Analysis method, method for producing cyclic nucleic acid, and nucleic acid

Abstract

Provided is an analysis method comprising generating complementary strands of one or more target nucleic acids in a state in which a nucleic acid splicing portion is bound to one end of each of the one or more target nucleic acids, splicing two or more generated complementary strands to each other via the nucleic acid splicing portion and forming a cyclic nucleic acid, and performing analysis using the cyclic nucleic acid.

Description

[0001] Cross-reference to related applications

[0002] This application claims the benefit of Japanese priority patent application JP2023-056365, filed on March 30, 2023, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to analytical methods, methods for manufacturing circular nucleic acids, and nucleic acids. More particularly, this disclosure relates to analytical methods for cell analysis, methods for manufacturing circular nucleic acids for cell analysis, and nucleic acids for cell analysis. Background Technology

[0004] Single-cell analysis is one of the most useful methods for analyzing cells or intracellular components. For example, single-cell analysis analyzes the nucleic acids, particularly mRNA (messenger ribonucleic acid), contained in each cell. Several techniques for performing single-cell analysis have been proposed to date.

[0005] The following PTL1 discloses a method for analyzing biological particles, comprising: a capture step, capturing biological particles via the biological particle capture portion at a surface where a molecule including a biological particle capture portion, a barcode sequence, and a cleavable linker is fixed thereto by a linker; a cleaving step, releasing the biological particles from the surface by cleaving the linker; and a separation step, separating the biological particles into a microspace.

[0006] Reference List

[0007] Patent documents

[0008] PTL1: PCT Patent Publication No. WO 2022 / 009642 Summary of the Invention

[0009] Technical issues

[0010] Single-cell analysis can analyze the nucleic acids present in each cell. For analysis, the following can be added to the target sequence: such as cell identifier sequences (e.g., cell barcodes) to identify the source of nucleic acids, molecular identifier sequences (e.g., molecular barcodes, unique molecular identifiers (UMIs), etc.) to identify the original number of nucleic acid molecules, and nucleic acid amplification sequences (e.g., amplification sequences, PCR (polymerase chain reaction) handles (handle sequences)).

[0011] However, when sequencing the target sequence, these added sequences may also need to be read, causing the reading of sequences other than the target sequence to partially consume the total number of reads (length x depth).

[0012] Furthermore, because PCR amplification of cDNA (complementary deoxyribonucleic acid) is exponential, the target nucleic acids initially present in large quantities within the cell tend to increase, and these target nucleic acids are easily detected. This can introduce bias into the analytical results. In other words, during the exponential amplification of nucleic acids, the small amounts of nucleic acids present in the cell are difficult to reflect in the analytical results.

[0013] Therefore, it is desirable to shorten the sequence provided to the target nucleic acid. Furthermore, in addition to shortening the sequence provided to the target nucleic acid, it is also desirable to reduce the aforementioned bias.

[0014] Solution to the problem

[0015] This disclosure provides an analytical method comprising: generating complementary strands of one or more target nucleic acids in a state in which a nucleic acid concatenating portion (nucleic acid linking portion, nucleic acid concatenating portion) is bound to one end of each of one or more target nucleic acids; splicing (linking, connecting, concatenating) two or more of the generated complementary strands together via the nucleic acid concatenating portion to form a circular nucleic acid; and performing analysis using the circular nucleic acid.

[0016] The nucleic acid splicing portion may include a target nucleic acid capturing portion, which is configured to capture the 3' end region of the target nucleic acid.

[0017] In the generation of complementary strands, nucleic acid splicing portions can be used as primers to generate complementary strands of target nucleic acids.

[0018] In the generation of complementary strands, double strands can be formed for each target nucleic acid and its complementary strand.

[0019] In the generation of circular nucleic acids, double strands can be spliced ​​together via nucleic acid splicing components.

[0020] The nucleic acid splicing portion may include a complementary strand capture portion configured to capture the 3' end region of the complementary strand.

[0021] In the generation of circular nucleic acids, the 5' end of one complementary strand and the 3' end of another complementary strand can be spliced ​​together, and this splicing can take place in a state in which the complementary strand capture portion of the nucleic acid splicing portion bound to one complementary strand and the 3' end region of the other complementary strand are bound together.

[0022] In the generation of circular nucleic acids, a single-stranded circular nucleic acid in which complementary strands are spliced ​​together can be obtained by forming a double-stranded circular nucleic acid and then removing the target nucleic acid from the double-stranded circular nucleic acid.

[0023] In the analysis, nucleic acid amplification reactions using circular nucleic acids can be performed.

[0024] Nucleic acid amplification reactions can be RCA (rolling circle amplification) or PCR.

[0025] The nucleic acid splicing portion may include: a target nucleic acid capture portion configured to capture the 3' end region of a target nucleic acid; a complementary strand capture portion configured to capture the 3' end region of a complementary strand generated in the process of generating the complementary strand; and a double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion to each other.

[0026] The target nucleic acid capture portion may have a poly-T sequence (multi-T sequence, poly-T sequence, poly-T sequence), and the complementary strand capture portion may have a base sequence complementary to the base sequence provided to the 3' end during reverse transcription by reverse transcriptase.

[0027] The double-stranded portion can have a restriction enzyme recognition sequence.

[0028] The double-stranded portion can have a non-natural base sequence.

[0029] The double-stranded portion can have a base sequence with an error-correcting function.

[0030] The analytical method can be an analytical method for performing single-cell analysis, and can use nucleic acid splicing parts that include different double-stranded parts for each cell.

[0031] The analytical method may include disrupting cells and generating complementary nucleic acids from the target nucleic acids contained within the cells.

[0032] Cell destruction can occur within a space allocated to each cell.

[0033] The nucleic acid splicing part can be fixed on the substrate.

[0034] In addition, this disclosure also provides a method for manufacturing circular nucleic acids, comprising: generating complementary strands of one or more target nucleic acids in a state in which nucleic acid splicing portions are bound to one end of each of one or more target nucleic acids, and splicing two or more generated complementary strands together via the nucleic acid splicing portions to form circular nucleic acids.

[0035] Furthermore, this disclosure also provides a nucleic acid comprising: a target nucleic acid capture portion configured to capture the 3' terminal region of a target nucleic acid; a complementary strand capture portion configured to capture the 3' terminal region of a complementary strand generated by generating a complementary strand of the target nucleic acid; and a double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion to each other.

[0036] The target nucleic acid capture portion can be single-stranded, and the complementary strand capture portion can also be single-stranded.

[0037] In addition, this nucleic acid can be used to generate circular nucleic acids. Attached Figure Description

[0038] [ Figure 1A ] Figure 1A This is a schematic diagram illustrating an example of the structure of a nucleic acid splicing part.

[0039] [ Figure 1B ] Figure 1B This is a diagram showing an example of a sequence group with error correction capabilities.

[0040] [ Figure 1C ] Figure 1C This is a diagram illustrating an example of the structure of a nucleic acid splicing portion.

[0041] [ Figure 1D ] Figure 1D This is a diagram illustrating an example of the structure of a nucleic acid splicing portion.

[0042] [ Figure 1E ] Figure 1E This is a schematic diagram illustrating a variation (variant, alteration, modification) of the structure of the nucleic acid splicing part.

[0043] [ Figure 1F ] Figure 1F This is a schematic diagram showing a variation of the structure of the nucleic acid splicing part.

[0044] [ Figure 2A ] Figure 2A This is a schematic diagram illustrating an example of the circular nucleic acid generation process using nucleic acid splicing components.

[0045] [ Figure 2B ] Figure 2B This is a schematic diagram to help explain the structure of mRNA.

[0046] [ Figure 2C ] Figure 2C This is a schematic diagram illustrating an example of sequencing to aid in understanding.

[0047] [ Figure 2D ] Figure 2D This is a conceptual diagram of the nucleic acid splicing components used in the first variant of the circular nucleic acid generation process.

[0048] [ Figure 2E ] Figure 2E This is a schematic diagram of the first variant that helps explain the process of generating circular nucleic acids using the nucleic acid splicing part.

[0049] [ Figure 2F ] Figure 2F This is a conceptual diagram of the nucleic acid splicing components used in the second variant of the circular nucleic acid generation process.

[0050] [ Figure 2G ] Figure 2G This is a schematic diagram of a second variation that helps explain the process of generating circular nucleic acids using the nucleic acid splicing part.

[0051] [ Figure 2H ] Figure 2H This is a conceptual diagram of the nucleic acid splicing components used in the third variant of the circular nucleic acid generation process.

[0052] [ Figure 2I ] Figure 2I This is a schematic diagram illustrating the third variation of the circular nucleic acid generation process that uses the nucleic acid splicing part.

[0053] [ Figure 3 ] Figure 3 A schematic diagram is depicted to help explain the bias in exponential amplification.

[0054] [ Figure 4A ] Figure 4A This is a diagram showing an example of a restriction site.

[0055] [ Figure 4B ] Figure 4B This is a diagram that helps explain the generation of instances of tagged shortreads.

[0056] [ Figure 5 ] Figure 5 A schematic diagram illustrating an example of an operation performed in a single-cell analysis method is depicted.

[0057] [ Figure 6A ] Figure 6A This is a schematic diagram illustrating an example of the structure of a complex.

[0058] [ Figure 6B ] Figure 6B This is a schematic diagram illustrating an example of the structure of the complex.

[0059] [ Figure 6C ] Figure 6C This is a schematic diagram illustrating an example of manufacturing a composite.

[0060] [ Figure 6D ] Figure 6D This is a schematic diagram illustrating an example of an operation performed in a variant of a single-cell analysis method.

[0061] [ Figure 6E ] Figure 6E This is a conceptual diagram of the nucleic acid splicing portion used in a variant of the single-cell analysis method.

[0062] [ Figure 7 ] Figure 7 A schematic diagram illustrating an example of an operation performed in a single-cell analysis method is depicted.

[0063] [ Figure 8 ] Figure 8 A schematic diagram illustrating an example of an operation performed in a single-cell analysis method is depicted.

[0064] [ Figure 9 ] Figure 9 A schematic diagram illustrating an example of an operation performed in a single-cell analysis method is depicted.

[0065] [ Figure 10 ] Figure 10 A schematic diagram illustrating an example of an operation performed in a single-cell analysis method is depicted.

[0066] [ Figure 11 ] Figure 11 A schematic diagram illustrating an example of nucleic acid generated via RCA processing is depicted.

[0067] [ Figure 12 ] Figure 12 This is a schematic diagram of an example of wells used for particle separation steps.

[0068] [ Figure 13A ] Figure 13A This is a schematic diagram illustrating an example of an apparatus used for performing a particle separation step.

[0069] [ Figure 13B ] Figure 13BThis is a schematic diagram illustrating an example of an apparatus used for performing a particle separation step.

[0070] [ Figure 14A ] Figure 14A This is a schematic diagram illustrating an example of a device used to form an emulsion.

[0071] [ Figure 14B ] Figure 14B This is a schematic diagram illustrating an example of a chip used to form an emulsion.

[0072] [ Figure 14C ] Figure 14C This is a conceptual diagram of an antibody-nucleic acid complex used when single-cell analysis and analysis of secreted molecules on the cell surface are performed simultaneously.

[0073] [ Figure 14D ] Figure 14D This is a schematic diagram illustrating an example of separating cells on a chip used to form an emulsion, whereby cells to be subjected to single-cell analysis and analysis of secretory molecules on the cell surface are separated.

[0074] [ Figure 14E ] Figure 14E This is a schematic diagram illustrating examples of operations performed in single-cell analysis and analysis of molecules secreted on the cell surface.

[0075] [ Figure 15 ] Figure 15 A schematic diagram illustrating an example of a chip used to form an emulsion is provided.

[0076] [ Figure 16 ] Figure 16 This is a block diagram that helps explain an example of a device used to form an emulsion.

[0077] [ Figure 17 ] Figure 17 An example flowchart of the operations performed on the emulsion is shown.

[0078] [ Figure 18A ] Figure 18A This is a schematic diagram showing an enlarged view of the connection flow passage and its vicinity.

[0079] [ Figure 18B ] Figure 18B This is a schematic diagram showing an enlarged view of the connecting flow channel and its vicinity.

[0080] [ Figure 19 ] Figure 19 This is a schematic diagram illustrating an example where a priming sequence (primer sequence, initiation sequence) is provided. Detailed Implementation

[0081] Preferred embodiments for carrying out this disclosure will be described below. It should be noted that the embodiments described below represent representative embodiments of this disclosure, and the scope of this disclosure is not limited to these embodiments. Incidentally, this disclosure will be described in the following order.

[0082] 1. First Implementation Method (Analysis Method)

[0083] 2. Second Implementation Method (Circular Nucleic Acid Manufacturing Method)

[0084] 3. Third Implementation Method (Nucleic Acid)

[0085] 1. First Implementation Method (Analysis Method)

[0086] (1) Basic Concepts

[0087] (question)

[0088] When the sequence of the identifier provided to the target sequence is shortened, identifier diversity (identifier variation) decreases, and the number of identifiers available for analyzing large numbers of cells and molecules becomes insufficient. Identifiers for single-stranded DNA are generated by the four base combinations of A / T / G / C, and their diversity is only 4. n (n = identifier length). 10 are needed within this range. 6 In the case of a single identifier, n=10. Furthermore, in practical analysis, error correction mechanisms are often incorporated, causing n to increase further. In addition, simply put, each time the length is shortened by 1, the diversity decreases to 1 / 4. Therefore, shortening the identifier length is not an ideal method.

[0089] Another approach would be to reduce the number of identifiers mentioned above. However, each identifier has a function, making it difficult to simply eliminate them.

[0090] For example, in the case of eliminating cell identifiers (cell barcodes), it is conceivable to analyze pooled (concentrated) cells via NGS (next-generation sequencing). However, in this case, it is difficult to classify the molecules to be analyzed for each cell. Furthermore, although it is conceivable to perform NGS on each cell individually, analyzing a large number of cells involves high costs.

[0091] In cases where molecular identifiers (molecular barcodes, UMIs) are eliminated, the molecules must be non-replicable, or the molecules that need to be replicated must share a common sequence. Not all mRNAs have distinct sequences, and multiple mRNAs may have the same sequence. Therefore, in cases where replication occurs after cDNA synthesis, it is impossible to distinguish between the original quantity and the quantity obtained from replication unless a sequence such as a UMI is provided.

[0092] In the absence of the amplification sequence (amplification sequence, PCR handle (handle sequence, ligation sequence, handle)), it is difficult to increase the molecules converted into cDNA. For amplification to occur without the amplification sequence, where the target sequence may be known, molecules with unknown sequences cannot be detected.

[0093] Furthermore, for amplification methods using PCR as exponential amplification, molecules with higher frequencies tend to increase, while molecules with lower frequencies are less likely to increase. Therefore, molecules with high frequencies tend to be easier to detect.

[0094] (Overview of this disclosure)

[0095] The analytical method according to embodiments of this disclosure uses circular nucleic acids in which complementary strands of two or more target nucleic acids are spliced ​​together via nucleic acid splicing portions.

[0096] In the analytical method, the nucleic acid splice region can serve as a cell identifier and also as a molecular identifier. Furthermore, the nucleic acid splice region can serve as an amplification sequence. Additionally, the length of the base sequence constituting the nucleic acid splice region can be shortened. That is, in the analytical method according to the embodiments of this disclosure, the length of the sequence added to the target nucleic acid can be shortened.

[0097] Here, target nucleic acid refers to the nucleic acid that will be captured by the nucleic acid splicing portion described later.

[0098] Furthermore, in circular nucleic acids, two or more target nucleic acids or sequences derived from target nucleic acids (including sequences complementary to the target nucleic acid) can be spliced ​​together. For example, one or more target nucleic acids or sequences derived from those target nucleic acids that are present in small amounts within the cell and one or more target nucleic acids or sequences derived from those target nucleic acids that are present in large amounts within the cell can be spliced ​​together. When such target nucleic acids with low frequency of presence are spliced ​​together with target nucleic acids with high frequency of presence, the probability of detecting target nucleic acids with low frequency of presence can be increased. Therefore, the bias described above can be reduced.

[0099] In one embodiment, the analytical method according to embodiments of the present disclosure includes: a complementary nucleic acid generation step, wherein complementary strands of one or more target nucleic acids are generated in a state in which nucleic acid splicing portions are bound to one end of each of one or more target nucleic acids; and a circular nucleic acid generation step, wherein two or more generated complementary strands are spliced ​​together via the nucleic acid splicing portions to form a circular nucleic acid. The analytical method may further include an analytical step of using the circular nucleic acid for analysis. In analytical methods using the thus generated circular nucleic acid, as described above, the length of the sequence provided to the target nucleic acid can be shortened. Furthermore, bias can be reduced.

[0100] The analysis method will be described below with reference to the accompanying drawings.

[0101] (2) Example 1 (Bulk analysis)

[0102] (2-1) Nucleic acid splicing part

[0103] The nucleic acid splicing portion used in the analytical method according to embodiments of this disclosure will be described first. In this specification, the portion involving the splicing of two or more complementary strands of a nucleic acid splicing portion is also referred to as a splice. Figure 1A An example of the structure of a nucleic acid splicing portion is shown in the figure. The nucleic acid splicing portion 10 shown in the figure includes: a target nucleic acid capturing portion 11 configured to capture the 3' end region of the target nucleic acid, a complementary strand capturing portion 12 configured to capture the 3' end region of the complementary strand of the target nucleic acid, and a double-stranded portion 13 connecting the target nucleic acid capturing portion 11 and the complementary strand capturing portion to each other.

[0104] Furthermore, the nucleic acid splicing portion used in the analytical method according to the embodiments of this disclosure can also be employed. Figure 1E or Figure 1F The structure shown is as depicted. (As in...) Figure 1A In the structural examples shown in these figures, the nucleic acid splicing portion 10 includes a target nucleic acid capture portion 11 and a complementary strand capture portion 12. The target nucleic acid capture portion 11 is configured to capture the 3' terminal region of the target nucleic acid, and the complementary strand capture portion 12 is configured to capture the terminal region of a sequence derived from the target nucleic acid, such as the 3' terminal region of the complementary strand of the target nucleic acid. On the other hand, the nucleic acid splicing portion 10 shown in these figures does not have a double-stranded portion 13. Incidentally, Figure 1F The nucleic acid splicing portion 10 shown in the image, besides... Figure 1E In addition to the structure, it also includes a sequence appendage 14.

[0105] In this technology, by using the resulting nucleic acid splicing portion, the sequence of the target nucleic acid or the circular nucleic acid having a sequence derived from the target nucleic acid can be shortened, and detection bias can be reduced.

[0106] The target nucleic acid capture portion 11 is configured, for example, to capture the 3' terminal region of the target nucleic acid. In the case where the target nucleic acid is mRNA, for example, the mRNA has a poly-A tail in its 3' terminal region. Therefore, the target nucleic acid capture portion 11 can be configured to capture the poly-A tail. Specifically, the target nucleic acid capture portion 11 can be a base sequence configured to capture the poly-A tail.

[0107] To enable the target nucleic acid capture portion 11 to capture the poly-A tail, the target nucleic acid capture portion may, for example, have a poly-T sequence (multi-T sequence, poly-T sequence, poly-T sequence). The length of the poly-T sequence may be, for example, 10 to 50 bases, and preferably 15 to 30 bases. That is, the poly-T sequence may, for example, include 10 to 50 T bases, and preferably 15 to 30 T bases. The target nucleic acid capture portion 11 may consist only of the poly-T sequence.

[0108] Furthermore, the target nucleic acid capture portion 11 can be single-stranded DNA or RNA. This facilitates binding to the target nucleic acid, and is particularly beneficial for complementary binding.

[0109] Incidentally, the length of the poly-T sequence can be even longer or even shorter, and can be varied, for example, depending on the length of the poly-A tail of the target nucleic acid.

[0110] Furthermore, when the RNA does not have a poly-T sequence or the target RNA sequence is known, a random sequence (random primer, random hexamer, etc.) or a sequence that specifically binds to the target RNA can be used as the target nucleic acid capture portion. The length of the random sequence can be, for example, 6 to 20 bases, and preferably 6 to 10 bases. The length of the sequence that specifically binds to the target RNA can be 10 to 40 bases, or preferably 15 to 35 bases.

[0111] Incidentally, examples of sequences complementary to the target RNA are shown in Tables 1 and 2 below. Table 1 shows examples of base sequences that specifically bind to ERBB2 (ERBB2_probe 1 to ERBB2_probe 19; these correspond to sequence IDs 109 to 127). Table 2 shows examples of base sequences that specifically bind to XPO1 (XPO1_probe 1 to XPO1_probe 13; these correspond to sequence IDs 128 to 140).

[0112] The target nucleic acid capture portion may contain at least one base sequence that specifically binds to the target RNA. In the presence of multiple different target RNAs, the target nucleic acid capture portion may contain at least one base sequence selected from the sequence group that specifically binds to each target RNA. In the presence of a single target RNA, the target nucleic acid capture portion may contain at least one base sequence selected from the sequence group that specifically binds to that single target RNA.

[0113] Sequences that specifically bind to the target RNA can be designed using methods similar to PCR primers and FISH (fluorescence in situ hybridization) probes. To briefly illustrate this, sequence sets of all possible combinations of a set length are generated, and for each sequence included in the sequence set, the stability of binding to the target sequence is calculated using the nearest neighbor method. Sequence sets that bind at 37°C, for example, -5 kcal / mol or lower, preferably -28 kcal / mol or lower, are extracted from the sequence sets. Further, sequence sets with a GC content of 40% to 60% are extracted from the extracted sequence sets. Optionally, sequences that meet one or more additional screening criteria are extracted, such as an A content of less than 28%, exclusion of sequences with four or more consecutive bases, a C content of 22% to 28%, etc. This process selects sequence sets that specifically bind to the target RNA.

[0114] [Table 1]

[0115] [Table 2]

[0116] The complementary strand capture portion 12 is configured, for example, to capture the terminal regions (e.g., the 3' terminal regions of the complementary strands of the target nucleic acid or nucleic acids having sequences derived from the target nucleic acid). Specifically, in the analytical method according to embodiments of this disclosure, the complementary strand capture portion of a nucleic acid splicing portion can capture the 3' terminal regions of the complementary strands of target nucleic acids (complementary strands generated from the cDNA synthesis of other target nucleic acids) other than those captured by the target nucleic acid capture portion of a nucleic acid splicing portion. Thus, two or more target nucleic acids or complementary strands of sequences derived from the target nucleic acid can be spliced ​​together via the nucleic acid splicing portion.

[0117] Here, "assembly via nucleic acid splicing portion" is not limited to the case of splicing via nucleic acid splicing portion, but also includes the case of splicing via sequence derived from nucleic acid splicing portion.

[0118] In the analytical method according to embodiments of the present disclosure, the complementary nucleic acid generation step is a step of generating two or more complementary strands to be spliced ​​together in a subsequent (next) circular nucleic acid generation step. More specifically, complementary strands of one or more target nucleic acids are generated in a state where the nucleic acid splicing portion is bound to one end of each of one or more target nucleic acids.

[0119] Incidentally, as will be described later, the complementary nucleic acid generation step can generate a nucleic acid having a sequence derived from the target nucleic acid, the complementary strand of the generated target nucleic acid serving as a template, and can provide a sequence captured by the complementary strand capture portion contained in the nucleic acid splicing portion.

[0120] For example, in the case where the target nucleic acid is mRNA, the complementary strand produced by reverse transcription of the mRNA has a CCC sequence (C: cytosine), which is generated at its 3' end by the reverse transcriptase performing the reverse transcription. Therefore, the complementary strand capture portion 12 can be configured to capture the CCC sequence. Specifically, the complementary strand capture portion 12 can be a base sequence configured to capture the CCC sequence.

[0121] Therefore, the complementary strand capture portion can have a base sequence that is complementary to the base sequence provided to the 3' end during reverse transcription by reverse transcriptase.

[0122] On the other hand, when using a nucleic acid splicing portion that does not have a double-stranded portion, the complementary strand capture portion included in the nucleic acid splicing portion can be an optional sequence, and a nucleic acid with a sequence derived from the target nucleic acid can be generated by using a primer having a sequence at the 5' end complementary to the sequence of the complementary strand capture portion. In this case, the complementary strand capture portion of the nucleic acid splicing portion located at the 5' end of the complementary strand (cDNA) generated by reverse transcription of mRNA can capture a sequence complementary to the sequence of the aforementioned complementary strand capture portion, wherein the complementary sequence originates from a primer present at the 5' end of a nucleic acid with a sequence derived from another target nucleic acid. Therefore, two or more complementary strands are spliced ​​together via the nucleic acid splicing portion.

[0123] When using this nucleic acid splicing portion, the sequence of the complementary strand capture portion is not limited to the GGG sequence, and can be formed into sequences of any length by combining the four bases of A, T, C, and G. When the sequence of the complementary strand capture portion is extended, circular nucleic acids can be formed when two or more generated complementary strands are spliced ​​together via the nucleic acid splicing portion to form circular nucleic acids, while reducing the failure to capture the generated complementary strands. Therefore, it can be expected, for example, that the probability of detecting complementary strands from sequences originating from mRNAs that occur at low frequencies will increase, and the accuracy of the analysis will thus be improved.

[0124] For example, the complementary strand capture portion 12 of the capture CCC sequence includes a GGG sequence. The GGG sequence can be DNA or RNA. That is, the GGG sequence can be GGG or rGrGrG (r: ribonucleotide, G: guanine).

[0125] Furthermore, the complementary strand capture portion 12 can be single-stranded DNA or RNA. This facilitates binding to the complementary strand.

[0126] In addition to the GGG sequence, the complementary strand capture portion may also include a self-binding repression sequence Hn or Nn. Here, H is a base other than G, i.e., H is A, T, or C. N is A, T, G, or C. n is the number of H or N, and can be, for example, an integer of 1 or greater. n can be, for example, any integer from 1 to 8. In the case where there are supposedly about 20,000 mRNAs, the mRNAs can be covered by such a numerical range. In some embodiments, n can be, for example, 1, 2, 3, 4, or 5, and can further be 1 or 2. Where n is 2 or greater, each H or N constituting the self-binding repression sequence can be selected individually and randomly. The complementary strand capture portion may, for example, have a base sequence of GGGH, GGGN, GGGHN, GGGNH, GGGHH, or GGGNN. The complementary strand capture portion may be DNA or may be RNA.

[0127] The double-stranded portion 13 is the portion that connects the target nucleic acid capture portion 11 and the complementary strand capture portion 12 to each other. The double-stranded portion 13 can be formed from double-stranded DNA, double-stranded RNA, or a hybrid of DNA and RNA. Preferably, the double-stranded portion 13 is DNA. This prevents degradation during RNA digestion, as described later, and promotes the formation of circular nucleic acids. Furthermore, the double-stranded portion 13 is readily usable as a primer in nucleic acid amplification.

[0128] The target nucleic acid capture portion 11 is spliced ​​to the 3' end of one strand of the double-stranded portion 13. The complementary strand capture portion 12 is spliced ​​to the 3' end of the other strand of the double-stranded portion 13. Because this structure is provided, circular nucleic acids can be formed by splicing the complementary strands, as described later.

[0129] The double-stranded portion 13 may include a sequence containing a random combination of four bases: A, T, C, and G. The random sequence preferably includes a sequence set with error correction functionality. Error-correcting sequence sets include, for example, sequence-Levenshtein codes and filled / truncated right-end edit (FREE) barcodes. Methods for generating these sequence sets and error correction mechanisms using these sequence sets are described in Buschmann and Bystrykh BMC Bioinformatics 2013, 14:272 and Proc NatlAcad Sci USA. 2018 Jul 3; 115 (27): E6217-E6226. Those skilled in the art can appropriately generate and use error-correcting sequence sets by referring to portions of these documents. Furthermore, in addition to the two sequence sets described above, sequence sets with error correction functionality known in the art are known, such as Levenshtein codes, Hamming codes, and Reed-Solomon codes, and any of these may be used in this disclosure. Incidentally, sequence sets with error-correcting capabilities can also be referred to as Indel-correcting DNA barcodes. Furthermore, software for generating such sequence sets with error-correcting capabilities, as well as software for performing error correction using these sequence sets, is known to those skilled in the art. Those skilled in the art can prepare and use sequence sets with error-correcting capabilities using such software. Therefore, the extracted sequence set can identify the original sequence even when a few base read errors (insertions, deletions, or substitutions) occur during sequencing.

[0130] The mechanism of error-correcting sequences and examples of their generation methods will be described below.

[0131] The mechanism of a sequence with error correction capability will be described below. However, the following description represents typical examples, and the sequence with error correction capability applicable in this disclosure is not limited to sequences based on these mechanisms.

[0132] The Hamming distance is considered to increase by 1 when one of the barcode sequences is replaced. For example, "TCT," where one of the sequences in "ACT" is replaced, has a distance of 1 (A replaced by T), there is a distance of 1 between "TCT" and "TAT" (C replaced by A), and there is a distance of 1 between "TAT" and "TAC" (T replaced by C). In the case of "ACT" and "TAC," the total distance is 3. For example, in the case where "ACT," "GTG," "TAC," and "CGA" are prepared as barcodes, their distances to each other are 3. In the case where the sequencing result is "ACG," "ACG" is different from the prepared barcode, therefore, an error is considered to have occurred. This distance is used to estimate which sequence has an error. The distance between the read "ACG" and the four prepared sequences is calculated. Under the assumption of a uniform error frequency, the most likely barcode is estimated to be "ACT," which has the shortest distance. Therefore, it is possible to detect and correct code that has been replaced by another code with Hamming distance. This may require at least 2... The length of k+1 is used to correct k errors.

[0133] The Levenshtein distance allows for handling not only substitutions but also insertions and deletions. For each substitution, insertion, or deletion, the distance is considered to increase by 1. For example, the distance between "GCG" and "GC" is considered 1 (G deletion), the distance between "GC" and "GA" is considered 1 (C replaced by A), and the distance between "GA" and "AGA" is considered 1 (A insertion). As a sum, the distance between "GCG" and "AGA" is 3. For example, sequences "GCG", "TTT", "AGA", and "CAC" with a mutual distance of 3 are prepared as barcodes. In the case where sequencing yields "GC", "GC" differs from the prepared barcode, and therefore, an error is considered to have occurred. The distance between the obtained "GC" and the prepared barcode is calculated. The sequence with the shortest distance is estimated as the most likely barcode. In the example above, "GCG", with an estimated distance of 1, is the barcode. This method is only applicable when the barcode length is known in advance.

[0134] By treating the distance between barcodes A and B of arbitrary length as the number of substitutions, insertions, and deletions, and by obtaining A through these operations once performed: reducing A to the same length as B, or adding the same bases until A has the same length as B, the sequence-Levenshtein distance can handle arbitrary lengths.

[0135] FREE (filled / truncated right end editbarcodes) is based on the Needleman-Wunsch algorithm to correct for substitutions, insertions, and deletions. Like a checkerboard pattern, the two sequences that need alignment are arranged vertically and horizontally. The two sequences are compared to each other. For example, if the bases at each position match (match), +2 is set. If the bases do not match (mismatch), -1 is set. If no base is present (gap), -2 is set. Alignments are generated by calculating for each unit (cell) and then backtracking the arrows in descending order of score. For example, in the case where “ATTGC” and “ATGC” are compared, “ATTGC” is aligned to “AT-GC” (a deletion or insertion of T).

[0136] As illustrated in the examples above, according to embodiments of this disclosure, a sequence with error correction functionality can be configured to detect erroneous base sequences in the event that an error has occurred in the base sequence identified by sequencing. The sequence with error correction functionality can, for example, be configured to detect differences between the sequence identified by sequencing and a sequence including a pre-prepared barcode. The pre-prepared barcode can, for example, be a sequence identified based on a distance associated with a change (e.g., substitution, insertion, deletion, etc.). This distance can be a distance based on predetermined rules, such as Hamming distance, Lewinstein distance, or sequence-Lewinstein distance, as described above. Furthermore, the pre-prepared barcode can, for example, be configured to identify changes through alignment in a FREE as described above. Particularly preferably, the sequence with error correction functionality can be configured to detect errors in the event that an error has occurred in the sequence and to predict the most likely barcode. As described above, prediction can be made based on predetermined rules according to the type of sequence with error correction functionality. For example, as described above, the estimation can be based on the distance between the sequence identified by sequencing and the pre-prepared barcode, or it can be based on the comparison between the sequence identified by sequencing and the pre-prepared barcode.

[0137] The following describes methods for generating sequences with error correction capabilities. However, the following description represents typical examples, and sequences with error correction capabilities applicable to this disclosure are not limited to those generated by these methods.

[0138] For example, Conway's lexicographic code algorithm is used to generate sequences with error correction capabilities. This algorithm generates all sequences of a set length and then selects sequences with a distance of d from each other. First, a sequence is unconditionally selected from the sequence group. Next, the distances to the selected sequence are calculated for the remaining sequence groups. Sequences with distances less than d are excluded. Sequences with distances equal to or greater than d are selected. When similar processing is performed on the remaining sequence groups, sequences with distances equal to or greater than d from the selected sequence are selected. Similar processing is performed on all sequence groups until no alternatives exist.

[0139] Therefore, a sequence with error correction capability can be a sequence generated by a predetermined algorithm. The predetermined algorithm can be an algorithm that selects sequences that meet predetermined criteria related to distance. Examples of distance are described above. The predetermined criteria can be selected based on the type of sequence with error correction capability that will be used.

[0140] Furthermore, for example, a dictionary-type code generation method can generate sequences with error correction capabilities. In this method, sequences of a set length are generated alphabetically, and the newly generated sequence is registered as a valid sequence when it does not overlap with the decoded sequences of candidate sequences. For example, when considering the case of correcting m errors in "CTCA", barcodes (e.g., CTGA, CCA, CTGC, etc.) are stored in the decoding sphere (decode field) DecodeSphere ("CTCA") of "CTCA" when m or fewer insertions / deletions / replacements have occurred. When another barcode candidate with a length of 4 bases, for example, barcode candidate "AACC", appears, and DecodeSphere ("AACC") does not overlap with DecodeSphere ("CTCA"), AACC is registered as a valid barcode. The number of correctable errors m and the barcode sequence length n are limited and may require at least 2 The length of m+1.

[0141] Therefore, a sequence with error correction functionality can be a sequence generated using a dictionary-type code generation method. In this method, as described above, barcodes are registered based on whether the barcodes stored in the decoding sphere overlap, and a sequence with error correction functionality can be generated from the registered barcodes.

[0142] Each sequence included in a sequence group with error correction functionality can, for example, be a sequence that is not complementary to existing RNA. For instance, from the viewpoint of improving sequencing efficiency, each sequence included in a sequence group with error correction functionality may have a guanine and cytosine content of 40% to 60%. Furthermore, from the above viewpoint, each sequence included in a sequence group with error correction functionality can be a sequence that does not have three or more consecutive homopolymer sequences. Further, from the above viewpoint, each sequence included in a sequence group with error correction functionality can be a sequence that does not have two or more self-complementary sequences. Sequences that at least meet these conditions can be used as sequences included in a sequence group with error correction functionality. The conditions that each sequence included in a sequence group with error correction functionality must meet can be appropriately varied depending on the type of sequence.

[0143] In this specification, a sequence with error correction capability may be represented by "(N)i" (where N is an optional base (A, T, G, or C), and i is the number of bases) and may have error correction capability. i may be, for example, 3 or greater, 4 or greater, or 5 or greater, may be further 10 or greater, and may be further 15 or greater. Furthermore, i may be, for example, 200 or less, 150 or less, or 100 or less, and may be further 50 or less. The length of the sequence with error correction capability can be appropriately varied depending on the number of sequences to be prepared (the number of barcodes). To extract one million or more sets of sequences with the capability to correct errors of one base, for example, double-stranded portions (especially sequences with error correction capability) may need to have a length of 16 or more bases. Figure 1B The sequence groups shown are cited, for example, as examples of sequence groups with error-correcting capabilities. SEQ ID Nos. 1 to 50 shown in the figure represent examples of sequences with 16mer error-correcting capabilities. SEQ ID Nos. 51 to 100 represent examples of sequences with 17mer error-correcting capabilities. Incidentally, it should be understood that, as mentioned above, in the case of one million or more sequence groups with error-correcting capabilities, for example, in the case of 16mer, this figure shows examples of a very small fraction of sequence groups. The sequence groups listed in the figure have a left end as a 3' end and a right end as a 5' end.

[0144] Furthermore, two or more of these extracted error-correcting sequence groups can be combined to increase the variety. Error-correcting sequences can be appropriately generated by those skilled in the art, and for example, sequences include those generated by the methods described above. However, the error-correcting sequences are not necessarily limited to these. That is, the double-stranded portion can have two or more error-correcting sequences.

[0145] As will be described in the examples of the analytical methods described later, the previously described random sequences can be different for each region in which nucleic acid splicing portions are arranged. That is, in the analytical method, when multiple regions in which nucleic acid splicing portions are arranged are used, the random sequences possessed by the multiple nucleic acid splicing portions arranged in a region each have the same base sequence, but the base sequences of the random sequences can be different between regions. Therefore, the previously described random sequences can be used as identifiers, or for example as cell identifiers, and can further be used as location information identifiers.

[0146] Preferably, in the two base sequence strands constituting the double-stranded portion 13, the strand coupled to the target nucleic acid capture portion 11 (especially the strand with poly-T) has its 5' end modified by phosphorylation. This allows for more reliable ligation processing, which will be described later.

[0147] Preferably, in the two base sequence strands constituting the double-stranded portion 13, the strand not connected to the target nucleic acid capture portion 11 (especially the strand without poly-T) has a 3' end connected to the complementary strand capture portion.

[0148] The double-stranded portion may preferably have a non-natural base sequence. In particular, random sequences may include non-natural base sequences. A non-natural base sequence refers to a base sequence that does not exist naturally. Those skilled in the art can appropriately design such base sequences. By using non-natural base sequences, unwanted double-strand formation and sequence detection errors can be suppressed, for example.

[0149] The double-stranded portion 13 may further include a priming sequence. The priming sequence may, for example, be a base sequence used as a primer in the nucleic acid amplification process described later. Those skilled in the art can appropriately select the sequence based on, for example, the type of nucleic acid amplification process and / or the type of enzyme used in the nucleic acid amplification process.

[0150] As described above, in the analytical method according to embodiments of the present disclosure, when using multiple regions in which nucleic acid splicing portions are arranged, the initiating sequences of the multiple nucleic acid splicing portions arranged in one region each have the same base sequence. Furthermore, the initiating sequences between regions may also be identical. Therefore, the amplification reaction occurs simultaneously from multiple circular nucleic acids having nucleic acid splicing portions with the same initiating sequence during a single amplification process.

[0151] The nucleic acid splicing part includes, for example: Figure 1F In the case of sequence additional portion 14 in the illustrated example, sequence additional portion 14 is positioned between target nucleic acid capture portion 11 and complementary strand capture portion 12. Sequence additional portion 14 may include DNA, may include RNA, or may include a hybrid of DNA and RNA.

[0152] The sequence appendix 14 may comprise a sequence consisting of a random combination of four bases: A, T, C, and G. The sequence appendix 14 may, for example, be provided with a random sequence as a cell identifier sequence (cell barcode) for identifying the origin of the nucleic acid. Alternatively, the sequence appendix 14 may be provided with a sequence set that has the functionality to be provided to the aforementioned double-stranded portion.

[0153] The nucleic acid splicing portion 10 (particularly the double-stranded portion 13 or the sequence appendage portion 14) may or may not include the UMI. When the UMI is omitted, the base sequence of the nucleic acid splicing portion can be shortened.

[0154] An example of the structure of nucleic acid splicing part 10 is shown in Figure 1C As shown in the image.

[0155] The nucleic acid splicing portions shown in a1 and a2 of the figure (corresponding to sequence IDs 101 to 104) have a poly-T sequence as the target nucleic acid capture portion (on the left side of the figure), a GGG sequence as the complementary strand capture portion (on the right side of the figure), and a random sequence as the double-stranded portion shown in the figure.

[0156] The nucleic acid splicing portions shown in b1 and b2 of the figure (corresponding to sequence IDs 105 to 108) have a poly-T sequence (on the left side of the figure) as the target nucleic acid capture portion and a GGG sequence (on the right side of the figure) as the complementary strand capture portion, and in addition to the random sequence, they also have a priming sequence (underlined portion) as the double-stranded portion. The priming sequence is the initiation site in the nucleic acid amplification process of the circular nucleic acid, which will be described later. For example, in the case where the nucleic acid amplification process is RCA, an RCA priming sequence can be used, and in the case where the nucleic acid amplification process is PCR, a PCR priming sequence can be used. The base sequences of these priming sequences can be suitably selected by those skilled in the art, as described above.

[0157] That is, in one implementation, such as Figure 1D As shown, the nucleic acid splicing portion 10 may have a first strand and a second strand, in which a target nucleic acid capture portion (capture sequence) and a random sequence (random) are spliced ​​together, and in the second strand a GGG sequence (or the aforementioned GGG (Hn and / or Nn) sequence) and a random sequence (random') complementary to the random sequence are spliced ​​together, and the nucleic acid splicing portion 10 may be configured such that the first strand and the second strand bind nucleic acids to each other through two complementary random sequences.

[0158] The target nucleic acid capture portion may include a poly-T sequence, for example, as described above, may include a random sequence (random primer, random hexamer, etc.), or may include a sequence (target primer) that specifically binds to the target RNA, the sequence specifically binding to the target RNA.

[0159] The two complementary random sequences correspond to the double-stranded portion described above. The GGG sequence corresponds to the complementary strand capture portion described above. For the first strand, the 5' end is on the random sequence side, and the 3' end is on the poly-T sequence side. For the second strand, the 5' end is on the random sequence side, and the 3' end is on the GGG sequence side.

[0160] (2-2) The process of circular nucleic acid formation

[0161] In the following text, see references Figure 2A and Figure 2BThe process of generating circular nucleic acids using nucleic acid splicing portions will be described, and nucleic acid amplification using circular nucleic acids will be further described. More specifically, in the circular nucleic acid generation process shown in the figure, two mRNAs are reverse transcribed to generate two cDNAs complementary to their respective mRNAs, and then these two cDNAs are spliced ​​together to generate a circular nucleic acid. The steps performed in the circular nucleic acid generation process are as follows. Incidentally, although two different mRNAs are spliced ​​together in this example, two or more identical mRNAs can also be spliced ​​together in this disclosure.

[0162] like Figure 2A As shown at the top, we assume there are two types of mRNA (mRNA1 and mRNA2). mRNA1 and mRNA2 are the target nucleic acids.

[0163] like Figure 2B As shown, mRNA1 includes the translation region CDS1 and further includes a polyA sequence polyA1 at its 3' end. Similarly, mRNA2 includes the translation region CDS1 and further includes a polyA sequence polyA2 at its 3' end.

[0164] exist Figure 2A In step S11 shown, the 3' end of mRNA1 is captured by the nucleic acid splicing portion 10. In the case where the 3' end of mRNA1 has a poly-A tail, the poly-T sequence 11 of the nucleic acid splicing portion captures the poly-A tail, or in particular, binds complementaryly to the poly-A tail. The 3' end of the mRNA is thus captured by the nucleic acid splicing portion (specifically, the target nucleic acid capture portion).

[0165] Similar to mRNA1, the 3' end of mRNA2 is also captured by nucleic acid splicing regions (especially target nucleic acid capture regions).

[0166] In step S12 of this diagram, cDNA of mRNA1 is synthesized. cDNA synthesis can be performed, for example, by reverse transcriptase. The nucleic acid splicing portion is used as a primer for cDNA synthesis. Therefore, as shown in the diagram, a hybrid of mRNA1 and cDNA1, H1, is formed.

[0167] Similarly, for mRNA2, cDNA2 is synthesized via reverse transcriptase, thus forming a hybrid of mRNA2 and cDNA2, H2.

[0168] In step S2, where cDNA synthesis is performed via reverse transcriptase, a CCC sequence is formed at the 3' end of the synthesized cDNA, as shown in the figure. This CCC sequence is used for the following circular nucleic acid formation, as described later.

[0169] Therefore, in the method according to the embodiments of the present disclosure, the complementary nucleic acid generation step of generating complementary strands of one or more target nucleic acids can be performed in a state in which the nucleic acid splicing portion is bound to one end of each of the one or more target nucleic acids.

[0170] In the complementary strand generation step, the complementary strand of the target nucleic acid can be generated using nucleic acid splicing portions as primers. In this step, double strands of each target nucleic acid and its complementary strand can be formed, and thus, hybrids of, for example, mRNA and cDNA can be formed, as described above.

[0171] In step S13 of the figure, the heterozygotes H1 and H2 generated in step S2 are spliced ​​together. This splicing is based on the complementary binding between the rGrGrG sequence of heterozygote H1 and the CCC sequence of heterozygote H2.

[0172] In step S14 of this diagram, a single-stranded circular nucleic acid is formed using heterozygotes H1 and H2. Specifically, the following steps are performed to form the single-stranded circular nucleic acid.

[0173] First, heterozygote H1 has a CCC sequence at its end opposite to the end of the rGrGrG sequence used for splicing in step S13. This CCC sequence is a single-stranded portion. Heterozygote H2 has an rGrGrG sequence at its end opposite to the end of the CCC sequence used for splicing in step S13. This rGrGrG sequence is also a single-stranded portion. Therefore, the CCC sequence of heterozygote H1 and the rGrGrG sequence of heterozygote H2 complement each other, thereby forming a double-stranded circular nucleic acid.

[0174] Therefore, in the circular nucleic acid generation step included in the method according to the embodiments of this disclosure, the double strands can be spliced ​​together via nucleic acid splicing portions.

[0175] Furthermore, in the circular nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand are spliced ​​together, and this splicing can be performed with the complementary strand capture portion of the nucleic acid splicing portion bound to the one complementary strand and the 3' end region of the other complementary strand bound to each other.

[0176] In double-stranded circular nucleic acids, there is a nick between the 5' end of the strand linked to cDNA1 and the 3' end of cDNA2 generated by reverse transcription of mRNA2 in the double strand of the nucleic acid splice portion that binds to mRNA1.

[0177] Similarly, in circular nucleic acids, there is also a gap between the 5' end of the strand linked to cDNA2 in the double strand of the nucleic acid splice portion that binds to mRNA2 and the 3' end of cDNA1 generated by reverse transcription of mRNA1.

[0178] Therefore, in step S14, ligation to eliminate these gaps is performed while the double-stranded circular nucleic acid is formed. Thus, cDNA1 and cDNA2 are set in a bound state, and the circular nucleic acid state can be maintained even when mRNA degradation, as described later, is performed.

[0179] In step S14, after ligation, mRNA degradation is performed. This degradation can be carried out using, for example, RNase, particularly RNase H. Thus, as shown in the figure, a single-stranded circular nucleic acid RN is formed.

[0180] Therefore, in the circular nucleic acid generation step, a double-stranded circular nucleic acid can be formed, and then the target nucleic acid (e.g., mRNA) can be removed from the double-stranded circular nucleic acid to provide a single-stranded circular nucleic acid in which complementary strands are spliced ​​together.

[0181] In step S15, primers are added to the single-stranded circular nucleic acid (RN). In this figure, primers are used that are configured to bind to the portion of the double-stranded portion that already constitutes the nucleic acid splice and the CCC sequence. That is, the primers are configured to cover the ligation point.

[0182] In step S16, RCA is performed using DNA polymerase starting with primers. The DNA polymerase thereby synthesizes, for example, a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid.

[0183] The base sequence of the resulting single-stranded linear nucleic acid is then decoded. Decoding can be performed using techniques known in this field.

[0184] Long-read sequence technology can be used as a sequence analysis technique. For example, the single-stranded cDNA mentioned above can be sequenced using nanopore sequencing (Oxford Nanopore Technologies plc).

[0185] Furthermore, these base sequences can be sequenced using high-fidelity sequencing (HiFi sequencing) (Pacific Biosciences, Inc.). In this case, such as... Figure 2CAs shown, a double-stranded structure is generated after the step of providing single-stranded cDNA via RCA extension using a poly-A tail or a poly-C tail. The circular nucleic acid obtained by further providing an SMRTbell (trademarked) linker to the double-stranded structure is sequenced. Here, the sequence of the nucleic acid splice portion is known. Therefore, by distinguishing the sequenced sequences by the known nucleic acid splice portion after sequencing, the original mRNA sequence can be identified and the number of original mRNAs can be counted. By decoding the recurring mRNA sequences, even in the case of multi-RCA, molecules produced by replication can be considered the same and counted. That is, identification of whether the quantity is the quantity of original mRNA or the quantity produced by replication can be performed without using UMI. Furthermore, in the case of using rGrGrGNN or GGGNN, identification can be based on the position of random NN occurrences.

[0186] Furthermore, by performing long-read sequencing on complementary sequences of recurring mRNAs, read errors can be corrected even if they occur during sequencing. In other words, as mentioned above, error correction is possible by splicing two or more mRNAs. This can lead to improved decoding accuracy.

[0187] In cases where rGrGrG is used as the complementary strand capture portion of the nucleic acid splicing part, the rGrGrG is degraded by RNase H. Furthermore, in cases where the complementary strand of the double-stranded portion in the nucleic acid splicing part (i.e., the strand connected to the complementary strand capture portion) is synthesized from ribonucleotides, the complementary strand is also degraded by RNase H. Therefore, in step S15 above (i.e., before RCA in step S16), a primer is added. This primer can, for example, be a primer having the same sequence as the complementary strand of the double-stranded portion.

[0188] Furthermore, in cases where GGG is used as the complementary strand capture portion and the complementary strand of the double-stranded portion in the nucleic acid splicing portion is synthesized from nucleotides, these are not degraded by RNase H. In this case, GGG and the complementary strand can be used as primers.

[0189] In the above Figure 2A In the example shown, a circular nucleic acid is formed using two mRNAs. However, a circular nucleic acid can be formed using three or more mRNAs. That is, a circular nucleic acid comprising cDNAs complementary to three or more mRNAs can be similarly formed. The circular nucleic acid can then be subjected to nucleic acid amplification processes, such as RCA, as described above.

[0190] As described above Figure 2A As shown in the diagram, during the generation of circular nucleic acids, two or more cDNAs are spliced ​​together, and for example, cDNA corresponding to low-expressed mRNA and cDNA corresponding to high-expressed mRNA can be spliced ​​together. This can improve the detection efficiency of low-expressed mRNA.

[0191] Furthermore, in the case of PCR, nucleic acids are amplified exponentially. Therefore, the amplification process amplifies far more mRNA that was present in large quantities before amplification than the mRNA that was present in small amounts before amplification. Consequently, the mRNA that was present in small amounts before amplification is not easily detected. In other words, the PCR method introduces a bias due to amplification (this bias is also known as amplification bias).

[0192] On the other hand, nucleic acid amplification based on the RCA method is not exponential amplification, thus reducing bias. That is, in one implementation, nucleic acid amplification using circular nucleic acids can be performed using the RCA method.

[0193] Incidentally, in some embodiments of the analytical method according to the present disclosure, a PCR method can be used. This is because, even when using a PCR method, bias can be reduced by performing PCR on circular nucleic acids. This will be described below.

[0194] Typically, after cDNA synthesis, a tag is attached to the nucleic acid amplified in a PCR method. In PCR, when sequences are generated exponentially, a unique molecular identifier (UMI) is usually provided during reverse transcription to identify the origin of the generated sequence. Here, the number of UMI types may need to be equal to or greater than the number of molecular types to be distinguished (e.g., the number of molecular types initially present in each cell). Therefore, a UMI typically includes a number of bases (nucleotides) equal to or greater than 6 (e.g., it could be 6 to 10 or equal to or greater than 10). The UMI sequence is decoded during sequencing. However, these sequences are not included in the target nucleic acid and may not initially require sequence decoding. Therefore, it is preferable to be able to exclude these sequences.

[0195] Furthermore, the PCR method exponentially amplifies nucleic acids, and therefore introduces bias, as molecules expressed in large quantities tend to increase and thus those molecules are more likely to be detected. For example, in... Figure 3 As shown in A, for example, in the case of a PCR method performed on a sample in which there is one target nucleic acid M1 and the number of target nucleic acids M2 is m (7 in the figure), the reverse transcription product of target nucleic acid M1 is amplified to 2. n The target nucleic acid M2 was amplified to (2 n )× m.

[0196] In the case of performing a PCR method on nucleic acids in which two or more target nucleic acids are spliced ​​together according to embodiments of the present disclosure, for example, a molecule expressed in a low amount is spliced ​​to a molecule expressed in a high amount, and the spliced ​​nucleic acids are subjected to a PCR method. This is expected to improve the detection efficiency of low-expression molecules. For example, consider the following case where, for reference... Figure 3 The sample described in A is subjected to splicing processing according to embodiments of the present disclosure, thereby, for example, generating a splice product in which one reverse transcription product of target nucleic acid M1 and three reverse transcription products of target nucleic acid M2 are spliced ​​together, and generating a splice product in which four reverse transcription products of target nucleic acid M2 are spliced ​​together, such as Figure 3 As shown in B. In this case, each splice product was amplified to 2. n Therefore, with Figure 3 Compared to case A, the difference between the number of amplified products of target nucleic acid M1 and the number of amplified products of target nucleic acid M2 is reduced.

[0197] Therefore, the analytical method according to the embodiments of this disclosure also reduces amplification bias when using the PCR method instead of the RCA method.

[0198] As described above, the method according to embodiments of this disclosure may include an analytical step of analyzing using circular nucleic acids. In this analytical step, a nucleic acid amplification reaction using circular nucleic acids may be performed. For example, the nucleic acid amplification reaction may be RCA or PCR.

[0199] (2-3) First variant of the circular nucleic acid generation method

[0200] The circular nucleic acids that can be used in the analytical methods according to embodiments of this disclosure are not particularly limited, as long as the circular nucleic acids are generated by splicing two or more complementary strands generated by the above-described complementary nucleic acid generation steps together via a nucleic acid splicing portion. Optionally, methods can be combined to generate circular nucleic acids.

[0201] The following description, with reference to the accompanying drawings, illustrates variations of the circular nucleic acid generation process. It should be noted that the circular nucleic acid generation process described in this specification is an example and is not limited to the pattern described herein.

[0202] Figure 2D This is a conceptual diagram of the nucleic acid splicing components used in the first variant of the circular nucleic acid generation method.

[0203] exist Figure 2DIn this context, <2D-α> is the nucleic acid splicing portion of the first variant used in the circular nucleic acid generation process. <2D-β> is the primer used when generating a nucleic acid having a sequence derived from the target nucleic acid in the circular nucleic acid generation process according to this variant. <2D-γ> is an RNA oligomer used to protect the complementary strand capture portion contained in the nucleic acid splicing portion during the generation of a nucleic acid having a sequence derived from the target nucleic acid.

[0204] The <2D-α> nucleic acid splicing portion includes a target nucleic acid capture portion (the poly-T in the figure) capable of capturing the poly-A sequence present at the 3' end of the mRNA, and a complementary strand capture portion (the splice in the figure). Incidentally, although in Figure 2D Although not depicted in the examples shown, the nucleic acid splicing portion may include a sequence appendage between the target nucleic acid capture portion and the complementary strand capture portion. The sequence of the complementary strand capture portion included in the nucleic acid splicing portion used in this variant can be formed as an optional sequence. Furthermore, in the case where the nucleic acid splicing portion includes a sequence appendage, an optional sequence corresponding to the function to be provided to the sequence appendage can be provided as the sequence of the sequence appendage.

[0205] The primers shown in <2D-β> include: a poly-T sequence complementary to the poly-A sequence added to the cDNA by terminal transferase in step S213 described later, and a sequence complementary to the sequence of the complementary strand capture portion contained in the nucleic acid splicing portion of <2D-α>. Therefore, when generating nucleic acids having sequences derived from the target nucleic acid, a starting point is set, while the cDNA generated during the circular nucleic acid generation process according to this variant is used as a template, and due to the sequence complementary to the sequence of the complementary strand capture portion, two or more generated nucleic acids can be appropriately spliced ​​together via the nucleic acid splicing portion.

[0206] The RNA oligomer of <2D-γ> comprises RNA. Furthermore, the sequence of the RNA oligomer is complementary to the sequence of the complementary strand capture portion contained in the nucleic acid splicing region of <2D-α>. Therefore, the RNA oligomer can be suitably hybridized to the 5' end of the cDNA generated during the circular nucleic acid generation process according to this variant.

[0207] In a first variation of the circular nucleic acid generation process, a poly-A tailing method is used in the complementary nucleic acid generation step, and nucleic acids with sequences derived from the target nucleic acid are generated by using primers that provide a sequence to be captured by the complementary strand capture portion contained in the nucleic acid splicing portion, with the complementary strand of the generated target nucleic acid as a template.

[0208] The following will refer to Figure 2E Describe it.

[0209] exist Figure 2EIn step S211, the nucleic acid splicing portion shown in <2D-α> is used, and the target nucleic acid capture portion contained in the nucleic acid splicing portion captures the 3' end of the mRNA. Except for the different nucleic acid splicing portion used, step S211 is performed in a manner similar to the circular nucleic acid generation process described above (2-2). Conditions similar to those applicable to the circular nucleic acid generation process can also be appropriately applied to this variant.

[0210] Regarding step S212, in which... Figure 2E The mRNA sequence in the sample is used as a template to generate cDNA. Similar conditions that can be applied to the circular nucleic acid generation process described above (2-2) can also be appropriately applied to this variant.

[0211] exist Figure 2E In step S213, the RNA-DNA complex generated in step S212 is degraded by RNase H to serve as a cDNA template for mRNA.

[0212] Subsequently, the poly-A sequence is added to the 3' end of the cDNA by processing with a terminal transferase. Therefore, even without reverse transcription using reverse transcriptase at the 5' end of the mRNA and without a CCC sequence provided, the primers shown in <2D-β> can be appropriately hybridized with the cDNA by the poly-T sequence provided in the next step S214.

[0213] exist Figure 2E In step S214, the primer of <2D-β> hybridizes with the 3' end of the cDNA generated in step S213, and the RNA oligomer of <2D-γ> hybridizes with the 5' end of the cDNA.

[0214] The order of hybridization is not restricted at this point. For example, the RNA oligomers can be hybridized first, or hybridization can be performed while the concentration of the RNA oligomers is higher than the concentration of the primers.

[0215] After that, Figure 2E In step S215, a nucleic acid with a sequence derived from the target nucleic acid is generated using a DNA polymerase without chain substitution activity, starting with a <2D-β> primer and using cDNA as a template. Incidentally, the nucleic acid generation reaction by the DNA polymerase without chain substitution activity ends at the site of RNA oligomer hybridization because the <2D-γ> RNA oligomer hybridizes to the 5' end of the cDNA.

[0216] exist Figure 2EIn step S216, the RNA-DNA complex generated in step S215 is treated with RNase H. Therefore, the RNA oligomer is degraded, and a DNA-DNA complex comprising the complementary strand of the target nucleic acid is obtained.

[0217] exist Figure 2E In step S217, two or more DNA-DNA complexes containing the complementary strands of the obtained target nucleic acid are spliced ​​together via the complementary strand capture portion of the nucleic acid splicing portion and a sequence complementary to the sequence of the complementary strand capture portion, the complementary sequence being derived from a <2D-β> primer. As a condition for this step, in this variant, conditions similar to those applicable to the circular nucleic acid generation process described above (2-2) may also be appropriately applied.

[0218] The splicing reaction shown in step S217 forms a single-stranded circular nucleic acid, as in the circular nucleic acid generation process described in (2-2) above, that is, as... Figure 2A The step S14 shown in the diagram is illustrated. Incidentally, optional conditions may be appropriately applied regarding the conditions used to form this single-stranded circular nucleic acid.

[0219] In a first variation of the circular nucleic acid generation method, a poly-A-tailing method is used in the complementary nucleic acid generation step, and primers are provided to capture the sequence to be captured by the complementary strand capture portion contained in the nucleic acid splicing portion. Therefore, even without reverse transcription of the 5' end of the mRNA using reverse transcriptase and without providing a CCC sequence, for example, a sequence complementary to the sequence of the complementary strand capture portion of the nucleic acid splicing portion can be provided. Thus, when a circular nucleic acid is formed by splicing two or more generated complementary strands together via the nucleic acid splicing portion, the failure to capture the generated complementary strand can be reduced. Therefore, it can be expected that the probability of detecting complementary strands, even those originating from mRNA sequences occurring at low frequencies, will increase, and analytical accuracy will be improved as a result.

[0220] Incidentally, combinations of DNA-DNA complexes spliced ​​together by the splicing reaction shown in step S217 cannot be selected. Therefore, the single-stranded circular nucleic acid formed in step S217 is a nucleic acid derived from cDNA sequences generated using the mRNA sequence as a template, randomly spliced ​​together. Given the existence of many types of spliced ​​cDNAs, and the fact that the spliced ​​cDNA sequences are substantially unique, single-stranded circular nucleic acids can be identified by the splicing sequence of the cDNAs.

[0221] After that, although Figure 2EAs not described above, in the circular nucleic acid generation process of (2-2) described above, for example, primers such as <2D-β> are added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized by RCA using DNA polymerase with the primer as the starting point. Incidentally, <2D-β> can be suitably used as the primer to be used at this time. However, the primer to be used is not limited to this. Furthermore, regarding these conditions, conditions similar to those applicable to the generation process of the circular nucleic acid generation process of (2-2) described above can also be suitably applied in this variant. Regarding the decoding of the base sequence of the obtained single-stranded linear nucleic acid, for example, methods similar to those applicable to the circular nucleic acid generation process of (2-2) described above can also be suitably applied in this variant.

[0222] (2-4) Second variation of the circular nucleic acid generation method

[0223] Figure 2F This is a conceptual diagram of the second variant of nucleic acid splicing parts used in the circular nucleic acid generation process.

[0224] exist Figure 2F In this context, <2F-α> is the nucleic acid splicing portion used in the first variant of the circular nucleic acid generation process. <2F-β> is the primer used when generating nucleic acids with sequences derived from the target nucleic acid during the circular nucleic acid generation process in this variant. <2F-γ> is the primer used in PCR, which uses the DNA-RNA complex formed in this variant as a template.

[0225] The <2F-.N nucleic acid splice portion includes a target nucleic acid capture portion (the poly-T in the figure) capable of capturing the poly-A sequence present at the 3' end of the mRNA and a complementary strand capture portion (the splice in the figure). A sequence appendage portion (the cell barcode in the figure) is provided between the target nucleic acid capture portion and the complementary strand capture portion. Incidentally, a pattern in which the nucleic acid splice portion used in this variant does not have a sequence appendage portion can also be used. The sequence of the complementary strand capture portion included in the nucleic acid splice portion used in this variant can be formed as an optional sequence. Furthermore, in the case where the nucleic acid splice portion includes a sequence appendage portion, an optional sequence corresponding to the function to be provided to the sequence appendage portion can be provided as the sequence of the sequence appendage portion.

[0226] The primers shown in <2F-β> comprise: a poly-T sequence complementary to the poly-A sequence added to the cDNA by terminal transferase in step S313 described later, and a sequence complementary to the sequence of the complementary strand capture portion contained in the nucleic acid splicing portion of <2F-α>. Incidentally, in the primers, the sequence complementary to the sequence of the complementary strand capture portion contained in the aforementioned nucleic acid splicing portion is formed from RNA. When using cDNA generated by the circular nucleic acid generation process according to this variant as a template to generate a nucleic acid having a sequence derived from the target nucleic acid, the primers shown in <2F-β> serve as a starting point and as a template in PCR, which will be described later, thereby providing the sequence of the complementary strand capture portion contained in the nucleic acid splicing portion to the 3' end of the nucleic acid synthesized in PCR.

[0227] The primers shown in <2F-γ> are formed from RNA. Furthermore, the primer sequences are complementary to the sequences of the complementary strand capture portions contained in the nucleic acid splicing regions of <2F-α>. Therefore, the starting point for nucleic acid extension reactions is set in PCR using the DNA-RNA complex formed in this variant as a template.

[0228] Furthermore, in a second variation of the circular nucleic acid generation process, a poly-A tailing method is used in the complementary nucleic acid generation step, and nucleic acids with sequences derived from the target nucleic acid are generated by using primers that provide a sequence to be captured by the complementary strand capture portion contained in the nucleic acid splicing portion, with the complementary strand of the generated target nucleic acid as a template.

[0229] The following will refer to Figure 2G Describe it.

[0230] Figure 2G Steps S311, S312, and S313 are similar to steps S211, S212, and S213 in the first variant of the circular nucleic acid generation method described above. Conditions similar to those applicable to the circular nucleic acid generation process can also be appropriately applied to this variant.

[0231] exist Figure 2G In step S314, a nucleic acid with a sequence derived from the target nucleic acid is generated using DNA polymerase with a primer of <2F-β> as the starting point and cDNA as the template. A sequence complementary to the sequence of the complementary strand capture portion contained in the nucleic acid splicing region of <2F-α> can be provided to the 5' end of the generated nucleic acid using a primer of <2F-β>.

[0232] exist Figure 2GIn step S315, PCR is performed using the primers shown in <2F-γ> formed from RNA, while the generated nucleic acid having a sequence derived from the target nucleic acid is used as a template. Incidentally, as mentioned above, the aforementioned nucleic acid used as a template has a sequence at its 5' end complementary to the sequence of the complementary strand capture portion contained in the nucleic acid splicing portion, which is derived from primer <2F-β> and formed from RNA. Therefore, in PCR, a DNA polymerase with reverse transcription activity, such as Tth DNA polymerase, can be appropriately used.

[0233] exist Figure 2G In step S316, the RNA-DNA complex generated in step S315 is treated with RNase H. Therefore, the RNA oligomer is degraded, and a DNA-DNA complex comprising the complementary strand of the target nucleic acid is obtained.

[0234] exist Figure 2G In step S317, two or more DNA-DNA complexes containing the complementary strands of the obtained target nucleic acid are spliced ​​together via the complementary strand capture portion of the nucleic acid splicing portion and a sequence complementary to the sequence of the complementary strand capture portion, the complementary sequence being derived from primer <2F-β>. As a condition for this step, in this variant, conditions similar to those applicable to the circular nucleic acid generation process in (2-2) or (2-3) above may also be appropriately applied.

[0235] The splicing reaction shown in step S317 forms a single-stranded circular nucleic acid, as in the circular nucleic acid generation process of (2-2) or (2-3) above, that is, as shown in step S14 in Figure 2. Incidentally, the conditions for forming this single-stranded circular nucleic acid, similar to those applicable to the circular nucleic acid generation process of (2-2) or (2-3) above, can also be appropriately applied to this variant.

[0236] After that, despite Figure 2G Not described herein, but as in the circular nucleic acid generation process of (2-2) or (2-3) above, primers are added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized via RCA using DNA polymerase with the primers as the starting point. Regarding these conditions, in this variant, conditions similar to those applicable to the circular nucleic acid generation process of (2-2) or (2-3) above may also be appropriately applied.

[0237] (2-5) The third variation of the circular nucleic acid generation method

[0238] Figure 2H This is a conceptual diagram of the nucleic acid splicing components used in the third variant of the circular nucleic acid generation process.

[0239] exist Figure 2H In this context, <2H-α> is the nucleic acid splicing part used in the first variant of the circular nucleic acid generation process. <2H-β> is the primer used when generating nucleic acids with sequences derived from the target nucleic acid during the circular nucleic acid generation process in this variant.

[0240] Similar to the <2F-α> nucleic acid splice portion, the <2H-α> nucleic acid splice portion includes a target nucleic acid capture portion (the poly-T in the figure) capable of capturing the poly-A sequence present at the 3' end of the mRNA, and a complementary strand capture portion (the splice in the figure). A sequence appendage (the cell barcode in the figure) is provided between the target nucleic acid capture portion and the complementary strand capture portion. However, the nucleic acid splice portion of the <2H- column differs from that of the <2F- portion in that the complementary strand capture portion of the nucleic acid splice portion at the <2H- position is formed from RNA.

[0241] Incidentally, a pattern in which the nucleic acid splicing portion used in this variant does not have a sequence appendix can also be used. The sequence of the complementary strand capture portion included in the nucleic acid splicing portion used in this variant can be formed as an optional sequence. Furthermore, in the case where the nucleic acid splicing portion includes a sequence appendix, an optional sequence corresponding to the function to be provided to the sequence appendix can be provided as the sequence appendix.

[0242] Similar to the primers shown in <2F-β>, the primers shown in <2H-β> include a poly-T sequence complementary to the poly-A sequence added to the cDNA by terminal transferase in step S413 described later, and a sequence complementary to the sequence of the complementary strand capture portion included in the nucleic acid splicing portion of <2H-α>. However, this primer differs from the primers shown in <2F-β> in that the sequence complementary to the sequence of the complementary strand capture portion included in the aforementioned nucleic acid splicing portion is formed from the DNA in this primer.

[0243] When using cDNA generated by the circular nucleic acid generation process according to this variant as a template to generate nucleic acids having sequences derived from the target nucleic acid, the primer shown in <2H-β> serves as the starting point, and the sequence complementary to the sequence of the complementary strand capture portion enables two or more generated nucleic acids to be properly spliced ​​together with each other via the nucleic acid splicing portion.

[0244] Furthermore, in a third variation of the circular nucleic acid generation process, a poly-A tailing method is used in the complementary nucleic acid generation step, and nucleic acids with sequences derived from the target nucleic acid are generated by using primers that provide a sequence to be captured by the complementary strand capture portion contained in the nucleic acid splicing portion, with the complementary strand of the generated target nucleic acid as a template.

[0245] The following will refer to Figure 2IDescribe it.

[0246] Figure 2I Steps S411, S412, and S413 are similar to steps S211, S212, and S213 in the first variant of the circular nucleic acid generation method described above. Conditions similar to those applicable to the circular nucleic acid generation process can also be appropriately applied to this variant.

[0247] exist Figure 2I In step S414, using a primer with <2H-β> as a starting point and cDNA as a template, a DNA polymerase without reverse transcription activity generates a nucleic acid with a sequence derived from the target nucleic acid. A sequence complementary to the sequence of the complementary strand capture portion contained in the <2H-α> nucleic acid splice portion can be provided to the 5' end of the generated nucleic acid using a <2H-β> primer. Furthermore, as described above, the complementary strand capture portion contained in the <2H-α> nucleic acid splice portion is formed from RNA, and the DNA polymerase used in step S414 does not have reverse transcription activity. Therefore, the nucleic acid extension reaction stops before the complementary strand capture portion contained in the <2H-α> nucleic acid splice portion. This yields a DNA-DNA complex containing the complementary strand of the target nucleic acid.

[0248] exist Figure 2I In step S415, two or more DNA-DNA complexes containing the complementary strands of the obtained target nucleic acid are spliced ​​together via the complementary strand capture portion of the nucleic acid splicing portion and a sequence complementary to the sequence of the complementary strand capture portion, the complementary sequence being derived from primer <2H-β>. As a condition for this step, in this variant, conditions similar to those applicable to the circular nucleic acid generation process in (2-2), (2-3), or (2-4) described above may also be appropriately applied.

[0249] The splicing reaction shown in step S415 forms a single-stranded circular nucleic acid, as in the circular nucleic acid generation process described in (2-2), (2-3), or (2-4) above, that is, as shown in step S14 of Figure 2. Incidentally, the conditions for forming this single-stranded circular nucleic acid, similar to those applicable to the circular nucleic acid generation processes described in (2-2), (2-3), or (2-4) above, can also be appropriately applied to this variant.

[0250] After that, despite Figure 2GNot described herein, but in the circular nucleic acid generation process described in (2-2), (2-3), or (2-4) above, primers are added to the formed single-stranded circular nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized via RCA using DNA polymerase with the primers as the starting point. Regarding these conditions, in this variant, conditions similar to those applicable to the generation process in the circular nucleic acid generation process described in (2-2), (2-3), or (2-4) above may also be appropriately applied.

[0251] (3) Example 2 (Use of restriction enzyme recognition sites)

[0252] According to embodiments of this disclosure, the restriction enzyme recognition sequence can be incorporated into the nucleic acid splicing portion. The restriction enzyme recognition sequence can, for example, be incorporated into the double-stranded portion. Therefore, by restriction enzyme treatment, the amplification product (e.g., amplification product generated by RCA or PCR) of the spliced ​​product (particularly, circular nucleic acid) assembled according to embodiments of this disclosure can be cleaved at the location of the restriction enzyme recognition sequence. Therefore, the amplification product can be easily analyzed. For example, the sequence generated by cleavage can be tagged for sequencing (particularly tagged for short-read sequencing). In cases where Hind III is used as the restriction enzyme, for example, [the following text is incomplete and requires further context: "to use Hind III as the restriction enzyme, for example, to use Hind III as the restriction enzyme ... Figure 4A The restriction site shown introduces the double-stranded portion. As indicated by the dashed line in the figure, the double-stranded DNA is cut, and a tag for sequencing is added here.

[0253] Figure 4B An example of generating tagged short reads is shown in the figure.

[0254] As shown in the figure, in step S31, the complementary strand CN of the circular nucleic acid GN generated according to the embodiments of this disclosure is synthesized by DNA polymerase. This generates double-stranded DNA.

[0255] In step S32, the double-stranded DNA is cleaved at the restriction site RN present in the double-stranded portion using a restriction enzyme. The cleavage generates a double-stranded DNA fragment containing one cDNA.

[0256] In step S33, tags T1 through T4 for sequencing are added to both ends of each double-stranded DNA fragment. For example, these tags can be configured to specifically recognize and bind to sequences and / or structures at sites cleaved by restriction enzymes. The types of these tags can be appropriately selected by those skilled in the art based on the sequencing method. For example, for the provided short-read sequencing, tags Read1, Read2, sample index (i5), sample index (i7), P5, and P7 are added. Sequencing is then performed using the provided sequencing apparatus. The sequence (and number of double-stranded fragments) of each double-stranded DNA fragment is thus identified.

[0257] (4) Example 3 (Single-cell analysis)

[0258] For example, the analytical method according to embodiments of this disclosure can be configured as a single-cell analysis method. That is, this disclosure also provides a single-cell analysis method. Preferably, nucleic acid splicing portions with different double-stranded parts for each cell can be used for single-cell analysis.

[0259] The following will refer to Figure 5 Describe the operations performed in single-cell analysis methods.

[0260] For single-cell analysis, as shown in Figure A, a nucleic acid splicing portion 10 is disposed on the surface (particularly a two-dimensional plane) of a substrate 40. In the figure, as described above, the nucleic acid splicing portion includes at least a target nucleic acid capture portion configured to capture the 3' end region of a target nucleic acid, a complementary strand capture portion configured to capture the 3' end region of a complementary strand generated by the complementary strand of the target nucleic acid, and a double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion to each other.

[0261] Here, it is desirable for nucleic acid splice portions with different sequences to be spliced ​​to their respective cells, and for nucleic acid splice portions with the same sequence to bind to a single cell. Therefore, the surface of the substrate 40 can be divided. Then, nucleic acid splice portions with the same sequence can be fixed to one region, and nucleic acid splice portions with different sequences can be fixed to their respective regions. Specifically, the sequence difference can be a difference in the double-stranded portion.

[0262] As shown in the figure, for example, the surface of the substrate can be divided into regions A1 and A2. Multiple nucleic acid splice portions with the same base sequence can be fixed in region A1, and multiple nucleic acid splice portions with the same base sequence can also be fixed in region A2. Then, the base sequences of the nucleic acid splice portions fixed in region A1 and the nucleic acid splice portions fixed in region A2 are different, especially the base sequences in the double-stranded portions.

[0263] It should be noted that in this disclosure, the surface on which the nucleic acid splice portions are fixed can be, for example, a flat surface of a substrate, a well, a plate, etc., as described above, but not limited to these. Alternatively, the surface on which the nucleic acid splice portions are fixed can be, for example, a surface of a bead. In the case of a bead, the bead corresponds to the corresponding region described above. For example, a cell can be captured by a bead. In this case, multiple nucleic acid splice portions having the same base sequence can be fixed onto a bead. The nucleic acids spliced ​​onto the portions of the corresponding beads can then be, for example, different from each other in random sequences. The nucleic acids spliced ​​onto the portions of the corresponding beads can each include the same target nucleic acid capture portion and / or the same complementary strand capture portion, and may further include the same priming sequence.

[0264] (Including the complex of nucleic acid splicing parts)

[0265] The cell-capturing portion for capturing cells and / or the fixation portion for immobilizing on a substrate can be bound to the nucleic acid splicing portion. The cell-capturing portion can be an antibody or a lipid. Furthermore, various barcode sequences can be bound to the nucleic acid splicing portion. The nucleic acid splicing portion can exist as an element of a complex that includes the cell-capturing portion and / or the fixation portion. References will be made below. Figures 6A to 6C Describe an instance of the complex.

[0266] Figure 6A An example of a schematic structure showing a nucleic acid splice portion bound to an antibody as a cell capture portion is shown.

[0267] The nucleic acid splicing portion 50 (the portion surrounded by dashed lines) can exist as part of a composite 100 fixed to the substrate 61. The composite 100 includes: a cell-capturing portion 63 configured to capture cells; the nucleic acid splicing portion 50 for capturing target nucleic acids within the cells; and a fixing portion for fixing the nucleic acid splicing portion 50 to the substrate. In the figures, structures indicated by reference numerals 54a, 54b, 55a, 55b, 56, 57, 58, 59, 60-1, and 62 correspond to the fixing portion. However, the structure of the fixing portion is not limited to this. The fixing portion may not need to have all the elements indicated by these reference numerals, and may have other structures as long as the fixing portion can fix the nucleic acid splicing portion to a predetermined surface.

[0268] The constituent elements of composite 100 will be described below.

[0269] First, the figure shows four structures 60-1 (nucleic acids in the figure) present in region A1, and illustrates the components such as nucleic acid splicing portions that bind only to one of these structures 60-1. It should be understood that this is a simplified form for ease of understanding. In reality, there are numerous structures 60-1 in region A1, and components such as nucleic acid splicing portions similarly bind to the corresponding structures.

[0270] Furthermore, as in region A1 and region A2, there are also a large number of structures 60-2, and components such as nucleic acid splicing parts are combined with their respective structures.

[0271] Furthermore, the substrate 61 is not limited to the two regions shown in the figure, and may have a number of regions corresponding to the number of cells to be captured.

[0272] The cell-capturing portion 63 can be a compound configured to capture desired cells.

[0273] In one embodiment, the cell-capturing portion 63 may be, for example, an antibody, as shown in the figure. This antibody may be, for example, an antibody configured to capture molecules present on the cell surface (particularly surface markers, etc.). Those skilled in the art can appropriately select the antibody depending on the type of cells to be captured.

[0274] In another embodiment, the cell-capturing portion may be, for example, lipids, such as... Figure 6B As shown. The complex in the figure is similar to Figure 6A The complex in the figure, except for the complex shown in the figure, has lipids that replace the antibody as the cell-capturing portion 63. These lipids can be lipids configured to capture cells. The type of lipid can be appropriately selected by those skilled in the art.

[0275] The nucleic acid splicing portion 50 includes a target nucleic acid capture portion 51 for capturing the 3' end region of the target nucleic acid, a complementary strand capture portion 52 (GGG in the figure) for capturing the 3' end region of the complementary strand of the target nucleic acid, and a double-stranded portion 53 (53a, 53b, 53c, 53d) connecting the target nucleic acid capture portion 51 and the complementary strand capture portion. These are the same as the target nucleic acid capture portion 11, the complementary strand capture portion 12, and the double-stranded portion 13 described above, and their description also applies in this example.

[0276] The target nucleic acid capture portion 51 can be a poly-T sequence, as described in the description above.

[0277] The complementary strand capture portion 52 can be a GGG sequence, as described above.

[0278] Regarding the target nucleic acid capture portion 51 and the complementary strand capture portion 52, all nucleic acid splicing portions present on the substrate 61 may have the same target nucleic acid capture portion 51 and the same complementary strand capture portion 52.

[0279] The double-stranded portion 53 includes random sequences 53a and 53b, and triggering sequences 53c and 53d. Random sequences 53a and 53b are complementary to each other. Furthermore, triggering sequences 53c and 53d are also complementary to each other.

[0280] Random sequences 53a and 53b have base sequences different for their respective regions. That is, the random sequences included in the nucleic acid splice portion fixed in region A1 each have the same base sequence. The random sequences included in the nucleic acid splice portion fixed in region A2 also each have the same base sequence. Then, the random sequence present in region A1 has a base sequence different from the random sequence present in region A2.

[0281] Regarding the initiating sequences 53c and 53d, all nucleic acid splicing portions present on the substrate 61 can, for example, have the same base sequence as the initiating sequences 53c and 53d. Therefore, circular nucleic acids can be generated as a whole by using the initiating sequences.

[0282] Complex 100 may have cleavage sites 54 (54a and 54b). Cleavage site 54 may, for example, be a restriction enzyme recognition site. Specifically, cleavage site 54 may be a site in which multiple restriction enzyme recognition sites are arranged consecutively. For example, cleavage site 54 may be a site in which 1 to 10 restriction enzyme recognition sites are arranged consecutively. Specifically, cleavage site 54 may be a site in which two to eight restriction enzyme recognition sites are arranged consecutively. More specifically, cleavage site 54 may be a site in which four to six restriction enzyme recognition sites (e.g., five restriction enzyme recognition sites) are arranged consecutively. The number of bases in each restriction enzyme recognition site may be, for example, 4 to 10, particularly 4 to 8. The restriction enzyme may be an endonuclease. Therefore, by providing multiple restriction enzyme recognition sites arranged in a conjoined pattern, the likelihood of cleavage can be increased.

[0283] Where cleavage site 54 is a restriction enzyme recognition site, cleavage site 54 can be configured as a double-stranded nucleic acid. One strand of the double-stranded nucleic acid can be spliced ​​with nucleic acid splicing portion 50 (specifically, target nucleic acid capture portion 51). The other strand of the double-stranded nucleic acid can be coupled to a barcode sequence, as described later.

[0284] The base length of cleavage site 54 can be, for example, 10 to 50 bases, particularly 20 to 40 bases.

[0285] For restriction enzyme recognition site cleavage, use the appropriate restriction endonuclease according to each sequence (http: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100003632). 1 U of restriction enzyme activity is, in principle, the amount of enzyme required to completely degrade 1 μg of λDNA within one hour at 37°C in 50 μl of each enzyme reaction solution. Adjust the amount of enzyme according to the amount of restriction enzyme recognition sequence.

[0286] The composite 100 may include location information barcode sequence portions 55 (55a and 55b). For example, the location information barcode sequence portion 55 may also be referred to as an array barcode sequence portion.

[0287] Location information barcode sequence portions 55a and 55b have different base sequences for each region. That is, the location information barcode sequence portions fixed in region A1 each have the same base sequence. The location information barcode sequence portions fixed in region A2 also each have the same base sequence. However, the location information barcode sequence portion present in region A1 has a base sequence different from the base sequence of the location information barcode sequence portion present in region A2.

[0288] Based on the location information corresponding to the base sequence of the location information barcode sequence portion, cells captured in each region can be associated with, for example, an image of each cell. The image can be obtained through an image acquisition device such as a microscope, for example, after cells have been captured in each region, but before the cells are removed from each region by a cut at a cutting site.

[0289] The basic length of the location information barcode sequence portion 55 can be adjusted such that the number of variations in the location information barcode sequence portion 55 is equal to or greater than the number of regions on the substrate (or equal to or greater than the number of units to be captured). For example, the base length can be equal to or greater than 10 bases, particularly equal to or greater than 12 bases, more particularly equal to or greater than 14 bases, or even more particularly equal to or greater than 16 bases. For example, the base length can be equal to or less than 100 bases, particularly equal to or less than 70 bases, more particularly equal to or less than 50 bases, and can be, for example, equal to or less than 30 bases.

[0290] The composite 100 may include a fixed barcode sequence portion 58.

[0291] The fixed barcode sequence portion 58 has a different base sequence for each region. That is, the fixed barcode sequence portions fixed in region A1 each have the same base sequence. The fixed barcode sequence portions fixed in region A2 also each have the same base sequence. Then, the fixed barcode sequence portion present in region A1 has a base sequence that is different from the base sequence of the fixed barcode sequence portion present in region A2.

[0292] The fixed barcode sequence portion 58 has a base sequence complementary to nucleic acid 60-1 pre-fixed on a substrate. Nucleic acid 60-1 is present only in a specific region. Therefore, the fixed barcode sequence specifically binds to nucleic acid 60-1. Then, the complex 100 with the fixed barcode sequence is fixed in the specific region.

[0293] The base length of the fixed barcode sequence portion 58 can be adjusted such that the number of variations in the fixed barcode sequence portion 58 is equal to or greater than the number of regions on the substrate (or equal to or greater than the number of cells to be captured). For example, the base length can be equal to or greater than 10 bases, particularly equal to or greater than 12 bases, more particularly equal to or greater than 14 bases, or even more particularly equal to or greater than 16 bases. For example, the base length can be equal to or less than 100 bases, particularly equal to or less than 70 bases, more particularly equal to or less than 50 bases, and can be, for example, equal to or less than 30 bases.

[0294] Complex 100 may include initiation portions 57 and 59. Initiation portion 57 has a base sequence for synthesizing a nucleic acid strand including the aforementioned barcode sequence portion and the aforementioned restriction enzyme recognition site. For example, the base length of each initiation portion is 10 to 30 bases, particularly 15 to 25 bases, more particularly 15 to 20 bases.

[0295] As described above, complex 100 may include a nucleic acid splicing portion, a cell-capturing portion for capturing cells, and a fixation portion for fixing the nucleic acid splicing portion at a specific location. The fixation portion may include the restriction enzyme recognition site and / or barcode sequence portion described above. Furthermore, regarding complex 100, the fixation barcode sequence portion 58 serves as a structure for fixing the complex at a specific location. However, the location information barcode portion may be configured as a structure for fixing the complex at a specific location.

[0296] As described above, complex 100 can bind to nucleic acid 60-1 immobilized on a substrate. Nucleic acid 60-1 can have different base sequences for different regions. Complex 100 is immobilized on the substrate through binding between nucleic acid 60-1 and immobilized barcode portion 58. That is, the nucleic acid splice portion can be immobilized on the substrate.

[0297] Nucleic acids 60 (60-1 and 60-2) can be attached to the substrate 61 via connectors 62. As connectors 62, materials known in the art can be used and can be appropriately selected by those skilled in the art.

[0298] In one embodiment, connector 62 may be a connector that can be cleaved by stimulation, and for example, a connector that can be cleaved by light or chemical stimulation. The light stimulation is particularly suitable for selectively applying stimulation to a specific location during the cleaving step described later.

[0299] When using this cleavable linker, it is not necessary to provide a cleavage site 54 including the restriction enzyme recognition site described above, but a cleavage site 54 including the restriction enzyme recognition site described above can be provided.

[0300] When using this cleavable linker, the strand spliced ​​to the nucleic acid splicing portion (specifically, the strand spliced ​​to the target nucleic acid capture portion) can be directly fixed to the substrate 61 via the linker (that is, without the intervention of the nucleic acid 60).

[0301] For example, a cleavable linker may include one selected from: arylcarbonylmethyl, nitroaryl, coumarin-4-ylmethyl, arylmethyl, a metal-containing group, and other groups, for example, that are cleavable linkers by optical stimulation. For example, groups described in "Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy", Chem. Rev. 2013, 113, 119-191, may be used as these groups.

[0302] For example, the arylcarbonylmethyl group can be benzoylmethyl, o-alkylbenzoyl, or p-hydroxybenzoyl. For example, the nitroaryl group can be o-nitrobenzyl, o-nitro-2-phenylethoxycarbonyl, or o-nitroaniline. For example, the arylmethyl group may or may not have a hydroxyl group introduced therein.

[0303] In the case of a cleavable linker that is optically cleavable, the linker is preferably cleaved by light with a wavelength of 360 nm or longer. This linker is preferably one capable of being cleaved at an energy not exceeding 0.5 μJ / μm² (see Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. Jan 2015; 12(1):23-6. doi:10.1038 / nmeth.3219). Using a linker cleaved by light with the aforementioned wavelength or energy can reduce cell damage (especially cleavage of DNA or RNA, etc.) that may occur when optical stimulation is applied.

[0304] Particularly preferably, the cleavable connector can be a connector that can be cleaved by light in a short wavelength region, specifically light in the wavelength region of 360 to 410 nm, or it can be a connector that can be cleaved by light in the near-infrared region or the infrared region, specifically light in the wavelength region of 800 nm and longer. If the cleavable connector is effectively cleaved by light having wavelengths in the visible light region, the surface to be analyzed may be difficult to process. Therefore, the connector is preferably a connector that can be cleaved by light in the aforementioned short wavelength region or by light in the aforementioned near-infrared region or the infrared region.

[0305] For example, cleavable linkers can include disulfide bonds as linkers that can be cleaved by chemical stimulation. For disulfide bond cleavage, reducing agents such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or 2-mercaptoethanol are used. For example, in the case of using TCEP, TCEP is reacted at 50 mM for approximately 15 minutes.

[0306] The composite 100 may include a plurality of cutable connectors. Preferably, the plurality of connectors can be spliced ​​together. With a cutting probability of 0.8 for a single connector, for example, by splicing three such connectors together, the cutting probability is increased to 0.992 (=1-0.2). 3 ).

[0307] (Including the manufacturing method of the complex containing the nucleic acid splicing component)

[0308] The following will refer to Figure 6C Describe the method for manufacturing the composite.

[0309] For complex 100, a first pool (library) comprising a first strand (particularly a single strand) of a target nucleic acid capture portion including a nucleic acid splicing portion and a second pool comprising a second strand (particularly a single strand) of a barcode sequence portion are prepared. A double-stranded structure is formed by combining the strands of the respective pools with each other. Here, the first strand comprises one of the two strands constituting the double-stranded portion of the nucleic acid splicing portion.

[0310] For double-stranded structures, a third strand (particularly a single strand) is prepared, comprising a complementary strand capture portion of the nucleic acid splicing portion. The third strand includes the other of the two strands constituting the double-stranded portion of the nucleic acid splicing portion. The third strand is then combined with a double-stranded structure obtained by binding the first and second strands together. This yields complex 100.

[0311] Figure 6C Oligo-pool 1 describes the process for obtaining the first strand. Oligo-pool 2 describes the process for obtaining the second strand.

[0312] (Oligonucleotide pool 1)

[0313] As shown in oligonucleotide pool 1, firstly, a single-stranded nucleic acid N1 (DNA or RNA) is prepared. The single-stranded nucleic acid N1 includes two initiation sites (initiators) and a sequence that forms the double-stranded portion (linker). The sequence that forms the target nucleic acid capture portion (p-T), the sequence that forms the cleavage site (cleavage site), and the sequence that forms the positional information barcode sequence portion between these triggering sites (array 1 barcode). ).

[0314] Here, marked with " The sequence of "" (that is, the sequence that forms the double-stranded part (the splice)) ) and the sequence that forms the location information barcode sequence (array 1 barcode) These have different base sequences for each region. Therefore, multiple single-stranded nucleic acids with different sequences in these regions but identical sequences in other parts are prepared as oligonucleotide pool 1.

[0315] Next, PCR is performed using two initiation sites. Specifically, as shown in the figure, for example, primers with a T7 promoter that bind to one initiation site and a reverse primer that binds to the other initiation site can be prepared, and PCR can be performed using these two primers. Double-stranded nucleic acid is obtained by PCR.

[0316] This double-stranded nucleic acid is transcribed in vitro (IVT). This yields an RNA transcript.

[0317] RNA transcripts are reverse transcribed (RT) using a reverse primer that binds to the initiation site, as shown in the figure. This generates cDNA corresponding to the RNA transcript.

[0318] After cDNA synthesis, an RNA digestion process is performed. This process removes RNA and retains only cDNA. This yields a single-stranded nucleic acid, which includes sequences forming the double-stranded portion, sequences forming the target nucleic acid capture portion, sequences forming the cleavage site, and sequences forming the positional information barcode portion.

[0319] (Oligonucleotide pool 2)

[0320] As shown in oligonucleotide pool 2, firstly, a single-stranded nucleic acid N2 (DNA or RNA) is prepared, which includes two initiation sites (initiation) and a sequence forming a cleavage site (cleavage site), and a sequence forming a position information barcode sequence portion (array 1# barcode). ) and the sequence that forms a fixed barcode sequence portion between these triggering sites (array 2# barcode) Incidentally, "#" indicates complementarity. For example, array 1 barcode. With array 1# barcode Complementary, and array 2 barcodes With array 2# barcode Complementary.

[0321] Here, marked with " The sequence (that is, the sequence that forms the location information barcode sequence part (array 1 / array 1# barcode)) ) and the sequence that forms the fixed barcode sequence (array 2# barcode) These have different base sequences for each region. Therefore, multiple single-stranded nucleic acids with different sequences in these regions but identical sequences in other parts are prepared as oligonucleotide pool 2.

[0322] Next, PCR is performed using two initiation sites. Specifically, as shown in the figure, for example, primers with a T7 promoter that bind to one initiation site and a reverse primer that binds to the other initiation site can be prepared, and PCR can be performed using these two primers. Double-stranded nucleic acid is obtained by PCR.

[0323] This double-stranded nucleic acid is transcribed in vitro (IVT). This yields RNA transcripts.

[0324] RNA transcripts are reverse transcribed (RT) using a reverse primer that binds to the initiation site, as shown in the figure. This generates cDNA corresponding to the RNA transcript.

[0325] After cDNA synthesis, RNA digestion is performed. This process removes RNA and retains only cDNA. This yields a single-stranded nucleic acid, which includes sequences forming cleavage sites, sequences forming positional barcode sequences, and sequences forming fixed barcode sequences.

[0326] As shown in the center of the figure, single-stranded nucleic acids synthesized in oligonucleotide pool 1 and oligonucleotide pool 2 bind to each other based on complementarity in the positional information barcode sequence portion and the cleavage site. This yields a conjugate. The single strand including the complementary strand capture portion and the other strand of the two strands forming the double-stranded portion further bind to the conjugate. This yields complex 100. Therefore, complex 100 can include three nucleic acid strands. Furthermore, binding can be based on complementarity between the two strands forming the double-stranded portion.

[0327] Incidentally, although the cell-capturing portion (antibody) can bind to the single strand in advance as shown in the figure, the cell-capturing portion can bind to the complex after the complex of the three nucleic acid strands has been formed.

[0328] Furthermore, the complex 100 includes a fixed barcode portion, as described above. Based on the complementarity between the fixed barcode portion and the nucleic acid 60-1 pre-positioned on the substrate, the specific complex will be fixed in a specific location.

[0329] As described above, for complex 100, a first pool of a first strand (particularly a single strand) comprising a target nucleic acid capture portion including a nucleic acid splicing portion and a second pool of a second strand (particularly a single strand) comprising a barcode sequence portion are prepared. A double-stranded structure is formed by combining the strands of the respective pools with each other. Here, the first strand comprises one of the two strands constituting the double-stranded portion of the nucleic acid splicing portion.

[0330] For double-stranded structures, a third strand (particularly a single strand) is prepared, comprising a complementary strand capture portion of the nucleic acid splicing portion. The third strand includes the other of the two strands constituting the double-stranded portion of the nucleic acid splicing portion. The third strand is then combined with a double-stranded structure obtained by binding the first and second strands together. This yields complex 100.

[0331] In one embodiment, the cell-capturing portion may be pre-bound to the third strand. In this case, the third strand is combined with a double strand obtained by binding the first and second strands together. This yields a complex having the cell-capturing portion.

[0332] In another embodiment, the cell-capturing portion may not be pre-bound to the third strand. In this case, the cell-capturing portion may be further bound to a complex obtained by binding the third strand to a double strand formed by binding the first and second strands together.

[0333] While the complex and nucleic acid splice region can be prepared by the manufacturing method described above, the manufacturing method is not limited to this. Depending on the structure of the complex and nucleic acid splice region, the manufacturing method can be appropriately modified.

[0334] In the following text, return to Figure 5 The description.

[0335] In step S41, cells are provided onto a substrate having a surface on which the composite is fixed. Cell-capturing portions of the composite capture the cells. As a result, as shown in Figure B, one cell is captured in one region. Incidentally, although only one chain with a complementary chain capturing portion is shown in Figure B, this is for simplicity and omission of figures. In practice, each composite may have a chain with a complementary chain capturing portion. Chains with complementary chain capturing portions may also be similarly omitted in other figures mentioned in this specification.

[0336] In step S42, for example, cleavage site 54 in the complex is cleaved. This cleavage can be performed by a restriction enzyme that recognizes the cleavage site.

[0337] Furthermore, in cases where the complex contains the cleavable connectors described above, treatments (photostimulation or chemical stimulation) for cleaving the connectors can be performed.

[0338] Following restriction enzyme treatment, the residue is degraded by an exonuclease. Thus, the target nucleic acid capture portion 51 becomes the 3' end region. Preferably, the exonuclease can be exonuclease III (E. coli) and can degrade from the 3' end of double-stranded DNA having a 5' overhang, a blunt end, or a 3' overhang with fewer than four bases.

[0339] The cutting process detaches each cell from the substrate, and each cell has a nucleic acid splice portion that binds to it via a cell capture portion. The detached cells are then separated one-by-one into microspaces. These microspaces can be spaces within emulsion particles or spaces within pores, as described later.

[0340] Then, each cell is destroyed in a separated state. For example, destruction can be achieved through cell lysis.

[0341] Through this disruption, the target nucleic acid within the cell is captured by the target nucleic acid capture portion that constitutes the nucleic acid splicing part. Therefore, as Figure 5 As shown in C, a conjugate of the target nucleic acid and the nucleic acid splicing portion is generated. That is, a hybrid of the target nucleic acid and the nucleic acid splicing portion is generated.

[0342] Because cell destruction occurs in a separated state, the target nucleic acids within the cell do not leave the tiny space. Therefore, nucleic acids spliced ​​together with portions having the same base sequence can be bound to all target nucleic acids originating from a single cell. Furthermore, as mentioned above, the nucleic acids spliced ​​together in each cell differ from each other in their double-stranded portions. Therefore, the base sequences of the cell and the nucleic acid splice portions (especially the double-stranded portions) are correlated with each other in a one-to-one relationship. This allows for single-cell analysis. The methods used for separation will be described separately later.

[0343] Therefore, the analytical method according to the embodiments of this disclosure may include a cell destruction step that destroys cells, and a complementary nucleic acid generation step that may be performed on target nucleic acids included in the cells.

[0344] Step S43 is cDNA synthesis, which is consistent with the description above. Figure 2A The same applies to step S12. The description of step S12 also applies to step S43.

[0345] When step S43 is performed, cDNA with a CCC sequence at the end of the generated strand is generated, such as... Figure 5 As shown in D.

[0346] Step S44 is the same as described in the above description. Figure 2A The same nucleic acid splicing as step S13 in the previous step. The description of step S13 also applies to step S44.

[0347] During step S44, two or more cDNAs are spliced ​​together via complementary strand capture portions, such as Figure 5 As shown in E.

[0348] Step S45 is the same as described above. Figure 2A The same circular nucleic acid formation, ligation, and primer addition as those in steps S14 and S15. The descriptions of steps S14 and S15 also apply to step S4 and S5.

[0349] When step S45 is performed, a circular nucleic acid with added primers is formed, such as... Figure 5 As shown in F.

[0350] Step S46 is the same as described above. Figure 2A The nucleic acid amplification in step S16 is the same as that in step S46. The description of step S16 also applies to step S46.

[0351] When performing step S46, as follows Figure 5 As shown in G, nucleic acids are amplified. The amplified nucleic acids are then used for single-cell analysis. For example, the nucleic acid sequence is sequenced, and the sequencing results are used to analyze each cell.

[0352] (Separated into tiny spaces)

[0353] The following will describe examples of methods used for separation.

[0354] For example, the microspace can be a space within an emulsion particle or a space within a pore. This separation isolates a cell from which one or more nucleic acid splice portions having the same double-stranded partial base sequence bind within an emulsion particle or a pore. The cell destruction step can then be performed within the microspace (that is, the space allocated for each cell).

[0355] In one embodiment, the separation step may include a determining step of deciding whether to separate cells into a microspace, and a particle separation step of separating cells determined to be separated in the determining step into the microspace. This allows for the separation of only target cells. Therefore, for example, cells other than target cells can be excluded from the analytical target, thereby improving analytical efficiency.

[0356] For example, this determination can be based on light generated by the cell (e.g., scattered light and / or autofluorescence, etc.), light generated from substances bound to the cell, or a formal image. For example, substances bound to the cell can be antibodies bound to the cell (especially antibodies labeled with fluorescent dyes). For example, scattered light generated from the cell can be forward scattered light and / or side scattered light. Doublet detection can be performed by detecting the signal height and / or area values ​​obtained from the scattered light. Single-cell determination based on formal image information is also possible. Whether a cell is a dead cell can be determined by the scattered light and / or formal image or fluorescence after staining with a dead cell staining reagent. Dead cells can thus be removed. In this disclosure, the determination step can be performed immediately before the separation step. This allows for the reliable separation of only single cells bound to the nucleic acid splice portion.

[0357] In another embodiment, the particle separation step can be performed without the determining step. The number of steps in the method according to embodiments of this disclosure can be reduced by omitting the determining step.

[0358] In yet another embodiment, this determination step can be performed during connector cutting. For example, cells anchored to the substrate can be observed, and connector cutting can be performed only on cells that need to be separated into tiny spaces. In this case, equipment such as a cell sorter may not be necessary.

[0359] The determination and particle separation steps will be described below.

[0360] (Determine the steps)

[0361] The determination step determines whether the cells released from the substrate will separate into the microspace. This determination can be based on light generated from the cell or light generated from the substance bound to the cell, as described above.

[0362] For example, the determination steps may include an irradiation step of irradiating the cells with light and a detection step of detecting the light produced by the irradiation.

[0363] The irradiation step can be performed, for example, by a light irradiation unit. For instance, the light irradiation unit may include a light source that emits light. Furthermore, the light irradiation unit may include an objective lens that focuses light onto the cells. Those skilled in the art can appropriately select the light source according to the analytical purpose. For example, the light source may be a laser diode, an SHG (second harmonic generation) laser, a solid-state laser, a gas laser, a high-brightness LED, or a halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light irradiation unit may include other optical elements as needed.

[0364] For example, the detection step can be performed by a detection unit that detects light generated from cells or substances bound to cells. For the detection unit, the light generated from cells or substances bound to cells, irradiated by a light irradiation unit, can be, for example, scattered light and / or fluorescence. For example, the detection unit may include a converging lens and a detector that converges light generated from biological particles. PMTs (photomultiplier tubes), photodiodes, CCDs, CMOS, etc., can be used as detectors. However, the detector is not limited to these. In addition to the converging lens and detector, the detection unit may also include other optical elements as needed. For example, the detection unit may further include a beam splitter. Optical components constituting the beam splitter include, for example, gratings, prisms, and filters. Due to the beam splitter, light of the wavelength to be detected can be detected, for example, in a state separated from light of other wavelengths. The detection unit can convert the detected light into an analog electrical signal via photoelectric conversion. The detection unit can further convert the analog electrical signal into a digital electrical signal via AD conversion.

[0365] The determination step can be performed by a determination unit, which can determine whether to perform cell determination based on the light detected in the detection step. For example, the determination unit can be implemented using an information processing device such as a general-purpose computer, and in particular, a processing unit included in the information processing device.

[0366] (Particle separation step)

[0367] The separation step includes a particle separation step that separates cells into microspaces. A microspace can refer to a space with a size capable of accommodating one of the aforementioned analytical targets. The size can be appropriately determined based on factors such as cell size. A microspace can be large enough to accommodate two or more cells that are analytical targets. However, in this case, in addition to the case where one cell is accommodated in one microspace, it is also possible for two or more cells to be accommodated in one microspace. Cells in a microspace accommodating two or more cells can be excluded from the analyte in the lysis step described later, or they can be excluded from the analytical target in the analytical step described later.

[0368] Furthermore, in the destruction step described later, for example, a conjugate of nucleic acid splicing portions and target nucleic acids can be generated. Preferably, the multiple microspaces are separated from each other, such that a conjugate generated in one microspace does not move to another microspace. Examples of such separated microspaces include spaces within pores and spaces within emulsion particles. That is, in a preferred embodiment, the microspace can be a space within a pore or a space within an emulsion particle. Hereinafter, examples of the particle separation step when the microspace is a space within a pore and examples of the particle separation step when the microspace is a space within an emulsion particle will be described.

[0369] (The situation of the space inside the hole)

[0370] exist Figure 12 The diagram illustrates an example of a pore used for the particle separation step. As shown, for example, multiple pores 400, each sized to accommodate a single cell, can be formed in the surface of a substrate 401. The liquid from the cell, released from the substrate by the aforementioned cutting, can be applied to the surface of the substrate 401 via, for example, an optional nozzle 402. Thus, the cell 403 is separated into the space within the pore 400, as shown. Therefore, a cell can enter the space within a pore; that is, the cell can be separated into a tiny space.

[0371] In the example shown in the figure, where a liquid comprising multiple cells is applied to a substrate in which pores are formed, the particle separation step can be performed without the determination step described above, or it can be performed after the determination step.

[0372] Furthermore, when performing the above-described determination steps, a device such as a cell sorter or single-cell dispenser can be used, which places a biological particle into a well. The device can also be used to separate cells into a substrate (e.g., a plate) in which multiple pores are formed. Commercially available devices can be used as the apparatus. For example, the device may include a light irradiation unit that irradiates cells with light, a detection unit that detects light from the cells, a determination unit that determines whether to place a cell into a well based on the detected light, and a dispensing unit that dispenses the determined cells into the wells.

[0373] The light irradiation unit and the detection unit perform a detection step. Then, the determination unit performs a determination step. The dispensing unit includes, for example, a microfluidic chip having nozzles for forming droplets containing cells.

[0374] The device positions the microfluidic chip and places a cell-containing droplet into a predetermined orifice based on the determination result of the determining unit. Alternatively, the device controls the direction of movement of the cell-containing droplet extracted from the nozzle by applying a charge to the droplet, based on the determination result of the determining unit. As a control, a droplet containing one cell is placed in the predetermined orifice. Thus, one cell is dispensed into one orifice.

[0375] For example, such as Figure 13AAs shown, a droplet containing cells is removed from a nozzle 502 of the microfluidic chip provided to the device. A light irradiation unit 504 emits light (e.g., laser L) towards the biological particles in the droplet. A detection unit 505 then performs a detection step and thereby detects the light (fluorescence F). A determination unit (not shown) then performs a determination step based on the detected light. Based on the determination result, a dispensing unit controls the direction of droplet movement by applying a charge to the droplet. As a control, a droplet containing the target biological particle is collected into a predetermined pore. Thus, one biological particle is dispensed into one pore.

[0376] By performing a determination step, it is possible, for example, to identify the cell population to which the biological particle belongs, to identify barcoded biological particles, or to identify droplets containing singlet biological particles based on the detection signal. This allows for the collection of only droplets containing the target biological particle. As a result, data exclusion is not required in the analysis steps described later, improving analytical efficiency.

[0377] The number of holes provided in a substrate (plate) can be, for example, 1 to 1000, particularly 10 to 800, and more particularly 30 to 500. However, the number of holes can be suitably selected by those skilled in the art.

[0378] (The situation within the space of emulsion particles)

[0379] For example, emulsion particles can be generated using microflow channels. For instance, the flow channels include a flow channel through which a first liquid forming the dispersion of the emulsion flows and a flow channel through which a second liquid forming the dispersion medium flows. Cells may be contained in the first liquid. The device also includes a region through which the two liquids contact each other to form an emulsion. Referring below... Figure 13B Examples describing microflow channels.

[0380] The microfluidic channel shown in the figure includes: a flow channel 601 through which a first liquid containing cells flows; and flow channels 602-1 and 602-2 through which a second liquid flows. The first liquid forms dispersoids. The second liquid forms the dispersion medium for the emulsion. Flow channels 601 and 602-1 and 602-2 merge with each other. Emulsion particles form at the merging point. Cells 603 are then separated within the emulsion particles. The size of the emulsion particles can be controlled, for example, by controlling the flow rate of these flow channels.

[0381] For an emulsion to form, the first and second liquids must be immiscible. For example, the first liquid can be a hydrophilic liquid and the second liquid can be a hydrophobic liquid, or vice versa.

[0382] Furthermore, the microflow channels shown in the figure may include flow channels 604 for introducing cell-destructive substances into the emulsion particles. When the microflow channels are configured such that flow channels 604 merge with flow channels 601 immediately in front of the confluence point, cell destruction by cell-destructive substances can be prevented before emulsion particles are formed.

[0383] Next, we will refer to Figure 14A and Figure 14B Examples of apparatuses for more efficiently forming emulsions comprising emulsion particles, each containing a cell, are described. This emulsion-forming apparatus can separate a cell into an emulsion particle, thereby reducing the number of empty emulsion particles with a very high probability. Furthermore, this emulsion-forming apparatus can increase the probability of separating a cell into an emulsion particle.

[0384] Figure 14A This is an example of a microflow channel chip (hereinafter also referred to as a “microchip”) used to form emulsion particles in a device.

[0385] The microchip 150 shown in the figure includes: a main flow channel 155 through which biological particles (especially cells) flow; and a collection flow channel 159 through which target particles among the biological particles are collected. The microchip 150 is provided with a particle sorting section 157. Figure 15 An enlarged view of the particle sorting section 157 is shown in Figure A. As shown in Figure A, the particle sorting section 157 includes a connecting flow channel 170 that connects the main flow channel 155 and the collection flow channel 159 to each other. A liquid supply flow channel 161, which supplies liquid to the connecting flow channel 170, is connected to the connecting flow channel 170. As described above, the microchip 150 has a flow channel structure including the main flow channel 155, the collection flow channel 159, the connecting flow channel 170, and the liquid supply flow channel 161.

[0386] Figure 14B This is supplementary explanation. Figure 14A The diagram shows the formation of emulsion particles in the microchip 150 and the separation of biological particles within the formed emulsion particles.

[0387] In addition, such as Figure 14A As shown, the microchip 150 constitutes part of the biological particle sorting device 200, which, in addition to the microchip, also includes a light irradiation unit 191, a detection unit 192, and a control unit 193. Figure 16 As shown, the control unit 193 may include a signal processing unit 194, a determination unit 195, and a sorting control unit 196. This bioparticle sorting device 200 is used as the emulsion forming apparatus described above.

[0388] like Figure 17 As shown, in order to form an emulsion comprising emulsion particles containing a target cell, the following steps can be performed in the microchip 150: a flow step S201 of supplying a first liquid containing cells into a main flow channel 155; a determination step S202 of determining whether the cells flowing through the main flow channel 155 are the target particles for collection; and a collection step S203 of collecting the target particles into a collection flow channel 159. The determination step S202 corresponds to the determination step described above. The collection step S203 corresponds to the particle separation step described above.

[0389] The steps are explained below.

[0390] (Flow steps)

[0391] In flow step S201, a first liquid comprising cells flows through the main flow channel 155. The first liquid flows from the confluence portion 162 to the particle sorting portion 157 within the main flow channel 155. The first liquid can be a laminar flow formed by a sample liquid comprising cells and a sheath fluid, and particularly can be a laminar flow in which the sample liquid is surrounded by a sheath fluid. The flow channel structure for forming the laminar flow will be described below.

[0392] Incidentally, the sheath fluid may include, for example, cell-disrupting components or cell-lysing components. This component is thus captured within the emulsion particles, allowing the cells within these particles to be destroyed during the disruption step described later. The cell-lysing component may be a cell-lysing enzyme, and may be, for example, proteinase K. For example, after capturing cells in emulsion particles containing proteinase K, the emulsion particles are placed at a predetermined temperature (e.g., 37°C to 56°C) for, for example, 1 hour or less, particularly less than 1 hour. The cells are thus lysed. Incidentally, proteinase K is active even at 37°C or lower. However, when using such lower temperatures, overnight incubation may be performed, for example, considering the reduced cell-lysing properties of proteinase K. Furthermore, the sheath fluid may include surfactants (e.g., SDS, Sarkosyl, Tween 20, Triton X-100, etc.). Surfactants can enhance the activity of proteinase K.

[0393] Furthermore, the sheath fluid may not contain cell-destructive components. In this case, the cells may be physically destroyed. Physical destruction methods can include, for example, optical treatment (e.g., optical cell lysis) or thermal treatment (e.g., cell lysis by heating). For instance, optical treatment can be performed by irradiating the emulsion particles with a laser to form plasma or cavitation bubbles within the particles. Thermal particle destruction can be achieved by heating the emulsion particles.

[0394] The microchip 150 is provided with a sample liquid inlet 151 and a sheath fluid inlet 153. Sample liquid containing cells and sheath fluid not containing cells are introduced into the sample liquid flow channel 152 and the sheath fluid flow channel 154 from these inlets, respectively.

[0395] Microchip 150 has a flow channel structure in which a sample flow channel 152 through which sample liquid flows and a sheath fluid flow channel 154 through which sheath fluid flows merge at a confluence 162 to form a main flow channel 155. The sample liquid and sheath fluid merge at the confluence 162 to form, for example, laminar flow where the sample liquid is surrounded by sheath fluid. Figure 14B The diagram illustrates the formation of laminar flow. As shown, laminar flow is formed such that the sample liquid introduced from the sample flow channel 152 is surrounded by the sheath fluid introduced from the sheath fluid flow channel 154.

[0396] Preferably, the cells are arranged substantially in a row in the laminar flow. As shown, for example, cells P can be arranged substantially in a row in the sample liquid. Therefore, the flow channel structure in this disclosure forms a laminar flow comprising cells flowing in a substantially rowed state.

[0397] The laminar flow proceeds through the main flow channel 155 to the particle sorting section 157. Preferably, the cells flow within the main flow channel 155 in a row. Therefore, during light irradiation in the detection area 156, which will be described below, the light generated by irradiating one particle and the light generated by irradiating another particle can be easily distinguished from each other.

[0398] (Determine the steps)

[0399] Step S202 determines whether the cells flowing through the main flow channel 155 are the target particles for collection. This determination can be performed by determination unit 195. Determination unit 195 can make the determination based on the light generated by irradiating the cells with light through light irradiation unit 191. An example of determination step S202 will be described in more detail below.

[0400] In the determination step S202, the light irradiation unit 191 irradiates the cells flowing through the main flow channel 155 (specifically the detection region 156) in the microchip 150 with light (e.g., excitation light, etc.), and the detection unit 192 detects the light generated by the light irradiation. Based on the characteristics of the light detected by the detection unit 192, the determination unit 195, including the control unit 193, determines whether the biological particle is the target particle for collection. For example, the determination unit 195 may make the determination based on scattered light, based on fluorescence, or based on an image (e.g., one or more of dark-field images, bright-field images, and phase difference images, etc.). In the collection step S203 described later, the control unit 193 controls the flow in the microchip 150 to collect the target particles into the collection flow channel 159.

[0401] The light irradiation unit 191 irradiates cells flowing within a flow channel in the microchip 150 with light (e.g., excitation light). The light irradiation unit 191 may include a light source emitting light and an objective lens that focuses the excitation light onto particles flowing through a detection region. Those skilled in the art can appropriately select the light source according to the analytical purpose. For example, the light source may be a laser diode, an SHG laser, a solid-state laser, a gas laser, a high-brightness LED, or a halogen lamp, or a combination of two or more of these. In addition to the light source and objective lens, the light irradiation unit may include other optical elements as needed.

[0402] (Collection steps)

[0403] In collection step S203, cells identified as target particles in determination step S202 are collected into collection flow channel 159. In collection step S203, the target particles, while contained within a first liquid, are collected into a second liquid immiscible with the first liquid within the collection flow channel. As a result, an emulsion can be formed within the collection flow channel 159, where the second liquid is the dispersion medium and the first liquid is the dispersion, and each emulsion particle in the emulsion includes one target particle. Therefore, the cells designated as targets are separated within the space within the emulsion particles.

[0404] like Figure 14B As shown, for example, in a state where it is contained in a first liquid (represented by white), the target particle P is collected into a second liquid (represented by gray). Thus, an emulsion particle 190 is formed, and a target particle P is separated within the space of an emulsion particle 190.

[0405] The collection steps will be described in more detail below.

[0406] A collection step S203 is performed in the particle sorting section 157 of the microchip 150. In the particle sorting section 157, laminar flow separation that has passed through the main flow channel 155 results in flow into two waste flow channels 158. Although in Figure 14A The particle sorting section 157 depicted has two waste flow channels 158, but the number of branch flow channels is not limited to two. For example, the particle sorting section 157 may be provided with one or more (e.g., two, three, four, etc.) branch flow channels. The branch flow channels may be configured as follows: Figure 14A It can branch out in the shape of the letter Y on a plane, or it can be configured to branch in three dimensions.

[0407] In the particle sorting section 157, only when the target particles are collected, a flow is formed from the main flow channel 155 through the connecting flow channel 170 into the collection flow channel 159, and the target particles are collected into the collection flow channel 159. Figure 15 Figure A shows an enlarged view of the particle sorting section 157. As shown in Figure A, the main flow channel 155 and the collection flow channel 159 are configured to communicate with each other via a connecting flow channel 170 on the same axis as the main flow channel 155. As shown in Figure B, target particles flow into the collection flow channel 159 through the connecting flow channel 170. As shown in Figure C, particles that are not target particles flow into the waste flow channel 158.

[0408] Figure 18A and Figure 18B An enlarged view of the connection flow channel 170 and its vicinity is shown in the figure. Figure 18A This is a schematic perspective view of the flow channel 170 and its vicinity. Figure 18B This is a schematic cross-sectional view in a plane passing through the centerline of the liquid supply flow channel 161 and the centerline of the connecting flow channel 170. The connecting flow channel 170 includes a flow channel 170a on the detection area 156 side (hereinafter also referred to as the upstream connecting flow channel 170a), a flow channel 170b on the collection flow channel 159 side (hereinafter also referred to as the downstream connecting flow channel 170b), and a connecting portion 170c for connecting the connecting flow channel 170 and the liquid supply flow channel 161. The liquid supply flow channel 161 is arranged substantially perpendicular to the flow channel axis of the connecting flow channel 170. Although in Figure 18A and 18B Two liquid supply flow channels 161 may be provided facing each other at approximately the center of the connecting flow channel 170, but only one liquid supply flow channel may be provided.

[0409] like Figure 18BAs indicated by the arrows, the second liquid is supplied from the two liquid supply flow channels 161 to the connecting flow channel 170. The second liquid flows from the connecting portion 170c to both the upstream connecting flow channel 170a and the downstream connecting flow channel 170b.

[0410] Without a collection step, the second liquid flows as follows.

[0411] The second liquid flowing upstream through the connecting flow channel 170a exits from the connecting surface of the connecting flow channel 170, which is connected to the main flow channel 155, and then splits and flows into two waste flow channels 158. Because the second liquid exits from the connecting surface, it prevents the first liquid and particles that do not need to be collected in the collection flow channel 159 from entering the collection flow channel 159 through the connecting flow channel 170.

[0412] The second liquid, which has already flowed to the downstream side connecting flow channel 170b, flows into the collecting flow channel 159. Therefore, the interior of the collecting flow channel 159 is filled with the second liquid, which, for example, becomes a dispersion medium for forming an emulsion.

[0413] During the collection step, the second liquid can also be supplied to the connecting channel 170 from both liquid supply channels 161. However, pressure changes within the collection flow channel 159 (specifically, negative pressure generated within the collection flow channel 159) create a flow from the main flow channel 155 through the connecting flow channel 170 to the collection flow channel 159. That is, a flow is formed sequentially from the main flow channel 155 to the collection flow channel 159 through the upstream connecting flow channel 170a, the connecting portion 170c, and the downstream connecting flow channel 170b. Therefore, the target particles are collected into the second liquid within the collection flow channel 159 while encapsulated in the first liquid. During the collection step, for example, an emulsion can be formed within the collection flow channel 159 or in a container connected to the collection flow channel end 163 via the flow channel.

[0414] In collection step S203, due to pressure changes within the collection flow channel 159, the target particles are collected into the collection flow channel via the connecting flow channel. For example, collection can be performed by generating a negative pressure within the collection flow channel 159, as described above. For example, a negative pressure can be generated by deforming the wall defining the collection flow channel 159 using an actuator 197 (specifically, a piezoelectric actuator) attached to the outside of the microchip 150. The negative pressure can create a flow into the collection flow channel 159. The actuator 197 can be attached to the outside of the microchip 150 to generate negative pressure, for example, by deforming the wall of the collection flow channel 159. The deformation of the wall can change the internal space of the collection flow channel 159, thereby generating negative pressure. For example, the actuator 197 can be a piezoelectric actuator. When the target particles are drawn into the collection flow channel 159, a laminar sample liquid or a combination of a laminar sample liquid and a sheath liquid can flow into the collection flow channel 159. Therefore, the target particles are sorted in the particle sorting section 157 and collected into the collection flow channel 159.

[0415] The target particles are collected within the collection flow channel 159 in a state of being encapsulated in the first liquid, into a second liquid that is immiscible with the first liquid. Therefore, as described above, an emulsion of the second liquid as a dispersion medium and the first liquid as a dispersion is formed within the collection flow channel 159.

[0416] The connecting flow channel 170 is provided with a liquid supply flow channel 161 to prevent biological particles that are not the target particles from entering the collection flow channel 159 through the connecting flow channel 170. A second liquid that is immiscible with the liquid (sample liquid and sheath fluid) flowing through the main flow channel 155 is introduced into the connecting flow channel 170 from the liquid supply flow channel 161.

[0417] A portion of the second liquid introduced into the connecting flow channel 170 forms the fluid flowing from the connecting flow channel 170 to the main flow channel 155. This prevents biological particles other than the target particles from entering the collection flow channel 159. Because the first liquid, which has already flowed through the main flow channel 155, flows towards the waste flow channel 158, the second liquid formed by the flow from the connecting flow channel 170 to the main flow channel 155 flows in the waste flow channel 158, just like the first liquid, but not within the main flow channel 155.

[0418] Incidentally, the remaining portion of the second liquid introduced into the flow channel 170 flows into the collection flow channel 159. Therefore, the interior of the collection flow channel 159 can be filled with the second liquid.

[0419] The interior of the collecting flow channel 159 may be filled with a second liquid that is immiscible with the first liquid. To fill the interior of the collecting flow channel 159 with the second liquid, the second liquid may be supplied from the liquid supply flow channel 161 to the connecting flow channel 170. This supply causes the second liquid to flow from the connecting flow channel 170 to the collecting flow channel 159. Therefore, the interior of the collecting flow channel 159 may be filled with the second liquid.

[0420] The laminar flow that has flowed into the waste flow channel 158 can be discharged to the outside of the microchip at the waste flow channel end 160. In addition, the target particles collected in the collection flow channel 159 can be discharged to the outside of the microchip at the collection flow channel end 161.

[0421] A container (not shown) may be connected to a collection flow channel end 163 via a flow channel such as a tube. An emulsion comprising the target particles being collected, wherein the first liquid is a dispersion and the second liquid is a dispersion medium, is collected into the container. Therefore, the bioparticle sorting device may include a flow channel for collecting the emulsion comprising the target particles into the container.

[0422] Furthermore, when the collection operation is performed with the collection flow channel end 163 closed, multiple emulsion particles can be retained within the collection flow channel 159. After the collection operation is completed, assays, such as single-cell analysis, can continue within the collection flow channel 159. For example, a disruption step, described later, can be performed within the collection flow channel 159. This disruption step then enables binding between the target-capturing molecules and the target substance.

[0423] Incidentally, such as Figure 14A As shown, in the microchip, there are, for example, two or more inlets and / or outlets for introducing liquid and discharging liquid, preferably all of which can be formed in one surface. As shown, the collecting flow channel end 163 and the two branch flow channel ends 160 are both formed on the surface formed by the sample liquid inlet 151 and the sheath fluid inlet 153. Furthermore, the introducing flow channel inlet 164 for introducing liquid into the introducing flow channel 161 is also formed on this surface. Therefore, in the microchip, liquid is introduced through all its inlets and discharged through its outlets on a single surface. This facilitates attachment of the chip to a biological particle sorting device.

[0424] Incidentally, in the figure, a portion of the sheath fluid flow channel 154 is indicated by a dashed line. This dashed portion is located below the sample fluid flow channel 152, indicated by the solid line (a position shifted in the direction of the optical axis indicated by the arrow). Where the flow channels indicated by the dashed lines and the flow channels indicated by the solid lines intersect, these flow channels are not interconnected. This description also applies to the dashed portion of the collection flow channel 159 and the branch flow channel 158 intersecting with it.

[0425] Furthermore, the liquid supply flow channel supplies liquid (particularly the second liquid) to the connecting flow channel. As a result, fluid flows from the connection point between the liquid supply flow channel and the connecting flow channel to the main flow channel within the connecting flow channel. This prevents liquid flowing through the main flow channel from entering the connecting flow channel, and also prevents particles other than the target particle from flowing through the connecting flow channel into the collection flow channel. As described above, during the collection step, for example, the negative pressure generated within the collection flow channel causes the first liquid containing one target particle to be collected through the connecting flow channel into the second liquid within the collection flow channel. As a result, emulsion particles containing the target particle are formed in the second liquid.

[0426] Furthermore, for example, by driving the piezoelectric actuator at an appropriate time (e.g., the time point at which the biological particles identified as the collection target particles arrive at the particle sorting section 157 in the determination step), the hydrophilic water solution containing the collection target particles is collected into the collection flow channel 159. This forms emulsion particles. For example, when determining whether a particle is the collection target particle using the peak signal and area signal in the determination step, it can also be determined whether the particle is a single particle (single peak), a particle containing two biological particles bound together (double peak), or a particle containing three biological particles bound together (triple peak). Therefore, the formation of emulsion particles containing two or more cells within a single emulsion particle can be avoided. Thus, emulsion particles containing one cell can be formed with high probability and high efficiency. Furthermore, because the formation of emulsion particles containing two or more cells bound together can be avoided, the operation of removing the binding material of two or more cells by means such as a cell sorter before the emulsion formation operation can be omitted.

[0427] (4-2) Variations of single-cell analysis

[0428] In single-cell analysis using the analytical method according to embodiments of the present disclosure, the circular nucleic acid generation process used in the analytical method is not particularly limited, and optional methods can be combined with the circular nucleic acid generation process, as long as the circular nucleic acid generation process generates circular nucleic acids by splicing two or more complementary strands generated by the above-described complementary nucleic acid generation step via a nucleic acid splicing portion.

[0429] Figure 6D As shown, as a variant of single-cell analysis, Figure 5 The circular nucleic acid generation process in the single-cell analysis shown is carried out through the first variant of the circular nucleic acid generation process (2-3). However, Figure 6E The nucleic acid splicing portion shown is used in Figure 6D The nucleic acid splicing part used in the generation of circular nucleic acids. Figure 6E The nucleic acid splicing portion shown differs from the nucleic acid splicing portion shown in <2D-α> used in the description of the first variant of the circular nucleic acid generation method in (2-3) in that... Figure 6E The nucleic acid splice shown has a sequence appendage (cell barcode) between the target nucleic acid capture portion (the poly-T in the figure) and the complementary strand capture portion (the splice in the figure).

[0430] Also in Figure 6D In step S610, the analysis is performed based on the single-cell analysis described above. Figure 5 Similar to step S42, in cases where each cell has a nucleic acid splicing portion bound via a cell capture portion, each cell released from the substrate is individually released into a microspace. Subsequently, each unit is destroyed in a separated state. As for these conditions, in this variant, conditions similar to those applicable to the single-cell analysis described above can also be appropriately applied. Incidentally, for the binding of the nucleic acid splicing portion via the cell capture portion in this case, the nucleic acid splicing portion and the cell capture portion can bind directly to each other, and as... Figure 6D As shown, the nucleic acid splicing portion can bind to the cell capture portion via a nucleic acid having a sequence complementary to the sequence of the nucleic acid splicing portion. When the nucleic acid splicing portion binds to the cell capture portion via a nucleic acid having a sequence complementary to the sequence of the nucleic acid splicing portion, the cell capture portion can be prevented from becoming a steric hindrance during the subsequent formation of circular nucleic acids.

[0431] Through this destruction, such as Figure 6D As shown in step S611, the target nucleic acid capture portion contained in the nucleic acid splicing portion shown in Figure 6F captures the 3' end of the mRNA in the disrupted cell. The subsequent steps are similar to those steps in the first variant of the circular nucleic acid generation process (2-3), and conditions similar to those applicable to the circular nucleic acid generation process may also be appropriately applied in this variant.

[0432] It should be noted that although the example of the circular nucleic acid generation process in single-cell analysis using the first variant of the circular nucleic acid generation process (2-3) is considered a variant of single-cell analysis, this is merely an example, and in single-cell analysis using the analytical method according to the embodiments of this disclosure, as a circular nucleic acid generation process, including the examples disclosed in this specification, a method can be appropriately used to generate circular nucleic acids by splicing two or more complementary strands generated by the complementary nucleic acid generation step together via a nucleic acid splicing portion.

[0433] (4-3) Examples of single-cell analysis

[0434] The expression analysis of mRNA in single cells using the analytical methods according to examples of this disclosure can be combined with other optional analyses, provided that the desired analytical features are not significantly impaired.

[0435] For example, while analyzing intracellular mRNA, analysis of molecules expressed intracellularly, molecules expressed on the cell surface, or secreted molecules captured on the cell surface can be performed using antibody-nucleic acid molecule complexes. In these complexes, the antibody binds to a nucleic acid molecule containing an antibody identifier sequence (antibody barcode) for identifying the source of the antibody, and a poly-A sequence captured as a target nucleic acid capture portion contained within the nucleic acid splicing region. Figure 14C As shown in the attached figures. The following is a detailed explanation with reference to the accompanying drawings. Incidentally, regarding the antibody-nucleic acid molecular complex described above, as... Figure 14C In the example shown, the nucleic acid molecule contained in the antibody-nucleic acid molecule complex may include an initiation sequence (amplification primer) in addition to the antibody identifier sequence and the polyadenylate (polyA) sequence. Furthermore, the optional nucleic acid splicing portions disclosed in this specification may be suitable for use as nucleic acid splicing portions in the examples of this application.

[0436] exist Figure 14CIn the antibody-nucleic acid molecular complexes described herein, the antibodies contained within the complex are those that specifically bind to molecules expressed intracellularly, molecules expressed on the cell surface, or secreted molecules captured on the cell surface. Furthermore, a unique sequence for each antibody is provided as an antibody identifier sequence for the nucleic acid molecules contained in the complex. That is, in cases where multiple secreted molecules are present on the cell surface to be analyzed, various antibody-nucleic acid molecular complexes are prepared according to these types. Furthermore, the antibody identifier sequence may have the aforementioned error-correcting function. Although Figure 14 shows an example of using antibody-nucleic acid molecular complexes, the substances forming complexes with nucleic acid molecules are not limited to antibodies, as long as the substance has the function of binding to molecules expressed intracellularly, molecules expressed on the cell surface, or secreted molecules captured on the cell surface. For example, antibody fragments (Fab, scFv, VHH, microantibodies, etc.), aptamers, molecularly imprinted polymers, etc., can be used. These complexes that bind to nucleic acid molecules can be used in the examples of this application.

[0437] The antibody-nucleic acid molecular complex prepared above binds to cells having molecules that serve as antigens expressed on their surfaces or secreted molecules that are captured on their surfaces due to antigen-antibody reactions. On the other hand, if the molecules that serve as antigens are not expressed intracellularly, expressed on the surface, or captured on the surface, the antibody-nucleic acid molecular complex does not bind to the cell.

[0438] Cells that bind to antibodies corresponding to molecules expressed intracellularly, molecules expressed on the cell surface, or secreted molecules captured on the surface are obtained by performing an antigen-antibody reaction on cells bound via the cell capture portion and the nucleic acid splicing portion using the antibody-nucleic acid molecule complex prepared above.

[0439] Next, the cells obtained from the antigen-antibody reaction are separated into a tiny space. Figure 14D An example is shown in which cells are separated into microspaces using a chip for forming an emulsion. However, there is no limitation to this method, and optional methods for separating cells into microspaces, including the examples disclosed in this specification, may be appropriately employed.

[0440] Incidentally, Figure 14D The example illustrates an instance where an antibody-nucleic acid molecule complex binds to a cell bound to a nucleic acid splicing portion via a cell capture portion, and this cell serves as a separated cell within the space of the emulsion particle. However, there are also cases where multiple antibody-nucleic acid molecule complexes bind to a single cell. Furthermore, as a condition for separating cells generated by an antigen-antibody reaction into the space within the emulsion particle, such as... Figure 14DAs shown, conditions similar to those applicable in the aforementioned methods (in the case of space within emulsion particles) can also be appropriately applied in the examples of this application.

[0441] Next, each cell is destroyed under conditions similar to those applicable to the single-cell analysis or its variants described above.

[0442] Through this destruction, such as Figure 14E As shown in step S1411, the target nucleic acid capture portion contained in the nucleic acid splicing portion captures the 3' end poly-A sequence of mRNA in the disrupted cells or the 3' end poly-A sequence of nucleic acid molecules contained in the antibody-nucleic acid molecule complex. Subsequent steps are similar to those steps in the circular nucleic acid generation process of (2-2). When these steps are performed, a single-stranded circular nucleic acid is synthesized, and a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid is synthesized. Incidentally, conditions similar to those applicable to the circular nucleic acid generation process can also be suitably applied in the examples of this application.

[0443] The single-stranded circular nucleic acids obtained as described above include not only sequences derived from mRNA but also antibody identifier sequences contained within the nucleic acid molecules of antibody-nucleic acid complexes. Therefore, by decoding the base sequences of single-stranded linear nucleic acids obtained using single-stranded circular nucleic acids as templates, the expression and secretion conditions of molecules acting as antigens, which bind to antibodies recognized by the antibody identifier sequences, can be analyzed. In other words, in addition to analyzing the expression of mRNA in cells isolated in small spaces, the expression and secretion conditions of expression and secretion molecules on the cell surface can be analyzed simultaneously.

[0444] Incidentally, for example, antibody recognition sequences can be more easily identified by providing antibody identifier sequences with different base sequence lengths for each antibody. In this case, for example, the base sequence of the antibody identifier sequence can be adjusted in the range of 5 to 25 bases.

[0445] Incidentally, the decoding of the base sequence of the obtained single-stranded linear nucleic acid, for example, can also be appropriately applied to this variant using a method similar to the circular nucleic acid generation process described above (2-2).

[0446] Furthermore, although examples of circular nucleic acid generation processes in which circular nucleic acid generation processes via (2-2) have been shown as examples of applications in single-cell analysis, these are merely examples, and in single-cell analysis using analytical methods according to embodiments of this disclosure, as examples of circular nucleic acid generation processes, including those disclosed in this specification, methods for generating circular nucleic acids by splicing two or more complementary strands generated by complementary nucleic acid generation steps together via nucleic acid splicing portions can be suitably used.

[0447] (5) Example 4 (Nucleic acid splicing part through phosphorylation modification)

[0448] In the two base sequence strands of the double-stranded portion constituting the nucleic acid splicing region, the strand coupled to the target nucleic acid capture region (especially the strand with poly-T) may have its 5' end modified by phosphorylation. This allows for reliable ligation processing as described later. This will refer to... Figure 7 Describe it.

[0449] As shown in Figure A, cells are captured by nucleic acid splicing portions fixed on a substrate.

[0450] In step S51, as described above... Figure 5 As described, cells with nucleic acid splice portions bound thereto are set to be released from a substrate and separated into microspaces (e.g., emulsion particles). The cells are disrupted in this separated state. Thus, as shown in Figure B, mRNA is released into the microspaces.

[0451] In step S52, the mRNA released into the microspace by disrupting the cell binds to the nucleic acid splicing portion (enclosed by the dashed line) to form a hybrid, as shown in Figure C. The hybrid is generated through complementary binding between the target nucleic acid capture portion (specifically the poly-T sequence) of the nucleic acid splicing portion and the poly-A tail of the mRNA.

[0452] Here, phosphorylation is used to modify the 5' terminal P of the two base sequences in the nucleic acid splicing region that are coupled to the target nucleic acid capture region (especially the strand with poly-T). In the figure, phosphorylation is represented by a circle. Phosphorylation can be used to more reliably perform the ligation step in subsequent stages.

[0453] Step S53 is cDNA synthesis, which is consistent with the description above. Figure 2A The same applies to step S12. The description of step S12 also applies to step S53.

[0454] When step S53 is performed, cDNA with a CCC sequence at the end of the generated strand is generated, such as... Figure 7 As shown in D.

[0455] Step S54 is nucleic acid assembly, which is consistent with the description above. Figure 2A The same applies to step S13. The description of step S13 also applies to step S54.

[0456] During step S54, two or more cDNA strands are spliced ​​together via complementary strand capture portions, such as Figure 7 As shown in E.

[0457] Step S55 is the same as described above. Figure 2AThe same circular nucleic acid formation, ligation, and primer addition as in steps S14 and S15. The descriptions of steps S14 and S15 also apply to step S55.

[0458] When step S55 is performed, a circular nucleic acid containing primers is formed, such as... Figure 7 As shown in F.

[0459] Here, because the 5' end of the nucleic acid splice portion is phosphorylated as described above, the ligation is more reliable. This allows for more efficient formation of circular nucleic acids.

[0460] Step S56 is the same as described above. Figure 2A The same nucleic acid amplification occurs in step S16. The description of step S16 also applies to step S56.

[0461] When step S56 is performed, the nucleic acid is amplified, such as Figure 7 As shown in G. The nucleic acids amplified in this way are used for single-cell analysis. For example, the nucleic acid sequence is sequenced, and each cell is analyzed using the sequencing results.

[0462] (6) Example 5 (including the nucleic acid splicing part of the self-binding inhibition sequence)

[0463] The complementary strand capture portion of the nucleic acid splice is not limited to the GGG sequence, and may include self-binding repression sequences (e.g., Hn and / or Nn) in addition to GGG. Here, H is a base other than G, that is, H is A, T, or C. N is A, T, G, or C. n is the number of Hs or Ns, and may be, for example, an integer of 1 or greater. For example, n may be an integer from 1 to 5. When n is 2 or greater, the H or N constituting the self-binding repression sequence may be chosen individually and randomly.

[0464] For example, when the complementary strand trapping region is a base sequence having a self-binding repression sequence with one base other than the GGG sequence, the complementary strand trapping region may have a base sequence of GGGH or GGGN. When the self-binding repression sequence has two bases, the complementary strand trapping region may have a base sequence of GGGHN, GGGNH, GGGNN, or GGGHH.

[0465] Figure 8 The image illustrates an example of the formation of circular nucleic acids when such a complementary strand capture portion is provided.

[0466] As shown in Figure A, the cell is captured by the nucleic acid splice portion fixed on the substrate.

[0467] In step S61, as described above... Figure 5As described, cells with nucleic acid splice portions bound thereto are released from a substrate and separated into microspaces (e.g., emulsion particles). The cells are disrupted in this separated state. Thus, mRNA is released into the microspaces, as shown in Figure B.

[0468] In step S62, the mRNA released into the microspace by disrupting the cell binds to the nucleic acid splicing portion to form a hybrid, as shown in Figure C. The hybrid is generated through complementary binding between the target nucleic acid capture portion (specifically the poly-T sequence) of the nucleic acid splicing portion and the poly-A tail of the mRNA.

[0469] Incidentally, phosphorylation modifies the 5' end of the two base sequences of the nucleic acid splicing region that are coupled to the target nucleic acid capture region (especially the strand with poly-T). In the figure, phosphorylation is represented by a circle. Phosphorylation can be used to more reliably perform subsequent ligation steps.

[0470] Step S63 is the same as described above. Figure 2A The same cDNA synthesis is performed in step S12. The description of step S12 also applies to step S63.

[0471] When step S63 is performed, cDNA with a CCC sequence at the end of the generated strand is generated, such as... Figure 8 As shown in D.

[0472] In step S64, RNA digestion and nucleic acid splicing are performed. Therefore, as shown in Figure E, the mRNA is digested and forms a single-stranded portion. The cDNA to be captured by the complementary strand capture portion 52 is the sequence H on the 5' end side of the CCC sequence at the 3' end of the single-stranded portion. The selection is based on the existence of H. Therefore, not all cDNA is captured by the complementary strand capture region, but only in cases where the complementary strand capture region is GGGH-3', for example, with 3'-CCCH. The cDNA is captured. Since H is one of the four ATGCs, the probability of forming a circular structure with only one cDNA is 1 / 4. As n (where n is an integer of 1 or greater) increases, the probability decreases and becomes (1 / 4). n Therefore, nucleic acids are generated by splicing two or more cDNAs together, with the two or more cDNAs binding together via the nucleic acid splicing portion.

[0473] Therefore, the formation of circular nucleic acids containing only one cDNA can be avoided. In other words, more circular nucleic acids in which two or more cDNAs are spliced ​​together can be generated.

[0474] In step S65, circular nucleic acid formation and ligation occur. Thus, as... Figure 8 As shown in F, circular nucleic acids are generated.

[0475] Here, because the 5' end of the nucleic acid splice portion is phosphorylated as described above, the ligation is more reliable. Therefore, circular nucleic acids can be formed more efficiently.

[0476] Step S66 is nucleic acid amplification, which is the same as described above. Figure 2A The nucleic acid amplification in step S16 is the same. The description of step S16 also applies to step S66.

[0477] When step S66 is performed, the nucleic acid is amplified, such as Figure 8 As shown in G. The nucleic acids amplified in this way are used for single-cell analysis. For example, the nucleic acid sequence is sequenced, and the sequencing results are used to analyze each cell.

[0478] (7) Example 6 (including the nucleic acid splicing part of the trigger sequence)

[0479] As described above, the double-stranded portion of the nucleic acid splice can have different base sequences for each region, and, for example, this base sequence can be a random sequence. The double-stranded portion can have a priming sequence other than a random sequence. The priming sequence can be the same in two or more regions, and, for example, the nucleic acid splice present in all regions can have the same priming sequence. Therefore, in nucleic acid amplification processing, nucleic acid amplification can be performed by using a universal priming sequence. Therefore, it is not necessary to add primers before performing nucleic acid amplification processing. This will refer to... Figure 9 Describe it.

[0480] As shown in Figure A, cells are captured by nucleic acid splicing portions fixed on a substrate.

[0481] In step S71, as described above... Figure 5 As described, cells with nucleic acid splice portions bound to them are released from a substrate and separated into tiny spaces (e.g., emulsion particles). The cells are disrupted in this separated state. Thus, as shown in Figure B, mRNA is released into the tiny spaces.

[0482] In step S72, the mRNA released into the microspace by the cell is disrupted to bind with the nucleic acid splicing portion to form a hybrid, such as... Figure 9 As shown in C, hybrids are generated through complementary binding between the target nucleic acid capture portion (specifically the poly-T sequence) of the nucleic acid splice region and the poly-A tail of the mRNA. The nucleic acid splice region has a priming sequence located in the double-stranded portion of the nucleic acid splice region.

[0483] Furthermore, phosphorylation is used to modify the 5' ends of the two base sequence strands of the nucleic acid splicing region that are coupled to the target nucleic acid capture region (especially the strand with poly-T). In the figure, phosphorylation is represented by circles. Phosphorylation can be used to more reliably perform subsequent ligation steps. Incidentally, phosphorylation modification can be omitted.

[0484] Step S73 is cDNA synthesis, which is consistent with the description above. Figure 2A The same applies to step S12. The description of step S12 also applies to step S73.

[0485] When step S73 is performed, cDNA with a CCC sequence at the end of the generated strand is generated, such as... Figure 9 As shown in D.

[0486] Step S74 is the same as described above. Figure 2A The nucleic acid splicing in step S13 is the same as that in step S74. The description of step S13 also applies to step S74.

[0487] During step S54, two or more cDNAs are spliced ​​together via complementary strand capture portions, such as Figure 9 As shown in E.

[0488] Step S75 is the same as described above. Figure 2A The same circular nucleic acids formed and ligated in step S14. The description of step S14 also applies to step S75.

[0489] When step S75 is performed, a circular nucleic acid with added primers is formed, such as Figure 9 As shown in F.

[0490] Here, because the 5' end of the nucleic acid splice portion is phosphorylated as described above, the ligation is more reliable. Therefore, circular nucleic acids can be formed more efficiently.

[0491] Furthermore, although primer addition is in reference Figure 2A The method described is used, but primer addition is not mandatory because the nucleic acid splicing portion has a priming sequence, as mentioned above.

[0492] Step S76 is the same as described above. Figure 2A The nucleic acid amplification in step S16 is the same as that in step S76. The description of step S16 also applies to step S76.

[0493] When step S76 is performed, the nucleic acid is amplified, such as Figure 9 As shown in G. The nucleic acids amplified in this way are used for single-cell analysis. For example, the nucleic acid sequence is sequenced, and the sequencing results are used to analyze each cell.

[0494] Figure 19 This is a schematic diagram illustrating an example where the nucleic acid splicing portion includes a trigger sequence. It can be confirmed that, in Figure 19 In the examples shown, unlike the nucleic acid splice portion without a trigger sequence shown in <19A>, the double-stranded portion of the nucleic acid splice portion in <19B> has a trigger sequence. Incidentally, although... Figure 19 The example shown represents an instance where the nucleic acid splice portion with a double-stranded portion has a trigger sequence, but nucleic acid splice portions without a double-stranded portion may also have a trigger sequence.

[0495] (8) Example 7 (including the nucleic acid splicing part of the self-binding inhibition sequence and the initiation sequence)

[0496] The nucleic acid splicing portion may include both the self-binding inhibition sequence and the initiation sequence described above. This inhibits the formation of circular nucleic acids containing only one cDNA and eliminates the need for primer addition before nucleic acid amplification. Figure 10 An example of forming a circular nucleic acid is shown in which such a complementary strand trap portion is provided.

[0497] As shown in Figure A, cells are captured by nucleic acid splicing portions fixed on a substrate.

[0498] In step S81, as described above... Figure 5 As described, cells with nucleic acid splice portions bound thereto are released from a substrate and separated into microspaces (e.g., emulsion particles). The cells are disrupted in this separated state. Therefore, mRNA is released into the microspaces, as shown in Figure B.

[0499] In step S82, the mRNA released into the microspace by disrupting the cell binds to the nucleic acid splice portion to form a hybrid, as shown in Figure C. The hybrid is generated through complementary binding between the target nucleic acid capture portion (specifically the poly-T sequence) of the nucleic acid splice portion and the poly-A tail of the mRNA. The nucleic acid splice portion has a priming sequence within the double-stranded portion of the nucleic acid splice portion.

[0500] Incidentally, phosphorylation modifies the 5' end of the two base sequences in the nucleic acid splicing region that are coupled (ligated) to the target nucleic acid capture region (especially the strand with poly-T). In the figure, phosphorylation is represented by a circle. Phosphorylation can be used to more reliably perform the ligation step in subsequent stages.

[0501] Step S83 is cDNA synthesis, which is the same as described above. Figure 2A The same applies to step S12. The description of step S12 also applies to step S83.

[0502] When step S83 is performed, cDNA with a CCC sequence at the end of the generated strand is generated, such as... Figure 10 As shown in D.

[0503] In step S84, RNA digestion and nucleic acid splicing are performed. Therefore, as shown in Figure E, the mRNA is digested and single-stranded portions are formed. Optional base H It is also exposed at the 3' end of the single-stranded portion before the CCC sequence of 3. That is, CCCH The sequence is present at the 3' end of the single-stranded portion. On the other hand, because the nucleic acid splice portion is DNA, it is not digested during RNA digestion. Therefore, the double-stranded portion coupled with the digested mRNA and GGGH remain. Then, GGGH is coupled with the complementary CCCH... This process involves the binding of two or more cDNAs together, resulting in a nucleic acid assembly where two or more cDNAs are spliced ​​together.

[0504] Therefore, the formation of circular nucleic acids containing only one cDNA can be avoided. In other words, more circular nucleic acids in which two or more cDNAs are spliced ​​together can be generated.

[0505] In step S85, circular nucleic acid formation and ligation occur. Therefore, as... Figure 8 As shown in F, circular nucleic acids are generated.

[0506] Here, because the 5' end of the nucleic acid splice portion is phosphorylated as described above, the ligation is more reliable. Therefore, circular nucleic acids can be formed more efficiently.

[0507] Furthermore, although primer addition is in reference Figure 2A The method described is used, but because the nucleic acid splicing portion has a priming sequence, primer addition is not necessary, as mentioned above.

[0508] Step S86 is nucleic acid amplification, which is the same as described above. Figure 2A The same applies to step S16. The description of step S16 also applies to step S66.

[0509] When step S86 is performed, the nucleic acid is amplified, such as Figure 10 As shown in G. The nucleic acids amplified in this way are used for single-cell analysis. For example, the nucleic acid sequence is sequenced, and the sequencing results are used to analyze each cell.

[0510] (9) Example 8 (Example of sequence result analysis)

[0511] In the analytical method according to embodiments of the present disclosure, nucleic acid amplification is performed using circular nucleic acids as templates. Here, in the circular nucleic acid, sequences corresponding to two or more target nucleic acids are spliced ​​together. For example, two or more cDNAs complementary to mRNA are spliced ​​together. Therefore, the product obtained in the nucleic acid amplification process has repeating units comprising sequences corresponding to two or more target nucleic acids.

[0512] For example, such as Figure 11 As shown in a, in the case where one of the formed circular nucleic acids has two cDNAs, nucleic acids in which repeating units consisting of two cDNAs spliced ​​together are obtained by RCA treatment of the circular nucleic acid.

[0513] Furthermore, in cases where one of the formed circular nucleic acids has three cDNAs, by performing RCA treatment on the circular nucleic acid, nucleic acids in which repeating units consisting of three cDNAs spliced ​​together are obtained.

[0514] As shown in Figure b, where the nucleic acid splicing portion also has a self-binding inhibition sequence, the product obtained in the nucleic acid amplification process has repeating units that include sequences corresponding to two or more target nucleic acids.

[0515] In the analytical method according to embodiments of the present disclosure, analysis based on repeat units can be performed. For example, analysis based on the type and / or quantity of target nucleic acid sequences contained in repeat units can be performed.

[0516] Nucleic acid amplification reduces the bias described above. Therefore, analytical results are obtained that appropriately account for the presence of target nucleic acids in small amounts within the cell.

[0517] (10) Example 9 (Example of generating circular nucleic acids and amplifying the generated circular nucleic acids)

[0518] like Figure 5 As shown, nucleic acid splicing portions with different base sequences are provided for the arrangement regions on the substrate. In addition to the different random sequences for each region, the double-stranded portion of the nucleic acid splicing portion has a common sequence for all regions (e.g., the initiation sequence described above). This common sequence can be used as an initiation sequence in nucleic acid amplification processes, as described above, or it can be used to bind to cell capture portions.

[0519] Nucleic acid splice parts are manufactured using synthetic techniques employing oligonucleotide pools as described above. Specifically, oligonucleotides with various base sequences are synthesized in bulk (en bloc), and subsequently subjected to PCR treatment, thereby obtaining a variety of nucleic acid splice parts with different base sequences at low cost and in large quantities. More specifically, IVT and RT (reverse transcription) are performed after PCR treatment. Furthermore, cell-capturing components such as antibodies or lipids bind to the ends of the nucleic acid splice parts.

[0520] Cell capture is performed using a substrate with the aforementioned nucleic acid splice portions arranged on it. After cell capture, the cells are released from the substrate using restriction enzymes, and each cell is sealed in a water-in-oil droplet. Cell lysis and mRNA capture via poly-T sequences occur within the droplets.

[0521] For cell lysis, use lysis reagents such as NP-40 Surfactant-Amps Detergent solution or IGEPEAL CA-630.

[0522] After capturing mRNA, a reverse transcription reaction is performed to synthesize cDNA from the mRNA. The reverse transcription reaction is carried out at 50°C for 50 minutes in the presence of dNTPs, 1 x Thermopol buffer (or 1 x RT buffer), SuperScript III, and RNasin plus (or RNase OUT).

[0523] Next, the GGG sequence included in the nucleic acid splicing portion binds complementary to the terminal CCC sequence provided during cDNA synthesis. This allows two or more cDNAs to splice together. After splicing, the RNA is degraded by RNase H and other enzymes. Then, ligation is performed at 37°C for 30 minutes or at 45°C for 45 minutes in the presence of 1 x Ampligase buffer, 50 μM dNTPs, 0.5 U / μL Ampligase, and 50 mM KCl. This yields circular nucleic acids. Incidentally, formamide can be added to the liquid used for the above ligation process at a concentration of approximately 20%.

[0524] RCA was performed using the obtained circular nucleic acid. RCA was performed at 30°C for 60 minutes in the presence of 1 U / μL Phi29 polymerase, 1 x Phi29 polymerase buffer, 0.25 mM dNTP, 0.2 μg / μL BSA, and 5% glycerol. This yielded an amplified product with repeating units complementary to the circular nucleic acid. The amplified product can be used for single-cell analysis. Specifically, analytical results were obtained that appropriately reflect the presence of small amounts of nucleic acid within the cells.

[0525] 2. Second Implementation Method (Circular Nucleic Acid Manufacturing Method)

[0526] This disclosure also provides a method for manufacturing circular nucleic acids. Circular nucleic acids are generated using the analytical method described in section 1 above. Therefore, the description of the method for generating circular nucleic acids also applies to the method for manufacturing circular nucleic acids according to embodiments of this disclosure.

[0527] In one embodiment, the manufacturing method includes a complementary nucleic acid generation step and a circular nucleic acid generation step. The complementary nucleic acid generation step generates complementary strands of one or more target nucleic acids in a state where the nucleic acid splicing portion is bound to one end of each of one or more target nucleic acids. The circular nucleic acid generation step splices two or more generated complementary strands together via the nucleic acid splicing portion to form a circular nucleic acid.

[0528] The manufacturing method according to embodiments of this disclosure can produce circular nucleic acids that can be used for cell analysis. The effects described in 1. above are achieved through the manufacturing method.

[0529] 3. Third Implementation Method (Nucleic Acid)

[0530] This disclosure also provides nucleic acids. Nucleic acids are nucleic acids corresponding to the nucleic acid splicing portions described in section 1 above. Therefore, the description of the nucleic acid splicing portions also applies to nucleic acids according to embodiments of this disclosure. Furthermore, this disclosure also provides complexes containing nucleic acids. The complex is complex 100 described in section 1 above, and its description also applies to these embodiments.

[0531] In one embodiment, the nucleic acid includes a target nucleic acid capture portion configured to capture the 3' terminal region of a target nucleic acid; a complementary strand capture portion configured to capture the 3' terminal region of a complementary strand generated by generating the complementary strand of the target nucleic acid; and a double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion to each other. Preferably, the target nucleic acid capture portion is single-stranded, and the complementary strand capture portion is single-stranded. Furthermore, the nucleic acid can be used to generate circular nucleic acids.

[0532] The nucleic acids according to embodiments of this disclosure can be used to generate the circular nucleic acids described above, and can also be further used for cell analysis. The effects described in 1 above are produced by the nucleic acids.

[0533] Incidentally, this disclosure can also adopt the following configurations.

[0534] <1>

[0535] An analytical method, comprising: In this process, the nucleic acid splicing portion is bound to one end of each of one or more target nucleic acids to generate a complementary strand of one or more target nucleic acids; This nucleic acid splicing portion splices two or more complementary strands together to form a circular nucleic acid; and The circular nucleic acid was used for analysis.

[0536] <2>

[0537] according to <1> The analytical methods, among which

[0538] The nucleic acid splicing portion includes a target nucleic acid capture portion configured to capture the 3' end region of the target nucleic acid.

[0539] <3>

[0540] according to <1> or <2> The analytical methods, among which, In the generation of complementary strands, the nucleic acid splicing portion is used as a primer to generate the complementary strand of the target nucleic acid.

[0541] <4>

[0542] according to <1> to <3> The analytical method for any one of them, where, In the generation of complementary strands, a double strand is formed for each target nucleic acid and its complementary strand.

[0543] <5>

[0544] according to <4> The analytical methods, among which, In the generation of circular nucleic acids, double strands are spliced ​​together via nucleic acid splicing.

[0545] <6>

[0546] according to <1> to <5> The analytical method for any one of them, among which

[0547] The nucleic acid splicing portion includes a complementary strand capture portion, which is configured to capture the 3' end region of the complementary strand.

[0548] <7>

[0549] according to <1> to <6> The analytical method for any one of them, where, In the formation of circular nucleic acids, the 5' end of one complementary strand and the 3' end of another complementary strand are spliced ​​together, and The splicing is performed with the complementary strand capture portion of the nucleic acid splice portion bound to one complementary strand and the 3' terminal region of the other complementary strand bound to each other.

[0550] <8>

[0551] according to <1> to <7> The analytical method for any one of them, where, In the generation of circular nucleic acids, a single-stranded circular nucleic acid in which complementary strands are spliced ​​together is obtained by forming a double-stranded circular nucleic acid and then removing the target nucleic acid from the double-stranded circular nucleic acid.

[0552] <9>

[0553] according to <1> to <8> The analytical method for any one of them, where, In this analysis, a nucleic acid amplification reaction using circular nucleic acids was performed.

[0554] <10>

[0555] according to <9> The analytical methods, among which

[0556] This nucleic acid amplification reaction includes rolling circle amplification or polymerase chain reaction.

[0557] <11>

[0558] according to <1> to <10> The analytical method for any one of them, among which

[0559] The nucleic acid assembly process includes: The target nucleic acid capture portion is configured to capture the 3' end region of the target nucleic acid. The complementary chain capture portion is configured to capture the 3' end region of the complementary chain generated during the complementary chain generation process, and The double-stranded portion connects the target nucleic acid capture portion and the complementary strand capture portion to each other.

[0560] <12>

[0561] according to <11> The analytical methods, among which

[0562] The target nucleic acid capture portion has a poly-T sequence, and

[0563] The complementary strand capture portion has a base sequence that is complementary to the base sequence provided to the 3' end during reverse transcription by reverse transcriptase.

[0564] <13>

[0565] according to <11> or <12> The analytical methods, among which

[0566] This double-stranded portion contains a restriction enzyme recognition sequence.

[0567] <14>

[0568] according to <11> to <13> The analytical method for any one of them, among which

[0569] The double-stranded portion has a non-natural base sequence.

[0570] <15>

[0571] according to <11> to <14> The analytical method for any one of them, among which

[0572] The double-stranded portion has a base sequence with error-correcting capabilities.

[0573] <16>

[0574] according to <11> to <15> The analytical method for any one of them, among which

[0575] The analytical methods include those used for single-cell analysis, and

[0576] Use nucleic acid splicing components that include different double-stranded parts for each cell.

[0577] <17>

[0578] according to <1> to <16> The analytical method for any one of them, among which

[0579] The analytical methods include destroying cells, and

[0580] Generate complementary nucleic acids from target nucleic acids contained in cells.

[0581] <18>

[0582] according to <16> or <17> The analytical methods, among which

[0583] Cell destruction occurs within a space allocated to each cell.

[0584] <19>

[0585] according to <1> to <18> The analytical method for any one of them, among which

[0586] The nucleic acid splicing part is fixed on the substrate.

[0587] <20>

[0588] A method for manufacturing circular nucleic acids, comprising: In a state where the nucleic acid splicing portion binds to one end of each of one or more target nucleic acids, a complementary strand of one or more target nucleic acids is generated; and This nucleic acid splicing part splices two or more complementary strands together to form a circular nucleic acid.

[0589] <21>

[0590] A nucleic acid, comprising: The target nucleic acid capture portion is configured to capture the 3' terminal region of the target nucleic acid; A complementary strand capture portion, configured to capture the 3' terminal region of the complementary strand generated by the complementary strand generation of the target nucleic acid; and The double-stranded portion connects the target nucleic acid capture portion and the complementary strand capture portion to each other.

[0591] <22>

[0592] according to <21> Nucleic acid, among which

[0593] The target nucleic acid capture portion comprises a single strand, and the complementary strand capture portion comprises a single strand.

[0594] <23>

[0595] according to <21> or <22> Nucleic acid, among which

[0596] This nucleic acid is used to generate circular nucleic acids.

[0597] Those skilled in the art will understand that various modifications, combinations, sub-combinations and alterations can be made according to design requirements and other factors, as long as they are within the scope of the appended claims or their equivalents.

[0598] Reference number list

[0599] 10: Nucleic acid assembly section

[0600] 11: Target Nucleic Acid Capture Section

[0601] 12: Complementary chain capture part

[0602] 13: Double-stranded portion

[0603] 14: Sequence appendix

[0604] 100: Complex.

Claims

1. An analysis method comprising: generating complementary strands of one or more target nucleic acids in a state in which a nucleic acid splicing moiety is bound to one end of each of the one or more target nucleic acids; splicing two or more generated complementary strands to each other via the nucleic acid splicing moiety and forming a circular nucleic acid; and performing analysis using the circular nucleic acid. 2.The analysis method according to claim 1, wherein the nucleic acid splicing moiety comprises a target nucleic acid capture moiety configured to capture a 3' terminal region of the target nucleic acid. 3.The analysis method according to claim 1, wherein, in the generation of the complementary strands, target nucleic acid complementary strand generation is performed using the nucleic acid splicing moiety as a primer. 4.The analysis method according to claim 1, wherein, in the generation of the complementary strands, double strands of each target nucleic acid and a complementary strand of each target nucleic acid are formed. 5.The analysis method according to claim 4, wherein, in the generation of the circular nucleic acid, the double strands are spliced via the nucleic acid splicing moiety. 6.The analysis method according to claim 1, wherein the nucleic acid splicing moiety comprises a complementary strand capture moiety configured to capture a 3' terminal region of the complementary strand. 7.The analysis method according to claim 1, wherein, in the generation of the circular nucleic acid, a 5' end of one complementary strand and a 3' end of another complementary strand are spliced to each other, and the splicing is performed in a state in which a complementary strand capture moiety of the nucleic acid splicing moiety bound to the one complementary strand and a 3' terminal region of the other complementary strand are bound to each other. 8.The analysis method according to claim 1, wherein, in the generation of the circular nucleic acid, a single-stranded circular nucleic acid in which the complementary strands are spliced to each other is obtained by forming a double-stranded circular nucleic acid and then removing the target nucleic acid from the double-stranded circular nucleic acid. 9.The analysis method according to claim 1, wherein, in the analysis, a nucleic acid amplification reaction using the circular nucleic acid is performed. 10.The analysis method according to claim 9, wherein the nucleic acid amplification reaction is rolling circle amplification or polymerase chain reaction. 11.The analysis method according to claim 1, wherein the nucleic acid splicing moiety comprises: a target nucleic acid capture moiety configured to capture a 3' terminal region of the target nucleic acid, a complementary strand capture moiety configured to capture a 3' terminal region of the complementary strand generated in the generation of the complementary strand, and a double strand portion connecting the target nucleic acid capture moiety and the complementary strand capture moiety to each other. 12.The analysis method according to claim 11, wherein the target nucleic acid capture moiety has a poly-T sequence, and the complementary strand capture moiety has a base sequence complementary to a base sequence provided to a 3' end when reverse transcription is performed by a reverse transcriptase. 13.The analysis method according to claim 11, wherein the double strand portion has a restriction enzyme recognition sequence. 14.The analysis method according to claim 11, wherein The double-stranded portion has a non-natural base sequence.

15. The analysis method according to claim 11, wherein The double-stranded portion has a base sequence with an error correction function.

16. The analysis method according to claim 11, wherein The analysis method is an analysis method for performing single-cell analysis, and A nucleic acid splicing portion including a different double-stranded portion for each cell is used.

17. The analysis method according to claim 1, wherein The analysis method includes disrupting cells, and Complementary nucleic acid generation is performed on the target nucleic acid contained in the cells.

18. The analysis method according to claim 17, wherein Disrupting the cells is performed within a space partitioned for each cell.

19. The analysis method according to claim 1, wherein The nucleic acid splicing portion is fixed to a substrate.

20. A circular nucleic acid manufacturing method, comprising: Generating a complementary strand of one or more target nucleic acids in a state in which a nucleic acid splicing portion binds to one end of each of the one or more target nucleic acids; and Splicing two or more generated complementary strands to each other via the nucleic acid splicing portion and forming a circular nucleic acid.

21. A nucleic acid, comprising: A target nucleic acid capture portion configured to capture a 3' end region of a target nucleic acid; A complementary strand capture portion configured to capture a 3' end region of a complementary strand generated by complementary strand generation of the target nucleic acid; and A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion to each other.

22. The nucleic acid according to claim 21, wherein The target nucleic acid capture portion is single-stranded, and the complementary strand capture portion is single-stranded.

23. The nucleic acid according to claim 21, wherein The nucleic acid is used to generate a circular nucleic acid.

Images