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

Abstract

This disclosure aims to shorten the sequence anointed on the target nucleic acid. In addition to shortening the sequence anointed on the target nucleic acid, this disclosure also aims to reduce the aforementioned bias. [Solution] This disclosure provides an analytical method comprising: a complementary nucleic acid generation step of generating complementary strands of one or more target nucleic acids, each with a nucleic acid linker attached to one end of each; a cyclic nucleic acid generation step of linking two or more generated complementary strands via the nucleic acid linkers to form a cyclic nucleic acid; and an analytical step of performing an analysis using the cyclic nucleic acid. This disclosure also provides nucleic acids used in the analytical method.

Description

Technical Field

[0001] This application claims priority to Japanese Patent Application JP2023 - 056365 filed on March 30, 2023, the entire content of which is incorporated herein by reference.

[0002] The present disclosure relates to an analysis method, a method for producing circular nucleic acids, and nucleic acids. More specifically, the present disclosure relates to an analysis method for cell analysis, a method for producing circular nucleic acids used for cell analysis, and nucleic acids used for cell analysis.

Background Art

[0003] Single - cell analysis is one of the very useful techniques for analyzing cells and intracellular components. In single - cell analysis, for example, nucleic acids contained in each cell, particularly mRNA, are analyzed. Several techniques have been proposed so far for performing single - cell analysis.

[0004] Non - Patent Document 1 below discloses a biological particle analysis method including a capture step of capturing biological particles via a biological particle capture portion on a surface to which a molecule including a biological particle capture portion, a barcode sequence, and a cleavable linker is fixed via the linker, a cleavage step of cleaving the linker to release the biological particles from the surface, and an isolation step of isolating the biological particles in a micro - space.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] In single-cell analysis, the nucleic acids present in each cell are sometimes analyzed. For this analysis, it is conceivable to add cell identifier sequences (e.g., cell barcode) to identify the origin of the nucleic acid, molecular identifier sequences (e.g., molecular barcode, Unique Molecular Identifier / UMI) to identify the original number of nucleic acid molecules, and nucleic acid amplification sequences (e.g., amplification sequence, PCR handle) to the target sequence. However, when sequencing the target sequence, these appended sequences also need to be read, and the total number of reads (length x depth) is taken up by reading sequences other than the target sequence. Furthermore, because PCR during cDNA amplification is exponential, target nucleic acids that are naturally present in large quantities in cells tend to increase, making them easier to detect. This can lead to bias in the analytical results. In other words, with exponential amplification of nucleic acids, nucleic acids that are present in small quantities in cells are less likely to be reflected in the analytical results.

[0007] Therefore, this disclosure aims to shorten the sequence anointed on the target nucleic acid. In addition to shortening the sequence anointed on the target nucleic acid, this disclosure also aims to reduce the aforementioned bias. [Means for solving the problem]

[0008] This disclosure is, A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, A cyclic nucleic acid generation step, which involves linking two or more complementary strands that have been generated via the nucleic acid linking portion to form a cyclic nucleic acid, and An analytical step in which an analysis is performed using the aforementioned cyclic nucleic acid, This provides an analytical method that includes [specific details]. The nucleic acid binding portion may have a target nucleic acid capture portion configured to capture the 3' terminal region of the target nucleic acid. In the complementary strand generation step, the nucleic acid linkage portion may be used as a primer to generate the complementary strand of the target nucleic acid. In the complementary strand generation step, a double strand may be formed between each target nucleic acid and its complementary strand. In the cyclic nucleic acid generation step, the two strands may be linked via the nucleic acid linking portion. The nucleic acid linker may have a complementary strand capture portion configured to capture the 3' terminal region of the complementary strand. In the cyclic nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand may be linked. The linkage may be performed with the complementary strand capture portion of the nucleic acid linker attached to one complementary strand bound to the 3' terminal region of the other complementary strand. In the aforementioned cyclic nucleic acid generation step, a double-stranded cyclic nucleic acid is formed, and the target nucleic acid is removed from the double-stranded cyclic nucleic acid to obtain a single-stranded cyclic nucleic acid in which the complementary strands are linked. In the analysis step, a nucleic acid amplification reaction using the cyclic nucleic acid may be performed. The nucleic acid amplification reaction may be RCA or PCR. The nucleic acid linking portion is A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary chain capturing unit configured to capture the 3' terminal region of the complementary chain generated in the complementary chain generation step, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, It may have. The target nucleic acid capture portion has a poly-T sequence, and The complementary chain capture region may have a nucleotide sequence that is complementary to the nucleotide sequence that is added to the 3' end during reverse transcription by reverse transcriptase. The double-stranded portion may have a restriction enzyme recognition sequence. The double-stranded portion may have a non-natural base sequence. The double-stranded portion may have a base sequence that has an error correction function. The aforementioned analytical method may be an analytical method for performing single-cell analysis. A nucleic acid linker having double-stranded portions that differ for each cell may be used. The analysis method may include a cell disruption step of disrupting cells, The complementary nucleic acid generation step may be performed on the target nucleic acid contained in the cell. The cell disruption step may be performed in a space partitioned for each cell. The nucleic acid linker may be immobilized on a substrate. Furthermore, the present disclosure provides a complementary nucleic acid generation step of generating a complementary strand of the one or more target nucleic acids in a state where a nucleic acid linker is bound to one end of each of the one or more target nucleic acids, and a circular nucleic acid generation step of linking two or more generated complementary strands via the nucleic acid linker to form a circular nucleic acid, and also provides a method for producing a circular nucleic acid including the above steps. Furthermore, the present disclosure provides a target nucleic acid capture unit configured to capture the 3'-terminal region of a target nucleic acid, a complementary strand capture unit configured to capture the 3'-terminal region of a complementary strand generated by generating a complementary strand of the target nucleic acid, a double-stranded portion connecting the target nucleic acid capture unit and the complementary strand capture unit, and also provides a nucleic acid having the above components. The target nucleic acid capture unit may be single-stranded, and the complementary strand capture unit may be single-stranded. The nucleic acid may be used to generate a circular nucleic acid.

Brief Description of the Drawings

[0009] [Figure 1A] It is a schematic diagram showing a configuration example of a nucleic acid linker. [Figure 1B] It is a diagram showing an example of a sequence group having an error correction function. [Figure 1C] It is a diagram showing a configuration example of a nucleic acid linker. [Figure 1D] It is a diagram showing a configuration example of a nucleic acid linker. [Figure 1E] It is a schematic diagram showing a modified example of the configuration of a nucleic acid linker. [Figure 1F] This is a schematic diagram showing a modified example of the structure of a nucleic acid linkage. [Figure 2A] This is a schematic diagram illustrating an example of a process for generating cyclic nucleic acids using nucleic acid ligators. [Figure 2B] This is a schematic diagram illustrating the structure of mRNA. [Figure 2C] This is a schematic diagram illustrating an example of sequencing. [Figure 2D] This is an illustrative diagram of the nucleic acid linkage and other components used in the first modified example of the cyclic nucleic acid production process. [Figure 2E] This is a schematic diagram illustrating the first modified example of the process for generating cyclic nucleic acids using nucleic acid ligators. [Figure 2F] This is an illustrative diagram of the nucleic acid linkage and other components used in a second modified example of the cyclic nucleic acid generation process. [Figure 2G] This is a schematic diagram illustrating a second modified example of the process for generating cyclic nucleic acids using nucleic acid linkers. [Figure 2H] This is an illustrative diagram of the nucleic acid linkage and other components used in a third modified example of the cyclic nucleic acid generation process. [Figure 2I] This is a schematic diagram illustrating a third modified example of the process for generating cyclic nucleic acids using nucleic acid ligators. [Figure 3] This is a schematic diagram illustrating the bias in exponential amplification. [Figure 4A] This figure shows an example of a restricted area. [Figure 4B] This is a schematic diagram illustrating an example of tagged short read generation. [Figure 5] This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis. [Figure 6A] This is a schematic diagram showing an example of the complex's structure. [Figure 6B] This is a schematic diagram showing an example of the complex's structure. [Figure 6C] This is a schematic diagram showing an example of the manufacturing process of the composite. [Figure 6D]This is a schematic diagram illustrating an example of the procedure performed in a modified single-cell analysis method. [Figure 6E] This is an illustrative diagram of the nucleic acid ligation site used in a modified version of the single-cell analysis method. [Figure 7] This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis. [Figure 8] This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis. [Figure 9] This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis. [Figure 10] This is a schematic diagram illustrating an example of the procedures performed in single-cell analysis. [Figure 11] A schematic diagram showing an example of nucleic acids produced by RCA processing. [Figure 12] This is a schematic diagram of an example of a well used to perform a particle isolation process. [Figure 13A] This is a schematic diagram showing an example of equipment used to perform a particle segregation process. [Figure 13B] This is a schematic diagram showing an example of equipment used to perform a particle segregation process. [Figure 14A] This is a schematic diagram illustrating an example of an apparatus for forming an emulsion. [Figure 14B] This is a schematic diagram illustrating an example of a tip for forming an emulsion. [Figure 14C] This is a diagram illustrating the antibody-nucleic acid molecule complex used when simultaneously performing single-cell analysis and analysis of secreted molecules on the cell surface. [Figure 14D] This is a schematic diagram illustrating an example of isolating cells for single-cell analysis and analysis of secreted molecules on the cell surface using a chip for forming emulsions. [Figure 14E] This is a schematic diagram illustrating examples of procedures performed in single-cell analysis and analysis of secreted molecules on the cell surface. [Figure 15] This is a schematic diagram illustrating an example of a tip for forming an emulsion. [Figure 16]This is a block diagram illustrating an example of an apparatus for forming an emulsion. [Figure 17] An example of a flowchart of the operations performed for emulsion formation is shown. [Figure 18A] This is a schematic diagram showing an enlarged view of the area near the connecting channel. [Figure 18B] This is a schematic diagram showing an enlarged view of the area near the connecting channel. [Figure 19] This is a schematic diagram illustrating an example of how to add a priming sequence. [Modes for carrying out the invention]

[0010] The following describes preferred forms for implementing this disclosure. The embodiments described below are representative of the disclosure, and the scope of this disclosure is not limited to these embodiments. The description of this disclosure will proceed in the following order. 1. First Embodiment (Analysis Method) 2. Second Embodiment (Method for Producing Cyclic Nucleic Acids) 3. Third Embodiment (Nucleic Acid)

[0011] 1. First Embodiment (Analysis Method)

[0012] (1) Basic concept

[0013] (assignment) Shortening the sequence of identifiers assigned to target sequences reduces the variety of identifiers, resulting in insufficient identifiers for analyzing a large number of cells and molecules. Identifiers composed of single-stranded DNA are made up of combinations of four bases: A / T / G / C, and their variations are simply 4 n (n = length of identifier) 6 When individual identifiers are needed, n=10, and in actual analysis, error correction mechanisms are often incorporated, making n even larger. Also, each time the length is reduced by one, the variation is simply reduced to 1 / 4. Therefore, methods that shorten the length of identifiers are undesirable.

[0014] While it might be possible to reduce the number of identifiers mentioned above, it's difficult to simply eliminate them because each identifier has a specific role. For example, to eliminate cell identifiers (cell barcodes), one could analyze pooled cells using next-generation sequencing (NGS). However, in this case, it is difficult to classify the analyzed molecules on a cell-by-cell basis. Alternatively, NGS could be performed on each individual cell, but this would be costly when analyzing a large number of cells. To eliminate molecular barcodes (UMIs), it is necessary to either prevent the molecule from being replicated or to ensure that the replicated molecules share a common sequence. Not all mRNAs have different sequences; multiple mRNAs with the same sequence are expressed. Therefore, if replication occurs after cDNA synthesis, it is impossible to distinguish between the original number and the number produced by replication unless a sequence such as a UMI is assigned. If the amplification sequence (PCR handle) is removed, it becomes difficult to amplify, for example, a cDNA-converted molecule. Amplification without an amplification sequence requires a known target sequence; however, molecules with unknown sequences cannot be detected. Furthermore, in amplification methods using PCR, which employs exponential amplification, molecules with higher frequencies are amplified more easily, while molecules with lower frequencies are amplified less easily. Therefore, high-frequency molecules are more easily detected.

[0015] (Summary of this disclosure) The analytical method according to this disclosure uses a cyclic nucleic acid in which the complementary strands of two or more target nucleic acids are linked via a nucleic acid linkage. In this analytical method, the nucleic acid ligator can function as a cell identifier and can also function as a molecular identifier. Furthermore, the nucleic acid ligator can function as an amplification sequence. Moreover, the length of the base sequence constituting the nucleic acid ligator can be shortened. That is, in the analytical method according to this disclosure, the length of the sequence added to the target nucleic acid can be shortened. Here, the target nucleic acid refers to the nucleic acid that is captured by the nucleic acid linkage site, which will be described later. Furthermore, the cyclic nucleic acid is formed by linking two or more target nucleic acids or sequences derived from such target nucleic acids (including sequences complementary to such sequences). For example, one or more target nucleic acids or sequences derived from such target nucleic acids that are present in small amounts in the cell may be linked to one or more target nucleic acids or sequences derived from such target nucleic acids that are present in large amounts in the cell. By linking a low-frequency target nucleic acid with a high-frequency target nucleic acid in this way, the possibility of detecting the low-frequency target nucleic acid can be increased. This reduces the bias described above.

[0016] In one embodiment, an analytical method according to the present disclosure includes a complementary nucleic acid generation step of generating complementary strands of one or more target nucleic acids, each with a nucleic acid linker attached to one end of each; and a cyclic nucleic acid generation step of linking the two or more generated complementary strands via the nucleic acid linkers to form a cyclic nucleic acid. The analytical method may further include an analytical step of performing an analysis using the cyclic nucleic acid. In an analytical method using the cyclic nucleic acid thus generated, the length of the sequence attached to the target nucleic acid can be shortened as described above. Furthermore, the bias can also be reduced. The analysis method described above will be explained below with reference to the diagrams.

[0017] (2) Example 1 (Bulk analysis)

[0018] (2-1) Nucleic acid linking part First, a nucleic acid ligator used in the analytical method according to this disclosure will be described. The portion of the nucleic acid ligator that is involved in the linking of two or more complementary strands is also referred to as a concatenator in this specification. An example of the configuration of the nucleic acid ligator is shown in Figure 1A. The nucleic acid ligator 10 shown in the figure includes a target nucleic acid capture portion 11 configured to capture the 3' terminal region of the target nucleic acid, a complementary strand capture portion 12 configured to capture the 3' terminal region of the complementary strand of the target nucleic acid, and a double-stranded portion 13 connecting the target nucleic acid capture portion 11 and the complementary strand capture portion. Furthermore, the nucleic acid ligator used in the analytical method according to this disclosure may have the configuration shown in Figure 1E or Figure 1F. The nucleic acid ligator 10 shown in these figures, similar to the configuration example in Figure 1A, comprises a target nucleic acid capture portion 11 configured to capture the 3' terminal region of the target nucleic acid, and a complementary strand capture portion 12 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, while not having a double-stranded portion 13. The nucleic acid ligator 10 shown in Figure 1F includes a sequence addition portion 14 in addition to the configuration in Figure 1E. In this technology, by using a nucleic acid linker configured in this way, it is possible to shorten the sequence introduced into the target nucleic acid or a circular nucleic acid having a sequence derived from the target nucleic acid, while also reducing the detection bias.

[0019] The target nucleic acid capture unit 11 is configured to capture, for example, the 3' terminal region of the target nucleic acid. For example, when the target nucleic acid is mRNA, since mRNA has a polyA tail at its 3' terminal region, the target nucleic acid capture unit 11 may be configured to capture the polyA tail, and in particular, it may be a base sequence configured to capture the polyA tail.

[0020] In order for the target nucleic acid capture portion 11 to capture the poly-A tail, the target nucleic acid capture portion may have, for example, a poly-T sequence. The length of the poly-T sequence may be, for example, 10 to 50 base pairs, preferably 15 to 30 base pairs. That is, the poly-T sequence may consist of, for example, 10 to 50 T sequences, preferably 15 to 30 T sequences. The target nucleic acid capture portion 11 may consist only of a poly-T sequence. Furthermore, the target nucleic acid capture portion 11 may be single-stranded DNA or RNA. This makes it easier for the target nucleic acid to bind, and in particular, easier for it to bind complementaryly. Furthermore, the length of the poly-T sequence may be longer or shorter, and may be modified, for example, by the length of the poly-A tail of the target nucleic acid. Furthermore, if the RNA does not have a poly-T sequence or the sequence of the target RNA is known, a random sequence (random primer, random hexamer, etc.) or a sequence that specifically binds to the target RNA may be used as the target nucleic acid capture site. The length of the random sequence may be, for example, 6 to 20 base pairs, preferably 6 to 10 base pairs. The length of the sequence that specifically binds to the target RNA may be 10 to 40 base pairs, preferably 15 to 35 base pairs.

[0021] Tables 1 and 2 below show examples of sequences complementary to the target RNA. Table 1 shows examples of nucleotide sequences that specifically bind to ERBB2 (ERBB2_Probe1 to ERBB2_Probe19, corresponding to sequence IDs 109 to 127). Table 2 shows examples of nucleotide sequences that specifically bind to XPO1 (XPO1_Probe1 to XPO1_Probe13, corresponding to sequence IDs 128 to 140). The target nucleic acid capture region may contain at least one nucleotide sequence that specifically binds to the target RNA. If there are multiple different target RNAs, the target nucleic acid capture region may contain at least one nucleotide sequence selected from the group of sequences that specifically bind to each target RNA. If there is only one target RNA, the target nucleic acid capture region may contain at least one nucleotide sequence selected from the group of sequences that specifically bind to that single target RNA. The design of sequences that specifically bind to target RNA can be carried out using methods similar to those for PCR primers and FISH (Fluorescence in situ hybridization) probes. Briefly speaking, a set of sequences is generated for all combinations of a set length, and the binding stability of each sequence in this set with the target sequence is calculated using the Nearest Neighbor method. From this set of sequences, a group of sequences that have a binding stability of, for example, -5 kcal / mol or less, preferably -28 kcal / mol or less, at 37°C is extracted. Furthermore, from this extracted group of sequences, a group of sequences with a GC content of 40% to 60% is further extracted. In addition, sequences that optionally satisfy one or more additional filters (conditions), such as an A content of less than 28%, exclusion of sequences of 4 or more bases, and a C content of 22% to 28%, are extracted. In this way, a group of sequences that specifically bind to target RNA is selected.

[0022] [Table 1]

[0023] [Table 2]

[0024] The complementary strand capture unit 12 is configured to capture, for example, the terminal region of a target nucleic acid or a nucleic acid having a sequence derived from a target nucleic acid (e.g., the 3' terminal region of the complementary strand of these nucleic acids). In particular, the complementary strand capture unit of one nucleic acid ligator can capture the 3' terminal region of a complementary strand of a target nucleic acid other than the target nucleic acid captured by the target nucleic acid capture unit of that one nucleic acid ligator (a complementary strand produced by the cDNA synthesis of that other target nucleic acid) in an analytical method according to this disclosure. This makes it possible to link two or more complementary strands of target nucleic acids or sequences derived from such target nucleic acids via the nucleic acid ligator. Here, "linking via nucleic acid ligatures" is not limited to cases where linking occurs via nucleic acid ligatures, but also includes cases where linking occurs via sequences derived from nucleic acid ligatures.

[0025] In the analytical method according to this disclosure, the complementary nucleic acid generation step is a step of generating two or more complementary strands to be linked in the subsequent cyclic nucleic acid generation step. More specifically, complementary strands of one or more target nucleic acids are generated with a nucleic acid linker attached to one end of each of the target nucleic acids. Furthermore, as will be described later, the complementary nucleic acid generation step may involve generating nucleic acids having sequences derived from the target nucleic acid using the complementary strand of the generated target nucleic acid as a template, or it may involve adding sequences that can be captured by the complementary strand capture portion of the nucleic acid linker. For example, when the target nucleic acid is mRNA, the complementary strand produced by reverse transcription of mRNA will have a CCC sequence (C: cytosine) generated at its 3' end by the reverse transcriptase that performs the reverse transcription. Therefore, the complementary strand capture portion 12 may be configured to capture the CCC sequence, and in particular, it may be a base sequence configured to capture the CCC sequence. Thus, the complementary chain capture region may have a nucleotide sequence that is complementary to the nucleotide sequence added to the 3' end during reverse transcription by reverse transcriptase. On the other hand, when using a nucleic acid ligator that does not have a double-stranded portion, the complementary strand capture portion of the nucleic acid ligator may be set to an arbitrary sequence, and a nucleic acid having a sequence derived from the target nucleic acid may be generated using a primer that has a sequence complementary to the sequence of the complementary strand capture portion on its 5' side. In this case, the complementary strand capture portion of the nucleic acid ligator located at the 5' end of the complementary strand (cDNA) produced by reverse transcription of mRNA can capture a sequence complementary to the sequence of the aforementioned complementary strand capture portion derived from the primer located at the 5' end of a nucleic acid having a sequence derived from another target nucleic acid, and two or more complementary strands are linked via the nucleic acid ligator. When such a nucleic acid ligator is used, the sequence of the complementary strand capture region is not limited to a GGG sequence, but can be a combination of the four bases A, T, C, and G to form a sequence of any length. When the sequence of the complementary strand capture region is made longer, when two or more generated complementary strands are linked via the nucleic acid ligator to form a circular nucleic acid, it is possible to form a circular nucleic acid while reducing the loss of generated complementary strands. As a result, it is expected that the probability of detecting complementary strands derived from sequences of mRNA that are expressed at low frequencies will increase, and analytical accuracy will improve.

[0026] The complementary strand capture unit 12 that captures the CCC sequence includes, for example, a GGG sequence. The GGG sequence may be DNA or RNA. That is, the GGG sequence may be GGG or rGrGrG (r: ribonucleotide, G: guanine). Furthermore, the complementary strand capture portion 12 may be single-stranded DNA or RNA. This facilitates binding to the complementary strand.

[0027] Furthermore, the complementary strand capture region may include a self-binding repression sequence Hn or Nn in addition to the GGG sequence. Here, H is a base other than G, i.e., A, T, or C. N is A, T, G, or C. n is the number of H or N, and may be an integer of 1 or more, for example. n may be an integer of any of 1 to 8, for example. Given that there are said to be about 20,000 types of mRNA, such a numerical range can cover the mRNA in question. In some embodiments, for example, n may be 1, 2, 3, 4, or 5, and may even be 1 or 2. When n is 2 or more, each H or N constituting the self-binding repression sequence may be independently and randomly selected from each other. The complementary strand capture region may have a base sequence such as GGGH, GGGN, GGGHN, GGGNH, GGGHH, or GGGNN. The complementary strand capture region may be DNA or RNA.

[0028] The double-stranded portion 13 is the portion connecting the target nucleic acid capture portion 11 and the complementary strand capture portion 12, and may 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 the RNA digestion process described later and facilitates the formation of circular nucleic acids. It is also easily usable as a primer in nucleic acid amplification.

[0029] A target nucleic acid capture portion 11 is attached to the 3' end of one of the double-stranded portions 13, and a complementary strand capture portion 12 is attached to the 3' end of the other strand. This structure allows for the formation of a cyclic nucleic acid by linking the generated complementary strands, as described later.

[0030] The double-stranded portion 13 may include a sequence composed of a random combination of the four bases A, T, C, and G, but this random sequence is preferably composed of a group of sequences having an error correction function. Examples of sequences having an error correction function include Sequence-Levenshtein code and filled / truncated right-end edit (FREE) barcodes. Methods for generating these sequences and error correction mechanisms using these sequences are described in Buschmann and Bystrykh BMC Bioinformatics 2013, 14:272 and Proc Natl Acad Sci US A. 2018 Jul 3; 115(27): E6217-E6226. Those skilled in the art can refer to these documents to generate and utilize sequences having an error correction function as appropriate. In addition to the two types of sequence groups described above, other sequences with error-correcting functions known in the art, such as Levenshtein codes, Hamming codes, or Reed-Solomon codes, are also known and may be used in this disclosure. Sequences with error-correcting functions may also be called indel-correcting DNA barcodes. Furthermore, those skilled in the art are aware of software that can be used to generate these sequences with error-correcting functions and to perform error correction using such sequences, and those skilled in the art can use such software to prepare and utilize sequences with error-correcting functions. As a result, sequences have been extracted that can identify the original sequence even if a few base pairs of read errors (insertion, deletion, or substitution) occur during sequencing.

[0031] The following describes the mechanism and generation method of sequences with error correction capabilities.

[0032] The following describes the mechanism of a sequence with error correction functionality. However, the following description is a representative example, and the sequences with error correction functionality applicable in this disclosure are not limited to sequences based on these mechanisms. In Hamming distance, each substitution in a barcode sequence increases the distance by one. For example, "TCT," which replaces "ACT," has a distance of 1 (substitution from A to T), "TCT" and "TAT" have a distance of 1 (substitution from C to A), "TAT" and "TAC" have a distance of 1 (substitution from T to C), and "ACT" and "TAC" have a distance of 3 (the sum of the two). For example, if you prepare barcodes such as "ACT," "GTG," "TAC," and "CGA," they will all have a distance of 3 from each other. If the result read by the sequence is "ACG," it is considered an error because it is different from the prepared barcode. Distance is used to infer which sequence was incorrect. Calculating the distance between the read "ACG" and the four prepared sequences, and assuming a uniform frequency of errors, the most likely barcode is presumed to be "ACT," which has the shortest distance. Thus, a code with a Hamming distance can detect and correct substitutions made by another code. To correct k errors, a length of at least 2*k+1 is required. Levenshtein distance can handle not only substitutions but also insertions and deletions. It considers the distance to increase by 1 for each substitution, insertion, or deletion of a base. For example, the distance between "GCG" and "GC" is 1 (deletion of G), the distance between "GC" and "GA" is 1 (substitution of C to A), and the distance between "GA" and "AGA" is 1 (insertion of A), resulting in a total distance of 3 between "GCG" and "AGA". For instance, prepare a set of barcode sequences "GCG", "TTT", "AGA", and "CAC" that have a distance of 3 from each other. If the result read by the sequencer is "GC", it is considered an error because it differs from the prepared barcode. The distance between the obtained "GC" and the prepared barcode is calculated, and the sequence with the shortest distance is inferred to be the most likely barcode. In the example above, "GCG", with a distance of 1, is inferred to be the barcode. This method is only applicable when the barcode length is known beforehand. In the Sequence-Levenshtein distance method, the distance between barcodes A and B of arbitrary length is defined as the number of substitutions, insertions, and deletions. Furthermore, arbitrary lengths can be handled by either trimming A until it is the same length as B, or adding the same bases to A until it is the same length as B, once. FREE (filled / truncated right-end edit barcodes) corrects substitutions, insertions, and deletions based on the Needleman-Wunsch algorithm. Two sequences to be aligned are arranged vertically and horizontally like a chessboard. The two sequences are compared, and for example, if the bases at each position match, a score of +2 is assigned; if they do not match, a score of -1 is assigned; and if a base is missing, a score of -2 is assigned. After calculating each cell, the alignment is created by moving backward with the arrows in descending order of score. For example, when comparing "ATTGC" and "ATGC", "ATTGC" is aligned to "AT-GC" (T deletion or insertion). As illustrated above, a sequence having an error correction function in accordance with this disclosure may be a nucleotide sequence configured to detect an error if it occurs in the nucleotide sequence identified by sequencing. The sequence having an error correction function may be configured to detect differences between, for example, a sequence identified by sequencing and a sequence composed of pre-prepared barcodes. The pre-prepared barcode may be a sequence identified based on distances related to mutations (e.g., substitutions, insertions, or deletions), and such distances may be distances based on predetermined rules, such as the Hamming distance, Levenshtein distance, or Sequence-Levenshtein distance, as described above. The pre-prepared barcode may also be configured to identify mutations by alignment, such as FREE as described above. Particularly preferably, the sequence having an error correction function may be configured to detect an error if it occurs in the sequence and to predict the most likely barcode. As described above, the inference may be performed based on predetermined rules depending on the type of sequence having an error correction function. For example, as described above, the inference may be based on the distance between the sequence identified by the sequence and a pre-prepared barcode, or it may be based on the alignment between the sequence identified by the sequence and a pre-prepared barcode.

[0033] A method for generating sequences with error correction functionality is described below. However, this description is a representative example, and the sequences with error correction functionality applicable in this disclosure are not limited to those generated by these methods. To generate sequences with error correction capabilities, for example, Conway's lexicographic code algorithm is employed. This algorithm generates all sequences for a given length and then selects sequences that have a distance d from each other. First, one sequence is unconditionally selected from the sequence set, and then the distance between the selected sequence and the remaining sequences is calculated. Sequences with a distance less than d are excluded, and sequences with a distance of d or greater are selected. The same process is performed on the remaining sequences until sequences with a distance of d or greater from the selected sequence are selected. This process is carried out for all sequences until there are no more suitable sequences. Thus, an array with error correction functionality may be an array generated by executing a predetermined algorithm. This predetermined algorithm may be an algorithm that selects an array that satisfies a predetermined criterion regarding distance. An example of this distance is as described above, and the predetermined criterion may be selected depending on the type of array with error correction functionality used. Furthermore, sequences with error correction functionality may be generated, for example, by a dictionary-style code generation method. In this generation method, sequences of a set length are generated in alphabetical order, and if the newly generated sequence does not overlap with the decoded sequence of the candidate sequence, it is registered as a valid sequence. For example, when considering the case of correcting m errors in "CTCA", the decodesphere DecodeSphere("CTCA") stores barcodes (e.g., CTGA, CCA, CTGC, ...) that occur when m or fewer insertions / deletions / substitutions occur. When another barcode candidate with a length of 4 bases, for example, barcode candidate "AACC", is encountered, if DecodeSphere("AACC") does not overlap with DecodeSphere("CTCA"), AACC is registered as a valid barcode. There are constraints on the number of correctable errors m and the barcode sequence length n, requiring a length of at least 2*m+1. Thus, the array having an error correction function may be an array generated by a dictionary-style code generation method. In this generation method, as described above, barcodes are registered based on whether or not they overlap with barcodes stored in the decode sphere, and the array having the error correction function may be generated from the registered barcodes.

[0034] Each sequence included in the error-correcting sequence group may be, for example, a sequence that is not complementary to existing RNA. For example, each sequence included in the error-correcting sequence group may have a guanine and cytosine content of, for example, 40% to 60%, from the viewpoint of sequencing efficiency. Also, each sequence included in the error-correcting sequence group may not have three or more consecutive homopolymer sequences, from the viewpoint mentioned above. Furthermore, each sequence included in the error-correcting sequence group may not have two or more self-complementary sequences, from the viewpoint mentioned above. Sequences that satisfy at least these conditions may be used as part of the error-correcting sequence group. The conditions that each sequence included in the error-correcting sequence group must satisfy may be appropriately changed depending on the type of sequence. Within this specification, a sequence having an error correction function is represented by "(N)i" (where N is any base (A, T, G, or C) and i is the number of bases) and may have an error correction function. i may be, for example, 3 or more, 4 or more, or 5 or more, and may also be 10 or more, or even 15 or more. Furthermore, i may be, for example, 200 or less, 150 or less, or 100 or less, or even 50 or less. The length of the sequence having an error correction function may be appropriately changed according to the number of sequences to be prepared (number of barcodes). For example, in order to extract more than 1 million sequences with a single-base error correction function, the double-stranded portion (especially the sequence having an error correction function) needs to be at least 16 bases long. An example of a sequence with an error correction function is shown in Figure 1B. SEQ IDs 1-50 shown in the figure are examples of 16-mer sequences with error correction functionality, and SEQ IDs 51-100 are examples of 17-mer sequences with error correction functionality. It should be understood that while there are over 1 million sequences with error correction functionality (for example, in the case of 16-mer sequences), those listed in the figure represent only a very small fraction. The sequences listed in the figure have a 3' end on the left and a 5' end on the right. Furthermore, it is possible to increase the variety by combining two or more of these extracted error-correction sequences. Sequences with error correction functions can be appropriately generated by those skilled in the art, and include, for example, those generated by the above generation method, but are not limited to these. That is, the double-stranded portion may have two or more sequences with error correction functions.

[0035] As will be explained in the example of the analysis method described later, the aforementioned random sequences may be different for each region in which nucleic acid ligatures are located. That is, in the analysis method, multiple regions in which nucleic acid ligatures are located are used, and the random sequences of multiple nucleic acid ligatures located in one region all have the same base sequence, but these base sequences may be different between regions. As a result, the aforementioned random sequences can be used as identifiers, for example, as cell identifiers, and furthermore, as location identifiers.

[0036] Preferably, the 5' end of the strand of the double-stranded portion 13 that is connected to the target nucleic acid capture portion 11 (particularly the strand containing poly(T)) is phosphorylated. This allows for more reliable execution of the ligation process described later. Preferably, the complementary strand capture unit is attached to the 3' end of one of the two base sequence strands constituting the double-stranded portion 13 that is not connected to the target nucleic acid capture unit 11 (particularly the strand without poly-T).

[0037] The double-stranded portion may preferably have a non-natural nucleotide sequence, and in particular, the random sequence may be composed of a non-natural nucleotide sequence. A non-natural nucleotide sequence means a nucleotide sequence that does not exist in nature. Those skilled in the art can appropriately design such a nucleotide sequence. By utilizing such a non-natural nucleotide sequence, for example, unnecessary double-strand formation and sequence detection errors can be suppressed.

[0038] The double-stranded portion 13 may further have a priming sequence. This priming sequence may be, for example, a base sequence that functions as a primer in the nucleic acid amplification process described later, and the sequence can be appropriately selected by a person skilled in the art depending on, for example, the type of nucleic acid amplification process and / or the type of enzyme used in the nucleic acid amplification process. As described above, in the analytical method according to this disclosure, multiple regions in which nucleic acid ligatures are located are used, and the priming sequences of the multiple nucleic acid ligatures located in one region all have the same base sequence, and furthermore, the priming sequences may be the same even between regions. As a result, in a single amplification process, amplification reactions occur simultaneously from multiple circular nucleic acids having nucleic acid ligatures with the same priming sequence.

[0039] If the nucleic acid ligator includes a sequence addition portion 14, as shown in the example in Figure 1F, the sequence addition portion 14 is positioned between the target nucleic acid capture portion 11 and the complementary strand capture portion 12. The sequence addition portion 14 may be formed from DNA, from RNA, or from a hybrid of DNA and RNA. The sequence appending portion 14 may include a sequence composed of a random combination of the four bases A, T, C, and G. For example, this random sequence may be attached as a cell barcode to identify the origin of the nucleic acid. Alternatively, the sequence appending portion 14 may be attached to a group of sequences that have the function of being attached to the double-stranded portion as described above.

[0040] The nucleic acid ligation region 10 (particularly the double-stranded region 13 and the sequence addition region 14) may contain UMI, but it may not contain UMI. By omitting UMI, the base sequence of the nucleic acid ligation region can be shortened.

[0041] An example of the configuration of the nucleic acid linking portion 10 is shown in Figure 1C. The nucleic acid ligation regions shown in a1 and a2 of the figure (corresponding to sequence IDs NO. 101 to 104) have a poly-T sequence (left side of the figure) as the target nucleic acid capture region, a GGG sequence (right side of the figure) as the complementary strand capture region, and a random sequence shown in the figure as the double-stranded region. The nucleic acid ligation regions shown in b1 and b2 of the figure (corresponding to sequence IDs NO. 105-108) have a poly-T sequence (left side of the figure) as the target nucleic acid capture region, a GGG sequence (right side of the figure) as the complementary strand capture region, and, in addition to the random sequence, a priming sequence (underlined portion) as the double-stranded portion. This priming sequence is the priming site in the nucleic acid amplification process for the circular nucleic acid described later. For example, if the nucleic acid amplification process is RCA, a priming sequence for RCA may be used, and if the nucleic acid amplification process is PCR, a priming sequence for PCR may be used. The base sequences of these priming sequences can be appropriately selected by those skilled in the art, as described above. In other words, in one embodiment, as shown in Figure 1D, the nucleic acid binding portion 10 may be configured as a nucleic acid having a first strand to which a target nucleic acid capture portion (capture sequence) and a random sequence (random) are linked, and a second strand to which a GGG sequence (or the GGG(Hn and / or Nn) sequence) and a random sequence (random') complementary to the random sequence are linked, and the first strand and the second strand are complementarily bound by the two complementary random sequences. The target nucleic acid capture portion may, for example, include a poly-T sequence as described above, or a random sequence (random primer, random hexamer, etc.), or a sequence that specifically binds to the target RNA (target primer). The two complementaryly linked random sequences correspond to the double-stranded portion described above. The GGG sequence corresponds to the complementary strand-capturing portion described above. With respect to the first strand, the random sequence side has a 5' end, and the poly-T sequence side has a 3' end. With respect to the second strand, the random sequence side has a 5' end, and the GGG sequence side has a 3' end.

[0042] (2-2) Process of generating circular nucleic acids The following describes the process of generating a circular nucleic acid using the nucleic acid ligator, with reference to Figures 2A and 2B, and further describes nucleic acid amplification using the circular nucleic acid. More specifically, in the circular nucleic acid generation process shown in the figures, two types of cDNA complementary to each mRNA are generated by reverse transcription of two types of mRNA, and these two types of cDNA are then ligated together to generate a circular nucleic acid. The steps performed in the circular nucleic acid generation process are as follows. In this example, two different mRNAs are ligated together, but in this disclosure, two or more identical mRNAs may be ligated together.

[0043] As shown in the upper part of Figure 2A, we assume the presence of two types of mRNA (mRNA1 and mRNA2). mRNA1 and mRNA2 are the target nucleic acids.

[0044] As shown in Figure 2B, mRNA1 contains the translational region CDS1 and also contains the polyA1 sequence at its 3' end. Similarly, mRNA2 contains the translational region CDS2 and also contains the polyA2 sequence at its 3' end.

[0045] In step S11 shown in Figure 2A, the 3' end of mRNA1 is captured by the nucleic acid ligator 10. The 3' end of mRNA1 has a poly-A tail, and the poly-T sequence 11 of the nucleic acid ligator captures the poly-A tail, particularly by complementary binding, thereby capturing the 3' end of mRNA by the nucleic acid ligator (particularly the target nucleic acid capture region). The 3' end of mRNA2, like mRNA1, is also captured by the nucleic acid ligation site (particularly the target nucleic acid capture site).

[0046] In step S12 of the figure, the cDNA of mRNA1 is synthesized. The synthesis of this cDNA may be carried out, for example, by reverse transcriptase. This cDNA synthesis is performed using the nucleic acid ligation site as a primer. As a result, a hybrid H1 of mRNA1 and cDNA1 is formed, as shown in the figure. Similarly, with mRNA2, cDNA2 is synthesized by reverse transcriptase, forming a hybrid H2 molecule of mRNA2 and cDNA2. In step S2, if cDNA synthesis is performed by reverse transcriptase, a CCC sequence is formed at the 3' end of the synthesized cDNA, as shown in the figure. As described later, this CCC sequence is used in the formation of the next circular nucleic acid. Thus, in a method according to this disclosure, a complementary nucleic acid generation step may be performed in which a nucleic acid linker is attached to one end of each of the one or more target nucleic acids, and a complementary strand of the one or more target nucleic acids is generated. In the complementary strand generation step, the nucleic acid linkage portion may be used as a primer to generate the complementary strand of the target nucleic acid. In the complementary strand generation step, a double strand may be formed between each target nucleic acid and its complementary strand, and as described above, for example, a hybrid of mRNA and cDNA may be formed.

[0047] In step S13 of the same figure, the hybrid H1 and hybrid H2 generated in step S2 are linked together. This linkage is achieved through complementary linkage between the rGrGrG sequence of hybrid H1 and the CCC sequence of hybrid H2.

[0048] In step S14 of the same figure, single-stranded cyclic nucleic acids are formed using hybrid H1 and hybrid H2. Specifically, the following steps are performed to form these single-stranded cyclic nucleic acids.

[0049] First, hybrid H1 has a CCC sequence at the end opposite to the end where the rGrGrG sequence used for ligation in step S13 is located, and this CCC sequence is a single-stranded portion. Hybrid H2 has an rGrGrG sequence at the end opposite to the end where the CCC sequence used for ligation in step S13 is located, and this is also a single-stranded portion. Therefore, a double-stranded circular nucleic acid is formed by the complementary binding of the CCC sequence of hybrid H1 and the rGrGrG sequence of hybrid H2. Thus, in the cyclic nucleic acid generation step included in the method according to this disclosure, the double strands may be linked via the nucleic acid linking portion. Furthermore, in the cyclic nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand may be linked, and this linkage may be performed with the complementary strand capture portion of the nucleic acid linking portion attached to the one complementary strand linked to the other complementary strand linked to the 3' terminal region of the other complementary strand linked to the one complementary strand linked to the other complementary strand linked to the one complementary strand.

[0050] In the aforementioned double-stranded circular nucleic acid, a nick exists between the 5' end of the strand connected to cDNA1 within the nucleic acid junction that was bound to mRNA1, and the 3' end of cDNA2, which was generated by the reverse transcription of mRNA2. Similarly, a nick exists between the 5' end of the strand connected to cDNA2 among the double strands of the nucleic acid linkage that were bound to mRNA2, and the 3' end of cDNA1 generated by the reverse transcription of mRNA1. Therefore, in step S14, ligation is performed to eliminate the nicks in the double-stranded circular nucleic acid while it is still formed. As a result, cDNA1 and cDNA2 become bound together, and the circular nucleic acid state is maintained even when mRNA degradation, as described later, occurs.

[0051] In step S14, mRNA degradation is performed after ligation. This degradation may be carried out using, for example, RNase, particularly RNase H. As a result, single-stranded cyclic nucleic acid RN is formed, as shown in the figure. Thus, in the circular nucleic acid generation step, a double-stranded circular nucleic acid is formed, and then the target nucleic acid (e.g., mRNA) is removed from the double-stranded circular nucleic acid to obtain a single-stranded circular nucleic acid with a complementary strand linked.

[0052] In step S15, a primer is attached to the single-stranded cyclic nucleic acid RN. In the figure, a primer is used that is configured to bind to the portion of the nucleic acid ligation site that constituted the double-stranded portion and to the CCC sequence. In other words, the primer is configured to cover the ligation point.

[0053] In step S16, Rolling Circle Amplification (RCA) is performed using DNA polymerase, starting from the primer. This DNA polymerase synthesizes, for example, a single-stranded linear nucleic acid complementary to the single-stranded circular nucleic acid.

[0054] The single-stranded linear nucleic acid formed in this manner has its base sequence deciphered. This sequencing may be performed using techniques known in the art. For example, Long Read Sequencing (LST) can be applied as such a sequence analysis technique. For instance, the aforementioned single-stranded cDNA can be sequenced using nanopore sequencing (Oxford Nanopore Technologies). Alternatively, the base sequence may be sequenced using HiFi sequencing (Pacific Biosciences). In this case, as shown in Figure 2C, a double-stranded structure is generated after a step is performed in which a poly(A) tail or poly(C) tail is attached to the single-stranded cDNA extended by RCA. Sequence is then performed on the circular nucleic acid obtained by further attaching an SMRTbell® adapter to the nucleic acid of this double-stranded structure. Here, since the sequence of the nucleic acid junction is known, after the sequencing, the original mRNA sequence can be identified and the number of original mRNAs can be counted by separating the sequenced sequence at the known nucleic acid junction. By decoding the repeatedly occurring mRNA sequence, even in the case of multi-RCA, the molecules produced by replication can be considered the same and counted. That is, it is possible to distinguish between the number of original mRNAs and the number produced by replication without using UMI. Furthermore, when rGrGrGNN or GGGNN is used, identification is also possible by the location of random NN occurrences. Furthermore, by performing long-read sequencing on repeatedly occurring mRNA complementary sequences, errors that occur during sequencing can be corrected. In other words, as described above, the ligation of two or more mRNAs makes it possible to correct such errors, which can lead to an improvement in decoding accuracy.

[0055] When rGrGrG is used as the complementary strand capture portion of the nucleic acid linkage, this rGrGrG is degraded by RNaseH treatment. Similarly, if the complementary strand of the double-stranded portion of the nucleic acid linkage (i.e., the strand connected to the complementary strand capture portion) is synthesized from ribonucleotides, this complementary strand is also degraded by RNaseH treatment. Therefore, a primer is added in step S15 (i.e., before RCA in step S16). This primer may be, for example, a primer having the same sequence as the complementary strand of the double-stranded portion. Furthermore, if GGG is used as the complementary strand capture portion and the complementary strand of the double-stranded portion in the nucleic acid linkage portion is synthesized from a nucleotide, these will not be degraded by RNaseH treatment. In this case, the GGG and the complementary strand may be used as primers.

[0056] In the example shown in Figure 2A above, a circular nucleic acid is formed using two mRNAs, but a circular nucleic acid may also be formed using three or more mRNAs. That is, a circular nucleic acid containing a complementary cDNA for each of the three or more mRNAs may be formed in the same way. Then, as described above, nucleic acid amplification treatment such as RCA may be performed on the circular nucleic acid.

[0057] As shown in Figure 2A above, by generating a circular nucleic acid, two or more cDNAs are linked within the circular nucleic acid. For example, a cDNA corresponding to low-expression mRNA can be linked to a cDNA corresponding to high-expression mRNA. This makes it possible to improve the detection efficiency of low-expression mRNA. Furthermore, in the PCR method, nucleic acids are amplified exponentially, so mRNAs that are present in large quantities before amplification are amplified far more than mRNAs that are present in small quantities before amplification. As a result, mRNAs that are present in small quantities before amplification are difficult to detect. In other words, the PCR method introduces amplification bias (also known as amplification bias). On the other hand, since nucleic acid amplification by the RCA method is not exponential amplification, this bias is reduced. In other words, in one embodiment, nucleic acid amplification using the cyclic nucleic acid may be performed by the RCA method.

[0058] In some embodiments of the analytical methods according to this disclosure, the PCR method may be used. This is because, even when the PCR method is used, the bias can be reduced by performing PCR on the cyclic nucleic acid. This will be explained below. Typically, after cDNA synthesis, the nucleic acids amplified by PCR are tagged. In PCR, sequences are generated exponentially, and it is common practice to assign a Unique Molecular Identifier (UMI) during reverse transcription to identify the origin of the generated sequences. Here, the number of UMI types must be greater than or equal to the number of molecules to be distinguished (for example, the number of molecules originally present in each cell). Therefore, UMIs usually consist of 6 or more base pairs (nucleotides) (for example, 6 to 10, or more than 10). The sequences of these UMIs are decoded during sequencing, but these sequences are not included in the target nucleic acid and do not inherently need to be sequenced, so it is preferable to exclude them. Furthermore, in the PCR method, nucleic acids are amplified exponentially, which tends to amplify molecules with high expression levels, resulting in a bias where these molecules are more easily detected. For example, as shown in Figure 3A, when the PCR method is performed on a sample in which there is one target nucleic acid M1 and m (7 in the figure) target nucleic acid M2 molecules, the reverse transcript of target nucleic acid M1 will be amplified by 2 n While it is amplified to, the target nucleic acid M2 is (2 n It is amplified to ) × m. When PCR is performed on nucleic acids that have been ligated with two or more target nucleic acids in accordance with this disclosure, for example, a molecule with low expression levels will be ligated with a molecule with high expression levels, and PCR will be performed on the ligated nucleic acid. This is expected to improve the detection efficiency of low-expression molecules. For example, consider a case where, by performing the ligation process in accordance with this disclosure on the sample described in Figure 3A, a ligated product is produced in which, for example, one reverse transcript of target nucleic acid M1 and three reverse transcripts of target nucleic acid M2 are ligated, and a ligated product is produced in which four reverse transcripts of target nucleic acid M2 are ligated, as shown in Figure 3B. In this case, each ligated product is 2 n As it is amplified in this way, the difference between the number of amplified products of target nucleic acid M1 and the number of amplified products of target nucleic acid M2 becomes smaller compared to case A in Figure 3. Thus, even when the PCR method is used instead of the RCA method in the analytical method described in this disclosure, amplification bias is reduced. As described above, the method according to this disclosure may include an analytical step of performing an analysis using the cyclic nucleic acid. In the analytical step, a nucleic acid amplification reaction using the cyclic nucleic acid may be performed, and the nucleic acid amplification reaction may be, for example, RCA or PCR.

[0059] (2-3) First modified example of the process of generating cyclic nucleic acids The cyclic nucleic acids that can be used in the analytical methods according to this disclosure are not particularly limited, as long as they are produced by linking two or more complementary strands generated by the complementary nucleic acid generation step described above via nucleic acid linkers, and can be produced by any combination of methods.

[0060] The following diagram illustrates a modified version of the cyclic nucleic acid production process. Note that the cyclic nucleic acid production process described herein is merely one example and is not limited to the form described herein.

[0061] Figure 2D is an illustrative diagram of the nucleic acid linkage and other components used in the first modified example of the circular nucleic acid production process. In Figure 2D, <2D-α> is a nucleic acid ligator used in the first modified version of the cyclic nucleic acid generation process. <2D-β> is a primer used in the cyclic nucleic acid generation process in this modified version to generate nucleic acids having a sequence derived from the target nucleic acid. <2D-γ> is an RNA oligomer used to protect the complementary strand capture sequence of the nucleic acid ligator when generating nucleic acids having a sequence derived from the target nucleic acid.

[0062] The nucleic acid ligator of <2D-α> has a target nucleic acid capture region (polyT in the figure) capable of capturing the polyA sequence located at the 3' end of mRNA, and a complementary strand capture region (concatenator in the figure). Although not shown in the example in Figure 2D, the nucleic acid ligator may also have a sequence addition region between the target nucleic acid capture region and the complementary strand capture region. The sequence of the complementary strand capture region of the nucleic acid ligator used in this modified example may be any sequence. Furthermore, if the nucleic acid ligator has a sequence addition region, any sequence corresponding to the function to be added to the sequence addition region can be assigned to it.

[0063] The primer shown in <2D-β> has 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 complementary strand capture sequence of the nucleic acid ligation site of <2D-α>. This allows the cDNA produced in the circular nucleic acid production process of this modified example to serve as a starting point for generating nucleic acids having sequences derived from the target nucleic acid, and the sequence complementary to the complementary strand capture sequence allows two or more generated nucleic acids to be suitably linked via the nucleic acid ligation site.

[0064] The <2D-γ> RNA oligomer is formed from RNA. Furthermore, the sequence of this RNA oligomer is complementary to the sequence of the complementary strand capture region of the <2D-α> nucleic acid ligation region. As a result, this RNA oligomer can be suitably hybridized to the 5' end of the cDNA produced in the circular nucleic acid generation process of this modified example.

[0065] In the first modified version of the cyclic nucleic acid generation process, the polyA tailing method is used in the complementary nucleic acid generation step. Using a primer that has been conjured with a sequence to be captured by the complementary strand capture portion of the nucleic acid ligator, the complementary strand of the generated target nucleic acid is used as a template to generate a nucleic acid having a sequence derived from the target nucleic acid. The following explanation will use Figure 2E.

[0066] In Figure 2E, in step S211, the nucleic acid ligator shown in <2D-α> is used, and the target nucleic acid capture portion of the nucleic acid ligator captures the 3' end of the mRNA. Step S211 is the same as the (2-2) circular nucleic acid production process described above, except for the difference in the nucleic acid ligator used, and the same conditions that can be applied to the circular nucleic acid production process can be suitably applied to this modified example as well.

[0067] In step S212, where cDNA is generated using the mRNA sequence in Figure 2E as a template, the same conditions that can be applied to the (2-2) circular nucleic acid generation process described above can also be suitably applied in this modified example.

[0068] In step S213 of Figure 2E, the RNA-DNA complex generated in step S212 is treated with RNaseH to degrade the mRNA that serves as the template for the cDNA. Subsequently, the cDNA is treated with terminal transferase to add a polyA sequence to its 3' end. This ensures that, for example, even if reverse transcription by reverse transcriptase does not reach the 5' end of the mRNA and the CCC sequence is not added, the primer shown in <2D-β> can be suitably hybridized to the cDNA in the next step S214 by the polyT sequence it possesses.

[0069] In step S214 of Figure 2E, a <2D-β> primer is hybridized to the 3' end of the cDNA generated in step S213, and a <2D-γ> RNA oligomer is hybridized to the 5' end. The order of hybridization in this case is not restricted; for example, the RNA oligomer may be hybridized first, or the concentration of the RNA oligomer may be higher than that of the primer and hybridized simultaneously.

[0070] Subsequently, in step S215 of Figure 2E, a DNA polymerase without strand displacement activity generates nucleic acids having a sequence derived from the target nucleic acid, using the cDNA as a template and starting with the <2D-β> primer. Since the <2D-γ> RNA oligomer is hybridized to the 5' end of the cDNA, the nucleic acid generation reaction by the DNA polymerase without strand displacement activity terminates at the position where the RNA oligomer is hybridized.

[0071] In step S216 of Figure 2E, the RNA-DNA complex generated in step S215 is treated with RNaseH, which degrades the RNA oligomer and yields a DNA-DNA complex containing the complementary strand of the target nucleic acid.

[0072] In step S217 of Figure 2E, two or more DNA-DNA complexes containing the complementary strand of the obtained target nucleic acid are linked via the complementary strand capture portion of the nucleic acid linkage and a sequence complementary to the sequence of the complementary strand capture portion derived from the <2D-β> primer. The conditions for this step can be similar to those applicable to the (2-2) cyclic nucleic acid production process described above, and these conditions can also be suitably applied in this modified example.

[0073] The coupling reaction shown in step S217 forms a single-stranded circular nucleic acid, similar to the (2-2) circular nucleic acid production process described above, and in the same manner as shown in step S14 in Figure 2A. Any conditions can be suitably applied to form this single-stranded circular nucleic acid.

[0074] In the first modified version of the circular nucleic acid generation process, the polyA tailing method is used in the complementary nucleic acid generation step. By using primers that have been conjugated with a sequence that is captured by the complementary strand capture region of the nucleic acid ligator, even if, for example, reverse transcription by reverse transcriptase does not extend to the 5' end of the mRNA and the CCC sequence is not conjugated, a sequence complementary to the complementary strand capture region of the nucleic acid ligator can be conjugated. Therefore, when two or more generated complementary strands are linked via the nucleic acid ligator to form a circular nucleic acid, it is possible to form a circular nucleic acid while reducing the loss of generated complementary strands. As a result, it is expected that the probability of detecting complementary strands derived from low-frequency expressed mRNA sequences will increase, and analytical accuracy will improve.

[0075] Furthermore, since the combination of DNA-DNA complexes to be linked in the linking reaction shown in step S217 cannot be selected, the single-stranded circular nucleic acid formed in step S217 will be a random link of sequences derived from cDNA generated using the mRNA sequence as a template. If there are many types of cDNAs to be linked and the order of the linked cDNAs does not substantially overlap, the single-stranded circular nucleic acid can be identified by the order of these linked cDNAs.

[0076] Subsequently, although not shown in Figure 2E, similar to the (2-2) circular nucleic acid generation process described above, a primer such as <2D-β> is 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, starting from the primer. While <2D-β> can be suitably used as the primer in this process, the primer used is not limited to this. Furthermore, the same conditions that can be applied to the generation process of the (2-2) circular nucleic acid generation process described above can also be suitably applied to this modified example. For example, the same method that can be applied to the (2-2) circular nucleic acid generation process described above can also be suitably applied to this modified example for sequencing the obtained single-stranded linear nucleic acid.

[0077] (2-4) Second variation of the process for generating cyclic nucleic acids Figure 2F is an illustrative diagram of the nucleic acid linkage and other components used in a second modified example of the cyclic nucleic acid production process. In Figure 2F, <2F-α> is the nucleic acid ligation site used in the first modified example of the circular nucleic acid generation process. <2F-β> is a primer used in the circular nucleic acid generation process in this modified example to generate nucleic acids having a sequence derived from the target nucleic acid. <2F-γ> is a primer used in PCR using the DNA-RNA complex formed in this modified example as a template.

[0078] The nucleic acid ligator of <2F-α> has a target nucleic acid capture portion (polyT in the figure) capable of capturing the polyA sequence located at the 3' end of mRNA, and a complementary strand capture portion (concatenator in the figure), with a sequence addition portion (cell barcode in the figure) provided between the target nucleic acid capture portion and the complementary strand capture portion. Note that the nucleic acid ligator used in this modification may also be in a form without the sequence addition portion. The sequence of the complementary strand capture portion of the nucleic acid ligator used in this modification may be any sequence. Furthermore, if the nucleic acid ligator has a sequence addition portion, any sequence corresponding to the function to be added to the sequence addition portion can be assigned to it.

[0079] The primer shown in <2F-β> has 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 complementary strand capture sequence of the nucleic acid ligation site of <2F-α>. In this primer, the sequence complementary to the complementary strand capture sequence of the nucleic acid ligation site is formed by RNA. The primer shown in <2F-β> serves as a starting point for generating nucleic acids having sequences derived from the target nucleic acid, using the cDNA produced in the circular nucleic acid generation process of this modified example as a template. Furthermore, in the PCR described later, it serves as a template, thereby attaching the complementary strand capture sequence of the nucleic acid ligation site to the 3' end of the nucleic acid synthesized by the PCR.

[0080] The primer shown in <2F-γ> is formed from RNA. Furthermore, the sequence of this primer is complementary to the sequence of the complementary strand capture region of the nucleic acid ligation region of <2F-α>. As a result, in PCR using the DNA-RNA complex formed in this modified example as a template, it serves as the starting point for the nucleic acid elongation reaction.

[0081] In the second modified version of the cyclic nucleic acid generation process, the polyA tailing method is used in the complementary nucleic acid generation step. Using a primer to which a sequence is captured by the complementary strand capture portion of the nucleic acid linker is attached, the complementary strand of the generated target nucleic acid is used as a template to generate nucleic acids having a sequence derived from the target nucleic acid. The following explanation will use Figure 2G.

[0082] In Figure 2G, steps S311, S312, and S313 are the same as steps S211, S212, and S213 in the first modified example of the cyclic nucleic acid production process described above, and the same conditions that can be applied to the cyclic nucleic acid production process can be suitably applied to this modified example as well.

[0083] In step S314 of Figure 2G, DNA polymerase is used to generate nucleic acids having a sequence derived from the target nucleic acid, starting with the <2F-β> primer and using the cDNA as a template. By using the <2F-β> primer, a sequence complementary to the complementary strand capture region of the <2F-α> nucleic acid ligation region can be added to the 5' end of the generated nucleic acid.

[0084] In step S315 of Figure 2G, PCR is performed using a nucleic acid having a sequence derived from the generated target nucleic acid as a template, with a primer shown as <2F-γ> formed from RNA. As mentioned above, the above nucleic acid used as a template has a 5' end derived from the <2F-β> primer and a sequence complementary to the complementary strand capture region of the nucleic acid ligation site formed from RNA. Therefore, a DNA polymerase with reverse transcription activity, such as Tth DNA polymerase, can be suitably used in this PCR.

[0085] In step S316 of Figure 2G, the RNA-DNA complex generated in step S315 is treated with RNaseH, which degrades the RNA oligomer and yields a DNA-DNA complex containing the complementary strand of the target nucleic acid.

[0086] In step S317 of Figure 2G, two or more DNA-DNA complexes, each containing the complementary strand of the obtained target nucleic acid, are linked via the complementary strand capture portion of the nucleic acid linkage and a sequence complementary to the sequence of the complementary strand capture portion derived from the <2F-β> primer. The conditions for this step can be similar to those applicable to the production process of the cyclic nucleic acid production process described in (2-2) or (2-3) above, and these conditions can also be suitably applied in this modified example.

[0087] The coupling reaction shown in step S317 forms a single-stranded circular nucleic acid, similar to the circular nucleic acid production process described in (2-2) or (2-3) above, and similar to the process shown in step S14 in Figure 2. The conditions for forming this single-stranded circular nucleic acid are also the same conditions that can be applied to the circular nucleic acid production process described in (2-2) or (2-3) above, and can be suitably applied in this modified example as well.

[0088] Subsequently, although not shown in Figure 2G, similar to the cyclic nucleic acid generation process described in (2-2) or (2-3) above, a primer is added to the formed single-stranded cyclic nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded cyclic nucleic acid is synthesized using DNA polymerase-mediated RCA starting from the primer. The same conditions that can be applied to the cyclic nucleic acid generation process described in (2-2) or (2-3) above can also be suitably applied in this modified example.

[0089] (2-5) A third variation of the cyclic nucleic acid generation process Figure 2H is an illustrative diagram of the nucleic acid linkage and other components used in a third modified example of the cyclic nucleic acid production process. In Figure 2H, <2H-α> is a nucleic acid ligation site used in the first modified example of the cyclic nucleic acid generation process. <2H-β> is a primer used in the cyclic nucleic acid generation process in this modified example to generate nucleic acids having a sequence derived from the target nucleic acid.

[0090] The nucleic acid junction of <2H-α>, like the nucleic acid junction of <2F-α>, has a target nucleic acid capture region (polyT in the figure) capable of capturing the polyA sequence located at the 3' end of mRNA, and a complementary strand capture region (concatenator in the figure), with a sequence addition region (cell barcode in the figure) provided between the target nucleic acid capture region and the complementary strand capture region. However, the nucleic acid junction of <2H-α> differs from that of <2F-α> in that the complementary strand capture region is formed of RNA. Furthermore, the nucleic acid ligator used in this modification may also be in a form without the sequence addition portion. The sequence of the complementary strand capture portion of the nucleic acid ligator used in this modification may be any sequence. Also, if the nucleic acid ligator has a sequence addition portion, any sequence corresponding to the function to be imparted to the sequence addition portion may be assigned to that sequence addition portion.

[0091] The primer shown as <2H-β>, like the primer shown as <2F-β>, has 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 complementary strand capture sequence of the nucleic acid ligation site of <2H-α>. However, in this primer, it differs from the primer shown as <2F-β> in that the sequence complementary to the complementary strand capture sequence of the nucleic acid ligation site is formed by DNA. The primer shown in <2H-β> serves as a starting point for generating nucleic acids having a sequence derived from the target nucleic acid, using the cDNA produced in the circular nucleic acid generation process of this modified example as a template. Furthermore, the sequence complementary to the complementary strand capture region allows for the suitable linking of two or more generated nucleic acids via the nucleic acid linking region.

[0092] In the third modified version of the cyclic nucleic acid generation process, the polyA tailing method is used in the complementary nucleic acid generation step. Using a primer to which a sequence is captured by the complementary strand capture portion of the nucleic acid linker is attached, the complementary strand of the generated target nucleic acid is used as a template to generate a nucleic acid having a sequence derived from the target nucleic acid. The following explanation will be given using Figure 2I.

[0093] In Figure 2I, steps S411, S412, and S413 are the same as steps S211, S212, and S213 in the first modified example of the cyclic nucleic acid production process described above, and the same conditions that can be applied to the cyclic nucleic acid production process can be suitably applied to this modified example as well.

[0094] In step S414 of Figure 2I, a DNA polymerase without reverse transcription activity is used, starting with a <2H-β> primer and using the cDNA as a template, to generate a nucleic acid having a sequence derived from the target nucleic acid. By using the <2H-β> primer, a sequence complementary to the complementary strand capture region of the <2H-α> nucleic acid ligator can be added to the 5' end of the generated nucleic acid. Furthermore, as mentioned above, the complementary strand capture region of the <2H-α> nucleic acid ligator is formed of RNA, and the DNA polymerase used in step S414 has no reverse transcription activity, so the nucleic acid elongation reaction stops before reaching the complementary strand capture region of the <2H-α> nucleic acid ligator. This results in a DNA-DNA complex containing the complementary strand of the target nucleic acid.

[0095] In step S415 of Figure 2I, two or more DNA-DNA complexes containing the complementary strand of the obtained target nucleic acid are linked via a complementary strand capture portion of the nucleic acid linkage and a sequence complementary to the sequence of the complementary strand capture portion derived from a <2H-β> primer. The conditions for this step can be suitably applied in this modified example to the same conditions that can be applied to the production process of the cyclic nucleic acid production process described in (2-2), (2-3), or (2-4) above.

[0096] The coupling reaction shown in step S415 forms a single-stranded circular nucleic acid, similar to the circular nucleic acid production processes described in (2-2), (2-3), or (2-4) above, and similar to the process shown in step S14 in Figure 2. The conditions for forming this single-stranded circular nucleic acid are also similar to those applicable to the circular nucleic acid production processes described in (2-2), (2-3), or (2-4) above, and can be suitably applied in this modified example as well.

[0097] Subsequently, although not shown in Figure 2G, similar to the cyclic nucleic acid generation process described in (2-2), (2-3), or (2-4) above, a primer is added to the formed single-stranded cyclic nucleic acid, and a single-stranded linear nucleic acid complementary to the single-stranded cyclic nucleic acid is synthesized using DNA polymerase-mediated RCA starting from the primer. The same conditions that can be applied to the cyclic nucleic acid generation process described in (2-2), (2-3), or (2-4) above can also be suitably applied in this modified example.

[0098] (3) Example 2 (Use of restriction enzyme recognition sites)

[0099] In accordance with this disclosure, a restriction enzyme recognition sequence may be incorporated into the nucleic acid ligation region. The restriction enzyme recognition sequence may be incorporated, for example, into the double-stranded region. This allows the amplified product (e.g., an amplified product by RCA or PCR) of the ligated product (particularly a cyclic nucleic acid) ligated according to this disclosure to be cleaved at the location of the restriction enzyme recognition sequence by restriction enzyme treatment. This makes it easier to analyze the amplified product. For example, the sequence resulting from the cleavage can be tagged for sequencing (particularly tagged for short-read sequencing). For example, when Hind III is used as the restriction enzyme, the restriction site shown in Figure 4A is introduced into the double-stranded region. As shown by the dashed line in the same figure, the double-stranded DNA is cleaved, and a sequencing tag is attached thereto.

[0100] Figure 4B shows an example of generating tagged short reads. As shown in the figure, in step S31, the complementary strand CN of the cyclic nucleic acid GN generated according to this disclosure is synthesized by DNA polymerase to produce double-stranded DNA. In step S32, the double-stranded DNA is cleaved at the restriction site RN located within the double-stranded region using a restriction enzyme. This cleavage generates a double-stranded DNA fragment containing one cDNA. In step S33, sequencing tags T1 to T4 are attached to both ends of each double-stranded DNA fragment. These tags may be configured to specifically recognize and bind to the sequence and / or structure of a site cleaved by restriction enzymes, for example. The types of tags may be appropriately selected by those skilled in the art depending on the sequencing method. For example, for short-read sequencing provided by Illumina, the tags Read1, Read2, Sample index(i5), Sample index(i7), P5, and P7 are attached. Sequencing is then performed using a sequencing instrument provided by Illumina. This identifies the sequence of each double-stranded DNA fragment (and the number of each double-stranded fragment).

[0101] (4) Example 3 (Single-cell analysis)

[0102] The analytical methods according to this disclosure may be configured, for example, as single-cell analysis methods. That is, this disclosure also provides single-cell analysis methods. Preferably, in such single-cell analysis, nucleic acid ligators having different double-stranded portions for each cell may be used. The procedures performed in this single-cell analysis method will be explained below with reference to Figure 5.

[0103] To perform the single-cell analysis method described above, the nucleic acid ligator 10 is positioned on the surface (particularly a two-dimensional plane) of the substrate 40, as shown in Figure A. In the Figure, the nucleic acid ligator comprises, as described above, at least a target nucleic acid capture portion 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 a double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion. Here, it is required that nucleic acid ligators having different sequences bind to each cell, and that nucleic acid ligators having the same sequence bind to each cell. Therefore, the surface of the substrate 40 may be divided, and nucleic acid ligators having the same sequence may be fixed to one region, and nucleic acid ligators having different sequences may be fixed to each region. In other words, the difference in sequence may be a difference in the double-stranded portion. For example, as shown in the figure, the surface of the substrate is divided into region A1 and region A2, and multiple nucleic acid ligatures having the same base sequence are immobilized in region A1, and multiple nucleic acid ligatures having the same base sequence may also be immobilized in region A2. The nucleic acid ligatures immobilized in region A1 and the nucleic acid ligatures immobilized in region A2 differ in their base sequences, and in particular in the base sequences of the two regions. In this disclosure, the surface to which the nucleic acid binding site is immobilized may be a flat surface such as a substrate, well, or plate as described above, but is not limited to these. Alternatively, it may be a surface such as a bead. In the case of beads, each bead corresponds to one of the regions described above. For example, one cell may be captured by one bead, in which case multiple nucleic acid binding sites having the same base sequence may be immobilized on one bead. Furthermore, the nucleic acid binding sites on each bead may be different from each other, for example, in a random sequence. Each nucleic acid binding site on each bead may have the same target nucleic acid capture site and / or complementary strand capture site, and may also have the same priming sequence.

[0104] (A complex containing nucleic acid linkages) The nucleic acid linker may have a cell-capturing portion for capturing cells and / or an immobilization portion for immobilizing it on a substrate. The cell-capturing portion may be an antibody or a lipid. Furthermore, various barcode sequences may be linked to the nucleic acid linker. The nucleic acid linker may exist as an element of a complex including these cell-capturing portions and / or immobilization portions. An example of such a complex will be described below with reference to Figures 6A to C.

[0105] Figure 6A shows a schematic example of the structure of a nucleic acid ligation site to which an antibody is bound as a cell-capturing site.

[0106] The nucleic acid binding portion 50 (the portion enclosed by the dashed line) may exist as part of a complex 100 immobilized on the substrate 61. The complex 100 has a cell capture portion 63 configured to capture cells, a nucleic acid binding portion 50 that captures target nucleic acids within the cells, and an immobilization portion for immobilizing the nucleic acid binding portion 50 on the substrate. In the figure, the structures indicated by reference numerals 54a, 54b, 55a, 55b, 56, 57, 58, 59, 60-1, and 62 correspond to the immobilization portion, but the configuration of the immobilization portion is not limited to these. The immobilization portion does not need to have all the elements indicated by these reference numerals, and may have other configurations as long as they can fix the nucleic acid binding portion to a predetermined surface. The components of the complex 100 are described below.

[0107] First, in the diagram, there are four structures 60-1 (represented as nucleic acids in the diagram) in region A1, and it is shown that only one of these structures 60-1 has components such as nucleic acid ligatures bound to it. Please understand that this is a simplification for the sake of clarity in the diagram. In reality, there are many structures 60-1 in region A1, and each of these structures has components such as nucleic acid ligatures bound to it in the same way. Furthermore, similar to region A1, region A2 also contains numerous structures 60-2, and each of these structures is similarly bound to components such as nucleic acid junctions. Furthermore, the substrate 61 may have a number of regions corresponding to the number of cells that are to be captured, not limited to the two regions shown in the figure.

[0108] The cell-capturing portion 63 may be a compound configured to capture a desired cell. In one embodiment, the cell capture unit 63 may be an antibody, for example, as shown in the figure. The antibody may be configured to capture molecules present on the cell surface (particularly surface markers, etc.). The antibody can be appropriately selected by a person skilled in the art depending on the type of cell to be captured. In other embodiments, the cell-capturing portion may be a lipid, as shown in Figure 6B, for example. The complex in Figure 6B is the same as in Figure 6A, except that it has a lipid as the cell-capturing portion 63 instead of an antibody. The lipid may be a lipid configured to capture cells, and its type can be appropriately selected by those skilled in the art.

[0109] The nucleic acid ligator 50 includes a target nucleic acid capture portion 51 configured to capture the 3' terminal region of the target nucleic acid, a complementary strand capture portion 52 (shown as GGG in the figure) configured to capture the 3' terminal region of the complementary strand of the target nucleic acid, and a double-stranded portion 53 (53a, 53b, 53c, and 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, complementary strand capture portion 12, and double-stranded portion 13 described above, and their descriptions also apply to this example.

[0110] The target nucleic acid capture region 51 may be a polyT sequence as described above. The complementary strand capture region 52 may also be a GGG sequence, as described above. With respect to the target nucleic acid capture portion 51 and the complementary strand capture portion 52, all nucleic acid binding portions present on the substrate 61 may have the same target nucleic acid capture portion 51 and complementary strand capture portion 52.

[0111] The double-stranded portion 53 has random sequences 53a and 53b, and priming sequences 53c and 53d. The random sequences 53a and 53b are complementary. The priming sequences 53c and 53d are also complementary. Random sequences 53a and 53b have different base sequences in each region. That is, the random sequences contained in the nucleic acid junction fixed in region A1 all have the same base sequence. The random sequences contained in the nucleic acid junction fixed in region A2 all have the same base sequence. Furthermore, the random sequences present in region A1 have different base sequences from the random sequences present in region A2. With respect to priming sequences 53c and 53d, for example, all nucleic acid ligation sites present on the substrate 61 may have priming sequences 53c and 53d with the same base sequence. This allows for the batch generation of circular nucleic acids using these priming sequences.

[0112] The complex 100 may have cleavage sites 54 (54a and 54b). The cleavage sites 54 may be, for example, restriction enzyme recognition sites, and more particularly, sites in which multiple restriction enzyme recognition sites are arranged in series. The cleavage sites 54 may be, for example, sites in which 1 to 10 restriction enzyme recognition sites are arranged in series, and more particularly, sites in which 2 to 8, more particularly 4 to 6, or for example, 5, restriction enzyme recognition sites are arranged in series. The number of bases of each restriction enzyme recognition site may be, for example, 4 to 10, and more particularly, 4 to 8. The restriction enzyme may be an endonuclease. By arranging multiple restriction enzyme recognition sites in series in this way, the possibility of cleavage can be increased. When the cleavage site 54 is a restriction enzyme recognition site, it may be configured as a double-stranded nucleic acid. One strand of the double-stranded nucleic acid may be connected to a nucleic acid linking site 50 (particularly a target nucleic acid capture site 51). The other strand of the double-stranded nucleic acid may be connected to a barcode sequence as described later. The base length of the cleavage site 54 may be, for example, 10 to 50 bases, and more particularly 20 to 40 bases.

[0113] For cleavage of restriction enzyme recognition sites, the appropriate restriction enzyme (http: / / catalog.takara-bio.co.jp / product / basic_info.php?unitid=U100003632) is used depending on the sequence. One unit of restriction enzyme activity is the amount of enzyme that completely degrades 1 μg of λDNA per hour at 37°C in 50 μl of each enzyme reaction solution, and the amount of enzyme is adjusted according to the amount of restriction enzyme recognition sequence.

[0114] The composite 100 may have a location information barcode array section 55 (55a and 55b). The location information barcode array section 55 may also be called, for example, an array barcode array section. The location information barcode sequence portions 55a and 55b have different base sequences in each region. That is, the location information barcode sequence portions fixed in region A1 all have the same base sequence. The location information barcode sequence portions fixed in region A2 all have the same base sequence. Furthermore, the location information barcode sequence portions present in region A1 have a different base sequence from the location information barcode sequence portions present in region A2. Based on the positional information corresponding to the base sequence of the positional information barcode sequence portion, it is possible to associate the cells captured in each region with, for example, an image of each cell. The image may be, for example, an image acquisition device such as a microscope, which is acquired after the cells have been captured in each region and before the cells have been released from each region by cleavage at the cleavage sites. The base length of the position information barcode sequence portion 55 may be adjusted so that the number of variations of the position information barcode sequence portion 55 is equal to or greater than the number of regions on the substrate (or the number of cells captured), for example, 10 bases or more, more particularly 12 or more, more particularly 14 or more, and even more particularly 16 or more. The base length may be, for example, 100 bases or less, more particularly 70 bases or less, more particularly 50 bases or less, and for example, 30 bases or less.

[0115] The composite 100 may have a barcode arrangement section 58 for immobilization. The immobilized barcode sequence portion 58 has a different base sequence for each region. That is, all immobilized barcode sequence portions immobilized in region A1 have the same base sequence. All immobilized barcode sequence portions immobilized in region A2 also have the same base sequence. Furthermore, the immobilized barcode sequence portion present in region A1 has a different base sequence from the immobilized barcode sequence portion present in region A2. The immobilization barcode sequence portion 58 has a base sequence complementary to the nucleic acid 60-1 pre-immobilized on the substrate, and the nucleic acid 60-1 is present only within a specific region. As a result, the immobilization barcode sequence specifically binds to the nucleic acid 60-1, and the complex 100 having the immobilization barcode sequence is immobilized within the specific region. The base length of the immobilization barcode sequence portion 58 may be adjusted so that the number of variations of the immobilization barcode sequence portion 58 is equal to or greater than the number of regions on the substrate (or greater than or equal to the number of cells captured), for example, 10 bases or more, more particularly 12 or more, more particularly 14 or more, and even more particularly 16 or more. The base length may be, for example, 100 bases or less, more particularly 70 bases or less, more particularly 50 bases or less, and for example, 30 bases or less.

[0116] The complex 100 may have priming regions 57 and 59. The priming region 57 has a base sequence used to synthesize a nucleic acid chain having the barcode sequence region and the restriction enzyme recognition site. The base length of each priming region is, for example, 10 to 30 bases, more particularly 15 to 25 bases, and more particularly 15 to 20 bases.

[0117] As described above, the complex 100 may have a nucleic acid linking portion, a cell capture portion for capturing cells, and an immobilization portion for immobilizing the nucleic acid linking portion at a specific position. The immobilization portion may include the restriction enzyme recognition site and / or barcode sequence portion described above. Furthermore, with respect to the complex 100, the immobilization barcode sequence portion 58 functions as a structure for immobilizing the complex at a specific position, but the position information barcode portion may be configured as a structure for immobilizing the complex at a specific position.

[0118] As described above, the complex 100 may be bound to nucleic acid 60-1 immobilized on the substrate. Nucleic acid 60-1 may have different base sequences in each region. The complex 100 is immobilized on the substrate by binding between nucleic acid 60-1 and the immobilization barcode portion 58. That is, the nucleic acid linkage portion may be immobilized on the substrate.

[0119] The nucleic acids 60 (60-1 and 60-2) may be immobilized on the substrate 61 via a linker 62. The linker 62 may be made of a material known in the art and can be appropriately selected by those skilled in the art.

[0120] In one embodiment, the linker 62 may be a linker that can be cleaved by stimulation, for example, a linker that can be cleaved by photostimulation or chemical stimulation. Photostimulation is particularly suitable for selectively stimulating a specific location in the cleavage process described later. When such a cleavable linker is used, the cleavage site 54 containing the restriction enzyme recognition site described above may or may not be provided. When such a cleavable linker is used, the chain connected to the nucleic acid binding portion (particularly the chain connected to the target nucleic acid capture portion) may be immobilized directly (i.e., without the nucleic acid 60) to the substrate 61 via the linker.

[0121] The cleavable linker may include, for example, a linker that can be cleaved by photostimulation, one selected from an arylcarbonylmethyl group, a nitroaryl group, a coumarin-4-ylmethyl group, an arylmethyl group, a metal-containing group, and other groups. These groups may include, for example, those described in Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy, Chem. Rev. 2013, 113, 119-191. For example, the arylcarbonylmethyl group may be a phenacyl group, an o-alkylphenacyl group, or a p-hydroxyphenacyl group. The nitroaryl group may be, for example, an o-nitrobenzyl group, an o-nitro-2-phenethyloxycarbonyl group, or an o-nitroanilide. The arylmethyl group may have a hydroxyl group introduced into it, or it may not have a hydroxyl group introduced.

[0122] If the linker that can be cleaved is a linker that can be cleaved by light stimulation, the linker may preferably be cleaved by light with a wavelength of 360 nm or longer. The linker preferably has a wavelength of 0.5 μJ / μm 2 The linker may be cleaved at the following energies (Light-sheet fluorescence microscopy for quantitative biology, Nat Methods. 2015 Jan;12(1):23-6. doi: 10.1038 / nmeth.3219.). By employing a linker cleaved at the above wavelengths of light or energies, it is possible to reduce cellular damage (especially DNA or RNA cleavage) that may occur when photostimulation is applied.

[0123] Particularly preferably, the cleavable linker may be a linker that is cleaved by light in the short wavelength region, specifically light in the wavelength region of 360 nm to 410 nm, or a linker that is cleaved by light in the near-infrared or infrared region, specifically light in the wavelength region of 800 nm or more. If the cleavable linker is a linker that is efficiently cut by light in the visible light region, handling of the analytical surface may become difficult. For this reason, it is preferable that the linker is a linker that is cleaved by light in the short wavelength region or light in the near-infrared or infrared region.

[0124] The cleavable linker may include, for example, a disulfide bond, as a linker that can be cleaved by chemical stimulation. To cleave the disulfide bond, reducing agents such as tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), or 2-mercaptoethanol are used. For example, when using TCEP, the reaction is carried out for about 15 minutes at a concentration of 50 mM.

[0125] The composite 100 may include multiple linkers that can be cleaved. Preferably, these multiple linkers may be connected in series. For example, if the cleavage probability of one linker is 0.8, then by connecting three such linkers in series, the cleavage probability becomes 0.992 (= 1 - 0.2). 3 ) will improve to that.

[0126] (Method for producing a complex containing nucleic acid linkages) The method for manufacturing the composite will be described below with reference to Figure 6C.

[0127] The complex 100 includes a first pool of a first strand (particularly single-stranded) having a target nucleic acid capture portion of the nucleic acid linking portion, and a second pool of a second strand (particularly single-stranded) having a barcode sequence portion, and the strands from each pool are joined to form a double-stranded structure. Here, the first strand has one of the two strands that constitute the double-stranded portion of the nucleic acid linking portion. A third strand (particularly a single strand) having a complementary strand-capturing portion of the nucleic acid linking portion is prepared for the double strand. This third strand has the other of the two strands that make up the double strand portion of the nucleic acid linking portion. Then, the third strand is bound to the double strand obtained by the linking of the first strand and the second strand to obtain the complex 100. In Figure 6C, the process for obtaining the first chain is shown on the Oligo-pool 1 side. The process for obtaining the second chain is shown on the Oligo-pool 2 side.

[0128] (Oligopool 1) As shown in oligopool 1, first, a single-stranded nucleic acid N1 (DNA or RNA) is prepared, having two priming sites, and between these priming sites, a sequence that forms the double-stranded portion (Concatenator*), a sequence that forms the target nucleic acid capture portion (PolyT), a sequence that forms the cleavage site (Cleavage site), and a sequence that forms the positional information barcode sequence portion (Array1 barcode*). Here, the sequences marked with an asterisk (*), namely the sequences that form the double-stranded portion (Concatenator*) and the sequences that form the positional barcode sequence portion (Array1 barcode*), have different base sequences for each region. Therefore, as oligopool 1, multiple types of single-stranded nucleic acids are prepared, in which these sequences are different but the sequences of other parts are the same.

[0129] Next, PCR is performed using the two priming sites. That is, for example, as shown in the figure, a primer having a T7 promoter and binding to one priming site, and a reverse primer binding to the other priming site are prepared, and PCR may be performed using these two types of primers. Double-stranded nucleic acid is obtained by this PCR.

[0130] In vitro transcription (IVT) is performed on the double-stranded nucleic acid in question. This yields an RNA transcript.

[0131] Reverse transcription (RT) is performed on the RNA transcript using a reverse primer that binds to the priming site, as shown in the figure. This generates cDNA corresponding to the RNA transcript.

[0132] After the cDNA synthesis, RNA digestion is performed. This process removes the RNA, leaving only the cDNA. In this way, a single-stranded nucleic acid is obtained having a sequence that forms the double-stranded portion, a sequence that forms the target nucleic acid capture portion, a sequence that forms the cleavage site, and a sequence that forms the positional information barcode sequence portion.

[0133] (Oligopool 2) As shown in Oligopool 2, first, a single-stranded nucleic acid N2 (DNA or RNA) is prepared, having two priming sites, a cleavage site between these priming sites, a sequence forming the positional barcode sequence (Array1# barcode*), and a sequence forming the immobilization barcode sequence (Array2# barcode*). Note that "#" indicates complementarity; for example, Array1 barcode* is complementary to Array1# barcode*, and Array2 barcode* is complementary to Array2# barcode*. Here, the sequences marked with "*", i.e., the sequences forming the positional information barcode sequence portion (Array1 / Array1# barcode*) and the sequences forming the immobilization barcode sequence portion (Array2# barcode*), have different base sequences for each region. Therefore, as oligopool 2, multiple types of single-stranded nucleic acids are prepared, in which these sequences are different but the sequences of other parts are the same.

[0134] Next, PCR is performed using the two priming sites. That is, for example, as shown in the figure, a primer having a T7 promoter and binding to one priming site, and a reverse primer binding to the other priming site are prepared, and PCR may be performed using these two types of primers. Double-stranded nucleic acid is obtained by this PCR.

[0135] In vitro transcription (IVT) is performed on the aforementioned double-stranded nucleic acid. This yields an RNA transcript.

[0136] Reverse transcription (RT) is performed on the RNA transcript using a reverse primer that binds to the priming site, as shown in the figure. This generates cDNA corresponding to the RNA transcript.

[0137] After the cDNA synthesis, RNA digestion is performed. This process removes the RNA, leaving only the cDNA. In this way, a single-stranded nucleic acid is obtained having a sequence that forms the cleavage site, a sequence that forms the positional barcode sequence, and a sequence that forms the immobilization barcode sequence.

[0138] As shown in the center of the figure, the single-stranded nucleic acid synthesized in oligopool 1 and the single-stranded nucleic acid synthesized in oligopool 2 are bound together based on complementarity in the positional barcode sequence portion and the cleavage site to obtain a conjugate. Further, a single strand having the complementary strand capture portion and the other strand of the two strands forming the double-stranded portion is bound to this conjugate to obtain a complex 100. Thus, the complex 100 may contain three nucleic acid strands. Furthermore, this binding may be performed based on complementarity between the two strands forming the double-stranded portion. As shown in the figure, a cell-capturing portion (antibody) may be pre-bound to the single strand, but the cell-capturing portion may also be bound to the complex of the three nucleic acid strands after the complex has been formed.

[0139] Furthermore, the composite 100 has an immobilization barcode portion as described above. Based on the complementarity between the immobilization barcode portion and the nucleic acid 60-1 pre-provided on the substrate, a specific composite is immobilized at a specific position.

[0140] As described above, the complex 100 is prepared by providing a first pool of a first strand (particularly single-stranded) having a target nucleic acid capture portion of the nucleic acid linking portion, and a second pool of a second strand (particularly single-stranded) having a barcode sequence portion, and by joining the strands of each pool, a double strand is formed. Here, the first strand has one of the two strands that constitute the double-stranded portion of the nucleic acid linking portion.

[0141] A third strand (particularly a single strand) having a complementary strand-capturing portion of the nucleic acid linking portion is prepared for the double strand. This third strand has the other of the two strands that make up the double strand portion of the nucleic acid linking portion. Then, the third strand is bound to the double strand obtained by the linking of the first strand and the second strand to obtain the complex 100.

[0142] In one embodiment, the cell-capturing portion may be pre-bound to the third chain. In this case, the third chain is bound to the double helix formed by the binding of the first and second chains, thereby obtaining a complex having the cell-capturing portion. In other embodiments, the cell-capturing portion does not need to be pre-bound to the third chain. In this case, the cell-capturing portion may be further bound to the complex obtained by the third chain being bound to the double helix formed by the binding of the first and second chains.

[0143] The complex and the nucleic acid linkage may be prepared by the manufacturing method described above, but the manufacturing method is not limited thereto. The manufacturing method for the complex and the nucleic acid linkage may be appropriately modified depending on their configuration.

[0144] Below, we will return to the explanation of Figure 5. In step S41, cells are supplied onto a substrate having a surface on which the complex is immobilized. The cell-capturing portion in the complex captures the cells. As a result, one cell is captured in one region, as shown in Figure B. Note that in Figure B, only one complex is shown to have a chain with a complementary chain-capturing portion, but this is an omission for the sake of simplifying the drawing. In reality, each complex may have a chain with a complementary chain-capturing portion. Similarly, in other figures referenced herein, chains with complementary chain-capturing portions may be omitted.

[0145] In step S42, for example, the cleavage site 54 in the complex is cleaved. This cleavage may be carried out by a restriction enzyme that recognizes the cleavage site. Furthermore, if the composite has the cleavable linker described above, a treatment (photostimulation or chemical stimulation) may be performed to cleave the linker. After restriction enzyme treatment, the residue is degraded with an exonuclease, so that the target nucleic acid capture region 51 becomes the 3' terminal region. The exonuclease is preferably Exonuclease III (E. coli), which can degrade double-stranded DNA from the 3' end, whether it has a 5' overhang, a blunt end, or a 3' overhang of less than 4 bases. This cleavage releases each cell from the substrate while maintaining its nucleic acid binding portion via the cell capture portion. The released cells are then isolated one by one into microspaces. These microspaces may be spaces within emulsion particles or spaces within wells, as described later. Then, in this isolated state, each cell is destroyed. This destruction may be, for example, a cell lysis treatment. As a result of this disruption, the target nucleic acid within the cell is captured by the target nucleic acid capture portion that constitutes the nucleic acid binding portion. This generates a conjugate of the target nucleic acid and the nucleic acid binding portion, as shown in Figure 5C. In other words, a hybrid of the target nucleic acid and the nucleic acid binding portion is created. By performing cell disruption in this isolated state, the target nucleic acids within the cells do not escape the microspace. Therefore, nucleic acid ligatures with the same base sequence can be attached to all target nucleic acids originating from a single cell. Furthermore, as mentioned above, the nucleic acid ligatures present in each cell differ from one another in their double-stranded portions. Therefore, a one-to-one relationship is established between the base sequences of the cells and the nucleic acid ligatures (especially the double-stranded portions). This enables single-cell analysis. The method for achieving this isolation will be explained separately later. Thus, the analytical method according to this disclosure may include a cell disruption step of destroying cells, and the complementary nucleic acid generation step may be performed on a target nucleic acid contained in the cells.

[0146] Step S43 is the same cDNA synthesis as step S12 in Figure 2A described above. The explanation for step S12 also applies to step S43. By performing step S43, a cDNA having a CCC sequence at the end of the generated strand is produced, as shown in Figure 5D.

[0147] Step S44 is the same nucleic acid ligation as step S13 in Figure 2A described above. The explanation for step S13 also applies to step S44. By performing step S44, two or more cDNAs are linked via the complementary strand capture region, as shown in Figure 5E.

[0148] Step S45 is the same cyclic nucleic acid formation, ligation, and primer addition as steps S14 and S15 in Figure 2A described above. The explanation for steps S14 and S15 also applies to step S45. By performing step S45, a primer-attached cyclic nucleic acid is formed, as shown in Figure 5F.

[0149] Step S46 is the same nucleic acid amplification as step S16 in Figure 2A described above. The explanation for step S16 also applies to step S46. By performing step S46, the nucleic acid is amplified as shown in Figure 5G. The thus amplified nucleic acid is used in single-cell analysis. For example, the nucleic acid sequence is sequenced, and each cell is analyzed using the sequencing results.

[0150] (Isolation into a microspace) Examples of the isolation methods described above are explained below.

[0151] The aforementioned microspace may be, for example, a space within an emulsion particle or a space within a well. By this isolation, one cell to which one or more nucleic acid linkers having the same double-stranded base sequence are bound is isolated within one emulsion particle or one well. The cell disruption step may then be carried out within a microspace (i.e., a space partitioned for each cell).

[0152] In one embodiment, the isolation step for performing the isolation may include a determination step for determining whether to isolate cells in a microspace, and a particle isolation step for isolating the cells determined to be isolated in the determination step into a microspace. This makes it possible to isolate only the target cells. Therefore, for example, unintended cells can be excluded from the analysis, thereby improving the efficiency of the analysis.

[0153] The discrimination may be performed, for example, based on light emitted from the cell (e.g., scattered light and / or autofluorescence), light emitted from a substance bound to the cell, or a morphological image. The substance bound to the cell may be, for example, an antibody bound to the cell (particularly a fluorescently labeled antibody). The scattered light emitted from the cell may be, for example, forward scattered light and / or side scattered light. Doublet detection can be performed from the signal height and / or area value obtained by scattered light detection. Single cell determination is also possible based on morphological image information. Whether a cell is dead can be determined from scattered light and / or a morphological image, or from fluorescence after staining with a dead cell staining reagent, thereby allowing for the removal of dead cells. In this disclosure, the discrimination step may be performed immediately before the isolation step, thereby ensuring the isolation of only single cells to which nucleic acid linkers are bound.

[0154] In other embodiments, the particle isolation step may be performed without performing the discrimination step. By omitting the discrimination step, the number of steps in the method according to this disclosure can be reduced. In yet another embodiment, the discrimination step may be performed during linker cleavage. For example, cells fixed on a substrate may be observed, and the linker cleavage process may be performed only on cells that should be isolated into a microspace. In this case, a device such as a cell sorter may not be used.

[0155] The discrimination process and the particle separation process will be described below.

[0156] (Discrimination process)

[0157] In the aforementioned discrimination step, a determination is made as to whether to isolate the cells released from the substrate into a microspace. This determination may be made based on light emitted from the cells or light emitted from substances bound to the cells, as described above.

[0158] The discrimination step may include, for example, an irradiation step of irradiating cells with light, and a detection step of detecting the light generated by the irradiation.

[0159] The irradiation step may be performed, for example, by a light irradiation unit that irradiates cells with light. The light irradiation unit may include, for example, a light source that emits light. The light irradiation unit may also include an objective lens that focuses light onto the cells. The light source may be appropriately selected by a person skilled in the art depending on the purpose of the analysis, and may be, for example, a laser diode, SHG laser, solid-state laser, gas laser, high-brightness LED, or 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.

[0160] The detection step may be performed by a detection unit that detects light generated from, for example, cells or substances bound to cells. The detection unit detects, for example, the light generated from cells or substances bound to cells by light irradiation by the light irradiation unit, which may be, for example, scattered light and / or fluorescence. The detection unit may include, for example, a focusing lens and a detector that focus light generated from biological particles. The detector may be, but is not limited to, PMTs, photodiodes, CCDs, and CMOSs. In addition to the focusing lens and detector, the detection unit may include other optical elements as needed. The detection unit may further include, for example, a spectrometer. Examples of optical components constituting the spectrometer include gratings, prisms, and optical filters. The spectrometer can, for example, separate and detect light of a wavelength to be detected from light of other wavelengths. The detection unit may convert the detected light into an analog electrical signal by photoelectric conversion. The detection unit may further convert the analog electrical signal into a digital electrical signal by AD conversion.

[0161] In the discrimination step, a determination unit may perform a determination process to determine whether a cell is distinguishable based on the light detected in the detection step. The processing by the determination unit can be implemented by an information processing device such as a general-purpose computer, and more particularly by a processing unit included in the information processing device.

[0162] (Particle isolation process)

[0163] The isolation step includes a particle isolation step of isolating cells into a microspace. The microspace may mean a space having dimensions that can accommodate one of the cells to be analyzed. The dimensions may be appropriately determined depending on factors such as the size of the cell. The microspace may have dimensions that can accommodate two or more cells to be analyzed, in which case, in addition to cases where one cell is contained in one microspace, there may also be cases where two or more cells are contained. Cells in a microspace containing two or more cells may be excluded from destruction in the destruction step described later, or may be excluded from analysis in the analysis step described later.

[0164] Furthermore, in the disruption step described later, for example, a conjugate of a nucleic acid linkage and a target nucleic acid may be generated. Preferably, the plurality of microspaces are separated from each other so that the conjugate generated in one microspace does not migrate to other microspaces. Examples of such separated microspaces include spaces within wells and spaces within emulsion particles. That is, in a preferred embodiment, the microspace may be a space within a well or a space within an emulsion particle. Below, examples of particle isolation steps when the microspace is a space within a well and examples of particle isolation steps when the microspace is a space within an emulsion particle will be described, respectively.

[0165] (In the case of space within a well)

[0166] A schematic diagram of an example of a well used to perform the particle isolation process is shown in Figure 12. As shown in the figure, a plurality of wells 400, each having dimensions capable of containing, for example, one cell, may be formed on the surface of the substrate 401. By applying a liquid containing the cells released from the substrate by the cleavage to the surface of the substrate 401, for example, from an arbitrary nozzle 402, the cells 403 are isolated in the space within the wells 400, as shown in the figure. In this way, one cell may be placed in the space within each well, and the cell may be isolated in a microspace.

[0167] When a liquid containing multiple cells is applied to a substrate with wells formed thereon, as in the example shown in the figure, the particle isolation step may be performed without carrying out the discrimination step described above, or the particle isolation step may be performed after the discrimination step has been carried out.

[0168] Furthermore, when performing the above discrimination process, a device that places one biological particle per well, such as a cell sorter or a single-cell dispenser, may be used. This device can also be used to isolate cells onto a substrate (such as a plate) with multiple wells. A commercially available device may be used as this device. This device may, for example, have a light irradiation unit that irradiates the cells with light, a detection unit that detects the light from the cells, a discrimination unit that determines whether to place the cells in the wells based on the detected light, and a distribution unit that distributes the cells determined to be placed in the wells into the wells.

[0169] The light irradiation unit and the detection unit perform the detection step, and the discrimination unit performs the discrimination step. The distribution unit includes, for example, a microfluidic chip having a nozzle that forms droplets containing cells.

[0170] The device manipulates the position of the microfluidic chip according to the determination result by the discrimination unit to place one cell-containing droplet into a predetermined well. Alternatively, the device controls the direction of travel of the cell-containing droplet exiting the nozzle using the charge applied to the droplet, according to the determination result by the discrimination unit. This control places one cell-containing droplet into a predetermined well. In this way, one cell is distributed into each well.

[0171] For example, as shown in Figure 13A, a cell-containing droplet is ejected from a nozzle 502 provided on the microfluidic chip of the apparatus. Light (e.g., laser light L) is irradiated onto the biological particles contained in the droplet by a light irradiation unit 504, and a detection process is performed by a detection unit 505, detecting light (fluorescence F). Then, a discrimination unit (not shown) performs a determination process based on the detected light. Then, according to the determination result, the distribution unit controls the direction of movement of the droplet using the charge attached to the droplet. Through this control, the droplet containing the target biological particles is collected in a predetermined well. In this way, one biological particle is distributed to each well.

[0172] By performing the aforementioned discrimination step, it is possible to identify, for example, the cell population to which the biological particles belong, the biological particles to which a barcode has been assigned, or the droplets containing singlet biological particles, according to the detection signal. This allows only the droplets containing the target biological particles to be recovered. As a result, it becomes unnecessary to exclude data in the analysis step described later, improving analytical efficiency.

[0173] The number of wells provided on a single substrate (plate) may be, for example, 1 to 1000, more particularly 10 to 800, and more particularly 30 to 500, but the number of wells may be appropriately selected by those skilled in the art.

[0174] (In the case of space within emulsion particles)

[0175] Emulsion particles can be generated, for example, using a microchannel. This channel includes, for example, a channel through which a first liquid flows, forming the dispersed phase of the emulsion, and a channel through which a second liquid flows, forming the dispersion medium. The first liquid may contain cells. The apparatus further includes a region where these two liquids come into contact to form an emulsion. An example of a microchannel is described below with reference to Figure 13B.

[0176] The microchannel shown in the figure includes channel 601 through which a first liquid containing cells flows, and channels 602-1 and 602-2 through which a second liquid flows. The first liquid forms emulsion particles (dispersed phase), and the second liquid forms the dispersion medium of the emulsion. Channel 601 and channels 602-1 and 602-2 merge, and emulsion particles are formed at this merging point. Cells 603 are then isolated inside these emulsion particles. For example, the size of the emulsion particles can be controlled by controlling the flow velocity of these channels.

[0177] In order to form an emulsion, the first liquid and the second liquid are immiscible with each other. For example, the first liquid may be hydrophilic and the second liquid may be hydrophobic, or vice versa.

[0178] Furthermore, the microchannel shown in the figure may include a channel 604 for introducing a cell-disrupting substance into the emulsion particles. By configuring the microchannel so that channel 604 merges with channel 601 immediately before the confluence point, it is possible to prevent cells from being destroyed by the cell-disrupting substance before the emulsion particles are formed.

[0179] Next, an example of a device for more efficiently forming emulsions containing emulsion particles with a single cell will be described with reference to Figures 14A and 14B. This emulsion forming device can isolate a single cell within a single emulsion particle with a very high probability, reducing the number of empty emulsion particles. Furthermore, this emulsion forming device also increases the probability of isolating a single cell within a single emulsion particle.

[0180] Figure 14A shows an example of a microfluidic chip (hereinafter also referred to as "microchip") used to form emulsion particles in the apparatus. The microchip 150 shown in the figure includes a main channel 155 through which biological particles (particularly cells) flow, and a recovery channel 159 through which particles to be recovered from the biological particles are recovered. The microchip 150 is provided with a particle sorting unit 157. An enlarged view of the particle sorting unit 157 is shown in Figure 15. As shown in A of the same figure, the particle sorting unit 157 includes a connecting channel 170 that connects the main channel 155 and the recovery channel 159. A liquid supply channel 161 capable of supplying liquid to the connecting channel 170 is connected to the connecting channel 170. As described above, the microchip 150 has a flow channel structure including the main channel 155, the recovery channel 159, the connecting channel 170, and the liquid supply channel 161. Figure 14B is a schematic diagram illustrating the formation of emulsion particles in the microchip 150 shown in Figure 14A, and the isolation of biological particles within the formed emulsion particles.

[0181] Furthermore, as shown in Figure 14A, the microchip 150, in addition to the microchip itself, constitutes part of the biological particle sorting device 200, which includes a light irradiation unit 191, a detection unit 192, and a control unit 193. The control unit 193 may include a signal processing unit 194, a determination unit 195, and a sorting control unit 196, as shown in Figure 16. The biological particle sorting device 200 is used as the emulsion forming device described above.

[0182] As shown in Figure 17, in order to form an emulsion containing emulsion particles containing one target cell, for example, in the microchip 150, a flow step S201 in which the first liquid containing the cell flows into the main channel 155, a discrimination step S202 in which it is determined whether the cell flowing in the main channel 155 is a particle to be recovered, and a recovery step S203 in which the particle to be recovered is recovered into the recovery channel 159 may be performed. The discrimination step S202 corresponds to the discrimination step described above. The recovery step S203 corresponds to the particle isolation step described above. The following explains each step.

[0183] (Flow process)

[0184] In the flow passage process S201, a first liquid containing cells is passed through the main channel 155. The first liquid flows through the main channel 155 from the confluence section 162 toward the particle sorting section 157. The first liquid may be a laminar flow formed from a sample liquid containing cells and a sheath liquid, and in particular, a laminar flow in which the sample liquid is surrounded by the sheath liquid. The channel structure for forming the laminar flow will be described below. The sheath solution may contain, for example, a cell-destroying component, such as a cell-lysing component. This allows the component to be incorporated into the emulsion particles, enabling cell destruction within the emulsion particles during the subsequent destruction step. The cell-lysing component may be a cell-lysing enzyme, such as proteinase K. For example, after capturing cells within emulsion particles containing proteinase K, the cells are lysed by placing the emulsion particles at a predetermined temperature (e.g., 37°C to 56°C) for, for example, less than 1 hour, particularly less than 1 hour. Although proteinase K is active even below 37°C, if such a lower temperature is used, the cell-lysing properties of proteinase K will decrease, and the cells may be incubated, for example, overnight. The sheath solution may also contain a surfactant (e.g., SDS, Sarkosyl, Tween 20, or Triton X-100). This surfactant can enhance the activity of proteinase K. Furthermore, the sheath fluid does not need to contain cell-destroying components. In this case, cells may be physically destroyed. As a physical destruction method, for example, optical treatment (e.g., optical cell lysis) or thermal treatment (e.g., thermal cell lysis) may be employed. Optical treatment may be performed, for example, by irradiating emulsion particles with laser light to form plasma or cavitation bubbles within the particles. Thermal particle destruction may be performed by heating the emulsion particles.

[0185] The microchip 150 is equipped with a sample fluid inlet 151 and a sheath fluid inlet 153. From these inlets, a sample fluid containing cells and a sheath fluid without cells are introduced into the sample fluid channel 152 and the sheath fluid channel 154, respectively.

[0186] The microchip 150 has a flow channel structure in which the sample channel 152 through which the sample liquid flows and the sheath liquid channel 154 through which the sheath liquid flows merge at a confluence 162 to form a main channel 155. The sample liquid and the sheath liquid merge at the confluence 162 to form a laminar flow in which, for example, the sample liquid is surrounded by the sheath liquid. A schematic diagram of the formation of this laminar flow is shown in Figure 14B. As shown in the figure, the laminar flow is formed by the sheath liquid introduced from the sheath liquid channel 154, surrounded by the sample liquid introduced from the sample channel 152. Preferably, the cells are arranged in a nearly single line in the laminar flow. For example, as shown in the figure, the cells P may be arranged in a nearly single line in the sample solution. Thus, in this disclosure, the flow channel structure forms a laminar flow containing cells flowing in a nearly single line.

[0187] The laminar flow flows through the main channel 155 toward the particle sorting section 157. Preferably, the cells flow in a single line within the main channel 155. This makes it easier to distinguish between the light generated by the irradiation of one microparticle and the light generated by the irradiation of other microparticles in the detection region 156 described below.

[0188] (Discrimination process)

[0189] In the discrimination step S202, it is determined whether the cells flowing through the main channel 155 are particles to be recovered. This discrimination can be performed by the discrimination unit 195. The discrimination unit 195 can perform this discrimination based on the light generated by the light irradiation unit 191 on the cells. An example of the discrimination step S202 will be described in more detail below.

[0190] In the discrimination step S202, the light irradiation unit 191 irradiates cells flowing in the main channel 155 (particularly the detection region 156) of the microchip 150 with light (e.g., excitation light), 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 included in the control unit 193 determines whether the biological particles are to be recovered. For example, the discrimination unit 195 can perform determination based on scattered light, determination based on fluorescence, or determination based on an image (e.g., one or more of dark-field images, bright-field images, and phase-contrast images). In the recovery step S203 described later, the control unit 193 controls the flow in the microchip 150 so that the particles to be recovered are recovered into the recovery channel 159.

[0191] The light irradiation unit 191 irradiates cells flowing through the channels in the microchip 150 with light (e.g., excitation light). The light irradiation unit 191 may include a light source that emits light and an objective lens that focuses the excitation light onto minute particles flowing through the detection area. The light source may be appropriately selected by those skilled in the art depending on the purpose of the analysis, and may be, for example, a laser diode, SHG laser, solid-state laser, gas laser, high-brightness LED, or 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.

[0192] (Recovery process)

[0193] In the recovery step S203, cells identified as target particles in the discrimination step S202 are recovered into the recovery channel 159. In the recovery step S203, the target particles are recovered into a second liquid in the recovery channel, which is immiscible with the first liquid, while still contained in the first liquid. This allows the formation of an emulsion in the recovery channel 159 with the second liquid as the dispersion medium and the first liquid as the dispersed phase, and each emulsion particle in the emulsion contains one target particle. This isolates the target cells within the space of the emulsion particles. For example, as shown in Figure 14B, the target particle P is recovered into the second liquid (shown in gray) while contained in the first liquid (shown in white). This forms emulsion particles 190, and one target particle P is isolated in the space within each emulsion particle 190. The recovery process will be explained in more detail below.

[0194] The recovery process S203 is performed in the particle sorting section 157 of the microchip 150. In the particle sorting section 157, the laminar flow that has flowed through the main channel 155 splits and flows into two waste channels 158. The particle sorting section 157 shown in Figure 14A has two waste channels 158, but the number of branch channels is not limited to two. The particle sorting section 157 may be provided with, for example, one or more (e.g., two, three, or four) branch channels. The branch channels may be configured to branch in a Y-shape on a single plane, as shown in Figure 14A, or they may be configured to branch in three dimensions.

[0195] In the particle sorting section 157, a flow is formed from the main channel 155 through the connecting channel 170 to the recovery channel 159 only when particles to be recovered are flowing in, and the particles to be recovered are recovered into the recovery channel 159. An enlarged view of the particle sorting section 157 is shown in Figure 15. As shown in A of the same figure, the main channel 155 and the recovery channel 159 are connected via the connecting channel 170, which is coaxial with the main channel 155. As shown in B of the same figure, the particles to be recovered flow through the connecting channel 170 to the recovery channel 159. Fine particles that are not to be recovered flow to the waste channel 158, as shown in C of the same figure.

[0196] An enlarged view of the vicinity of the connection channel 170 is shown in FIGS. 18A and 18B. FIG. 18A is a schematic perspective view of the vicinity of the connection channel 170. FIG. 18B is a schematic cross-sectional view in a plane passing through the center lines of the liquid supply channel 161 and the connection channel 170. The connection channel 170 includes a channel 170a on the detection region 156 side (hereinafter also referred to as the upstream connection channel 170a), a channel 170b on the recovery channel 159 side (hereinafter also referred to as the downstream connection channel 170b), and a connection portion 170c between the connection channel 170 and the liquid supply channel 161. The liquid supply channel 161 is provided so as to be substantially perpendicular to the axis of the channel of the connection channel 170. In FIGS. 18A and 18B, two liquid supply channels 161 are provided so as to face each other at a substantially central position of the connection channel 170, but only one liquid supply channel may be provided.

[0197] From the two liquid supply channels 161, the second liquid is supplied to the connection channel 170 as indicated by the arrows in FIG. 18B. The second liquid flows from the connection portion 170c into both the upstream connection channel 170a and the downstream connection channel 170b.

[0198] When the recovery process is not performed, the second liquid flows as follows. The second liquid that has flowed into the upstream connection channel 170a exits from the connection surface with the main channel 155 of the connection channel 170 and then flows separately into the two waste channels 158. By the second liquid exiting from the connection surface, it is possible to prevent the first liquid and fine particles that do not need to be recovered from entering the recovery channel 159 through the connection channel 170. The second liquid that has flowed into the downstream connection channel 170b flows into the recovery channel 159. As a result, the inside of the recovery channel 159 is filled with the second liquid, and the second liquid serves as a dispersion medium for, for example, emulsion formation.

[0199] Even when the recovery process is performed, the second liquid can be supplied from the two liquid supply channels 161 to the connection channel 170. However, due to pressure fluctuations in the recovery channel 159, particularly by generating a negative pressure in the recovery channel 159, a flow is formed that flows from the main channel 155 through the connection channel 170 to the recovery channel 159. That is, a flow is formed that flows from the main channel 155 through the upstream connection channel 170a, the connection portion 170c, and the downstream connection channel 170b in this order to the recovery channel 159. As a result, the particles to be recovered are recovered into the second liquid in the recovery channel 159 while being wrapped in the first liquid. By performing the recovery process, an emulsion, for example, can be formed in the recovery channel 159 or in a container connected to the end of the recovery channel 163, for example, via a channel.

[0200] In the recovery step S20叁, due to pressure fluctuations in the recovery channel 159, the particles to be recovered are recovered into the recovery channel through the connection channel. The recovery may be performed, for example, as described above, by generating a negative pressure in the recovery channel 159. The negative pressure can be generated, for example, by a wall defining the recovery channel 159 being deformed by an actuator 197 (particularly a piezo actuator) attached outside the microchip 150. The negative pressure can form the flow entering the recovery channel 159. To generate the negative pressure, the actuator 197 can be attached outside the microchip 150 so that, for example, the wall of the recovery channel 159 can be deformed. By the deformation of the wall, the internal space of the recovery channel 159 changes, and a negative pressure can be generated. The actuator 197 can be, for example, a piezo actuator. When the particles to be recovered are sucked into the recovery channel 159, the sample liquid constituting the laminar flow or the sample liquid and the sheath liquid constituting the laminar flow can also flow into the recovery channel 159. In this way, the particles to be recovered are sorted in the particle sorting unit 157 and recovered into the recovery channel 159. た通り、回収流路159内に負圧を発生させることによって行われてよい。当該負圧は、例えばマイクロチップ150の外部に取り付けられているアクチュエータ197(特にはピエゾアクチュエータ)により、回収流路159を規定する壁が変形されることにより生じうる。当該負圧によって、回収流路159へ入る当該流れが形成されうる。当該負圧を発生させるために、例えば、回収流路159の壁を変形させることができるように、アクチュエータ197がマイクロチップ外部に取り付けられうる。当該壁の変形によって、回収流路159の内空が変化されて、負圧が発生されうる。アクチュエータ197は、例えばピエゾアクチュエータでありうる。回収対象粒子が回収流路159へと吸い込まれる際には、前記層流を構成するサンプル液又は前記層流を構成するサンプル液及びシース液も、回収流路159へと流れうる。このようにして、回収対象粒子は、粒子分取部157において分取されて、回収流路159へと回収される。

[0201] The particles to be recovered are encapsulated in the first liquid and then recovered into a second liquid, which is immiscible with the first liquid, within the recovery channel 159. As a result, as described above, an emulsion is formed within the recovery channel 159, with the second liquid as the dispersion medium and the first liquid as the dispersed phase.

[0202] To prevent biological particles that are not to be recovered from entering the recovery channel 159 through the connecting channel 170, the connecting channel 170 is equipped with a liquid supply channel 161. A second liquid, which is immiscible with the liquid (sample liquid and sheath liquid) flowing through the main channel 155, is introduced into the connecting channel 170 from the liquid supply channel 161. A portion of the second liquid introduced into the connecting channel 170 forms a flow from the connecting channel 170 toward the main channel 155, preventing biological particles other than those to be recovered from entering the recovery channel 159. The second liquid formed by the flow from the connecting channel 170 toward the main channel 155 flows through the waste channel 158, just like the first liquid, without flowing through the main channel 155, due to the flow of the first liquid flowing through the main channel 155 toward the waste channel 158. Furthermore, any remaining second liquid introduced into the connecting channel 170 flows into the recovery channel 159. As a result, the recovery channel 159 can be filled with the second liquid.

[0203] The recovery channel 159 may be filled with a second liquid that is immiscible with the first liquid. In order to fill the recovery channel 159 with the second liquid, the second liquid may be supplied from the liquid supply channel 161 to the connecting channel 170. Upon this supply, the second liquid flows from the connecting channel 170 to the recovery channel 159, thereby filling the recovery channel 159 with the second liquid.

[0204] The laminar flow that flows into the waste channel 158 can be discharged outside the microchip at the end of the waste channel 160. Furthermore, the particles to be recovered that are collected into the recovery channel 159 can be discharged outside the microchip at the end of the recovery channel 161.

[0205] A container (not shown) may be connected to the end 163 of the recovery channel via a channel such as a tube. An emulsion in which the first liquid containing the particles to be recovered is used as the dispersion phase and the second liquid as the dispersion medium is recovered into the container. Thus, the biological particle sorting device may be equipped with a channel for recovering the emulsion containing the particles to be recovered into a container. Furthermore, by closing the end 163 of the recovery channel and performing the recovery operation, multiple emulsion particles can be retained within the recovery channel 159. After the completion of the recovery operation, assays such as single-cell analysis can be performed continuously within the recovery channel 159. For example, the disruption step described later may be performed within the recovery channel 159. In conjunction with the disruption step, binding of the target capture molecule to the target substance may occur.

[0206] As shown in Figure 14A, the microchip may have, for example, two or more inlets and / or outlets, preferably all inlets and / or outlets, formed on a single surface. As shown in the same figure, both the recovery channel end 163 and the two branch channel ends 160 are formed on the same surface as the sample liquid inlet 151 and the sheath liquid inlet 153. Furthermore, the introduction channel inlet 164 for introducing liquid into the introduction channel 161 is also formed on the same surface. In this way, all of the inlets for introducing liquid and the outlets for discharging liquid are formed on a single surface of the microchip. This facilitates the attachment of the chip to the biological particle sorting device. In the same figure, a portion of the sheath liquid channel 154 is shown with a dotted line. This portion is located at a lower position (shifted in the direction of the optical axis indicated by the arrow) than the sample liquid channel 152 shown with a solid line, and it is the point where the dotted channel and the solid channel intersect; these channels are not connected. This explanation also applies to a portion of the recovery channel 159 shown with a dotted line and the branch channel 158 that intersects with this portion.

[0207] Furthermore, the liquid supply channel supplies liquid (particularly the second liquid) to the connecting channel. This creates a flow within the connecting channel that flows from the connection point between the liquid supply channel and the connecting channel toward the main channel, preventing the liquid flowing in the main channel from entering the connecting channel and preventing fine particles other than the target particles from flowing through the connecting channel into the recovery channel. When performing the recovery process, as described above, for example, the negative pressure generated in the recovery channel causes the first liquid containing one target particle to be recovered through the connecting channel and into the second liquid in the recovery channel. As a result, emulsion particles containing one target particle are formed in the second liquid.

[0208] Furthermore, when a biological particle determined to be a particle to be recovered in the determination step is driven, for example, by activating a piezo actuator at an appropriate timing (for example, when it reaches the particle sorting unit 157), the hydrophilic solution containing the particle to be recovered is recovered into the recovery channel 159, and emulsion particles are formed. In the determination step, by using, for example, peak signals and area signals to determine whether a particle is a particle to be recovered, it is also possible to determine whether it is a single microparticle (singlet), a doublet (two biological particles bound together), or a triplet (three biological particles bound together). Therefore, it is possible to avoid the formation of emulsion particles containing two or more cells in a single emulsion particle. As a result, emulsion particles containing one cell can be formed with high probability and high efficiency. In addition, since it is possible to avoid the formation of emulsion particles containing two or more bound cells in this way, it is possible to omit the operation of removing bounds of two or more cells before the emulsion formation operation, for example, by using a cell sorter.

[0209] (4-2) Variations of single-cell analysis In single-cell analysis using the analytical method according to this disclosure, the process for generating cyclic nucleic acids used in the analytical method is not particularly limited, as long as it generates cyclic nucleic acids by linking two or more complementary strands generated by the complementary nucleic acid generation step described above via nucleic acid linkers, and any combination of methods can be used.

[0210] Figure 6D shows a modified version of the single-cell analysis, where the cyclic nucleic acid generation process in the single-cell analysis shown in Figure 5 is performed using the first modified version of the cyclic nucleic acid generation process (2-3). However, the nucleic acid ligator used in the cyclic nucleic acid generation process in Figure 6D is the same as the nucleic acid ligator shown in Figure 6E. The nucleic acid ligator shown in Figure 6E differs from the nucleic acid ligator shown as <2D-α> used in the explanation of the first modified version of the cyclic nucleic acid generation process (2-3) in that it has a sequence addition region (cell barcode in the figure) between the target nucleic acid capture region (poly-T in the figure) and the complementary strand capture region (concatenator in the figure).

[0211] In step S610 of Figure 6D, similar to step S42 in Figure 5 relating to the aforementioned single-cell analysis, each cell released from the substrate is isolated in a microspace while the nucleic acid ligator remains attached via the cell capture unit. Subsequently, each cell is destroyed in this isolated state. These conditions are the same as those applicable to the aforementioned single-cell analysis and can be suitably applied in this modified example. In this case, the binding of the nucleic acid ligator via the cell capture unit may be direct, or, as shown in Figure 6D, the nucleic acid ligator may be bound to the cell capture unit via a nucleic acid having a sequence complementary to the sequence of the nucleic acid ligator. When the nucleic acid ligator is bound to the cell capture unit via a nucleic acid having a sequence complementary to the sequence of the nucleic acid ligator, steric hindrance of the cell capture unit in the subsequent cyclic nucleic acid generation process can be avoided.

[0212] Following the aforementioned disruption, as shown in step S611 of Figure 6D, the target nucleic acid capture portion of the nucleic acid ligator shown in Figure 6F captures the 3' end of the mRNA in the disrupted cell. The subsequent steps are the same as those in the first modified example of the circular nucleic acid production process (2-3), and the same conditions that can be applied to the said circular nucleic acid production process can be suitably applied to this modified example as well.

[0213] As an example of a modified single-cell analysis, a method in which the cyclic nucleic acid generation process is carried out by the first modified cyclic nucleic acid generation process (2-3) is shown. However, this is merely an example, and in single-cell analysis using the analytical method according to this disclosure, a method can be suitably used as the cyclic nucleic acid generation process, including the example disclosed herein, in which a cyclic nucleic acid is generated by linking two or more complementary strands produced by a complementary nucleic acid generation process via a nucleic acid linker.

[0214] (4-3) Examples of applications of single-cell analysis Single-cell mRNA expression analysis using the analytical methods described herein can be used in combination with any other analysis, provided that the desired analytical characteristics are not significantly impaired.

[0215] For example, by using an antibody-nucleic acid molecule complex in which an antibody is bound to a nucleic acid molecule having an antibody identifier sequence (Antibody barcode) that identifies the origin of the antibody, and a poly(A) sequence that is captured by the target nucleic acid capture site of the nucleic acid ligator, as shown in Figure 14C, it is possible to analyze intracellular mRNA simultaneously with analyzing molecules expressed within cells, molecules expressed on the cell surface, or secreted molecules captured on the cell surface. This will be explained in detail below with reference to the figures. Note that the nucleic acid molecule in the antibody-nucleic acid molecule complex described above may also have a priming sequence (Amp. Primer) in addition to the antibody identifier sequence and poly(A) sequence, as in the example shown in Figure 14C. Furthermore, any nucleic acid ligator disclosed herein can be suitably used as the nucleic acid ligator used in this application example.

[0216] In the complex of the antibody-nucleic acid molecule shown in FIG. 14C, as the antibody comprised in the complex, an antibody that specifically binds to a molecule expressed intracellularly, a molecule expressed on the cell surface, or a secreted molecule captured on the cell surface is used. Further, as the antibody identifier sequence possessed by the nucleic acid molecule comprised in the complex, a sequence unique to each antibody is provided. That is, when there are a plurality of secreted molecules on the cell surface to be analyzed, a plurality of types of antibody-nucleic acid molecule complexes are adjusted according to the types thereof. Furthermore, the antibody identifier sequence may have the error correction function described above. In FIG. 14, an example of using the antibody-nucleic acid molecule complex was shown, but what forms a complex with the nucleic acid molecule is not limited to an antibody as long as it has a function of binding to a molecule expressed intracellularly, a molecule expressed on the cell surface, or a secreted molecule captured on the cell surface. For example, antibody fragments (such as Fab, scFv, VHH, Minobody, etc.), aptamers, molecular imprint polymers, etc. can be used. In this application example, complexes in which these are bound to nucleic acid molecules may also be used.

[0217] The above-adjusted antibody-nucleic acid molecule complex binds, by an antigen-antibody reaction, to cells on which the antigenic molecule is surface-expressed or the secreted molecule is surface-captured. On the other hand, when the antigenic molecule is not intracellularly expressed, surface-expressed, and not captured, the antibody-nucleic acid molecule complex does not bind to the cell.

[0218] By subjecting the cell to which the nucleic acid ligation part is bound via the above-described cell capture part to an antigen-antibody reaction using the above-adjusted antibody-nucleic acid molecule complex, cells to which an antibody corresponding to a molecule expressed intracellularly, a molecule expressed on the cell surface, or a secreted molecule captured on the surface is bound can be obtained.

[0219] Next, the cells that have undergone the antigen-antibody reaction are isolated into microspaces. FIG. 14D shows an example of isolating cells into microspaces using a chip for forming an emulsion, but the method is not limited thereto, and any method of isolating cells into microspaces, including the examples disclosed in this specification, can be preferably employed.

[0220] In the example shown in Figure 14D, a single antibody-nucleic acid molecule complex is bound to a cell that has a nucleic acid linkage site attached via a cell capture site, as a cell isolated in the space within the emulsion particle. However, multiple antibody-nucleic acid molecule complexes may also be bound to a single cell. Furthermore, as shown in Figure 14D, the conditions for isolating cells subjected to an antigen-antibody reaction in the space within the emulsion particle are the same conditions that can be applied using the method described above (in the case of space within emulsion particles), and these conditions can be suitably applied in this application example as well.

[0221] Next, each cell is destroyed under conditions similar to those applicable to the single-cell analysis or its modified form described above.

[0222] Following the aforementioned disruption, as shown in step S1411 of Figure 14E, the target nucleic acid capture portion of the nucleic acid ligator captures the 3' polyA sequence of mRNA in the disrupted cell or the 3' polyA sequence of the nucleic acid molecule in the antibody-nucleic acid molecule complex. The subsequent steps are the same as those for the (2-2) circular nucleic acid generation process, through which 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. The same conditions that can be applied to the circular nucleic acid generation process can also be suitably applied in this application example.

[0223] The single-stranded circular nucleic acid obtained as described above contains, in addition to the sequence derived from mRNA, the antibody identifier sequence of the nucleic acid molecule in the antibody-nucleic acid molecule complex. Therefore, by decoding the base sequence of the single-stranded linear nucleic acid obtained using the single-stranded circular nucleic acid as a template, it is possible to analyze the expression and secretion status of the antigen molecule to which the antibody identified by the antibody identifier sequence binds. In other words, in addition to analyzing the expression of mRNA in cells isolated in a microspace, it is possible to simultaneously analyze the expression and secretion status of molecules expressed and secreted on the surface of those cells.

[0224] Furthermore, assigning antibody identifier sequences with different base sequence lengths for each antibody can make it easier to identify the antibody identifier sequence. In this case, the base sequence of the antibody identifier sequence may be adjusted to a range of, for example, 5 to 25 bases.

[0225] Furthermore, the sequencing of the obtained single-stranded linear nucleic acid can be suitably performed using the same method as that used in the (2-2) circular nucleic acid generation process described above.

[0226] Furthermore, as an example of single-cell analysis application, a method for generating cyclic nucleic acids using the (2-2) cyclic nucleic acid generation process is shown. However, this is merely an example, and in single-cell analysis using the analytical method according to this disclosure, a method for generating cyclic nucleic acids, including the example disclosed herein, can be suitably used, which involves linking two or more complementary strands generated by a complementary nucleic acid generation process via a nucleic acid linker.

[0227] (5) Example 4 (Phosphorylation modification of nucleic acid ligatures)

[0228] Of the two base sequence strands constituting the double-stranded portion of the nucleic acid ligation region, the 5' end of the strand connected to the target nucleic acid capture region (particularly the strand containing poly(T)) may be phosphorylated. This allows for more reliable execution of the ligation process described later. This will be explained with reference to Figure 7.

[0229] As shown in Figure A, cells are captured by nucleic acid binding sites immobilized on the substrate.

[0230] In step S51, as explained above with respect to Figure 5, the cells to which the nucleic acid linkages have been attached are released from the substrate and isolated within a microspace (e.g., emulsion particles). While isolated in this state, the cells are destroyed. As a result, mRNA is released into the microspace, as shown in B of the same figure.

[0231] In step S52, mRNA released into the microspace by cell disruption binds to nucleic acid junctions (outlined by dashed lines), generating a hybrid as shown in C in the figure. This hybrid is generated by the complementary binding of the target nucleic acid capture region (particularly the poly-T sequence) of the nucleic acid junction and the poly-A tail of the mRNA.

[0232] Here, the 5' terminal P of the strand of the nucleic acid ligation region that is connected to the target nucleic acid capture region (particularly the strand containing poly(T)) is phosphorylated. In the figure, phosphorylation is indicated by a circular shape. This phosphorylation is useful for ensuring more reliable execution of the subsequent ligation step.

[0233] Step S53 is the same cDNA synthesis as step S12 in Figure 2A described above. The explanation for step S12 also applies to step S53. By performing step S53, cDNA having a CCC sequence at the end of the generated strand is produced, as shown in Figure 7D.

[0234] Step S54 is the same nucleic acid ligation as step S13 in Figure 2A described above. The explanation for step S13 also applies to step S54. By performing step S54, two or more cDNAs are linked via the complementary strand capture region, as shown in Figure 7E.

[0235] Step S55 is the same cyclic nucleic acid formation, ligation, and primer addition as steps S14 and S15 in Figure 2A described above. The explanation for steps S14 and S15 also applies to step S55. By performing step S55, a primer-attached cyclic nucleic acid is formed, as shown in Figure 7F. As described above, the phosphorylation of the 5' end of the nucleic acid ligation site ensures that the ligation is carried out more reliably. This allows for more efficient formation of cyclic nucleic acids.

[0236] Step S56 is the same nucleic acid amplification as step S16 in Figure 2A described above. The explanation for step S16 also applies to step S56. By performing step S56, the nucleic acid is amplified as shown in Figure 7G. The nucleic acid thus amplified is used in single-cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed using the sequencing results.

[0237] (6) Example 5 (Nucleic acid linkage region including self-binding inhibitory sequence)

[0238] The complementary strand capture region of the nucleic acid linkage is not limited to the GGG sequence, and may include a self-binding inhibiting sequence (e.g., Hn and / or Nn) in addition to GGG. Here, H is a base other than G, i.e., A, T, or C. N is A, T, G, or C. n is the number of H or N, and may be an integer of 1 or more, for example. n may be an integer of any of 1 to 5, for example. When n is 2 or more, each H or N constituting the self-binding inhibiting sequence may be selected independently and randomly from each other. For example, if the complementary strand capture region is a nucleotide sequence that has a GGG sequence in addition to a single-nucleotide self-binding inhibiting sequence, the complementary strand capture region may have the nucleotide sequence GGGH or GGGN. If the self-binding inhibiting sequence consists of two nucleotides, the complementary strand capture region may have the nucleotide sequence GGGHN, GGGNH, GGGNN, or GGGHH. An example of cyclic nucleic acid formation in cases where such a complementary chain capture region is present is shown in Figure 8.

[0239] As shown in Figure A, cells are captured by nucleic acid binding sites immobilized on the substrate.

[0240] In step S61, as explained above with respect to Figure 5, the cells to which the nucleic acid linkages have been bound are released from the substrate and isolated within a microspace (e.g., emulsion particles). While isolated in this state, the cells are destroyed. As a result, mRNA is released into the microspace, as shown in B of the same figure.

[0241] In step S62, mRNA released into the microspace by cell disruption binds to the nucleic acid junction, generating a hybrid as shown in C in the figure. This hybrid is generated by the complementary binding of the target nucleic acid capture region (particularly the poly-T sequence) of the nucleic acid junction and the poly-A tail of the mRNA.

[0242] Furthermore, the 5' end of the strand connected to the target nucleic acid capture site (particularly the strand containing poly(T)) of the two base sequence strands in the nucleic acid ligation region is phosphorylated. In the figure, phosphorylation is indicated by a circular shape. This phosphorylation is useful for ensuring more reliable execution of the subsequent ligation step.

[0243] Step S63 is the same cDNA synthesis as step S12 in Figure 2A described above. The explanation for step S12 also applies to step S63. By performing step S63, a cDNA having a CCC sequence at the end of the generated strand is produced, as shown in Figure 8D.

[0244] In step S64, RNA digestion and nucleic acid ligation take place. As a result, mRNA is digested, as shown in E in the figure, and a single-stranded portion is formed. The sequence H* at the 5' end of the 3' CCC sequence at the 3' end of this single-stranded portion selects the cDNA to be captured by the complementary strand capture region 52. That is, because of the presence of H*, not all cDNA is captured by the complementary strand capture region; for example, if the complementary strand capture region is GGGH-3', it captures cDNA containing 3'-CCCH*. Since there are four types of H (ATGC), the probability of circularization by only one cDNA is 1 / 4. As the n of Hn (where n is an integer greater than or equal to 1) increases, this probability decreases, becoming (1 / 4). n This results in the production of nucleic acids in which two or more cDNAs are linked together via nucleic acid linkage sites. In this way, it is possible to prevent the formation of circular nucleic acids containing only one cDNA. That is, it is possible to generate more circular nucleic acids in which two or more cDNAs are linked together.

[0245] In step S65, cyclic nucleic acid formation and ligation take place. This generates cyclic nucleic acids as shown in Figure 8F. As described above, the phosphorylation of the 5' end of the nucleic acid ligation site ensures that the ligation is carried out more reliably. This allows for more efficient formation of cyclic nucleic acids.

[0246] Step S66 is the same nucleic acid amplification as step S16 in Figure 2A described above. The explanation for step S16 also applies to step S66. By performing step S66, the nucleic acid is amplified as shown in Figure 8G. The thus amplified nucleic acid is used in single-cell analysis. For example, the nucleic acid sequence is sequenced, and each cell is analyzed using the sequencing results.

[0247] (7) Example 6 (Nucleic acid ligation region including priming sequence)

[0248] As described above, the double-stranded portion of the nucleic acid junction may have different base sequences in each region, for example, a random sequence. In addition to the random sequence, the double-stranded portion may have a priming sequence. The priming sequence may be the same in two or more regions, for example, nucleic acid junctions present in all regions may have the same priming sequence. This allows nucleic acid amplification to be performed using a common priming sequence. Therefore, it is not necessary to add primers before the nucleic acid amplification process. This will be explained with reference to Figure 9.

[0249] As shown in Figure A, cells are captured by nucleic acid binding sites immobilized on the substrate.

[0250] In step S71, as explained above with respect to Figure 5, the cells to which the nucleic acid linkages have been attached are released from the substrate and isolated within a microspace (e.g., emulsion particles). While isolated in this state, the cells are destroyed. As a result, mRNA is released into the microspace, as shown in B of the same figure.

[0251] In step S72, mRNA released into the microspace by cell disruption binds to the nucleic acid junction, generating a hybrid as shown in Figure 9C. This hybrid is generated by the complementary binding of the target nucleic acid capture region (particularly the poly-T sequence) of the nucleic acid junction and the poly-A tail of the mRNA. The nucleic acid junction has a priming sequence in its double-stranded portion.

[0252] Furthermore, the 5' end of the strand connected to the target nucleic acid capture site (particularly the strand containing poly(T)) of the two base sequence strands in the nucleic acid ligation region is phosphorylated. In the figure, phosphorylation is indicated by a circular shape. This phosphorylation is useful for more reliable execution of the subsequent ligation step. Note that phosphorylation modification is not required.

[0253] Step S73 is the same cDNA synthesis as step S12 in Figure 2A described above. The explanation for step S12 also applies to step S73. By performing step S73, cDNA having a CCC sequence at the end of the generated strand is produced, as shown in Figure 9D.

[0254] Step S74 is the same nucleic acid ligation as step S13 in Figure 2A described above. The explanation for step S13 also applies to step S74. By performing step S54, two or more cDNAs are linked via the complementary strand capture region, as shown in Figure 9E.

[0255] Step S75 is the same cyclic nucleic acid formation and ligation as step S14 in Figure 2A described above. The explanation for step S14 also applies to step S75. By performing step S75, a primer-attached cyclic nucleic acid is formed, as shown in Figure 9F. As described above, the phosphorylation of the 5' end of the nucleic acid ligation site ensures that the ligation is carried out more reliably. This allows for more efficient formation of cyclic nucleic acids. Furthermore, although primer addition is performed in the method described with respect to Figure 2A, primer addition is not necessary because the nucleic acid ligation site has a priming sequence as described above.

[0256] Step S76 is the same nucleic acid amplification as step S16 in Figure 2A described above. The explanation for step S16 also applies to step S76. By performing step S76, the nucleic acid is amplified as shown in Figure 9G. The nucleic acid thus amplified is used in single-cell analysis. For example, the sequence of the nucleic acid is sequenced, and each cell is analyzed using the sequencing results.

[0257] Figure 19 is a schematic diagram showing an example of adding a priming sequence to a nucleic acid ligament. In the example shown in Figure 19, it can be seen that the nucleic acid ligament shown in <19A> does not have a priming sequence, while the nucleic acid ligament shown in <19B> has a priming sequence added to the double-stranded portion. Although the example shown in Figure 19 shows the addition of a priming sequence to a nucleic acid ligament with a double-stranded portion, the priming sequence can also be added to a nucleic acid ligament that does not have a double-stranded portion.

[0258] (8) Example 7 (Nucleic acid linkage including self-binding inhibitory sequence and priming sequence)

[0259] The nucleic acid linking region may include both the self-binding inhibiting sequence and the priming sequence described above. This suppresses the formation of a circular nucleic acid having only one cDNA and eliminates the need for a primer addition step before nucleic acid amplification. An example of circular nucleic acid formation in the case of such a complementary strand capture region is shown in Figure 10.

[0260] As shown in Figure A, cells are captured by nucleic acid binding sites immobilized on the substrate.

[0261] In step S81, as explained above with respect to Figure 5, the cells to which the nucleic acid linkages have been attached are released from the substrate and isolated within a microspace (e.g., emulsion particles). While isolated in this state, the cells are destroyed. As a result, mRNA is released into the microspace, as shown in B of the same figure.

[0262] In step S82, mRNA released into the microspace by cell disruption binds to the nucleic acid junction, generating a hybrid as shown in C in the figure. This hybrid is generated by the complementary binding of the target nucleic acid capture region (particularly the poly-T sequence) of the nucleic acid junction and the poly-A tail of the mRNA. The nucleic acid junction has a priming sequence in its double-stranded portion.

[0263] Furthermore, the 5' end of the strand connected to the target nucleic acid capture site (particularly the strand containing poly(T)) of the two base sequence strands in the nucleic acid ligation region is phosphorylated. In the figure, phosphorylation is indicated by a circular shape. This phosphorylation is useful for ensuring more reliable execution of the subsequent ligation step.

[0264] Step S83 is the same cDNA synthesis as step S12 in Figure 2A described above. The explanation for step S12 also applies to step S83. By performing step S83, cDNA having a CCC sequence at the end of the generated strand is produced, as shown in Figure 10D.

[0265] In step S84, RNA digestion and nucleic acid ligation occur. As a result, the mRNA is digested, as shown in E in the figure, and a single-stranded portion is formed. An arbitrary base H* is also exposed before the 3' CCC sequence at the 3' end of the single-stranded portion. That is, a CCCH* sequence is present at the 3' end of the single-stranded portion. On the other hand, since the nucleic acid ligation region is DNA, it is not digested in the RNA digestion. As a result, the double-stranded portion and GGGH that were connected to the digested mRNA remain. Then, GGGH binds to the complementary CCCH*. This generates a nucleic acid in which two or more cDNAs are ligated together via the nucleic acid ligation region. In this way, it is possible to prevent the formation of circular nucleic acids containing only one cDNA. That is, it is possible to generate more circular nucleic acids in which two or more cDNAs are linked together.

[0266] In step S85, cyclic nucleic acid formation and ligation take place. This generates cyclic nucleic acids as shown in Figure 8F. As described above, the phosphorylation of the 5' end of the nucleic acid ligation site ensures that the ligation is carried out more reliably. This allows for more efficient formation of cyclic nucleic acids. Furthermore, although primer addition is performed in the method described with respect to Figure 2A, primer addition is not necessary because the nucleic acid ligation site has a priming sequence as described above.

[0267] Step S86 is the same nucleic acid amplification as step S16 in Figure 2A described above. The explanation for step S16 also applies to step S66. By performing step S86, the nucleic acid is amplified as shown in Figure 10G. The thus amplified nucleic acid is used in single-cell analysis. For example, the nucleic acid sequence is sequenced, and each cell is analyzed using the sequencing results.

[0268] (9) Example 8 (Example of sequence result analysis)

[0269] In the analytical method according to this disclosure, a nucleic acid amplification process is performed using a circular nucleic acid as a template. Here, the circular nucleic acid has sequences corresponding to two or more target nucleic acids ligated together. For example, two or more cDNAs complementary to mRNA are ligated together. Therefore, the product obtained in the nucleic acid amplification process has repeating units having sequences corresponding to two or more target nucleic acids.

[0270] For example, as shown in Figure 11a, if a single circular nucleic acid is formed and has two cDNAs, RCA treatment can be applied to the circular nucleic acid to obtain a nucleic acid in which repeating units formed by the linkage of these two cDNAs appear repeatedly. Furthermore, if a single circular nucleic acid is formed and contains three cDNAs, RCA treatment can be applied to the circular nucleic acid to obtain a nucleic acid in which repeating units, each consisting of three linked cDNAs, appear repeatedly. As shown in Figure b, even when the nucleic acid linkage portion has a self-binding inhibiting sequence, the product obtained in the nucleic acid amplification process has repeating units having sequences corresponding to two or more target nucleic acids. In the analytical methods pursuant to this disclosure, analysis may be performed based on the repeating units. For example, analysis may be performed based on the types and / or number of target nucleic acid sequences contained in the repeating units. The nucleic acid amplification process described above reduces the bias mentioned earlier. Therefore, analytical results that appropriately consider target nucleic acids, which are present in small quantities within cells, can be obtained.

[0271] (10) Example 9 (Example of the generation of circular nucleic acids and the amplification process of the generated circular nucleic acids)

[0272] Nucleic acid ligators having different base sequences are arranged in an arrayed region on the substrate as shown in Figure 5. The double-stranded portions of the nucleic acid ligators have a different random arrangement for each region, as well as a common sequence (for example, the priming sequence described above) in all regions. This common sequence may be used as a priming sequence in the nucleic acid amplification process as described above, or it may be used for binding to the cell capture portion.

[0273] The nucleic acid ligator is manufactured using the synthesis technology utilizing the oligopool described above. Specifically, oligonucleotides with various different base sequences are synthesized together, and then PCR is performed on these oligonucleotides to obtain a large quantity of nucleic acid ligators with different base sequences at low cost. More specifically, in vitro transcription (IVT) and reverse transcription (RT) are performed after the PCR process. Furthermore, a cell capture site, such as an antibody or lipid, is attached to the end of the nucleic acid ligator.

[0274] Cell capture is performed using a substrate on which the above-mentioned nucleic acid linkers are arrayed. After cell capture, the cells are released from the substrate using restriction enzymes, and each cell is encapsulated in a water-in-oil droplet. Cell lysis and mRNA capture by poly-T sequences are performed within the droplet. For the aforementioned cell lysis, dissolution reagents such as NP-40 Surfactant-Amps Detergent solution or IGEPAL CA-630 are used.

[0275] After mRNA capture, a reverse transcription reaction is performed to synthesize the cDNA of the mRNA. This reverse transcription reaction is carried out at 50°C for 50 minutes in the presence of dNTPs, 1x Thermopol buffer (or 1x RT buffer), SuperScript III, and RNasin plus (or RNase OUT).

[0276] Next, the GGG sequence contained in the nucleic acid ligation region binds complementaryally to the terminal CCC sequence added during cDNA synthesis, thereby ligating two or more cDNAs. After ligation, the RNA is degraded with RNase H or the like, and then ligation is performed in the presence of 1x Ampligase buffer, 50 μM dNTPs, 0.5 U / μL Ampligase, and 50 mM KCl at 37°C for 30 minutes or 45°C for 45 minutes. In this way, a circular nucleic acid is obtained. Formamide may be added to the liquid in which the above ligation is performed at a concentration of approximately 20%.

[0277] RCA is performed using the obtained cyclic nucleic acid. This RCA is carried out at 30°C for 60 minutes in the presence of 1 U / μL Phi29 polymerase, 1x Phi29 polymerase buffer, 0.25 mM dNTPs, 0.2 μg / μL BSA, and 5% glycerol. This yields an amplified product having repeating units complementary to the cyclic nucleic acid. This amplified product is useful for single-cell analysis, and in particular, it provides analytical results that appropriately reflect nucleic acids present in small numbers within cells.

[0278] 2. Second Embodiment (Method for Producing Cyclic Nucleic Acids)

[0279] This disclosure also provides a method for producing a cyclic nucleic acid. The cyclic nucleic acid is the cyclic nucleic acid produced in the analytical method described in Section 1 above. Therefore, the description of the method for producing the cyclic nucleic acid also applies to the method for producing a cyclic nucleic acid according to this disclosure. In one embodiment, the manufacturing method may include a complementary nucleic acid production step of generating complementary strands of one or more target nucleic acids, with nucleic acid linkers attached to one end of each of the target nucleic acids, and a cyclic nucleic acid production step of linking the two or more generated complementary strands via the nucleic acid linkers to form a cyclic nucleic acid. The manufacturing method according to this disclosure can produce cyclic nucleic acids useful for cell analysis. The manufacturing method produces the effects described in 1. above.

[0280] 3. Third Embodiment (Nucleic Acid)

[0281] This disclosure also provides nucleic acids. These nucleic acids correspond to the nucleic acid linkers described in Section 1 above. Therefore, the description of the nucleic acid linkers also applies to nucleic acids according to this disclosure. This disclosure also provides complexes having these nucleic acids. These complexes are complex 100 described in Section 1 above, and the description of them also applies to this embodiment. In one embodiment, the nucleic acid comprises 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 the 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. Preferably, the target nucleic acid capture portion is single-stranded, and the complementary strand capture portion is also single-stranded. The nucleic acid may also be used to generate a cyclic nucleic acid. The nucleic acids provided in this disclosure are useful for generating the cyclic nucleic acids described above, and are also useful for cell analysis. The effects described in section 1 above are achieved by these nucleic acids.

[0282] Furthermore, this disclosure may also take the following form. [1] A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, A cyclic nucleic acid generation step, which involves linking two or more complementary strands that have been generated via the nucleic acid linking portion to form a cyclic nucleic acid, and An analytical step in which an analysis is performed using the aforementioned cyclic nucleic acid, Analytical methods including those mentioned. [2] The analytical method according to [1], wherein the nucleic acid binding portion has a target nucleic acid capture portion configured to capture the 3' terminal region of the target nucleic acid. [3] The analytical method according to [1] or [2], wherein, in the complementary chain generation step, the nucleic acid linkage portion is used as a primer to generate a complementary chain of the target nucleic acid. [4] The analytical method according to any one of [1] to [3], wherein in the complementary strand generation step, a double strand is formed between each target nucleic acid and the complementary strand of each target nucleic acid. [5] The analytical method according to [4], wherein in the cyclic nucleic acid generation step, the double strands are linked via the nucleic acid linking portion. [6] The analytical method according to any one of [1] to [5], wherein the nucleic acid linker has a complementary strand capture portion configured to capture the 3' terminal region of the complementary strand. [7] In the aforementioned cyclic nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand are linked. The ligation is performed with the complementary strand capture portion of the nucleic acid ligation portion attached to one complementary strand bound to the 3' terminal region of the other complementary strand. The analysis method described in any one of [1] to [6]. [8] The analytical method according to any one of [1] to [7], wherein in the cyclic nucleic acid generation step, a double-stranded cyclic nucleic acid is formed, and the target nucleic acid is removed from the double-stranded cyclic nucleic acid to obtain a single-stranded cyclic nucleic acid in which the complementary strands are linked. [9] The analytical method according to any one of [1] to [8], wherein a nucleic acid amplification reaction using the cyclic nucleic acid is performed in the analytical step.

[10] The analytical method according to [9], wherein the nucleic acid amplification reaction is RCA or PCR.

[11] The nucleic acid linking portion is A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary chain capturing unit configured to capture the 3' terminal region of the complementary chain generated in the complementary chain generation step, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, The analytical method described in any one of [1] to

[10] , which has the following characteristics.

[12] The target nucleic acid capture portion has a poly-T sequence, and The complementary chain capture region has a base sequence that is complementary to the base sequence that is added to the 3' end during reverse transcription by reverse transcriptase. The analytical method described in

[11] .

[13] The analytical method according to

[11] or

[12] , wherein the double-stranded portion has a restriction enzyme recognition sequence.

[14] The analytical method according to any one of

[11] to

[13] , wherein the double-stranded portion has a non-natural base sequence.

[15] The analytical method according to any one of

[11] to

[14] , wherein the double-stranded portion has a base sequence that has an error correction function.

[16] The aforementioned analytical method is an analytical method for performing single-cell analysis. A nucleic acid ligator having a different double-stranded portion for each cell is used. The analysis method described in any one of

[11] to

[15] .

[17] The aforementioned analytical method includes a cell disruption step that destroys cells, The complementary nucleic acid generation step is performed on the target nucleic acid contained in the cell. The analysis method described in any one of [1] to

[16] .

[18] The analytical method described in

[17] , wherein the cell disruption step is performed in a space partitioned for each cell.

[19] The nucleic acid linker is immobilized on a substrate, as described in any one of [1] to

[18] .

[20] A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, and A cyclic nucleic acid generation step involves linking two or more generated complementary strands via the nucleic acid linking portion to form a cyclic nucleic acid. A method for producing cyclic nucleic acids, including 〔twenty one〕 A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary strand capture unit configured to capture the 3' terminal region of the complementary strand generated by the formation of a complementary strand of the target nucleic acid, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, Nucleic acids containing [the specified substance]. 〔twenty two〕 The nucleic acid according to

[21] , wherein the target nucleic acid capture portion is single-stranded, and the complementary strand capture portion is single-stranded. 〔twenty three〕 The nucleic acid is the nucleic acid according to

[21] or

[22] , used to produce a cyclic nucleic acid. [Explanation of Symbols]

[0283] 10 Nucleic acid junction 11 Target nucleic acid capture section 12 Complementary chain trapping section 13 Double-stranded section 14. Array addition section 100 complex

Claims

1. A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, and a complementary chain of the one or more target nucleic acids is generated. A cyclic nucleic acid generation step, which involves linking two or more generated complementary strands via the nucleic acid linking portion to form a cyclic nucleic acid, and An analytical step in which an analysis is performed using the aforementioned cyclic nucleic acid, Analytical methods including those mentioned.

2. The analytical method according to claim 1, wherein the nucleic acid binding portion has a target nucleic acid capture portion configured to capture the 3' terminal region of the target nucleic acid.

3. The analytical method according to claim 1, wherein in the complementary chain generation step, the nucleic acid linkage portion is used as a primer to generate a complementary chain of the target nucleic acid.

4. The analytical method according to claim 1, wherein in the complementary strand generation step, a double strand is formed between each target nucleic acid and the complementary strand of each target nucleic acid.

5. The analytical method according to claim 4, wherein in the cyclic nucleic acid generation step, the double strands are linked via the nucleic acid linking portion.

6. The analytical method according to claim 1, wherein the nucleic acid linking portion has a complementary strand capture portion configured to capture the 3' terminal region of the complementary strand.

7. In the cyclic nucleic acid generation step, the 5' end of one complementary strand and the 3' end of another complementary strand are linked. The ligation is performed with the complementary strand capture portion of the nucleic acid ligation portion attached to one complementary strand bound to the 3' terminal region of the other complementary strand. The analytical method according to claim 1.

8. The analytical method according to claim 1, wherein in the cyclic nucleic acid generation step, a double-stranded cyclic nucleic acid is formed, and a target nucleic acid is removed from the double-stranded cyclic nucleic acid to obtain a single-stranded cyclic nucleic acid in which the complementary strands are linked.

9. The analytical method according to claim 1, wherein a nucleic acid amplification reaction using the cyclic nucleic acid is performed in the analytical step.

10. The analytical method according to claim 9, wherein the nucleic acid amplification reaction is RCA or PCR.

11. The nucleic acid linking portion is A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary chain capturing unit configured to capture the 3' terminal region of the complementary chain generated in the complementary chain generation step, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, The analytical method according to claim 1, comprising:

12. The target nucleic acid capture portion has a poly-T sequence, and The complementary chain capture region has a base sequence that is complementary to the base sequence that is added to the 3' end during reverse transcription by reverse transcriptase. The analytical method according to claim 11.

13. The analytical method according to claim 11, wherein the double-stranded portion has a restriction enzyme recognition sequence.

14. The analytical method according to claim 11, wherein the double-stranded portion has a non-natural base sequence.

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

16. The aforementioned analytical method is an analytical method for performing single-cell analysis. A nucleic acid ligator having a different double-stranded portion for each cell is used. The analytical method according to claim 11.

17. The aforementioned analytical method includes a cell disruption step that destroys cells, The complementary nucleic acid generation step is performed on the target nucleic acid contained in the cell. The analytical method according to claim 1.

18. The analytical method according to claim 17, wherein the cell disruption step is performed in a space partitioned for each cell.

19. The analytical method according to claim 1, wherein the nucleic acid linking portion is immobilized on a substrate.

20. A complementary nucleic acid generation step in which a nucleic acid linker is attached to one end of each of one or more target nucleic acids, and a complementary nucleic acid generation step in which a complementary chain of the one or more target nucleic acids is generated, A cyclic nucleic acid generation step involves linking two or more generated complementary strands via the nucleic acid linking portion to form a cyclic nucleic acid. A method for producing cyclic nucleic acids, including

21. A target nucleic acid capture unit configured to capture the 3' terminal region of the target nucleic acid, A complementary strand capture unit configured to capture the 3' terminal region of the complementary strand generated by the formation of a complementary strand of the target nucleic acid, A double-stranded portion connecting the target nucleic acid capture portion and the complementary strand capture portion, Nucleic acids containing [the specified substance].

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 produce a cyclic nucleic acid.

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