光化学方法进行受控切割的位点。在某些实施方案中，所述切割位点是酶切位点。在某些实施方案中，所述切割位点是USER酶的酶切位点(UUU)。
在某些实施方案中，所述第一引物的切除区与所述载体序列的第一固定序列所包含的切割位点不同。
在某些实施方案中，所述扩增包括PCR。
核酸阵列及试剂盒
在第三方面，本发明提供了一种用于检测样品中核酸空间信息的核酸阵列，其包括表面连接有多种载体序列的固相支持物(例如芯片)，每种载体序列在所述阵列中占不同位置，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：定位序列和第一固定序列，
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应。
在某些实施方案中，所述每种载体序列(也即，包含相同定位序列的载体序列)在所述固相支持物表面所占的面积(也即，活性区域)的直径小于1微米，例如约900纳米，约800纳米，约700纳米，约600纳米，或约500纳米。
在某些实施方案中，所述核酸阵列进一步包含第一核酸分子，所述第一核酸分子从5’到3’的方向上包含：第一固定序列互补序列和定位序列互补序列，并且与所述载体序列的第一固定序列和定位序列杂交形成双链。易于理解的是，所述第一核酸分子中仅第一固定序列互补序列和定位序列互补序列与所述载体序列的相应序列互补形成双链，因此第一固定序列与载体序列所形成的双链是不完全双链，即部分双链结构。
在某些实施方案中，每种载体序列中的每个拷贝的载体序列均包含与其杂交的第一核酸分子。
在某些实施方案中，所述载体序列、第一核酸分子是单链核酸序列。在某些实施方案中，所述载体序列、第一核酸分子是单链DNA序列。
在一些实施方案中，所述核酸阵列进一步包含第二核酸分子，所述第二核酸分子与第一核酸分子连接，从而固定至所述核酸阵列，所述第二核酸分子包含捕获序列；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物。
在某些实施方案中，每个第一核酸分子均连接所述第二核酸分子。
在某些实施方案中，所述第二核酸分子的5’端与第一核酸分子的3’端连接。
在另一些实施方案中，所述核酸阵列进一步包含第二核酸分子，所述第二核酸分子与载体序列发生杂交，从而固定至所述核酸阵列。
在此类实施方案中，每个载体序列在其5’端进一步包含第二固定序列，所述第二固定序列允许与其互补的核苷酸序列发生退火；并且，
所述第二核酸分子从5’至3’方向上包含第二固定序列互补序列和捕获序列；所述第二固定序列互补序列与所述载体序列的第二固定序列杂交形成双链；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物。
在某些实施方案中，每种载体序列中的每个拷贝的载体序列均包含与其杂交的第二核酸分子。
在某些实施方案中，所述第二固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应(例如可作为桥式PCR引物的结合位点)。
在某些实施方案中，所述第二固定序列与定位序列邻接。
在某些实施方案中，所述第二核酸分子具有5’端修饰。在某些实施方案中，所述修饰是磷酸化或生物素修饰。
在某些实施方案中，所述第二核酸分子是单链核酸序列。在某些实施方案中，所述第二核酸分子是单链DNA序列。在某些实施方案中，所述第二核酸分子是单链RNA序列。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经扩增所形成的扩增产物，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增。
在某些实施方案中，所述多个拷贝的载体序列是由所述载体序列的多联体所形成的DNB。在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经滚环复制形成的DNB。
在某些实施方案中，所述多个拷贝的载体序列是由所述载体序列的克隆群形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经桥式PCR扩增形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经乳液PCR扩增形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经多重链置换扩增形成的DNA簇。
在某些实施方案中，所述第一核酸分子还包含唯一分子标识符(UMI)序列，所述UMI序列位于第一固定序列互补序列的5’端；或者，所述第二核酸分子还包含UMI序列，所述UMI序列位于捕获序列的5’端；
所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个，5-50个，5-20个，例如10个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种。
在某些实施方案中，所述固相支持物是芯片。在某些实施方案中，所述固相支持物能够用作测序平台，例如测序芯片。在某些实施方案中，所述固相支持物是高通量测序芯片，例如用于Illumina、MGI或Thermo Fisher测序平台的高通量测序芯片。
在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列。在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列。在某些实施方案中，所述poly-T寡核苷酸序列包括至少10个(例 如至少20个)脱氧胸腺嘧啶核苷残基。
在某些实施方案中，所述定位序列的长度大于1nt，例如大于5nt。在某些实施方案中，所述定位序列的长度为5-50nt，例如10-50nt，10-30nt，或20-30nt。在某些实施方案中，不同种载体序列所包含的定位序列的长度可以相同或不同。
在某些实施方案中，所述捕获序列的长度大于1nt。在某些实施方案中，所述捕获序列的长度为1-100nt，例如1-50nt，例如10-30nt。
在某些实施方案中，所述第一固定序列的长度大于1nt，例如大于10nt。在某些实施方案中，所述第一固定序列的长度为10-200nt。在某些实施方案中，所述第一固定序列的长度为20-100nt，例如20-50nt。
在某些实施方案中，所述第二固定序列的长度大于1nt，例如大于10nt。在某些实施方案中，所述第二固定序列的长度为10-200nt，例如10-100nt，10-50nt，10-30nt，或10-20nt。
在第四方面，本发明提供了一种用于检测样品中核酸空间信息的试剂盒，其包含：(i)第三方面所述的核酸阵列，其中，所述核酸阵列不包含第二核酸分子；以及，(ii)第二核酸分子，所述第二核酸分子从5’至3’方向上包含固定区和捕获序列；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物。
在一些实施方案中，所述试剂盒包含：(i)核酸阵列，其包括表面连接有多种载体序列的固相支持物(例如芯片)，每种载体序列在所述阵列中占不同位置，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：定位序列和第一固定序列，
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应；
所述核酸阵列还包含第一核酸分子，所述第一核酸分子从5’到3’的方向上包含：第一固定序列互补序列和定位序列互补序列，并且与所述载体序列的第一固定序列和定位序列杂交形成双链；
以及，(ii)第二核酸分子，所述第二核酸分子的固定区包含双链DNA序列。
易于理解的是，可以使用连接酶将(ii)所述的第二核酸分子连接至(i)所述的核酸阵列所 包含的第一核酸分子上。因此，在某些实施方案中，所述试剂盒进一步包含连接酶。
在另一些实施方案中，所述试剂盒包含：(i)核酸阵列，其包括表面连接有多种载体序列的固相支持物(例如芯片)，每种载体序列在所述阵列中占不同位置，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：第二固定序列、定位序列和第一固定序列，
所述第二固定序列允许与其互补的核苷酸序列发生退火；
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应；
所述核酸阵列还包含第一核酸分子，所述第一核酸分子从5’到3’的方向上包含：第一固定序列互补序列和定位序列互补序列，并且与所述载体序列的第一固定序列和定位序列杂交形成双链；
以及，(ii)第二核酸分子，所述第二核酸分子的固定区包含第二固定序列互补序列。
在某些实施方案中，所述第二固定序列与定位序列邻接。
易于理解的是，在允许退火的条件下，(ii)所述的第二核酸分子可与(i)所述的核酸阵列所包含的载体序列的互补区域杂交，进而可以使用连接酶将第二核酸分子连接至第一核酸分子上。因此，在某些实施方案中，所述试剂盒进一步包含连接酶。
在另一方面，本发明还涉及第三方面所述的核酸阵列或第四方面所述的试剂盒用于检测样品中核酸的空间信息的用途，或者在制备用于检测样品中核酸的空间信息的检测试剂中的用途。
在某些实施方案中，所述核酸的空间信息包括核酸的定位、分布和/或表达。
在某些实施方案中，所述样品是组织样品，例如含有细胞的组织样品。在某些实施方案中，所述样品是组织切片。在某些实施方案中，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
在第五方面，本发明还涉及一种用于检测样品中核酸的空间信息的核酸阵列，其包括表面连接有多种载体序列的固相支持物(例如芯片)，每种载体序列在所述阵列中占不同位置，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：捕获模板序列、定位序列和第一固定序列，其中，
所述捕获模板序列包含捕获序列的互补序列，所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；
所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应，并且，所述第一固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；
所述核酸阵列还包含第一核酸分子(亦可称为捕获探针)，所述第一核酸分子从5’到3’的方向上包含：结合区、切除区、以及载体序列互补区，
所述结合区包含能够与所述固相支持物表面连接的连接子；
所述切除区包含切割位点；
所述载体序列互补区包含能够与所述载体序列互补的序列，其从5’到3’的方向上包含：第一固定序列互补序列、定位序列互补序列以及捕获序列；并且，所述捕获序列具有3’自由端以使所述第一核酸分子能作为延伸引物；
并且，所述第一核酸分子的载体序列互补区与所述载体序列杂交形成双链。
在某些实施方案中，所述载体序列、第一核酸分子是单链核酸序列。在某些实施方案中，所述载体序列、第一核酸分子是单链DNA序列。
在某些实施方案中，每种载体序列中的每个拷贝的载体序列均包含与其杂交的上述第一核酸分子。
在某些实施方案中，所述第一核酸分子的连接子是能够与活化基团(例如NH 2)偶联的连接基团，所述固相支持物表面具有活化基团(例如NH 2)修饰。在某些实施方案中，所述连接子包括-SH、-DBCO或-NHS。在某些实施方案中，所述连接子是
Figure PCTCN2020090340-appb-000003
(DBCO)，所述固相支持物表面连接有
Figure PCTCN2020090340-appb-000004
(
Figure PCTCN2020090340-appb-000005
8-NHS ester)。
在某些实施方案中，所述第一固定序列包含的切割位点是切刻酶酶切位点。在某些实施方案中，所述切刻酶选自USER、BamHI、BmtI等。在某些示例性实施方案中，所述酶切位点如SEQ ID NO:14所示。
在某些实施方案中，所述第一核酸分子的切除区所包含的切割位点是可以通过化学、 酶或光化学方法进行受控切割的位点。在某些实施方案中，所述切割位点是酶切位点。在某些实施方案中，所述切割位点是USER酶的酶切位点(UUU)。
在某些实施方案中，所述第一核酸分子的切除区与所述载体序列的第一固定序列所包含的切割位点不同。
在某些实施方案中，所述核酸阵列通过第二方面所述的方法制备。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经扩增所形成的扩增产物，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增。
在某些实施方案中，所述多个拷贝的载体序列是由所述载体序列的多联体所形成的DNB。在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经滚环复制形成的DNB。
在某些实施方案中，所述多个拷贝的载体序列是由所述载体序列的克隆群形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经桥式PCR扩增形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经乳液PCR扩增形成的DNA簇。
在某些实施方案中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经多重链置换扩增形成的DNA簇。
在某些实施方案中，所述载体序列进一步包含位于所述捕获模板序列的下游、且位于所述第一固定序列的上游的UMI互补序列，其与UMI序列互补，所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个，5-50个，5-20个，例如10个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种；
并且，所述第一核酸分子的载体序列互补区进一步包含位于所述捕获序列上游、且位于所述第一固定序列互补序列的下游的UMI序列。
在某些实施方案中，所述UMI互补序列位于所述定位序列和捕获模板序列之间，或者位于所述第一固定序列与定位序列之间。
在某些实施方案中，每种载体序列(也即，包含相同定位序列的载体序列)中的每个拷贝的载体序列具备彼此不同的UMI互补序列。相应地，与每个拷贝的载体序列杂交的 第一核酸分子(捕获探针)也具备彼此不同的UMI序列。
在某些实施方案中，通过载体序列的第一固定序列所包含的切割位点将所述载体序列从核酸阵列中去除。在此类实施方案中，所述核酸阵列包括表面连接有多种捕获探针(第一核酸分子)的固相支持物(例如芯片)，每种捕获探针(第一核酸分子)在所述阵列中占不同位置，且定向为具有3’自由端以使所述捕获探针(第一核酸分子)能作为延伸引物，其中每种捕获探针(第一核酸分子)从5’到3’的方向上包含：结合区、切除区、定位序列互补序列以及捕获序列，其中，
所述结合区包含能够与所述固相支持物表面连接的连接子；
所述切除区包含切割位点；
所述定位序列与所述阵列上该种捕获探针的位置相对应；
所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(1a)能够捕获mRNA的寡核苷酸序列；和/或，(1b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列。
在某些实施方案中，所述每种捕获探针中的每条捕获探针(也即，包含相同定位序列/定位序列互补序列的捕获探针)具备彼此不同的UMI序列，所述UMI序列位于所述捕获序列的上游，并且位于所述切除区的下游。在一些实施方案中，所述UMI序列位于所述捕获序列的5’端，例如位于所述捕获序列与定位序列互补序列之间。在另一些实施方案中，所述UMI序列位于所述定位序列互补序列的5’端，例如位于所述切除区与定位序列互补序列之间。
在某些实施方案中，所述每种载体序列(也即，包含相同定位序列的载体序列)或每种捕获探针(也即，包含相同定位序列/定位序列互补序列的捕获探针)在所述固相支持物表面所占的面积(也即，活性区域)的直径小于1微米，例如约900纳米，约800纳米，约700纳米，约600纳米，或约500纳米。在某些实施方案中，所述每种载体序列或每种捕获探针的活性区域的直径为约500纳米。
在某些实施方案中，所述固相支持物是芯片。在某些实施方案中，所述固相支持物能够用作测序平台。在某些实施方案中，所述固相支持物是测序芯片(华大智造)，如BGISEQ-500平台。在某些实施方案中，所述固相支持物是高密度阵列芯片，例如可以如专利CN103180496B所描述的方法获得。
在某些实施方案中，所述第一固定序列的长度大于1bp，例如大于10bp，或大于20bp。在某些实施方案中，所述第一固定序列的长度为20-100bp，例如20-80bp。
在某些实施方案中，所述定位序列的长度大于1bp，例如大于10bp。在某些实施方案中，所述定位序列的长度为10-100bp，例如10-50bp，例如10-30bp，例如20bp。
在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列。在某些实施方案中，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列。在某些实施方案中，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
在某些实施方案中，所述捕获序列的长度大于1bp。在某些实施方案中，所述捕获序列的长度为1-100bp，例如10-50bp，例如10-30bp。
检测方法
在第六方面，本发明提供了一种检测样品中核酸的空间信息的方法，其包括以下步骤：
(1)提供第三方面所述的核酸阵列，或者，通过第一方面所述的方法获得核酸阵列；其中，
所述核酸阵列包含连接于固相支持物(例如芯片)表面的多种载体序列，每种载体序列在所述阵列中占有不同位置，并且每种载体序列包含多个拷贝的载体序列；
每个载体序列包含与其杂交的第一核酸分子和第二核酸分子，并且所述第一核酸分子和第二核酸分子彼此未连接；
所述第一核酸分子包含与所述阵列上该种载体序列的位置相对应的定位序列互补序列，
所述第二核酸分子包含能够捕获样品中的核酸的捕获序列；
(2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述第二核酸分子的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的载体序列的位置；
(3)(i)连接杂交于每个载体序列上的第一核酸分子和第二核酸分子(例如使用连接酶)；
在允许引物延伸的条件下，以连接的第一和第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；和/或，
以被捕获的核酸分子作为引物，以所述连接的第一和第二核酸分子为模板，进行引物延伸反应，以产生经延长的被捕获核酸分子，所述经延长的被捕获核酸分子具有所述定位 序列作为空间信息标记；
或者，(ii)在允许引物延伸的条件下，以所述第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生经延长的第二核酸分子，所述经延长的第二核酸分子包含被捕获核酸的互补序列；连接杂交于每个载体序列上的第一核酸分子和经延长的第二核酸分子(例如使用连接酶)，与所述被第一核酸分子连接的经延长的第二核酸分子具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；
(4)从阵列表面释放至少一部分空间信息标记的核酸分子，其中所述部分包括定位序列或其互补链以及被捕获核酸分子或其互补链；和
(5)直接或间接地分析步骤(4)中被释放的核酸分子的序列。
在此类实施方案中，在步骤(2)捕获靶核酸之前，所述核酸阵列上的第一核酸分子和第二核酸分子未连接。
在第七方面，本发明提供了一种检测样品中核酸的空间信息的方法，其包括以下步骤：
(1)提供第一方面所述的核酸阵列，或者，通过第三方面所述的方法获得核酸阵列；其中，
所述核酸阵列包含连接于固相支持物(例如芯片)表面的多种载体序列，每种载体序列在所述阵列中占有不同位置，并且每种载体序列包含多个拷贝的载体序列；
每个载体序列包含与其杂交的第一核酸分子，所述第一核酸分子连接第二核酸分子；
所述第一核酸分子包含与所述阵列上该种载体序列的位置相对应的定位序列互补序列，
所述第二核酸分子包含能够捕获样品中的核酸的捕获序列；
(2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述第二核酸分子的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的载体序列的位置；
(3)(iii)在允许引物延伸的条件下，以连接的第一和第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；和/或，
以被捕获的核酸分子作为引物，以所述连接的第一和第二核酸分子为模板，进行引物延伸反应，以产生经延长的被捕获核酸分子，所述经延长的被捕获核酸分子具有所述定位序列作为空间信息标记；
(4)从阵列表面释放至少一部分空间信息标记的核酸分子，其中所述部分包括定位序列或其互补链以及被捕获核酸分子或其互补链；和
(5)直接或间接地分析步骤(4)中被释放的核酸分子的序列。
在此类实施方案中，在步骤(2)捕获靶核酸之前，所述核酸阵列上的第一核酸分子与第二核酸分子已连接。
在第六或第七方面所述方法的某些实施方案中，所述多个拷贝的载体序列是由所述载体序列的多联体所形成的DNB，或者，所述多个拷贝的载体序列是由所述载体序列的克隆群形的DNA簇。
在第六或第七方面所述方法的某些实施方案中，所述载体序列、第一核酸分子是单链DNA。在某些实施方案中，所述第二核酸分子是单链DNA或单链RNA。
在第八方面，本发明提供了一种检测样品中核酸的空间信息的方法，其包括以下步骤：
(1)提供第五方面所述的核酸阵列，或者，通过第二方面所述的方法获得核酸阵列；其中，所述核酸阵列包含连接于固相支持物(例如芯片)表面的多种载体序列，每种载体序列在所述阵列中占有不同位置，并且每种载体序列包含多个拷贝的载体序列；
每个载体序列包含与其杂交的第一核酸分子，所述第一核酸分子包含与所述阵列上该种载体序列的位置相对应的定位序列互补序列以及能够捕获样品中的核酸的捕获序列；
(2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述第一核酸分子的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的第一核酸分子的位置；
(3)在允许引物延伸的条件下，以所述第一核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；
(4)从阵列表面释放至少一部分空间信息标记的核酸分子，其中所述部分包括定位序列或其互补链以及被捕获核酸分子或其互补链；和
(5)直接或间接地分析步骤(4)中被释放的核酸分子的序列。
在某些实施方案中，在步骤(2)之前，所述方法还包括对所述载体序列的第一固定序列所包含的切割位点进行切割，以消化所述载体序列，同时将所述第一核酸分子(捕获探针)连接于固相支持物(例如芯片)表面。在此类实施方案中，所述核酸阵列包含连接于固相 支持物(例如芯片)表面的多种捕获探针，每种捕获探针在所述阵列中占有不同位置，并且，所述捕获探针包含与所述阵列上该种捕获探针的位置相对应的定位序列互补序列以及能够捕获样品中的核酸的捕获序列；
(2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述捕获探针的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的捕获探针的位置；
(3)在允许引物延伸的条件下，以所述捕获探针作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，所产生的延伸产物包含作为空间信息标记的定位序列互补序列以及被捕获核酸分子的互补序列，从而产生空间信息标记的DNA分子；可选地，生成所述空间信息标记的DNA分子的互补链，和/或可选地，扩增所述空间信息标记的DNA分子；
(4)从阵列表面释放至少一部分空间信息标记的DNA分子和/或他们的互补链或扩增子，其中所述部分包括空间信息标记序列或其互补链；和
(5)直接或间接地分析步骤(4)中被释放的核酸分子的序列。
在第八方面所述方法的某些实施方案中，所述的第一核酸分子、捕获探针是DNA分子，例如单链DNA。
在第六至第八任一方面所述方法的某些实施方案中，所述核酸的空间信息包括核酸的定位、分布和/或表达。
在第六至第八任一方面所述方法的某些实施方案中，所述样品是组织样品，例如组织切片。在某些实施方案中，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
在第六至第八任一方面所述方法的某些实施方案中，所述方法用于非诊断用途。
在第六至第八任一方面所述方法的某些实施方案中，在步骤(5)中可以采用任何核酸分析方法。在某些实施方案中，该步骤可以包括测序。在某些实施方案中，可以采用序列特异性分析方法。例如，可以实施序列特异性扩增反应，比如通过使用引物，且该引物对所述定位域和/或一种特定的目标序列(例如，一种待测的特定目标DNA)具有特异性。因此，在某些实施方案中，该步骤可以包括序列特异性的PCR反应。
在第六至第八任一方面所述方法的某些实施方案中，步骤(5)所得到的序列分析信息可被用于获得样品中核酸的空间信息(即，位置信息)。在某些实施方案中，该空间信息可 从测得的序列分析信息的性质来推定，例如，其可以揭示一种特定核酸的存在，而该核酸可能在所测样品的环境中本身就具有空间信息意义，以及/或者可以结合阵列上样品的位置和测序信息来推定空间信息(例如，空间定位)。因此，本方法可以仅包括将序列分析信息与样品的某个位置相关联的步骤，例如，利用定位标记和其与样品上某位置间的关联性。在某些实施方案中，可以通过关联序列分析数据和样品图像来便利地获得空间信息。因此，在此类实施方案中，第六至第八任一方面所述方法还包括步骤(6)：将步骤(5)获得的序列分析信息与所述样品的图像相关联，其中该样品在步骤(3)之前或之后成像。在某些实施方案中，所述样品的成像是利用光、明视场、暗视场、相位差、荧光、镜像、干涉或共聚焦显微镜或其组合。
在第六方面所述方法的某些实施方案中，所述方法用于检测样品中的转录组。在此类实施方案中，在步骤(3)(i)中，以连接的第一和第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备第一核酸分子中所包含的定位序列互补序列作为空间信息标记，且可选地，扩增所述cDNA分子；或者，在步骤(3)(ii)中，以第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，连接杂交于每个载体序列上的第一核酸分子和所述cDNA分子(例如使用连接酶)，以产生具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记的cDNA分子，且可选地，扩增所述cDNA分子；以及，在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含空间信息序列或其互补链。在某些实施方案中，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
在第七方面所述方法的某些实施方案中，所述方法用于检测样品中的转录组。在此类实施方案中，在步骤(3)(iii)中，以连接的第一和第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备第一核酸分子中所包含的定位序列互补序列作为空间信息标记，且可选地，扩增所述cDNA分子；以及，在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含空间信息序列或其互补链。在某些实施方案中，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
在第八方面所述方法的某些实施方案中，所述方法用于检测样品中的转录组。在此类实施方案中，在步骤(3)中，以所述捕获探针为RT引物，从已捕获的RNA分子合成cDNA 分子，所述cDNA分子具备空间信息标记，且可选地，扩增所述cDNA分子；在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含空间信息标记序列或其互补链。在某些实施方案中，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
在第六至第八任一方面所述方法的某些实施方案中，在所述空间信息标记的核酸分子(例如DNA分子)或所述空间信息标记的cDNA分子从阵列表面释放之前或之后，生成所述互补链或所述cDNA第二链。
第二链DNA(例如cDNA)的合成步骤可以在阵列上原位进行，可以是单独的第二链合成步骤，也可以作为扩增反应的初始步骤。可选地，可以从阵列上释放DNA(例如cDNA)的第一链(也即，以被捕获核酸分子为模板所生成的链)，然后再进行第二链合成，例如，在溶液中进行反应，可以作为一个单独步骤或作为扩增反应中的一个步骤。
当第二链合成在阵列上(即原位)进行时，该方法可以包括一个可选的步骤，即在第二链合成前移除被捕获的核酸分子(如RNA)，例如可以采用RNA硝化酶(RNase)，如RNase H。此步骤的操作流程为本领域所熟知并有记载。但是，此步骤一般并无必要，且在大多数情况下，RNA会自然降解。从阵列上除去样品的步骤，一般也会移除阵列上的RNA。
在某些实施方案中，DNA(例如cDNA)的第二链在单一反应中生成，第二链合成可以通过本领域已知的任何适当的方法来实现。例如，从阵列上释放的cDNA第一链，随后使用随机引物(例如六聚体引物)和DNA聚合酶(例如链置换聚合酶)，以第一链为模板进行DNA合成反应。因此，在某些实施方案中，所述互补链或第二链的合成使用随机引物和链置换聚合酶。
在第六至第八任一方面所述方法的某些实施方案中，在序列分析前进一步包括对所述空间信息标记的核酸分子(例如DNA分子)或cDNA分子进行扩增的步骤。在某些实施方案中，所述扩增步骤在所述空间信息标记的核酸分子(例如DNA分子)或cDNA分子从阵列释放之后实施，或所述扩增步骤在阵列上原位实施(也即，在所述第一核酸分子和/或载体序列和/或捕获探针依然连接于固相支持物表面时)。在某些实施方案中，所述扩增步骤包括PCR。
在第六至第八任一方面所述方法的某些实施方案中，在步骤(4)中，所述分子通过如下方法从阵列表面释放：(i)核酸剪切；(ii)变性；和/或(iii)物理方法。在某些实施方案中，通过将热水或缓冲液应用至所述固相支持物来释放所述分子。
在某些实施方案中，在测序之前进一步包括对被释放的分子进行纯化的步骤。
在某些实施方案中，在样品与阵列接触之后且步骤(3)之前，进一步包括给样品补水的步骤。
在某些实施方案中，在步骤(4)之前，所述方法进一步包括洗涤阵列以除去残余样品(例如，组织)的步骤。
在某些实施方案中，所述阵列包含至少一个方向标记，来为阵列上的样品定向。
在某些实施方案中，在步骤(5)中，所述序列分析步骤包括测序步骤。在某些实施方案中，所述测序步骤包括基于可逆染色终止剂的测序反应。
本发明第六至第八任一方面所述的检测核酸空间信息的方法可用于RNA检测以及转录组分析、DNA检测及基因组分析等。空间信息对转录组学及基因组学相关研究具有重要意义，特别是在例如组织的不同细胞或区域中的转录组或基因组变异研究中有用，例如比较正常和病态细胞或组织，或研究疾病进程中的转录组或基因组变化等。
举例来说，阿尔茨海默症的病理生理学分析显示其病变过程涉及到神经元和胶质细胞的相互作用，而相关转录组和表观基因组研究也发现，阿尔兹海默症病人大脑中的神经元功能严重受损、先天免疫反应异常。然而，群体水平的研究是不能够揭示细胞间和细胞群内变化的复杂性，尤其是对于那些稀有的细胞类型。普通单细胞水平的研究也不能够区分同时期不同组织区域的特定细胞类型特征与神经退行性病变期间的细胞成分变化。因此，要想进一步揭示疾病的致病机制和发展模式迫切需要获取带有空间维度的单细胞转录组信息。
本发明第六至第八任一方面所述的检测核酸空间信息的方法可以通过连接于芯片的带有位置标记的捕获序列将大脑组织样本中不同区域的核酸分子固定至芯片，并进行测序，从而获得含有准确位置信息的转录组结果，以实现对阿尔茨海默病病变期间不同区域的特定细胞类型的变化的检测。特别地，由于本发明芯片上DNB或DNA簇的活性区域低至纳米级，而细胞直径约为12um，因此本发明的芯片能够实现亚细胞分辨率的空间定位信息。
本发明还包含以下示例性实施方案：
项目1.一种产生核酸阵列的方法，所述核酸阵列用于检测样品中生物分子(例如核酸)的空间信息，所述方法包括以下步骤：
(1)提供环状核酸模板，所述环状核酸模板包含一种捕获探针的模板序列，所述模板序列在5’至3’方向上包含接头区、空间标签区及捕获区；其中，
所述接头区包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；
所述空间标签区包含空间标签序列，所述空间标签序列对应于该种捕获探针在阵列上的位置；
所述捕获区包含能够捕获样品中生物分子(例如核酸)的捕获序列；其中，所述捕获序列包括：(1a)能够捕获mRNA的寡核苷酸序列；和/或，(1b)随机或简并的寡核苷酸序列；或，(c)针对特定靶分子(例如靶核酸)的特异性序列；
(2)以所述环状核酸模板为模板进行滚环扩增(RCA)，以获得由所述模板序列的互补序列(即，模板互补序列)的多联体所形成的纳米球(DNB)；
(3)将所述DNB连接于固相载体(例如芯片)表面；
(4)提供探针引物，并以所述DNB所包含的模板互补序列为模板，进行引物延伸反应，以产生延伸产物，其中与所述模板互补序列杂交的链即为捕获探针；可选地，扩增所述延伸产物；所述探针引物在5’至3’方向上包含结合区、切除区和引物接头区；其中，
所述结合区包含能够与所述固相载体表面连接的连接子；
所述切除区包含切割位点；
所述引物接头区与所述DNB所包含的模板互补序列的接头区序列(也即，所述模板序列接头区的互补序列)的全部或部分互补，且具有3’自由端，以使所述探针引物能作为引物起始延伸反应；优选地，所述引物接头区包含所述模板序列的接头区的序列或其片段；
(5)将所述探针引物与所述固相载体表面连接；其中，步骤(4)与(5)以任意顺序进行；
(6)对接头区所包含的切割位点进行切割，以消化所述DNB，使得步骤(4)中的延伸产物与形成延伸产物的模板DNB分离，从而将捕获探针连接于固相载体(例如芯片)表面；
优选地，所述环状核酸模板、DNB以及捕获探针是DNA；
优选地，在步骤(1)中提供多种环状核酸模板，每种环状核酸模板包含各不相同的捕获探针的模板序列，从而获得表面连接有多种捕获探针的固相载体(例如芯片)。
项目2.项目1的方法，其中，所述接头区所包含的切割位点是切刻酶酶切位点；
优选地，所述切刻酶选自USER、BamHI、BmtI。
项目3.项目1或2的方法，其中，所述接头区还包含测序引物杂交区和/或扩增引物杂交区；其中，所述测序引物杂交区允许测序引物退火并起始测序反应，所述扩增引物杂交区允许扩增引物退火并起始延伸及扩增反应。
项目4.项目1-3任一项的方法，其中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列；
优选地，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列；
优选地，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
项目5.项目1-4任一项的方法，其中，所述模板序列还包含位于所述捕获区上游、且位于所述接头区下游的探针标签区，所述探针标签区包含由修饰碱基组成的探针标签互补序列，所述修饰碱基能够与多种主要碱基(例如，C、G、A、T、U)形成氢键互补配对；
优选地，所述探针标签区位于所述空间标签区和捕获区之间，或者位于所述接头区与空间标签区之间；
优选地，所述探针标签互补序列包含多个(例如至少10个)Inosine。
项目6.项目1-5任一项的方法，其具备以下特征中的一项或多项：
(i)所述接头区的长度大于1bp，例如大于10bp，或大于20bp；优选地，所述接头区的长度为20-100bp；
(ii)所述空间标签区的长度大于1bp，例如大于10bp；优选地，所述空间标签区的长度为10-100bp；
(iii)所述捕获区的长度大于1bp；优选地，所述捕获区的长度为1-100bp；
(iv)所述探针标签区的长度大于1bp，例如大于5bp；优选地，所述探针标签区的长度为5-100bp。
项目7.项目1-6任一项的方法，其中，所述固相载体是芯片；
优选地，所述固相载体能够用作测序平台，例如测序芯片。
项目8.项目1-7任一项的方法，其中，在步骤(4)中，在进行引物延伸反应的同时，对所述空间标签序列的互补序列进行测序，从而获得相应的捕获探针所包含的空间标签序列的序列信息。
项目9.项目1-7任一项的方法，其中，在步骤(4)之前，还包括对所述DNB所包含的空间标签序列的互补序列进行测序的步骤；
优选地，在完成测序之后，通过洗涤以去除因测序而被添加至合成链中的dNTP。
项目10.项目1-9任一项的方法，其中，所述连接子是能够与活化基团(例如NH 2)偶联的连接基团，所述固相载体表面具有活化基团(例如NH 2)修饰；
优选地，所述连接子包括-SH、-DBCO或-NHS；
Figure PCTCN2020090340-appb-000006
项目11.项目1-10任一项的方法，其中，所述切除区所包含的切割位点是可以通过化学、酶或光化学方法进行受控切割的位点；
优选地，所述切割位点是酶切位点；
优选地，所述切除区与所述接头区所包含的切割位点不同。
项目12.项目1-11任一项的方法，其中，在所述扩增包括PCR。
项目13.通过项目1-12任一项的方法制备的核酸阵列。
项目14.一种用于检测样品中生物分子(例如核酸)的空间信息的核酸阵列，其包括表面连接有多种捕获探针的固相载体(例如芯片)，每种捕获探针在所述阵列中占不同位置，且定向为具有3’自由端以使所述捕获探针能作为延伸引物，其中每种捕获探针从5’到3’的方向上包含：结合区、切除区、空间标签序列以及捕获序列，其中，
所述结合区包含能够与所述固相载体表面连接的连接子；
所述切除区包含切割位点；
所述空间标签序列与所述阵列上该种捕获探针的位置相对应；
所述捕获序列能够与待捕获生物分子(例如核酸)的全部或部分杂交，其包括：(1a)能够捕获mRNA的寡核苷酸序列；和/或，(1b)随机或简并的寡核苷酸序列；或，(c)针对特定靶分子(例如靶核酸)的特异性序列。
项目15.项目14的核酸阵列，其中，所述每种捕获探针中的每条捕获探针(也即，包含相同空间标签序列的捕获探针)具备彼此不同的探针标签序列，所述探针标签序列位于所述捕获序列的上游，并且位于所述切割区的下游；
优选地，所述探针标签序列位于所述捕获序列与空间标签序列之间，或者，位于所述切割区与空间标签序列之间。
项目16.项目14或15的核酸阵列，其中，所述每种捕获探针(也即，包含相同空间标签序列的捕获探针)在所述固相载体表面所占的面积(也即，活性区域)的直径小于1微米；
优选地，所述每种捕获探针的活性区域的直径为约500纳米。
项目17.项目14-16任一项的核酸阵列，其中，所述固相载体是芯片；
优选地，所述固相载体能够用作测序平台，例如测序芯片。
项目18.项目14-17任一项的核酸阵列，其中，所述核酸阵列通过项目1-12任一项的方法制备。
项目19.一种检测样品中生物分子的空间信息的方法，其包括以下步骤：
(1)提供项目13-18任一项的核酸阵列，或者，通过项目1-12任一项的方法获得核酸阵列；所述核酸阵列包含连接于固相载体(例如芯片)表面的多种捕获探针，每种捕获探针在所述阵列中占有不同位置，并且，所述捕获探针包含与所述阵列上该种捕获探针的位置相对应的空间标签序列以及能够捕获样品中的生物分子的捕获序列；
(2)将所述核酸阵列与待测样品接触，以使得所述捕获探针的捕获序列与待测样品中的生物分子结合，从而使得该生物分子的位置被对应至核酸阵列上的捕获探针的位置，并且产生空间标签标记的生物分子；
(3)从阵列表面释放空间标签标记的生物分子；和
(4)直接或间接地分析步骤(3)中被释放的生物分子的序列。
项目20.一种检测样品中核酸的空间信息的方法，其包括以下步骤：
(1)提供项目13-18任一项所述的核酸阵列，或者，通过项目1-12任一项的方法获得核酸阵列；所述核酸阵列包含连接于固相载体(例如芯片)表面的多种捕获探针，每种捕获探针在所述阵列中占有不同位置，并且，所述捕获探针包含与所述阵列上该种捕获探针的位置相对应的空间标签序列以及能够捕获样品中的核酸的捕获序列；
(2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述捕获探针的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的捕获探针的位置；
(3)在允许引物延伸的条件下，以所述捕获探针作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，所产生的延伸产物包含空间标签序列以及被捕获核酸分子的互补序列，从而产生空间标签标记的DNA分子；可选地，生成所述空间标签标记的DNA分子的互补链，和/或可选地，扩增所述空间标签标记的DNA分子；
(4)从阵列表面释放至少一部分空间标签标记的DNA分子和/或他们的互补链或扩增子，其中所述部分包括空间标签序列或其互补链；和
(5)直接或间接地分析步骤(4)中被释放的核酸分子的序列；
优选地，所述核酸的空间信息包括核酸的定位、分布和/或表达；
优选地，所述捕获探针是DNA分子；
优选地，所述样品是组织样品，例如组织切片；
优选地，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
项目21.项目20的方法，在步骤(5)中，所述序列分析包括测序或序列特异性的PCR反应。
项目22.项目20或21的方法，所述方法还包括步骤(6)：将步骤(5)获得的序列分析信息与所述样品的图像相关联，其中该样品在步骤(3)之前或之后成像。
项目23.项目20-22任一项的方法，所述方法用于检测样品中的转录组，其中：
在步骤(3)中，以所述捕获探针为RT引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备空间标签标记，且可选地，扩增所述cDNA分子；
在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含空间标签序列或其互补链；
优选地，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
项目24.项目20-23任一项的方法，其中，在所述空间标签标记的DNA分子或所述空间标签标记的cDNA分子从阵列表面释放之前或之后，生成所述互补链或所述cDNA第二链；
优选地，所述互补链或第二链的合成使用随机引物和链置换聚合酶。
项目25.项目20-24任一项的方法，其中，在序列分析前进一步包括对所述空间标签标记的DNA分子或cDNA分子进行扩增的步骤；
优选地，所述扩增步骤在所述空间标签标记的DNA或cDNA分子从阵列释放之后实施，或所述扩增步骤在阵列上原位实施；
优选地，所述扩增步骤包括PCR。
项目26.项目20-25任一项的方法，其中，在序列分析之前进一步包括对被释放的分子进行纯化的步骤。
项目27.项目20-26任一项的方法，在步骤(4)之前，所述方法进一步包括洗涤阵列以除去残余样品(例如，组织)的步骤。
项目28.项目20-27任一项的方法，在步骤(4)中，所述分子通过如下方法从阵列表面 释放：(i)核酸剪切；(ii)变性；和/或(iii)物理方法；
优选地，所述分子通过酶切从捕获探针的切除区释放。
项目29.项目20-28任一项的方法，在步骤(6)中，所述样品的成像是利用光、明视场、暗视场、相位差、荧光、镜像、干涉或共聚焦显微镜或其组合。
有益效果
本发明提供了一种全新的用于检测核酸空间信息的阵列及其制备方法，该核酸阵列应用于核酸空间信息检测时，可以同时实现高精度亚细胞定位和高通量组织定位。本发明的阵列及基于该阵列的检测方法在细胞定位，亚细胞定位，细胞器定位，细胞互作，细胞器互作，分子通路研究，疾病诊断等方面具备重大应用价值。
附图说明
图1示出了实施例2中捕获mRNA后进行cDNA合成的结构示意图。
图2示出了实施例2中从芯片上释放的分子的结构示意图。
图3显示了实施例2中2100检测cDNA片段分布的结果。
图4显示了实施例3中cDNA测序所得到的一链25bp序列与捕获芯片上定位序列的fq的匹配结果。
图5显示了实施例3中组织切片中mRNA的表达情况图。
图6显示了实施例4中一个示例性实施方案的探针引物及DNB中所包含的载体序列结构示意图。
图7示出了实施例4中芯片上所连接的探针结构示意图。
图8显示了实施例5中对所捕获的核酸分子进行cDNA合成后的结构示意图。
图9显示了实施例5中从芯片上所释放的分子的结构示意图。
图10显示了实施例5中2100检测cDNA片段分布的结果。
图11显示了实施例6中cDNA测序所得到的一链20bp序列与捕获芯片上定位序列的fq的匹配结果。
图12显示了实施例6中组织切片中mRNA的表达情况图。
具体实施方式
现参照下列意在举例说明本发明(而非限定本发明)的实施例来描述本发明。
除非特别指明，否则基本上按照本领域内熟知的以及在各种参考文献中描述的常规方法进行实施例中描述的实验和方法。另外，实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。本领域技术人员知晓，实施例以举例方式描述本发明，且不意欲限制本发明所要求保护的范围。本文中提及的全部公开案和其他参考资料以其全文通过引用合并入本文。
实施例1.捕获芯片的制备-1
1、设计并合成下述DNA文库序列。委托华大六合进行序列合成。
5’磷酸化-AAGTCGGAGGCCAAGCGGTCTTAGGAAGACAA(接头A，SEQ ID NO:1)NNNNNNNNNNNNNNNNNNNNNNNNN(定位序列互补序列，N表示任意碱基，例如C、G、A或T)CTGATAAGGTCGCCA(第二固定序列互补序列，SEQ ID NO:2)CAACTCCTTGGCTCACAGAACGACATGGCTACGATCCGACTT(接头B，SEQ ID NO:3)-3’。其中，接头A包含第一固定序列互补序列的部分序列以及环化位点，接头B包含第一固定序列互补序列的另一部分、酶切位点、环化位点。
2、文库原位扩增
DNA纳米球(DNB)制备：配置下面的反应体系40ul，投入80fmol上述DNA文库，DNB引物序列为GGCCTCCGACTTAAGTCGGATCGT(SEQ ID NO:4)，由华大六合合成。
成分 体积(ul) 终浓度
DNA文库序列 80fmol(X)  
10×phi29 buffer(BGI自产) 4 1X
DNB引物序列10uM 4 1uM
分子水 32-x  
将上述反应体积放置于PCR仪反应，反应条件如下：95℃ 3min，40℃ 3min；反应结束后，放于冰上，加入40ul DNBSEQ测序试剂盒中DNB制备所需的混合酶I，2ul混合酶II，及1ul ATP(母液100mM，Thermo Fisher)，0.1ul T4 ligase(BGI自产)，混匀后，将上述反应体系至于PCR仪30℃，反应20min，形成DNB。将所述的DNB按照BGISEQ500 SE50试剂盒所述的方法将DNB装载至BGISEQ500测序芯片上。
3、定位序列的测序解码：按照BGISEQ500 SE50测序试剂盒说明书，对定位序列进行解码测序，设置25bp的测序长度。测序形成的fq文件储存待用。
4、捕获序列固定：委托华大六合合成下面DNA序列：5’磷酸化-CTGATAAGGTCGCCA(第二固定序列互补序列，SEQ ID NO:5)NNNNNNNNNN(UMI)TTTTTTTTTTTTTTTTTTTVN(捕获序列，SEQ ID NO:6)-3’，其中，N表示任意碱基(例如，C、G、A或T)。将测序芯片从测序仪取下，向芯片中泵入BGISEQ500SE50试剂盒中的7号孔位的切除试剂(保证试剂覆盖整张芯片，无气泡产生)，将芯片置于60℃，反应10min。反应完后，在测序芯片中泵入适量的5×SSC(上海生工购买)，使SSC充分置换芯片中上一个试剂。用5×SSC稀释捕获序列至1uM，加入适量稀释好的捕获序列至芯片，使芯片中充满捕获序列。将芯片置于常温反应30min左右，使捕获序列充分杂交至DNB上。
5、芯片切块：将制备好的芯片切成若干小片，切片大小根据实验需要进行调整，将芯片浸泡在50mM pH8.0的tris buffer中，制备4℃待用。
实施例2.组织mRNA捕获及cDNA合成
1.冰冻组织切片。按照冰冻切片的标准流程，制作小鼠的小脑组织切片。
2.mRNA捕获。根据切片组织的大小，取合适大小的实施例1制备的芯片，放置于室温，至芯片上的液体挥发完后，在切片机内利用切片组织和芯片的温差，将切片组织贴在捕获芯片上。将贴好的组织切片放置室温，在芯片上加入5×SSC反应液(充分覆盖贴有组织的区域)，30℃反应30min后，使组织内的mRNA与芯片上的捕获区域充分杂交反应。
3.cDNA合成。使用5×SSC室温清洗芯片两次，配置如下逆转录酶反应体系200ul，将反应液加到芯片上，充分覆盖，42℃反应90min-180min，mRNA将以polyT为引物进行cDNA合成，mRNA的3端带上TSO标签(AAGTCGGAGGCCAAGCGGTC/rG//rG//iXNA_G/)(SEQ ID NO:7)，用于cDNA互补链的合成。上述过程的结构示意图如图1所示。
Figure PCTCN2020090340-appb-000007
Figure PCTCN2020090340-appb-000008
4.空间定位区与捕获区连接。cDNA合成后，用5×SSC清洗芯片两次，配置如下反应体系1ml，向芯片中泵入合适的体积，保证芯片中充满下列连接反应液，将图1中所示的nick补齐。室温反应30min。反应结束后，使用5×SSC清洗芯片，温度55℃，清洗3遍，每遍5min。
成分 体积(ul) 浓度
10×T4 ligase buffer(BGI自产) 100 1×
T4 ligase(600U/ul,BGI自产) 100 60u/ul
甘油(阿拉丁) 10 10％
分子水 790  
5.cDNA释放。cDNA一链在芯片合成后，在芯片上加入适量的formamide溶液，并在55℃下反应10min，将cDNA链从芯片上释放下来。所释放的分子具有如图2所示的结构。收集从芯片上释放的反应液，使用2×XP磁珠纯化cDNA一链，最后用45ul TE buffer(Thermo fisher)回收产物。使用qubit ssDNA检测试剂盒对cDNA单链进行定量检测。
6.cDNA扩增。配置如下反应体系100ul：
Figure PCTCN2020090340-appb-000009
将上述反应体系至于PCR仪，设置如下反应程序，95℃ 3min，11循环(98℃ 20s，58℃ 20s，72℃ 3min)，72℃5min，4℃ ∞。反应结束后，用XP beads进行磁珠纯化回收。使用qubit试剂盒dsDNA浓度定量，2100检测cDNA片段分布。2100检测结果如图3所示，cDNA长度正常。
实施例3.cDNA建库测序
1.Tn5打断。根据cDNA浓度，取20ng cDNA，加入0.5uM Tn5打断酶及相应buffer(Tn5打断酶包被方法按照stLFR建库试剂盒操作)，混匀配成20ul的反应体系，在55℃反应10min后，加入5ul 0.1％SDS混匀室温5min结束Tn5打断步骤。
2.PCR扩增。配置如下反应体系100ul：
Figure PCTCN2020090340-appb-000010
混匀后置于PCR仪，设置如下程序95℃ 3min，11循环(98℃ 20s，58℃ 20s，72℃ 3min)，72℃ 5min，4℃ ∞。反应结束后，用XP beads进行磁珠纯化回收。使用qubit试剂盒dsDNA浓度定量。
3.测序。取上述打断后的扩增产物80fmol，进行DNB制备。配置如下40ul反应体系：
Figure PCTCN2020090340-appb-000011
将上述反应体积放置于PCR仪反应，反应条件如下：95℃ 3min，40℃ 3min；反应结束后，放于冰上，加入40ul DNBSEQ测序试剂盒中DNB制备所需的混合酶I，2ul混合酶II，及1ul ATP(母液100mM，Thermo Fisher)，0.1ul T4 ligase(BGI自产)，混匀后，将上述反应体系至于PCR仪30℃，反应20min，形成DNB。将所述的DNB按照MGISEQ2000上PE50试剂盒所述的方法将DNB装载至MGISEQ2000的测序芯片上，并按照相关说明书进行测序，选择PE50测序模型，其中一链测序分成两段测序25bp后进行15cycle暗反应后，再测10bp UMI序列，二链设置50bp。
数据分析
1.通过比对将cDNA测序所得到的一链25bp序列，与捕获芯片上定位序列的fq(实施例1中步骤3所得的测序结果)进行匹配。匹配结果如图4所示，亮区是cDNA测序的25bp与捕获芯片完全匹配的区域，此区域代表捕获芯片上用于组织捕获的区域。说明捕获芯片可以使用空间定位区域对组织捕获区进行精确定位。
2.对cDNA测序匹配到捕获芯片的DNB进一步分析，将这些DNB reads的二链测序结果cDNA(反应组织中的mRNA表达)与小鼠的基因组进行比对分析。将比对上小鼠基因组的DNB，通过25bp的测序结果将小鼠的mRNA信息比对到捕获芯片上，如图5所示，左边显示的是所分析的组织切片中mRNA的表达全貌，该全貌图表明，此捕获芯片可以分析出组织中mRNA的表达差异。此图右边是随机取小鼠小脑中表达的一个基因在组织中的表达水平，表明此芯片可以分析出组织样品中某个基因在整个组织中的表达差异。
实施例4.捕获芯片的制备-2
1.设计并合成下述DNA文库序列。委托华大六合进行序列合成。
5’磷酸化-GAACGACATGGCTTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG(固定序列1，SEQ ID NO:12)NNNNNNNNNNNNNNNNNNNN(定位序列模板序列，N表示任意碱基，例如，C、G、A或T)IIIIIIIIII(UMI模板序列，I为Inosine)TTTTTTTTTTTTTTTTTTTTT(捕获序列，SEQ ID NO:13)CCTCAGC(酶切位点，SEQ ID NO:14)CCTTGGCTCACA(固定序列2，SEQ ID NO:15)。其中，固定序列1包含第一固定序列互补序列的部分序列以及环化位点，固定序列2包含第一固定序列互补序列的部分序列以及环化位点。
2.文库原位扩增
DNA纳米球(DNB)制备：配置下面的反应体系40ul，投入80fmol上述DNA文库，DNB引物序列为GACATGGCTACGTGTGAGCCAAGG(SEQ ID NO:16)，由华大六合合成。
成分 体积(ul) 终浓度
DNA文库序列 80fmol(×)  
10×phi29 buffer(BGI自产) 4 1×
DNB引物序列10uM 4 1uM
分子水 32-x  
将上述反应体积放置于PCR仪反应，反应条件如下：95℃ 3min，40℃ 3min；反应结束后，放于冰上，加入40ul DNBSEQ测序试剂盒中DNB制备所需的混合酶I，2ul混合酶II，及1ul ATP(母液100mM，Thermo Fisher)，0.1ul T4 ligase(BGI自产)，混匀后，将上述反应体系至于PCR仪30℃，反应20min，形成DNB。将所述的DNB按照BGISEQ500 SE50试剂盒所述的方法将DNB装载至BGISEQ500测序芯片上。
3.空间信息解码
(1)芯片表面修饰：
将上述BGISEQ-500平台芯片表面连接
Figure PCTCN2020090340-appb-000012
8-NHS ester，结构如下：
Figure PCTCN2020090340-appb-000013
按照下列方法进行芯片表面修饰：NHS-PEG8-Azido(564.58g/mol)浓度为45μM，配制100ml方法：
试剂 用量 单位
NHS-PEG8-Azido 2.54 mg
1×PBS(pH 7.4) 100 ml
-20℃保存，避免反复冻融。
DBCO-primer浓度为1uM，用PBS稀释
(2)引物探针偶联：
华大六合定制合成以下引物探针序列：
DBCO(连接基团)-UUU(USER酶切位点)TTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG(SEQ ID NO:17，该序列包含第一固定序列互补序列、PCR扩增位点序列、中间序列)。将1uM的上述引物 探针序列使用PBS稀释通入修饰有azido的芯片上，室温反应1h或过夜。
(3)空间信息解码。按照BGISEQ500 SE50测序试剂盒说明书，对空间信息序列进行解码测序，设置30bp的测序长度(前20bp为空间信息序列，后10bp为探针标签序列)。测序形成的fq文件储存待用。
(4)捕获区合成：
配制dTTP及Hifi聚合酶的混合液，以DNB为模板，含有空间定位区的探针序列为引物，dTTP为底物，延伸出oligo dT序列。
4.含有空间信息的探针释放
配制1uM Spatial_RNA_BbvCI引物(用5×SSC稀释)，引物序列CCTCAGCCAACTCCT(SEQ ID NO:18)由华大六合合成；室温杂交30min；配制BbvCI切除体系(1.5ml)：15ul RE+150ul 10×CS Buffer+1335ul ddH 2O，通入空间定位区解码后的芯片，在37℃反应1h或过夜，加入用测序试剂盒(华大智造)中的WB2洗两次，再使用formamide在55℃下反应15min，最后使用WB2冲洗两次。所得探针示意图如图7所示，探针序列如下所示：
UUU(切除区)TTTTTCCCGTAGCCATGTCGTTCTGCGCCTTCCCGATG(第一固定序列互补序列，SEQ ID NO:19)NNNNNNNNNNNNNNNNNNN(定位序列互补序列，其与步骤一中的DNA文库序列中的定位序列模板序列相同)NNNNNNNNNN(UMI序列，其是以步骤一中的UMI模板序列为模板获得的随机碱基序列的互补序列)TTTTTTTTTTTTTTTTTTTTT(捕获序列，SEQ ID NO:20)。
5.芯片切块
将制备好的捕获芯片切成若干小片，切片大小根据实验需要进行调整，将芯片浸泡在50mM pH8.0的tris buffer中，4℃待用。
实施例5.组织mRNA捕获及cDNA合成
1.冰冻组织切片。按照冰冻切片的标准流程，制作小鼠的小脑组织切片。
2.mRNA捕获。根据切片组织的大小，取合适大小的实施例4制备获得的芯片，放置于室温，至芯片上的液体挥发完后，在切片机内利用切片组织和芯片的温差，将切片组织贴在捕获芯片上。将贴好的组织切片放置室温，在芯片上加入5×SSC反应液(充分覆盖贴有组织的区域)，30℃反应30min后，使组织内的mRNA与芯片上的捕获区域充分杂交反应。
3.cDNA合成。使用5×SSC室温清洗芯片两次，配置如下逆转录酶反应体系200ul，将反应液加到芯片上，充分覆盖，42℃反应90min-180min，mRNA将以polyT为引物进行cDNA合成，mRNA的3端带上TSO标签(CGTAGCCATGTCGTTCTGCG/rG//rG//iXNA_G/)(SEQ ID NO:21)，用于cDNA互补链的合成。上述过程的结构示意图如图8所示。
Figure PCTCN2020090340-appb-000014
4.cDNA释放。cDNA一链在芯片合成后，配制user酶反应体系，并按照USER酶的使用说明书进行反应。所释放的分子具有如图9所示的结构。收集从芯片上释放的反应液，使用2×XP磁珠纯化cDNA一链，最后用45ul TE buffer(Thermo fisher)回收产物。
5.cDNA扩增。配置如下反应体系100ul：
Figure PCTCN2020090340-appb-000015
Figure PCTCN2020090340-appb-000016
将上述反应体系至于PCR仪，设置如下反应程序，95℃ 3min，11循环(98℃ 20s，58℃ 20s，72℃ 3min)，72℃ 5min，4℃ ∞。反应结束后，用XP beads进行磁珠纯化回收。使用qubit试剂盒dsDNA浓度定量，2100检测cDNA片段分布。2100检测结果如图10所示，cDNA长度正常。
实施例6.cDNA建库测序
1.Tn5打断。根据cDNA浓度，取20ng cDNA，加入0.5uM Tn5打断酶及相应buffer(Tn5打断酶包被方法按照stLFR建库试剂盒操作)，混匀配成20ul的反应体系，在55℃反应10min后，加入5ul 0.1％SDS混匀室温5min结束Tn5打断步骤。
2.PCR扩增。配置如下反应体系100ul：
Figure PCTCN2020090340-appb-000017
混匀后置于PCR仪，设置如下程序95℃ 3min，11循环(98℃ 20s，58℃ 20s，72℃ 3min)，72℃ 5min，4℃ ∞。反应结束后，用XP beads进行磁珠纯化回收。使用qubit试剂盒dsDNA浓度定量。
3.测序。取上述打断后的扩增产物80fmol，进行DNB制备。配置如下40ul反应体系：
Figure PCTCN2020090340-appb-000018
Figure PCTCN2020090340-appb-000019
将上述反应体积放置于PCR仪反应，反应条件如下：95℃ 3min，40℃ 3min；反应结束后，放于冰上，加入40ul DNBSEQ测序试剂盒中DNB制备所需的混合酶I，2ul混合酶II，及1ul ATP(母液100mM，Thermo Fisher)，0.1ul T4 ligase(BGI自产)，混匀后，将上述反应体系至于PCR仪30℃，反应20min，形成DNB。将所述的DNB按照MGISEQ2000上PE50试剂盒所述的方法将DNB装载至MGISEQ2000的测序芯片上，并按照相关说明书进行测序，选择customer测序模式，其中中一链测序分成两段测序20bp后进行10bp探针标签序列测序，二链设置50bp。
数据分析
1.通过比对将cDNA测序所得到的一链20bp序列，与芯片上空间信息序列的fq(实施例四中步骤3所得的测序结果)进行匹配。匹配结果如图11所示，亮区是cDNA测序的20bp与捕获芯片完全匹配的区域，此区域代表捕获芯片上用于组织捕获的区域。说明捕获芯片可以使用空间定位区域对组织捕获区进行精确定位。
2.对cDNA测序匹配到捕获芯片的DNB进一步分析，将这些DNB reads的二链测序结果cDNA(反应组织中的mRNA表达)与小鼠的基因组进行比对分析。将比对上小鼠基因组的DNB，通过20bp的测序结果将小鼠的mRNA信息比对到捕获芯片上，如图12所示，左边显示的是所分析的组织切片中mRNA的表达全貌，该全貌图表明，此捕获芯片可以分析出组织中mRNA的表达差异。此图右边是随机取小鼠小脑中表达的一个基因在组织中的表达水平，表明此芯片可以分析出组织样品中某个基因在整个组织中的表达差异。
尽管本发明的具体实施方式已经得到详细的描述，但本领域技术人员将理解：根据已经公布的所有教导，可以对细节进行各种修改和变动，并且这些改变均在本发明的保护范围之内。本发明的全部分为由所附权利要求及其任何等同物给出。

Claims (49)

  1. 一种用于检测样品中核酸空间信息的核酸阵列，其包括表面连接有多种载体序列的固相支持物(例如芯片)，每种载体序列在所述阵列中占不同位置，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：定位序列和第一固定序列，其中，
    所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
    所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应。
  2. 权利要求1所述的核酸阵列，其中，所述核酸阵列进一步包含第一核酸分子，所述第一核酸分子从5’到3’的方向上包含：第一固定序列互补序列和定位序列互补序列，并且与所述载体序列的第一固定序列和定位序列杂交形成双链；
    优选地，每种载体序列中的每个拷贝的载体序列均包含与其杂交的第一核酸分子。
  3. 权利要求1或2所述的核酸阵列，其中，所述核酸阵列进一步包含第二核酸分子，所述第二核酸分子与第一核酸分子连接，所述第二核酸分子包含捕获序列；
    所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物；
    优选地，每个第一核酸分子均连接所述第二核酸分子。
  4. 权利要求1或2所述的核酸阵列，其中，每个载体序列在其5’端进一步包含第二固定序列，所述第二固定序列允许与其互补的核苷酸序列发生退火；
    所述第二固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应。
  5. 权利要求4所述的核酸阵列，其中，所述核酸阵列进一步包含第二核酸分子，所述第二核酸分子从5’至3’方向上包含第二固定序列互补序列和捕获序列；
    所述第二固定序列互补序列与所述载体序列的第二固定序列杂交形成双链；
    所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异 性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物；
    优选地，每种载体序列中的每个拷贝的载体序列均包含与其杂交的第二核酸分子。
  6. 权利要求1-5任一项所述的核酸阵列，其中，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经扩增所形成的扩增产物，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增；
    优选地，所述多个拷贝的载体序列是由所述载体序列的多联体所形成的DNB；优选地，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经滚环复制形成的DNB；
    优选地，所述多个拷贝的载体序列是由所述载体序列的克隆群形成的DNA簇；
    例如，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经桥式PCR扩增形成的DNA簇；
    例如，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经乳液PCR扩增形成的DNA簇；
    例如，所述多个拷贝的载体序列是以所述载体序列的互补序列为模板经多重链置换扩增形成的DNA簇。
  7. 权利要求1-6任一项所述的核酸阵列，其中，所述第一核酸分子还包含唯一分子标识符(UMI)序列，所述UMI序列位于第一固定序列互补序列的5’端；
    所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种；
    优选地，每个第一核酸分子所包含的UMI序列彼此不同。
  8. 权利要求1-6任一项所述的核酸阵列，其中，所述第二核酸分子还包含UMI序列，所述UMI序列位于捕获序列的5’端；
    所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种；
    优选地，每个第二核酸分子所包含的UMI序列彼此不同。
  9. 权利要求1-8任一项所述的核酸阵列，其中，所述固相支持物是芯片；
    优选地，所述固相支持物能够用作测序平台，例如测序芯片；
    优选地，所述固相支持物是高通量测序芯片，例如用于Illumina、MGI或ThermoFisher测序平台的高通量测序芯片。
  10. 权利要求1-9任一项所述的核酸阵列，其中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列；
    优选地，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列；
    优选地，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
  11. 权利要求1所述的核酸阵列，其中，所述载体序列进一步包含位于所述定位序列上游的捕获模板序列，所述捕获模板序列包含捕获序列的互补序列，所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；
    并且，所述载体序列的第一固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；
    并且，所述核酸阵列进一步包含第一核酸分子，所述第一核酸分子从5’到3’的方向上包含：结合区、切除区、以及载体序列互补区，
    所述结合区包含能够与所述固相支持物表面连接的连接子；
    所述切除区包含切割位点；
    所述载体序列互补区包含能够与所述载体序列互补的序列，其从5’到3’的方向上包含：第一固定序列互补序列、定位序列互补序列以及捕获序列；并且，所述捕获序列具有3’自由端以使所述第一核酸分子能作为延伸引物；
    并且，所述第一核酸分子的载体序列互补区与所述载体序列杂交形成双链；
    优选地，每种载体序列中的每个拷贝的载体序列均包含与其杂交的第一核酸分子。
  12. 权利要求11所述的核酸阵列，其中，所述载体序列进一步包含位于所述捕获模板序列的下游、且位于所述第一固定序列的上游的UMI互补序列，其与UMI序列互补，所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种；
    并且，所述第一核酸分子的载体序列互补区进一步包含位于所述捕获序列上游、且位 于所述第一固定序列互补序列的下游的UMI序列；
    优选地，所述UMI互补序列位于所述定位序列和捕获模板序列之间，或者位于所述第一固定序列与定位序列之间；
    优选地，每种载体序列(也即，包含相同定位序列的载体序列)中的每个拷贝的载体序列具备彼此不同的UMI互补序列。
  13. 权利要求11或12所述的核酸阵列，其中，所述第一核酸分子的连接子是能够与活化基团(例如NH 2)偶联的连接基团，所述固相支持物表面具有活化基团(例如NH 2)修饰；
    优选地，所述连接子包括-SH、-DBCO或-NHS；
    优选地，所述连接子是
    Figure PCTCN2020090340-appb-100001
    (DBCO)，所述固相支持物表面连接有
    Figure PCTCN2020090340-appb-100002
    (
    Figure PCTCN2020090340-appb-100003
    ester)。
  14. 权利要求11-13任一项所述的方法，其中，所述第一核酸分子的切除区所包含的切割位点是可以通过化学、酶或光化学方法进行受控切割的位点；
    优选地，所述第一核酸分子的切除区所包含的切割位点是酶切位点；
    优选地，所述第一核酸分子的切除区与所述载体序列的第一固定序列所包含的切割位点不同。
  15. 权利要求11-14任一项所述的核酸阵列，其中，所述固相支持物是芯片；
    优选地，所述固相支持物能够用作测序平台，例如测序芯片；
    优选地，所述固相支持物是高通量测序芯片，例如用于Illumina、MGI或Thermo Fisher测序平台的高通量测序芯片。
  16. 权利要求11-15任一项所述的核酸阵列，其中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列；
    优选地，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列；
    优选地，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核 苷残基。
  17. 一种试剂盒，其包含：(i)权利要求1-10任一项所述的核酸阵列，其中，所述核酸阵列不包含第二核酸分子；以及，(ii)第二核酸分子，所述第二核酸分子从5’至3’方向上包含固定区和捕获序列；
    所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物。
  18. 权利要求15所述的试剂盒，其中，所述试剂盒包含权利要求2所述的核酸阵列，并且所述第二核酸分子的固定区包含双链核酸序列(例如双链DNA序列)。
  19. 权利要求15所述的试剂盒，其中，所述试剂盒包含权利要求4所述的核酸阵列，并且所述第二核酸分子的固定区包含第二固定序列互补序列。
  20. 权利要求15-17任一项所述的试剂盒，其中，当所述核酸阵列中包含的第一核酸分子不包含UMI序列时，所述第二核酸分子还包含UMI序列，所述UMI序列位于捕获序列的5’端；
    所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种。
  21. 一种产生用于检测生物样品中核酸的空间信息的核酸阵列的方法，其包括以下步骤：
    (1)提供多种载体序列，每种载体序列包含多个拷贝的载体序列，所述载体序列从5’到3’的方向上包含：定位序列和第一固定序列，
    所述定位序列具有与该种载体序列在阵列上的位置相对应的独一无二的核苷酸序列；
    所述第一固定序列允许与其互补的核苷酸序列发生退火并起始延伸反应；
    (2)将所述多种载体序列连接于固相支持物(例如芯片)表面；
    (3)提供第一引物，并以所述载体序列为模板，进行引物延伸反应，使得所述载体序列的第一固定序列和定位序列区域形成双链，其中与所述载体序列杂交的链即为第一核酸 分子，所述第一核酸分子从5’至3’方向上包含第一固定序列和定位序列的互补序列；其中，所述第一引物在其3’端包含第一固定序列互补区，所述第一固定序列互补区包含所述第一固定序列的互补序列或其片段，且具有3’自由端。
  22. 权利要求21所述的方法，其中，在步骤(1)中通过以下步骤提供多种载体序列：
    (i)提供多种载体模板序列，所述载体模板序列包含所述载体序列的互补序列；
    (ii)以每种载体模板序列为模板，进行核酸扩增反应，以获得每种载体模板序列的扩增产物，所述扩增产物包含多个拷贝的载体序列；
    优选地，所述扩增选自滚环复制(RCA)、桥式PCR扩增、多重链置换扩增(MDA)或乳液PCR扩增；
    优选地，进行滚环复制，以获得由所述载体序列的多联体所形成的DNB；
    优选地，进行桥式PCR扩增、乳液PCR扩增或多重链置换扩增，以获得由所述载体序列的克隆群形的DNA簇。
  23. 权利要求21或22所述的方法，其中，所述方法进一步包括以下步骤：
    (4)提供第二核酸分子，所述第二核酸分子包含捕获序列；
    所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物，
    (5)将所述第二核酸分子与所述第一核酸分子连接(例如，使用连接酶将所述第二核酸分子与第一核酸分子连接)。
  24. 权利要求23所述的方法，其中，所述第二核酸分子从5’至3’方向上包含固定区和捕获序列，所述固定区包含双链DNA序列。
  25. 权利要求23所述的方法，其中，每个载体序列在其5’端进一步包含第二固定序列，所述第二固定序列允许与其互补的核苷酸序列发生退火；所述方法进一步包括以下步骤：
    (4)提供第二核酸分子，所述第二核酸分子从5’至3’方向上包含第二固定序列互补序列和捕获序列；
    所述第二固定序列互补序列允许与其互补的核苷酸序列发生杂交；
    所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述捕获序列具有3’自由端以使所述第二核酸分子能作为延伸引物；
    (5)在允许退火的条件下使所述第二固定序列互补序列与第二固定序列发生杂交，从而将所述第二核酸分子连接至所述载体序列；
    (6)任选地，将分别杂交在载体序列上的第一核酸分子和第二核酸分子连接(例如，使用连接酶将所述第二核酸分子与第一核酸分子连接)。
  26. 权利要求23-25任一项所述的方法，其中，在步骤(3)中，所述第一引物在其第一固定序列互补区的5’端还包含唯一分子标识符(UMI)序列，从而使得所述第一核酸分子在其第一固定序列互补序列的5’端包含该UMI序列；或者，在步骤(4)中，所述第二核酸分子还包含UMI序列，所述UMI序列位于捕获序列的5’端；
    所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种。
  27. 权利要求22所述的方法，其中，
    在步骤(1)中，所述载体序列进一步包含位于所述定位序列上游的捕获模板序列，所述捕获模板序列包含捕获序列的互补序列，所述捕获序列能够与待捕获核酸的全部或部分杂交，其包括：(a)能够捕获mRNA的寡核苷酸序列；和/或，(b)随机或简并的寡核苷酸序列；或，(c)针对特定靶核酸的特异性序列；并且，所述载体序列的第一固定序列还包含切割位点，所述切割可以选自切刻酶(nicking enzyme)酶切、USER酶切、光切除、化学切除或CRISPR切除；
    在步骤(3)中，使得所述载体序列的第一固定序列、定位序列、以及捕获模板序列区域形成双链，从而使得所述第一核酸分子从5’至3’方向上包含第一固定序列互补序列、定位序列互补序列和捕获序列；其中，
    所述第一引物在5’至3’方向上包含结合区、切除区和第一固定序列互补区，所述结合区包含能够与所述固相支持物表面连接的连接子，所述切除区包含切割位点；
    并且，所述方法进一步包括以下步骤：
    (4)将所述第一引物与所述固相支持物表面连接；其中，步骤(3)与(4)以任意顺序进行；
    (5)任选地对所述载体序列的第一固定序列所包含的切割位点进行切割，以消化所述 载体序列，使得步骤(3)中的延伸产物与形成延伸产物的模板(即载体序列)分离，从而将所述第一核酸分子连接于固相支持物(例如芯片)表面；
    优选地，每种载体序列是由多个拷贝的载体序列的多联体所形成的DNB。
  28. 权利要求27所述的方法，其中，在步骤(1)中，所述第一固定序列包含的切割位点是切刻酶酶切位点；
    优选地，所述切刻酶选自USER、BamHI、BmtI。
  29. 权利要求27或28所述的方法，其中，在步骤(1)中，所述载体序列进一步包含位于所述捕获模板序列的下游、且位于所述第一固定序列的上游的UMI互补序列，其与UMI序列互补，所述UMI序列是由至少1个(例如至少2个，至少3个，至少4个，或至少5个；例如5-100个)N组成的核苷酸序列，每个N各自独立地为A、C、G和T中的任何一种；
    并且，在步骤(3)中，使得所述载体序列的第一固定序列、定位序列、捕获模板序列、以及UMI互补序列区域形成双链，从而使得所述第一核酸分子从5’至3’方向上包含第一固定序列互补序列、定位序列互补序列和捕获序列、以及所述位于所述捕获序列上游、且位于所述第一固定序列互补序列的下游的UMI序列；
    优选地，所述UMI互补序列位于所述定位序列和捕获模板序列之间，或者位于所述第一固定序列与定位序列之间。
  30. 权利要求27-29任一项的方法，其中，所述第一引物的连接子是能够与活化基团(例如NH 2)偶联的连接基团，所述固相支持物表面具有活化基团(例如NH 2)修饰；
    优选地，所述连接子包括-SH、-DBCO或-NHS；
    优选地，所述连接子是
    Figure PCTCN2020090340-appb-100004
    (DBCO)，所述固相支持物表面连接有
    Figure PCTCN2020090340-appb-100005
    (
    Figure PCTCN2020090340-appb-100006
    ester)。
  31. 权利要求27-30任一项的方法，其中，所述第一引物的切除区所包含的切割位点是可以通过化学、酶或光化学方法进行受控切割的位点；
    优选地，所述第一引物的切除区所包含的切割位点是酶切位点；
    优选地，所述第一引物的切除区与所述载体序列的第一固定序列所包含的切割位点不同。
  32. 权利要求21-31任一项所述的方法，其中，所述能够捕获mRNA的寡核苷酸序列包括能够与mRNA的poly-A尾杂交的序列；
    优选地，所述能够捕获mRNA的寡核苷酸序列包括poly-T寡核苷酸序列；
    优选地，所述poly-T寡核苷酸序列包括至少10个(例如至少20个)脱氧胸腺嘧啶核苷残基。
  33. 权利要求21-32任一项所述的方法，其中，所述固相支持物是芯片；
    优选地，所述固相支持物能够用作测序平台，例如测序芯片；
    优选地，所述固相支持物是高通量测序芯片，例如用于Illumina、MGI或Thermo Fisher测序平台的高通量测序芯片。
  34. 权利要求21-33任一项所述的方法，其中，在步骤(3)中，在进行延伸反应的同时，对所述载体序列进行测序，从而获得所述载体序列所包含的定位序列的序列信息。
  35. 权利要求21-34任一项所述的方法，其中，在步骤(3)之前，还包括对所述载体序列进行测序的步骤；
    优选地，在完成测序之后，通过洗涤以去除因测序而被添加至合成链中的dNTP。
  36. 通过权利要求23-26任一项所述的方法制备的核酸阵列；
    优选地，所述核酸阵列如权利要求3、5-10任一项中定义。
  37. 通过权利要求27-31任一项所述的方法制备的核酸阵列；
    优选地，所述核酸阵列如权利要求11-16任一项中定义。
  38. 一种检测样品中核酸的空间信息的方法，其包括以下步骤：
    (1)提供权利要求3或5所述的核酸阵列，或者，通过权利要求23-26任一项的方法 获得核酸阵列；其中，
    所述核酸阵列包含连接于固相支持物(例如芯片)表面的多种载体序列，每种载体序列在所述阵列中占有不同位置，并且每种载体序列包含多个拷贝的载体序列；
    每个载体序列包含与其杂交的第一核酸分子，所述第一核酸分子连接第二核酸分子，或者，每个载体序列包含与其杂交的第一核酸分子和第二核酸分子；
    所述第一核酸分子包含与所述阵列上该种载体序列的位置相对应的定位序列互补序列，
    所述第二核酸分子包含能够捕获样品中的核酸的捕获序列；
    (2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述第二核酸分子的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的载体序列的位置；
    (3)(i)当所述第一核酸分子和第二核酸分子彼此未连接时，连接杂交于每个载体序列上的第一核酸分子和第二核酸分子(例如使用连接酶)；
    在允许引物延伸的条件下，以连接的第一和第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；和/或，
    以被捕获的核酸分子作为引物，以所述连接的第一和第二核酸分子为模板，进行引物延伸反应，以产生经延长的被捕获核酸分子，所述经延长的被捕获核酸分子具有所述定位序列作为空间信息标记；
    或者，(ii)当所述第一核酸分子和第二核酸分子彼此未连接时，在允许引物延伸的条件下，以所述第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生经延长的第二核酸分子，所述经延长的第二核酸分子包含被捕获核酸的互补序列；连接杂交于每个载体序列上的第一核酸分子和经延长的第二核酸分子(例如使用连接酶)，与所述被第一核酸分子连接的经延长的第二核酸分子具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；
    或者，(iii)当所述第一核酸分子和第二核酸分子彼此连接时，在允许引物延伸的条件下，以连接的第一和第二核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；和/或，
    以被捕获的核酸分子作为引物，以所述连接的第一和第二核酸分子为模板，进行引物 延伸反应，以产生经延长的被捕获核酸分子，所述经延长的被捕获核酸分子具有所述定位序列作为空间信息标记；
    (4)从阵列表面释放至少一部分空间信息标记的核酸分子，其中所述部分包括定位序列或其互补链以及被捕获核酸分子或其互补链；和
    (5)直接或间接地分析步骤(4)中被释放的核酸分子的序列；
    优选地，所述核酸的空间信息包括核酸的定位、分布和/或表达；
    优选地，所述样品是组织样品，例如组织切片；
    优选地，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
  39. 权利要求38所述的方法，所述方法用于检测样品中的转录组，其中：
    (a)在步骤(3)(i)中，以连接的第一和第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备第一核酸分子中所包含的定位序列互补序列作为空间信息标记，且可选地，扩增所述cDNA分子；或者，在步骤(3)(ii)中，以第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，连接杂交于每个载体序列上的第一核酸分子和所述cDNA分子(例如使用连接酶)，以产生具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记的cDNA分子，且可选地，扩增所述cDNA分子；或者，在步骤(3)(iii)中，以连接的第一和第二核酸分子作为反转录引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备第一核酸分子中所包含的定位序列互补序列作为空间信息标记，且可选地，扩增所述cDNA分子；
    和，
    (b)在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含定位序列或其互补链；
    优选地，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
  40. 一种检测样品中核酸的空间信息的方法，其包括以下步骤：
    (1)提供权利要求11-16任一项所述的核酸阵列，或者，通过权利要求27-31任一项所述的方法获得核酸阵列；其中，所述核酸阵列包含连接于固相支持物(例如芯片)表面的多种载体序列，每种载体序列在所述阵列中占有不同位置，并且每种载体序列包含多个拷 贝的载体序列；
    每个载体序列包含与其杂交的第一核酸分子，所述第一核酸分子包含与所述阵列上该种载体序列的位置相对应的定位序列互补序列以及能够捕获样品中的核酸的捕获序列；
    (2)将所述核酸阵列在允许退火的条件下与待测样品接触，以使得待测样品中的核酸与所述第一核酸分子的捕获序列发生退火，从而使得该核酸的位置被对应至核酸阵列上的第一核酸分子的位置；
    (3)在允许引物延伸的条件下，以所述第一核酸分子作为引物，以被捕获的核酸分子为模板，进行引物延伸反应，以产生延伸产物，与所述被捕获核酸分子杂交的链具有第一核酸分子中所包含的定位序列互补序列作为空间信息标记；
    (4)从阵列表面释放至少一部分空间信息标记的核酸分子，其中所述部分包括定位序列或其互补链以及被捕获核酸分子或其互补链；和
    (5)直接或间接地分析步骤(4)中被释放的核酸分子的序列；
    优选地，所述核酸的空间信息包括核酸的定位、分布和/或表达；
    优选地，所述样品是组织样品，例如组织切片；
    优选地，所述组织切片从固定组织制备，例如，以福尔马林固定石蜡包埋(FFPE)的组织或深度冷冻的组织。
  41. 权利要求40所述的方法，所述方法用于检测样品中的转录组，其中：
    在步骤(3)中，以所述第一核酸分子为RT引物，从已捕获的RNA分子合成cDNA分子，所述cDNA分子具备第一核酸分子中所包含的定位序列互补序列作为空间信息标记，且可选地，扩增所述cDNA分子；
    在步骤(4)中，从阵列表面释放至少一部分所述cDNA分子和/或其扩增子，其中所述被释放的核酸分子可以是cDNA分子的第一和/或第二链或其扩增子，且其中所述部分包含定位序列或其互补链；
    优选地，在步骤(1)中，所述捕获序列包含能够捕获mRNA的寡核苷酸序列。
  42. 权利要求38-41任一项所述的方法，其中，所述多个拷贝的载体序列是由所述载体序列的多联体所形成的DNB，或者，所述多个拷贝的载体序列是由所述载体序列的克隆群形的DNA簇。
  43. 权利要求38-42任一项所述的方法，在步骤(5)中，所述序列分析包括测序或序列特异性的PCR反应。
  44. 权利要求38-43任一项所述的方法，所述方法还包括步骤(6)：将步骤(5)获得的序列分析信息与所述样品的图像相关联，其中该样品在步骤(3)之前或之后成像；
    优选地，所述样品的成像是利用光、明视场、暗视场、相位差、荧光、镜像、干涉或共聚焦显微镜或其组合。
  45. 权利要求38-44任一项所述的方法，其中，在所述空间信息标记的核酸分子(例如DNA分子)或所述空间信息标记的cDNA分子从阵列表面释放之前或之后，生成所述互补链或所述cDNA第二链；
    优选地，所述互补链或第二链的合成使用随机引物和链置换聚合酶。
  46. 权利要求38-45任一项所述的方法，其中，在序列分析前进一步包括对所述空间信息标记的核酸分子(例如DNA分子)或cDNA分子进行扩增的步骤；
    优选地，所述扩增步骤在所述空间信息标记的核酸分子(例如DNA分子)或cDNA分子从阵列释放之后实施，或所述扩增步骤在阵列上原位实施；
    优选地，所述扩增步骤包括PCR。
  47. 权利要求38-46任一项所述的方法，其中，在序列分析之前进一步包括对被释放的核酸分子进行纯化的步骤。
  48. 权利要求38-47任一项所述的方法，在步骤(4)之前，所述方法进一步包括洗涤阵列以除去残余样品(例如，组织)的步骤。
  49. 权利要求38-48任一项所述的方法，在步骤(4)中，所述核酸分子通过如下方法从阵列表面释放：(i)核酸剪切；(ii)变性；和/或(iii)物理方法。
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