Method for detecting fluorescence

Abstract

The invention relates to a single-stranded oligonucleotide marked by a fluorescent substance and a quenching substance. A single-stranded oligonucleotide that forms a hairpin structure on the 5'side by means of self-hybridization, forms a three-layer chain structure on the 5 '-terminal portion when a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, and dissociates the fluorescent substance and the quenching substance by cleavage of a flap endonuclease in which the three-layer chain structure is recognized, thereby forming a single-stranded oligonucleotide having a hairpin structure on the 5 '-side and a single-stranded oligonucleotide having a nucleotide sequence complementary to the 3'-side. Fluorescent light is emitted under the irradiation of exciting light; the single-stranded oligonucleotide satisfies any one of the following (A) to (C): (A) a single-stranded oligonucleotide containing a non-complementary base pair inside the hairpin structure. And (B) a single-stranded oligonucleotide in which the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by four or more residues. And (C) a single-stranded oligonucleotide in which the fluorescent substance is bound to a portion other than a base.

Description

Technical Field

[0001] The present invention relates to a method for detecting fluorescence. This application claims priority based on Japanese Patent Application Nos. 2022-099031, 2022-099032, 2022-099033, and 2022-099035, each filed in Japan on June 20, 2022, and the contents of which are incorporated herein by reference. Background Art

[0002] As a method for detecting a trace amount of an analyte molecule, a method using FRET (Fluorescence resonance energy transfer) is known.

[0003] For example, in genetic diagnosis, there are many methods for accurately and rapidly detecting and quantifying target nucleic acids. Among them, Invasive Cleavage Assay (ICA) is a method that applies invasion reaction using a nuclease such as 5'-nuclease and FRET, and has excellent operability and reaction stability (e.g., Non-Patent Document 1).

[0004] Here, refer to Figure 1 ICA will be described. Figure 1 is a schematic diagram for explaining an example of ICA. Figure 1 In the example of , the presence of T (thymine) base 101 in the target nucleic acid 100 is detected. First, a flap probe 110 and an invader probe 120 complementary to the target nucleic acid 100 are hybridized. As a result, the invader probe 120 hybridizes to a portion of the target nucleic acid 100 adjacent to the position to which the flap probe 110 hybridizes. Furthermore, at least one base at the 3' end of the invader probe 120 invades the position of the 5' end of the region 141 where the flap probe 110 hybridizes with the target nucleic acid 100, forming a first triple-stranded structure 130.

[0005] Next, when flap endonuclease is reacted with the first triplex structure 130, the flap site 140 of the first triplex structure 130 is cut to generate the nucleic acid fragment 140. Next, the nucleic acid fragment 140 hybridizes with the nucleic acid fragment 150 to form the second triplex structure 160.

[0006] exist Figure 1 In the example of , the 5' end of the nucleic acid fragment 150 is bound to a fluorescent substance F, and a quenching substance Q is bound to the 3' side several bases away from the 5' end of the nucleic acid fragment 150. The fluorescent substance F and the quenching substance Q are located in close proximity in space. Therefore, the fluorescence emitted by the fluorescent substance F can be quenched by the quenching substance Q.

[0007] Next, when the flap endonuclease is reacted with the second three-layer chain structure 160, the flap site 170 of the second three-layer chain structure 160 is cut, generating a nucleic acid fragment 170. As a result, the fluorescent substance F is released from the quenching substance Q, and emits fluorescence under the irradiation of the excitation light. By detecting this fluorescence, the presence of the T (thymine) base 101 in the target nucleic acid 100 can be detected.

[0008] In this specification, a flap probe may be referred to as an allele probe, and an invader probe may be referred to as an invader oligonucleotide.

[0009] In the ICA described in non-patent document 1, a FRET box having a fluorescent substance and a quenching substance is used. In the absence of a target nucleic acid in the reaction system, the fluorescent substance is located near the space of the quenching substance. Thus, the fluorescence emission from the fluorescent substance is suppressed by the quenching substance. In the presence of a target nucleic acid in the reaction system, the ICA reaction is carried out under the action of a nucleic acid lyase, and the fluorescent substance is separated from the quenching substance. Thus, fluorescence is emitted from the fluorescent substance.

[0010] In ICA, multiple target nucleic acids can be detected simultaneously by using multiple probes with different fluorescence wavelengths corresponding to multiple target nucleic acids. For example, when the fluorescence is blue, green, yellow, orange, and red, five different target nucleic acids can be detected simultaneously in one measurement.

[0011] In addition, when ICA is performed in a microscopic space such as a micropore, the concentration of the analyte molecule in the reaction system can be concentrated. Thus, in a microscopic space, the time until the detection signal reaches a sufficient intensity can be shortened. For example, Patent Document 1 describes that gene testing can be performed by performing an enzyme reaction in a microscopic space with a volume of less than 1 pL.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent Application Publication No. 2004-309405

[0015] Non-patent literature

[0016] Non-patent document 1: Eis et al, An invasive cleavage assay for direct quantitation of specific RNAs, Nature Biotechnology, Vol. 19, 673-676 (2001). Summary of the invention

[0017] Problems to be solved by the invention

[0018] An object of the present invention is to provide a technology for improving reaction efficiency in a detection reaction of a trace amount of an analyte molecule.

[0019] For example, when using multiple fluorescent probes, the fluorescence intensity of blue and red is weak due to the characteristics of fluorescent molecules, so it is difficult to detect fluorescence. Therefore, in one embodiment, the present invention provides a detection method for further improving the fluorescence intensity emitted from a fluorescent probe having a fluorescent substance and a quenching substance in the presence of an analyte molecule.

[0020] In addition, the inventors found that in ICA, Figure 1 The formation of the three-layer chain structure formed by the hybridization of the nucleic acid fragment 140 and the nucleic acid fragment 150 (hereinafter sometimes referred to as the "fluorescent substrate") is hindered by the fluorescent substance F and the quenching substance Q labeled on the fluorescent substrate. If the formation of the three-layer chain structure is hindered, the reactivity of ICA is reduced. Therefore, in one embodiment, the present invention provides a fluorescent substrate for ICA that can efficiently form a three-layer chain structure.

[0021] In addition, the inventors found that Figure 1 In the exemplified ICA, in addition to the signal specific to the gene polymorphism or gene mutation, the background signal sometimes also rises. The rise of the background signal is related to the false positive determination, which can be said to be a risk factor for misjudgment in genetic diagnosis. Therefore, in one embodiment, the present invention provides a technology for suppressing the background rise in ICA.

[0022] In addition, the inventors found that Figure 1 In the exemplified ICA, there is room for improvement in the efficiency of cleavage of a fluorescent substrate by flap endonuclease. Therefore, in one embodiment, the present invention provides a technique for improving the efficiency of cleavage of a fluorescent substrate by flap endonuclease in ICA.

[0023] Means for solving problems

[0024] The present invention includes the following aspects.

[0025] [1] A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, forms a triple-stranded structure at the 5' end portion when hybridized with a single-stranded oligonucleotide having a base sequence complementary to the 3' side, and is cut by a flap endonuclease that recognizes the triple-stranded structure, freeing the fluorescent substance and the quenching substance to emit fluorescence under irradiation with excitation light; wherein the single-stranded oligonucleotide satisfies any one of the following (A) to (C):

[0026] (A) a single-stranded oligonucleotide comprising a non-complementary base pair in the interior of the hairpin structure;

[0027] (B) a single-stranded oligonucleotide in which the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by 4 residues or more;

[0028] (C) A single-stranded oligonucleotide in which the fluorescent substance is bound to a portion other than the base.

[0029] [2] The single-stranded oligonucleotide according to [1], wherein (A) is satisfied, the fluorescent substance is labeled on the nucleotide residue at the 5' end, and the quenching substance is labeled on the nucleotide residue located more on the 5' side among the nucleotide residues constituting the non-complementary base pair.

[0030] [3] The single-stranded oligonucleotide according to [1], wherein the base of the nucleotide residue at the 5′ terminal is a pyrimidine base.

[0031] [4] The single-stranded oligonucleotide according to [1], wherein the quencher is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0032] [5] The single-stranded oligonucleotide according to [1], wherein the fluorescent substance is labeled on the nucleotide residue at the 5' end, and when cut by flap nuclease, the oligonucleotide fragment containing the fluorescent substance is released, and the length of the oligonucleotide fragment is more than 2 residues.

[0033] [6] The single-stranded oligonucleotide according to [1], wherein a phosphate group is added to the 5′ end and the fluorescent substance is bound to the phosphate group.

[0034] [7] The single-stranded oligonucleotide according to [1], wherein the quencher substance is bound to a base of a nucleotide residue located at the 3′ side of the fluorescent substance.

[0035] [8] The single-stranded oligonucleotide according to [1], wherein the molecular weight of the fluorescent substance is 350 to 1100.

[0036] [9] The single-stranded oligonucleotide according to [1], wherein both the fluorescent substance and the quencher substance are present on one strand of the double-stranded oligonucleotide portion of the hairpin structure.

[0037]

[10] A method for detecting fluorescence from a matrix having a fluorescent substance, a quenching substance and a matrix body, comprising a step of causing the fluorescent substance to emit fluorescence by separating the fluorescent substance from the quenching substance in the matrix, wherein when the fluorescent substance is located in the spatial vicinity of the quenching substance, the fluorescence emission from the fluorescent substance is suppressed by the quenching substance, and the peak wavelength of the absorption spectrum of the quenching substance is located on the short wavelength side relative to the peak wavelength of the emission spectrum of the fluorescent substance.

[0038]

[11] The method according to

[10] , wherein the step of causing the fluorescent substance to emit fluorescence is performed in the presence of a target substance.

[0039]

[12] The method according to

[10] or

[11] , wherein the peak wavelength of the absorption spectrum of the quenching substance is 50 to 100 nm shorter than the peak wavelength of the emission spectrum of the fluorescent substance.

[0040]

[13] The method according to

[10] or

[11] , wherein the matrix body is nucleic acid.

[0041]

[14] The method according to

[10] or

[11] , wherein the peak wavelength of the emission spectrum of the fluorescent substance is 600 to 700 nm, and the peak wavelength of the absorption spectrum of the quenching substance is 550 to 640 nm.

[0042]

[15] The method according to

[14] , wherein in the step of causing the fluorescent substance to emit fluorescence, the fluorescent substance and the quenching substance in the matrix are separated by the action of a nuclease.

[0043]

[16] The method according to

[10] or

[11] , wherein the step of causing the fluorescent substance to emit fluorescence is performed in a microscopic space.

[0044]

[17] The method according to

[11] , wherein the target substance is nucleic acid.

[0045]

[18] The method according to

[10] or

[11] , wherein the fluorescence is further observed using a fluorescence microscope.

[0046] The present invention may also include the following aspects.

[0047] [A1] A method for detecting fluorescence from a matrix having a fluorescent substance, a quenching substance and a matrix body, comprising a step of causing the fluorescent substance to emit fluorescence by separating the fluorescent substance from the quenching substance in the matrix, wherein, when the fluorescent substance is located in the spatial vicinity of the quenching substance, the fluorescence emission from the fluorescent substance is suppressed by the quenching substance, and the peak wavelength of the absorption spectrum of the quenching substance is located on the short wavelength side relative to the peak wavelength of the emission spectrum of the fluorescent substance.

[0048] [A2] The method according to [A1], wherein the step of causing the fluorescent substance to emit fluorescence is performed in the presence of a target substance.

[0049] [A3] The method according to [A1] or [A2], wherein the peak wavelength of the absorption spectrum of the quenching substance is 50 to 100 nm shorter than the peak wavelength of the emission spectrum of the fluorescent substance.

[0050] [A4] The method according to any one of [A1] to [A3], wherein the substrate body is a nucleic acid.

[0051] [A5] The method according to any one of [A1] to [A4], wherein the peak wavelength of the emission spectrum of the fluorescent substance is 600 to 700 nm, and the peak wavelength of the absorption spectrum of the quenching substance is 550 to 640 nm.

[0052] [A6] The method according to [A4], wherein, in the step of causing the fluorescent substance to emit fluorescence, the fluorescent substance and the quenching substance in the matrix are separated by the action of a nuclease.

[0053] [A7] The method according to any one of [A1] to [A6], wherein the step of causing the fluorescent substance to emit fluorescence is performed in a micro space.

[0054] [A8] The method according to [A2], wherein the target substance is a nucleic acid.

[0055] [A9] The method according to any one of [A1] to [A8], wherein the fluorescence is further observed using a fluorescence microscope.

[0056] The present invention may also include the following aspects.

[0057] [B1] A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease that recognizes the triple-chain structure, and emit fluorescence under the irradiation of excitation light; wherein the fluorescent substance is bound to a portion other than the base.

[0058] [B2] The single-stranded oligonucleotide according to [B1], wherein a phosphate group is added to the 5′ end and the fluorescent substance is bound to the phosphate group.

[0059] [B3] The single-stranded oligonucleotide according to [B2], wherein the quencher substance is bound to a base of a nucleotide residue located at the 3′ side of the fluorescent substance.

[0060] [B4] The single-stranded oligonucleotide according to any one of [B1] to [B3], wherein the base of the nucleotide residue at the 5′ terminal is a pyrimidine base.

[0061] [B5] The single-stranded oligonucleotide according to any one of [B1] to [B4], wherein the quencher is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0062] [B6] The single-stranded oligonucleotide according to any one of [B1] to [B5], wherein the molecular weight of the fluorescent substance is 350 to 1100.

[0063] [B7] The single-stranded oligonucleotide according to any one of [B1] to [B6], wherein both the fluorescent substance and the quencher substance are present on one strand of the double-stranded oligonucleotide portion of the hairpin structure.

[0064] [B8] A method for improving the efficiency of forming a triple-stranded structure in a fluorescent substrate of a flap nuclease, wherein the fluorescent substrate is a first single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, and the method includes a step of contacting the first single-stranded oligonucleotide with a second single-stranded oligonucleotide having a base sequence complementary to the 3' side of the first single-stranded oligonucleotide, the first single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when the second single-stranded oligonucleotide hybridizes, a triple-stranded structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease, and emit fluorescence under the irradiation of excitation light; the fluorescent substance is bound to the portion of the first single-stranded oligonucleotide other than the base.

[0065] The present invention may also include the following aspects.

[0066] [C1] A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: it forms a hairpin structure on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease that recognizes the triple-chain structure, and emit fluorescence under the irradiation of excitation light; wherein the single-stranded oligonucleotide contains non-complementary base pairs inside the hairpin structure.

[0067] [C2] The single-stranded oligonucleotide according to [C1], wherein the fluorescent substance is labeled on the nucleotide residue at the 5' end, and the quenching substance is labeled on the nucleotide residue located more on the 5' side among the nucleotide residues constituting the non-complementary base pair.

[0068] [C3] The single-stranded oligonucleotide according to [C1] or [C2], wherein the base of the nucleotide residue at the 5′ terminal is a pyrimidine base.

[0069] [C4] The single-stranded oligonucleotide according to any one of [C1] to [C3], wherein the quencher is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0070] [C5] A method for inhibiting background rise in ICA, comprising the step of adding a fluorescent substrate to the reaction solution of ICA, wherein the fluorescent substrate is a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, and the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when hybridized with a single-stranded oligonucleotide having a base sequence complementary to the 3' side, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease that recognizes the triple-chain structure, and emit fluorescence under the irradiation of excitation light; wherein the single-stranded oligonucleotide contains non-complementary base pairs inside the hairpin structure.

[0071] The present invention may also include the following aspects.

[0072] [D1] A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease that recognizes the triple-chain structure, and emit fluorescence under the irradiation of excitation light; wherein the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by more than 4 residues.

[0073] [D2] The single-stranded oligonucleotide according to [D1], wherein the fluorescent substance is labeled on the nucleotide residue at the 5' end, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0074] [D3] The single-stranded oligonucleotide according to [D2], wherein the length of the oligonucleotide fragment is 2 residues or more.

[0075] [D4] The single-stranded oligonucleotide according to [D2] or [D3], wherein the base of the nucleotide residue at the 5′ terminal is a pyrimidine base.

[0076] [D5] A single-stranded oligonucleotide according to any one of [D2] to [D4], wherein the single-stranded oligonucleotide contains a non-complementary base pair inside the hairpin structure, and the quenching substance is labeled on a nucleotide residue located more on the 5' side among the nucleotide residues constituting the non-complementary base pair.

[0077] [D6] A method for improving the cutting efficiency when a nucleic acid triple-chain structure is cut by a flap nuclease, comprising a step of bringing the nucleic acid triple-chain structure into contact with the flap nuclease, wherein the nucleic acid triple-chain structure comprises a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance and a single-stranded oligonucleotide having a base sequence complementary to the 3' side, wherein the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when the single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease, and emit fluorescence under the irradiation of excitation light; wherein the nucleotide residues of the single-stranded oligonucleotide labeled with the fluorescent substance and the nucleotide residues labeled with the quenching substance are separated by more than 4 residues.

[0078] Effects of the Invention

[0079] According to the present invention, it is possible to provide a technique for improving the reaction efficiency in a detection reaction of a trace amount of an analyte molecule.

[0080] In one embodiment, the present invention provides a detection method for further improving the fluorescence intensity emitted from a fluorescent probe having a fluorescent substance and a quencher substance in the presence of an analyte molecule. The detection method is suitable for digital measurement using ICA.

[0081] In one embodiment, according to the present invention, a fluorescent substrate for ICA capable of efficiently forming a three-layer chain structure can be provided.

[0082] In one embodiment, according to the present invention, a technique for suppressing background rise in ICA can be provided.

[0083] In one embodiment, according to the present invention, a technique for improving the efficiency of cleavage of a fluorescent substrate by flap endonuclease in ICA can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 This is a schematic diagram for explaining an example of invasion lysis assay (ICA).

[0085] Figure 2 This is a schematic cross-sectional view for explaining a method of feeding a reagent solution to a fluid device.

[0086] Figure 3 It is a figure which shows schematically the 1st cracking structure.

[0087] Figure 4 It is a diagram schematically showing the second α-cleavage structure.

[0088] Figure 5 It is a diagram schematically showing the second β-cleavage structure.

[0089] Figure 6 This is a diagram schematically showing the reaction mechanism of ICA in Experimental Example A1.

[0090] Figure 7 This is a photograph of fluorescence obtained when the FRET cassette 4-1 was used in Experimental Example A1.

[0091] Figure 8 This is a photograph of fluorescence obtained when the FRET cassette 4-2 was used in Experimental Example A1.

[0092] Fig. 9 This is a photograph of fluorescence obtained when the FRET cassette 4-3 was used in Experimental Example A1.

[0093] Fig.10 It is a histogram of the signal intensity when the FRET cassette 4-1 was used in Experimental Example A1.

[0094] Fig.11It is a histogram of the signal intensity when the FRET cassette 4-2 was used in Experimental Example A1.

[0095] Fig.12 It is a histogram of the signal intensity when the FRET cassette 4-3 was used in Experimental Example A1.

[0096] Fig.13 This is a graph showing the S / N value in Experimental Example A1.

[0097] Fig.14 This is a graph showing the SN value in Experimental Example A1.

[0098] Fig.15 This is a graph showing the results when a fluorescent substrate in which a fluorescent substance is bound to a base is used in Experimental Example A1.

[0099] Fig.16 This is a graph showing the results when a fluorescent substrate in which a fluorescent substance is bound to a portion other than a base is used in Experimental Example A1.

[0100] Fig.17 This is a graph showing the results of Experimental Example B1.

[0101] Fig.18 This is a graph showing the results of Experimental Example B1.

[0102] Fig.19 This is a graph showing the results of Experimental Example B2.

[0103] Fig. 20 This is a graph showing the results of Experimental Example B2.

[0104] Fig.21 This is a graph showing the results of Experimental Example B3.

[0105] Fig. 22 This is a graph showing the results of Experimental Example B3.

[0106] Fig.23 This is a graph showing the results of Experimental Example B4.

[0107] Fig.24 This is a graph showing the results of Experimental Example B4.

[0108] Fig.25 This is a graph showing the results of Experimental Example B5.

[0109] Fig.26 This is a graph showing the results of Experimental Example C1.

[0110] Fig. 27 This is a graph showing the results of Experimental Example C1.

[0111] Fig.28This is a graph showing the results of Experimental Example D1.

[0112] Fig.29 This is a graph showing the results of Experimental Example D1.

[0113] Fig.30 This is a graph showing the results of Experimental Example D1.

[0114] Fig.31 This is a graph showing the results of Experimental Example D1. DETAILED DESCRIPTION

[0115] <Implementation A>

[0116] [Method for detecting fluorescence from a matrix having a fluorescent substance, a quenching substance, and a matrix body]

[0117] A method of a first aspect relates to a method for detecting fluorescence from a matrix having a fluorescent substance, a quenching substance, and a matrix body. The method of the first aspect includes a step of causing the fluorescent substance to emit fluorescence by separating the fluorescent substance from the quenching substance in the matrix.

[0118] (Matrix)

[0119] The matrix comprises a matrix body, a fluorescent substance and a quenching substance, wherein the fluorescent substance and the quenching substance are respectively combined on the matrix body.

[0120] The binding of the fluorescent substance to the matrix body is not particularly limited as long as the effect of the present invention can be exerted, and may be chemically bonded. The binding of the quenching substance to the matrix body is not particularly limited as long as the effect of the present invention can be exerted, and may be chemically bonded.

[0121] The matrix body is not particularly limited as long as it can bring into play the effect of the present invention, and can be natural or synthetic. As the matrix body, for example, polymers can be enumerated. As polymers, there is no particular limitation, and nucleic acids, nucleic acid analogs, polyamino acids, proteins, oligoamino acids, etc. can be enumerated.

[0122] As natural nucleic acid, for example, genomic DNA, mRNA, rRNA, hnRNA, miRNA, tRNA, etc. can be mentioned. Natural nucleic acid can be nucleic acid recovered from an organism, or nucleic acid recovered from water, organic matter, etc. that have been in contact with an organism. As a method for recovering natural nucleic acid, known methods such as the phenol / chloroform method can be mentioned.

[0123] Examples of synthetic nucleic acids include synthetic DNA, synthetic RNA, cDNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and the like.

[0124] The synthesis method of the synthesized nucleic acid is not particularly limited, and examples thereof include known chemical synthesis methods such as the β-cyanoethylphosphoramidite method and the DNA solid phase synthesis method, known nucleic acid amplification methods, reverse transcription reaction, etc. Examples of nucleic acid amplification methods include PCR, LAMP, SMAP, NASBA, and RCA.

[0125] (Fluorescent substance)

[0126] Examples of the fluorescent substance include red fluorescent substances such as ATTO643, ATTO633, Alexa Fluor 647, Cy5, HiLyteFluor 647, and ATTO663.

[0127] The peak wavelength of the fluorescence spectrum of the fluorescent substance is preferably 600 to 700 nm, more preferably 610 to 690 nm, further preferably 620 to 680 nm, particularly preferably 630 to 670 nm, and most preferably 640 to 660 nm.

[0128] (Quenching substance)

[0129] Examples of the quenching substance include Black Hole Quencher (BHQ) (registered trademark)-1, BHQ (registered trademark)-2, BHQ (registered trademark)-3, Fluorescence Quencher 1 (Tide Quencher 1, TQ1), Fluorescence Quencher 2 (Tide Quencher 2, TQ2), Fluorescence Quencher 2WS (Tide Quencher 2WS, TQ2WS), Fluorescence Quencher 3 (Tide Quencher 3, TQ3), Fluorescence Quencher 3WS (Tide Quencher 3WS, TQ3WS), Fluorescence Quencher 4 (Tide Quencher 4, TQ4), Fluorescence Quencher 4WS (Tide Quencher 4WS, TQ4WS), Fluorescence Quencher 5 (Tide Quencher 5, TQ5), Fluorescence Quencher 5WS (Tide Quencher 5WS, TQ5WS), fluorescence quencher 6WS (TideQuencher6WS, TQ6WS), fluorescence quencher 7WS (Tide Quencher 7WS, TQ7WS), QSY35, QSY7, QSY9, QSY21, Iowa Black FQ, Iowa Black RQ, etc., preferably one or more selected from BHQ-2, TQ4, TQ4WS, TQ5, TQ5WS, QSY21 and Iowa Black RQ, more preferably BHQ-2.

[0130] In one embodiment, the peak wavelength of the absorption spectrum of the quenching substance is preferably 550 to 640 nm, more preferably 560 to 630 nm, further preferably 570 to 620 nm, and particularly preferably 570 to 600 nm.

[0131] In one embodiment, the peak wavelength of the absorption spectrum of the quenching substance is located on the short wavelength side relative to the peak wavelength of the emission spectrum of the fluorescent substance. In this case, the peak wavelength of the absorption spectrum of the quenching substance is preferably 50 to 100 nm shorter than the peak wavelength of the emission spectrum of the fluorescent substance, more preferably 55 to 80 nm shorter, and further preferably 60 to 70 nm shorter.

[0132] In the matrix, when the fluorescent substance is located near the space of the quenching substance, the fluorescence emission from the fluorescent substance is suppressed by the quenching substance. Here, "fluorescence emission is suppressed" means the following.

[0133] In the absence of a quenching substance, the intensity of fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as A. In addition, in the case where the quenching substance exists in the vicinity of the space of the fluorescent substance, the intensity of fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as B. Here, "fluorescence emission is suppressed" means that the value of B / A is 40% or less.

[0134] The distance between the fluorescent substance and the quenching substance when the fluorescent substance is located near the space of the quenching substance is not particularly limited as long as the fluorescence emission from the fluorescent substance can be suppressed by the quenching substance, but is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 2 nm or less.

[0135] The method for separating the fluorescent substance and the quenching substance in the matrix is ​​not particularly limited as long as the fluorescent substance can emit fluorescence. For example, the matrix having the fluorescent substance and the quenching substance can be subjected to physical actions such as shearing, heating, cooling, and electromagnetic wave irradiation. Chemical actions such as exposure to chemical substances can also be applied. Biological actions such as enzyme reactions can also be applied.

[0136] The distance between the fluorescent substance and the quenching substance in a state where the fluorescent substance and the quenching substance are separated is not limited as long as fluorescence can be emitted from the fluorescent substance, but is preferably 5 nm or more, more preferably 10 nm or more, and further preferably 20 nm or more.

[0137] (Target substance)

[0138] The step of causing the fluorescent substance to emit fluorescence can be performed in the presence of a target substance. The target substance is not particularly limited as long as it is a substance that directly or indirectly separates the fluorescent substance from the quenching substance, and examples thereof include nucleic acids, enzymes, proteins such as antibodies, and nucleic acids are preferred.

[0139] When the target substance is a nucleic acid, in the step of causing the fluorescent substance to emit fluorescence, the fluorescent substance and the quenching substance in the matrix can be separated by the action of a nuclease.

[0140] (Small Space)

[0141] In the method of the first mode, the process of making the fluorescent substance emit fluorescence is preferably carried out in a small space. Specifically, it is preferred to use a device with a hole array, and the hole array has a plurality of small holes. Since the absolute number of the matrix in the small space is small, the number of fluorescent molecules as the cut-off reaction product in the previous method also decreases, and the fluorescence intensity decreases. In contrast, in the method of this embodiment, the reaction efficiency can be improved and the number of fluorescent molecules as the cut-off product can be increased. Therefore, the fluorescence intensity can be increased even in a small space. As a device, for example, the following devices can be used, but are not limited to this.

[0142] The shape, size and arrangement of the wells are not particularly limited, and it is preferred to use a well array consisting of wells that can accommodate a liquid containing a target nucleic acid used in the method of the present invention and a certain amount of a reagent solution used in the detection step. The wells may be used directly without treatment, or may be pre-treated in advance to immobilize an extraction reagent, a detection reagent such as an antibody, a specific binding substance, etc., or to cover the well opening with a lipid bilayer membrane, etc., on the inner wall of the well, depending on the purpose.

[0143] The device may have a flow path, and may also transport a liquid dispersed with target nucleic acid via the flow path. The shape, structure, capacity, etc. of the flow path are not particularly limited, and it is preferred to use a device having a flow path such that when transporting a liquid dispersed with a structure, the structure is introduced into each hole of the hole array, and when inserting a sealing liquid, each hole can be individually sealed to form a micro droplet.

[0144] Figure 2 is a schematic cross-sectional view of a device in one embodiment. Figure 2 As shown, the device 100 includes a substrate 104 and a cover material 101 .

[0145] The substrate 104 may be formed of a light-transmitting resin. The substrate 104 may be substantially transparent.

[0146] The substrate 104 has a plurality of holes 105. The holes 105 of the substrate 104 are open on the surface of the substrate 104. The shape, size and arrangement of the holes 105 are not particularly limited. Figure 2In the example shown, in the device 100, a plurality of holes 105 of the same shape and size capable of accommodating a reagent solution 107 (a liquid in which target nucleic acid is dispersed) are formed on a substrate 104. In addition, when particles are used in the detection method of the first embodiment, holes 105 of the same shape and size capable of accommodating one or more particles and capable of accommodating a certain amount of a reagent solution 107 containing particles may be formed on the substrate 104.

[0147] In the device 100 , the diameter of the holes 105 may be, for example, about 3 μm, and the depth of the holes 105 may be, for example, about 4.5 μm. Alternatively, the holes 105 may be arranged in a triangular lattice or a square lattice and formed on the substrate 104 .

[0148] The region including the plurality of holes 105 of the substrate 104 is filled with a reagent solution 107 to be analyzed. Inside this region, a flow path 106 is provided between the substrate 104 and the cover member 101 .

[0149] The cover material 101 may be melt-bonded or bonded to the base material 104. For example, the cover material 101 may be formed of a thermoplastic resin such as a cycloolefin polymer or a cycloolefin copolymer.

[0150] The substrate 104 is formed using, for example, a resin. The type of resin is not particularly limited, and it is preferred to use a resin that is resistant to the sealing liquid when forming reagents and droplets. In addition, in the case of fluorescent observation of the signal, it is preferred to select a resin with less autofluorescence. For example, cycloolefin polymers, cycloolefin copolymers, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesin, amorphous fluororesin, etc. can be cited. It should be noted that the materials shown as examples of the substrate 104 are just examples, and the materials are not limited to these.

[0151] A plurality of holes 105 may be formed on one surface of the substrate 104 in the thickness direction. As a forming method using a resin, in addition to injection molding, hot stamping, photo stamping, etc. may be used. In addition, when a fluororesin is used, for example, a layer of CYTOP (registered trademark) (Asahi Glass) may be provided on the substrate 104, and the microscopic holes formed on the CYTOP (registered trademark) become the holes 105.

[0152] The cover material 101 is formed so as to have a convex portion on the surface facing the substrate 104 when assembled. For example, a thermoplastic resin fluid can be formed into a plate with a convex portion by using a molding die. In addition, a liquid supply port 102 and a liquid waste port 103 can also be formed on the cover material 101.

[0153] When the lid 101 and the base 104 are formed as described above, the lid 101 and the base 104 overlap in such a way that the convex portion of the lid contacts the surface of the base 104 on the side where the hole 105 opens. In addition, the lid 101 and the base 104 are melt-bonded by laser melt bonding or the like while overlapping as described above.

[0154] The number of wells in the device is preferably 100,000 to 6,000,000. The total volume of the wells is preferably 0.1 to 10 μL.

[0155] (ICA)

[0156] The method of the first aspect can be applied to, for example, invasive cleavage assay (ICA).

[0157] The target substance in the method of the first mode can be a target nucleic acid in ICA. In ICA, two oligonucleotides that can hybridize with the target nucleic acid are used to form a cleavage structure with a specific overlapping structure. Then, the obtained cleavage structure is cut using an enzyme with 5' nuclease activity, and the cut fragment is detected, thereby detecting the target nucleic acid.

[0158] More specifically, ICA is a method in which two oligonucleotides are annealed to different adjacent sites on a target nucleic acid, and the 5' side of one oligonucleotide is annealed to overlap with the other oligonucleotide annealed to the target nucleic acid, thereby forming a cleavage structure having a specific overlapping structure.

[0159] The cleavage structure has a portion of another oligonucleotide invaded below one oligonucleotide. The invaded oligonucleotide is sometimes referred to as an invader oligonucleotide, and the overlapping oligonucleotide is sometimes referred to as an allele probe.

[0160] The enzyme with 5' nuclease activity recognizes the intrusion structure in the cleavage structure and cuts off the overlapping part on the 5' side of the allele probe. By designing the allele probe to be cut to have a melting point close to the temperature of the cutting reaction performed using the 5' nuclease activity, the part annealed with the target nucleic acid remaining after cutting is also dissociated from the target nucleic acid. Then, the allele probe before cutting is newly annealed to form a cleavage structure again, and the cutting reaction is repeated, and the fragment (flap fragment) after cutting is amplified. Therefore, by measuring the flap fragment, the target nucleic acid can be detected.

[0161] Various methods have been disclosed as methods for measuring flap fragments. For example, as a secondary reaction, for a detection nucleic acid different from the target nucleic acid, a flap fragment is used as an invader oligonucleotide and another oligonucleotide is used as an allele probe, and these are annealed separately to form a cleavage structure, and the allele probe is cut in the same manner as in the primary reaction, and the second flap fragment is amplified, and the second flap fragment can also be detected.

[0162] In the secondary reaction, the measurement can also be performed using a FRET (Fluorescence resonance energy transfer) reaction. Specifically, by allowing the flap fragment to act on a FRET box (a probe having a fluorescent substance and a quenching substance) as an intruder oligonucleotide to form a cleavage structure, the FRET box is cut off to separate the fluorescent substance from the quenching substance, and the generated fluorescence signal is measured, thereby enabling the flap fragment to be measured with high sensitivity. The measurement reaction of these flap fragments can be carried out simultaneously in the same reaction solution as the generation reaction of the flap fragment.

[0163] When ICA is performed in each well of the above-mentioned device, it is preferred that the number of target substances in each well of the device be one or less.

[0164] The method of this embodiment may further include the step of observing the fluorescence using a fluorescence microscope.

[0165] According to the method of the first aspect described above, in the presence of the analyte molecule, the fluorescence intensity emitted from the fluorescent probe having the fluorescent substance and the quencher substance is further increased.

[0166] As an embodiment of the method of the first embodiment, for example, the following embodiments can be cited. The method of this embodiment is to apply the substrate described in the first embodiment to Figure 1 The method of the ICA reaction is exemplified.

[0167] In the method of this embodiment, the "step of causing the fluorescent substance to emit fluorescence by separating the fluorescent substance and the quenching substance in the matrix" in the first method includes a step of contacting a first single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance and a second single-stranded oligonucleotide having a base sequence complementary to the 3' side of the first single-stranded oligonucleotide.

[0168] In the method of this embodiment, the first single-stranded oligonucleotide forms a hairpin structure on the 5' side through self-hybridization. When the second single-stranded oligonucleotide hybridizes, a three-layer chain structure is formed at the 5' end portion. The fluorescent substance and the quenching substance are freed by being cut by the nuclease and emit fluorescence under the irradiation of excitation light.

[0169] In the method of this embodiment, when the fluorescent substance is located in the spatial vicinity of the quencher substance, the fluorescent emission from the fluorescent substance is suppressed by the quencher substance.

[0170] In the method of this embodiment, the peak wavelength of the absorption spectrum of the quenching substance is located on the shorter wavelength side with respect to the peak wavelength of the emission spectrum of the fluorescent substance.

[0171] The method of this embodiment may include a step of detecting fluorescence. Fluorescence can be detected using a fluorescence microscope.

[0172] The first single-stranded oligonucleotide having a hairpin structure formed by self-hybridization at the 5′ side and labeled with a fluorescent substance and a quenching substance corresponds to Figure 1 The second single-stranded oligonucleotide having a base sequence complementary to the 3' side of the first single-stranded oligonucleotide is Figure 1 The three-layered chain structure formed at the 5' end portion corresponds to Figure 1 The second third-layer chain structure 160.

[0173] As described later in the examples, by making the fluorescent substance of the fluorescent substrate be combined with the part other than the base, a three-layer chain structure can be formed efficiently, which can improve the reactivity of ICA. The fluorescent substance being combined with the part other than the base means that the fluorescent substance is combined with the sugar residue, phosphate group, sugar residue or phosphate group in the base of the oligonucleotide forming the fluorescent substrate. Wherein, since the fluorescence intensity is further improved, the fluorescent substance is preferably combined with the phosphate group of the fluorescent substrate.

[0174] In this specification, the reactivity of ICA can be improved by the fluorescence signal detected by the result of ICA reaching a specified value in a shorter time. Alternatively, the fluorescence signal intensity detected by the result of ICA can reach a higher value. Alternatively, the fluorescence signal of the background detected by the result of ICA can be maintained at a lower value.

[0175] The single-stranded oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.

[0176] In a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, the number of bases between the nucleic acid bound to the fluorescent substance and the nucleic acid bound to the quenching substance is preferably more than 11 bases and less than 30 bases, more preferably more than 4 bases and less than 10 bases, and further preferably more than 1 base and less than 3 bases.

[0177] As the nuclease, an enzyme having 5' nuclease activity is preferred, a modified DNA polymerase having 5' nuclease activity but lacking polymerase activity is more preferred, and flap endonuclease is further preferred.

[0178] Examples of flap endonucleases include flap endonuclease 1 (NCBI accession number: WP_011012561.1, Holliday junction 5'flap endonuclease (GEN1)) (NCBI accession number: NP_001123481.3), and excision repair protein (NCBI accession number: AAC37533.1).

[0179] The single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance (sometimes referred to as a fluorescent substrate) preferably has a phosphate group added to the 5' end, and the fluorescent substance is bound to the phosphate group added to the 5' end.

[0180] As described later in the Examples, a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bound to the phosphate group added to the 5' end has higher ICA reactivity than a fluorescent substrate having a fluorescent substance bound to a base, and can form a triple-stranded structure more efficiently.

[0181] The following formula (A1) is a chemical formula representing an example of a fluorescent substrate in which a phosphate group is added to the 5' end and a fluorescent substance is bound to the phosphate group added to the 5' end. As shown in the following formula (A1), the fluorescent substance can be bound to the phosphate group added to the 5' end via a linking group composed of a hydrocarbon group having about 1 to 100 carbon atoms. The number of carbon atoms in the linking group is preferably about 1 to 30, and more preferably about 1 to 10.

[0182] By making the number of carbon atoms of the linking group greater than or equal to the lower limit, the distance between the fluorescent substance and the cutting position of the nucleic acid cleaving enzyme can be appropriately maintained. As a result, the cutting reaction performed by the nucleic acid cleaving enzyme is not easily hindered by the fluorescent substance, and the reaction efficiency performed by the nucleic acid cleaving enzyme is improved. By making the number of carbon atoms of the linking group less than or equal to the upper limit, the distance between the fluorescent substance and the quenching substance becomes shorter. As a result, the quenching effect of the quenching substance is improved.

[0183] [Chemical formula 1]

[0184]

[0185] The following formula (A2) is a chemical formula representing an example of a fluorescent substrate in which a fluorescent substance is bound to a base.

[0186] [Chemical formula 2]

[0187]

[0188] Alternatively, as an embodiment of the method of the first embodiment, for example, the following first embodiment can be cited. The first embodiment is a method for detecting a target nucleic acid. The method of the first embodiment is a method in which the substrate described in the first embodiment is applied to the above-mentioned ICA reaction.

[0189] (First Embodiment)

[0190] The method of the first embodiment comprises: step (a), forming a first cleavage structure using a target nucleic acid, a first oligonucleotide and a second oligonucleotide; step (b), cleaving the first oligonucleotide constituting the first cleavage structure using a cleavage agent having 5' nuclease activity to generate a third oligonucleotide consisting of a 5' side portion from the first oligonucleotide; step (c), forming a second α cleavage structure using a third oligonucleotide, a fourth oligonucleotide and a detection nucleic acid; step (d), cleaving the fourth oligonucleotide constituting the second α cleavage structure using a cleavage agent having 5' nuclease activity to generate a fifth oligonucleotide consisting of a 5' side portion from the fourth oligonucleotide; and step (e), detecting the fifth oligonucleotide.

[0191] Hereinafter, steps (a) to (e) will be described in detail with reference to the drawings. In the following steps, the first oligonucleotide corresponds to the above-mentioned allele probe, and the second oligonucleotide corresponds to the above-mentioned invader oligonucleotide. The detection nucleic acid corresponds to the above-mentioned FRET box.

[0192] 《Process (a)》

[0193] like Figure 3 As illustrated, in step (a), the first cleavage structure is formed using the target nucleic acid 1, the first oligonucleotide, and the second oligonucleotide.

[0194] The target nucleic acid 1 includes a first region (hereinafter sometimes referred to as T1 region), a second region (hereinafter sometimes referred to as T2 region) and a third region (hereinafter sometimes referred to as T3 region). The T1 region is adjacent to and downstream of the T2 region, and the T2 region is adjacent to and downstream of the T3 region.

[0195] The first oligonucleotide has a nucleotide sequence completely complementary to the T3 region at the 3' side portion, and the second oligonucleotide has a nucleotide sequence completely complementary to the T2 region at the 3' side portion and a nucleotide sequence completely complementary to the T1 region at the 5' side portion.

[0196] That is, the first oligonucleotide is an oligonucleotide that can hybridize with the T3 region of the target nucleic acid, and the second oligonucleotide is an oligonucleotide that can hybridize with the T1 region and the T2 region of the target nucleic acid. Therefore, by adding the target nucleic acid, the first oligonucleotide and the second oligonucleotide to the reaction solution and mixing them, when the second oligonucleotide anneals with the T1 region and the T2 region, and the first oligonucleotide anneals with the T3 region, a first cleavage structure can be obtained in which the 5' side portion of the region annealing to the T3 region in the first oligonucleotide overlaps with one or more nucleotide sequences in the 3' side portion of the second oligonucleotide annealing to the 2 region.

[0197] Figure 3 is a diagram schematically showing the first cracking structure. Figure 3 In the figure, "1" represents the target nucleic acid, "T1" to "T3" represent the T1 region to the T3 region, "n1" represents the first oligonucleotide, and "n2" represents the second oligonucleotide. Thus, the first cleavage structure adopts a branched structure in which the second oligonucleotide invades the bottom of the first oligonucleotide and the 5' side of the first oligonucleotide overlaps thereon.

[0198] 《Process (b)》

[0199] The first oligonucleotide constituting the first cleavage structure generated in step (a) is cleaved by a cleavage agent having 5' nuclease activity to generate a third oligonucleotide consisting of a portion derived from the 5' side of the first oligonucleotide.

[0200] The cleavage agent having 5' nuclease activity used herein recognizes the branch structure in the first cleavage structure, that is, the boundary portion between the 3' side portion of the first oligonucleotide annealing to the T3 region and the 5' side portion overlapping with the second oligonucleotide ( Figure 3 The oligonucleotide consisting of the 5' side portion of the region after annealing to the T3 region of the first oligonucleotide generated by the cleavage is used as the third oligonucleotide.

[0201] In step (b), after the cleavage reaction occurs, when the 3' side portion of the first oligonucleotide annealed to the T3 region dissociates from the target nucleic acid, the uncut first oligonucleotide anneals to the T3 region again to form a first cleavage structure. Thus, the invasion reaction is repeated without the need for operations such as thermal denaturation, and even trace amounts of target nucleic acid can be detected with high sensitivity.

[0202] The first oligonucleotide and the second oligonucleotide can be designed and synthesized by a conventional method considering the nucleotide sequence of the target nucleic acid, the type of the lysing agent used, the reaction temperature in the invasion reaction, the type of the reaction solution, etc. Here, when the Tm value of the second oligonucleotide is fully higher than the reaction temperature, the hybridization of the second oligonucleotide and the target nucleic acid is likely to become too strong. On the other hand, when the Tm value of the second oligonucleotide is too low than the reaction temperature, the annealing efficiency with the target nucleic acid will become too poor. Therefore, the Tm value of the second oligonucleotide is preferably about the reaction temperature.

[0203] In order to efficiently repeat the invasion reaction, the 3' side portion of the first oligonucleotide needs to be dissociated from the T3 region quickly after the cleavage reaction. Therefore, the Tm value of the region hybridizing to the T3 region of the first oligonucleotide is preferably sufficiently lower than the reaction temperature of the invasion reaction.

[0204] In addition, in the second oligonucleotide, the 3' end of the nucleotide sequence that is preferably completely complementary to the T2 region is the nucleotide at the second position from the 3' end of the second oligonucleotide. Thus, the 3' end of the second oligonucleotide overlaps with the nucleotide at the 5' end of the position annealed with the T3 region of the first oligonucleotide, so the first cleavage structure can be obtained efficiently. At this time, the nucleotide at the 3' end of the second oligonucleotide can be a nucleotide complementary to the corresponding nucleotide of the target nucleic acid, or it can be a non-complementary nucleotide, more preferably a non-complementary nucleotide. This is because the branched structure part in the first cleavage structure becomes more unstable, and the efficiency of the invasion reaction is further improved.

[0205] In the case where the target nucleic acid includes a specific nucleotide (target base) in a nucleotide sequence such as a gene polymorphism, the target base is preferably located in the T2 region or the T3 region in the target nucleic acid, preferably near the branch structure in the first cleavage structure, and more preferably in the T2 region near the branch structure. By designing the first oligonucleotide and the second oligonucleotide in a manner such that the target base comes near the branch structure, the detection accuracy of the target base can be improved.

[0206] The cleavage agent with 5' nuclease activity used in step (b) is not particularly limited as long as it can recognize the branch structure and has 5' nuclease activity, and is preferably a structure-specific enzyme. In addition, it can be an enzyme that only has 5' nuclease activity or an enzyme that has other enzyme activities. In addition, such a structure-specific enzyme can be an enzyme from any biological species or a synthetic enzyme.

[0207] As such a cleavage agent, an enzyme having 5' nuclease activity is preferred, a modified DNA polymerase having 5' nuclease activity but lacking polymerase activity is more preferred, and flap endonuclease is further preferred. The flap endonuclease is the same as described above.

[0208] The cleavage agent having 5' nuclease activity is preferably a thermostable enzyme having 5' nuclease activity. By using a thermostable enzyme, when using a target nucleic acid previously amplified by PCR, PCR and invasion reaction can be performed using the same reaction system.

[0209] In addition, the amount of the first oligonucleotide and the second oligonucleotide added to the reaction solution is not particularly limited, and it is preferred to add more of the first oligonucleotide than the second oligonucleotide. This is because, when the amount of the first oligonucleotide added is large, it is possible to expect to repeatedly perform the invasion reaction more efficiently. For example, it is preferred that the amount of the first oligonucleotide added is more than twice the amount of the second oligonucleotide added, more preferably about 2 to 100 times, further preferably about 5 to 50 times, and particularly preferably about 10 times.

[0210] It should be noted that the composition of the reaction solution is not particularly limited as long as it can form a cleavage structure and does not hinder the 5' nuclease activity of the cleavage agent used, and can be appropriately determined by considering the type of target nucleic acid, the type of the first oligonucleotide and / or the second oligonucleotide, the type of the cleavage agent used, etc. In addition, the reaction temperature can also be appropriately determined by considering the type of the cleavage agent used, the Tm value of the first oligonucleotide and / or the second oligonucleotide, etc.

[0211] 《Process (c)》

[0212] like Figure 4 As exemplified, in step (c), the second α-cleavage structure is formed by the third oligonucleotide and the fourth oligonucleotide produced in step (b) and the detection nucleic acid 2.

[0213] The detection nucleic acid comprises a first region (hereinafter sometimes referred to as D1 region), a second region (hereinafter sometimes referred to as D2 region) and a third region (hereinafter sometimes referred to as D3 region), wherein the D1 region is adjacent to and located downstream of the D2 region, and the D2 region is adjacent to and located downstream of the D3 region. In addition, the fourth oligonucleotide has a nucleotide sequence completely complementary to the third region of the detection nucleic acid at the 3' side portion, and the third oligonucleotide has a nucleotide sequence completely complementary to the second region of the detection nucleic acid at the 3' side portion, and has a nucleotide sequence completely complementary to the first region of the detection nucleic acid at the 5' side portion.

[0214] That is, the fourth oligonucleotide is an oligonucleotide that can hybridize with the D3 region of the detection nucleic acid, and the third oligonucleotide is an oligonucleotide that can hybridize with the D1 region and the D2 region of the detection nucleic acid. Therefore, by adding the detection nucleic acid, the third oligonucleotide and the fourth oligonucleotide to the reaction solution and mixing them, when the third oligonucleotide anneals with the D1 region and the D2 region, and the fourth oligonucleotide anneals with the D3 region, a second α-cleavage structure can be obtained in which the 5' side portion of the region annealed to the D3 region in the fourth oligonucleotide overlaps with one or more nucleotide sequences in the 3' side portion of the third oligonucleotide annealed to the D2 region.

[0215] Figure 4 It is a diagram schematically showing the second α-cleavage structure. Figure 4 In the figure, "2" represents the detection nucleic acid, "D1" to "D3" represent the D1 region to the D3 region, "n3" represents the third oligonucleotide, and "n4" represents the fourth oligonucleotide. In this way, the third oligonucleotide invades below the fourth oligonucleotide, and the 5' side of the fourth oligonucleotide forms a branched structure overlapping thereon. In the second α-cleavage structure, the fourth oligonucleotide functions as an allele probe, and the third oligonucleotide functions as an invader oligonucleotide.

[0216] 《Process (d)》

[0217] In step (d), the fourth oligonucleotide constituting the second α-cleavage structure generated in step (c) is cleaved by a cleavage agent having 5' nuclease activity to generate a fifth oligonucleotide consisting of a portion derived from the 5' side of the fourth oligonucleotide.

[0218] Specifically, in the same manner as in step (b), Figure 4 The boundary portion of the 3' side portion of the fourth oligonucleotide annealing to the D3 region and the 5' side portion overlapping with the third oligonucleotide indicated by the arrow in the figure is cut by a cleavage agent having 5' nuclease activity. The oligonucleotide consisting of the 5' side portion of the region annealing to the D3 region of the fourth oligonucleotide generated by the cutting is used as the fifth oligonucleotide.

[0219] The fourth oligonucleotide may be the substrate described in the first embodiment. For example, an oligonucleotide in which a fluorescent molecule functioning as a fluorescent substance is labeled at the 5' side portion (the portion to become the fifth oligonucleotide) and a fluorescent molecule functioning as a quenching substance is labeled at the site annealing to the D3 region may be used as the fourth oligonucleotide.

[0220] That is, in the fourth oligonucleotide, the fluorescent substance is located near the space of the quenching substance, and the fluorescence emission from the fluorescent substance is suppressed by the quenching substance. The peak wavelength of the absorption spectrum of the quenching substance is located on the shorter wavelength side relative to the peak wavelength of the emission spectrum of the fluorescent substance.

[0221] When the fourth oligonucleotide is labeled with a fluorescent substance and a quenching substance, the number of bases between the nucleic acid bound to the fluorescent substance and the nucleic acid bound to the quenching substance is preferably more than 11 bases and less than 30 bases, more preferably more than 4 bases and less than 10 bases, and further preferably more than 1 base and less than 3 bases.

[0222] By making the fluorescent substance of the fourth oligonucleotide be combined with the part other than the base, a three-layer chain structure can be formed efficiently, which can improve the reactivity of ICA. The fluorescent substance is combined with the part other than the base, which means that the fluorescent substance is combined with the sugar residue, phosphate group, sugar residue or phosphate group in the base of the oligonucleotide forming the fluorescent substrate. Among them, it is preferred that the fluorescent substance is combined with the phosphate group of the fluorescent substrate.

[0223] The fourth oligonucleotide (which may be referred to as a fluorescent substrate) preferably has a phosphate group added to the 5' end and a fluorescent substance is bound to the phosphate group added to the 5' end.

[0224] As described later in the Examples, a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bound to the phosphate group added to the 5' end has higher ICA reactivity than a fluorescent substrate having a fluorescent substance bound to a base, and can form a triple-stranded structure more efficiently.

[0225] The above formula (A1) is a chemical formula representing an example of a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bonded to the phosphate group added to the 5' end. As shown in the above formula (A1), the fluorescent substance can be bonded to the phosphate group added to the 5' end via a linking group composed of a hydrocarbon group having about 1 to 100 carbon atoms.

[0226] The number of carbon atoms of the linking group is preferably about 1 to 30, and more preferably about 1 to 10. By making the number of carbon atoms of the linking group above the lower limit, the distance between the fluorescent substance and the cutting site of the cleavage agent having 5' nuclease activity can be appropriately maintained. As a result, the cleavage reaction performed by the cleavage agent is not easily hindered by the fluorescent substance, and the efficiency of the reaction performed by the cleavage agent is improved. By making the number of carbon atoms of the linking group below the upper limit, the distance between the fluorescent substance and the quenching substance becomes shorter. As a result, the quenching effect of the quenching substance is improved.

[0227] The cleavage reaction in step (d) separates the fluorescent substance and the quenching substance in the fourth oligonucleotide, and fluorescence is emitted from the fluorescent substance.

[0228] 《Process (e)》

[0229] In step (e), fluorescence emitted from the fluorescent substance of the fifth oligonucleotide produced in step (d) is detected. Fluorescence can be observed using a fluorescence microscope.

[0230] Alternatively, as an embodiment of the method of the first embodiment, for example, the following second embodiment can be cited. The second embodiment is a method for detecting a target nucleic acid. The method of the second embodiment is a method in which the substrate described in the first embodiment is applied to the above-mentioned ICA reaction.

[0231] (Second Embodiment)

[0232] The second embodiment includes the following steps (f) to (h) after the above steps (a) to (b) instead of the steps (c) to (e).

[0233] In the following procedure, the first oligonucleotide corresponds to the allele probe described above, the second oligonucleotide corresponds to the invasive oligonucleotide described above, and the sixth oligonucleotide corresponds to the FRET box described above.

[0234] 《Process (f)》

[0235] like Figure 5 As exemplified, in step (f), the second β-cleavage structure is formed by the third oligonucleotide and the sixth oligonucleotide produced in step (b).

[0236] The sixth oligonucleotide comprises a first region (hereinafter sometimes referred to as the P6-1 region), a second region (hereinafter sometimes referred to as the P6-2 region) and a third region (hereinafter sometimes referred to as the P6-3 region), the P6-1 region is adjacent to and located downstream of the P6-2 region, the P6-2 region is adjacent to and located downstream of the P6-3 region, the P6-1 region has a nucleotide sequence that is completely complementary to the third oligonucleotide, and the P6-2 region has a nucleotide sequence that can form an intramolecular hairpin structure.

[0237] Figure 5 is a diagram schematically showing the second β-cleavage structure. Figure 5 In the figure, "n3" represents the third oligonucleotide, "n6" represents the sixth oligonucleotide, and "P6-1" to "P6-3" represent the P6-1 region to the P6-3 region. In this way, the third oligonucleotide invades below the sixth oligonucleotide, and the P6-3 region of the sixth oligonucleotide forms a branched structure overlapping thereon. In the second β-cleavage structure, the sixth oligonucleotide functions as an allele probe, and the third oligonucleotide functions as an invading oligonucleotide.

[0238] 《Process (g)》

[0239] In step (g), the sixth oligonucleotide constituting the second β cleavage structure generated in step (f) is cleaved by a cleavage agent having 5' nuclease activity to generate a seventh oligonucleotide consisting of the 5' side portion (ie, P6-3 region) derived from the sixth oligonucleotide.

[0240] Specifically, in the same manner as in step (b), Figure 5 The boundary portion of the P6-2 region of the sixth oligonucleotide indicated by the arrow and the P6-3 region of the 5' side portion overlapping with the third oligonucleotide is cut by a cleavage agent having 5' nuclease activity. The oligonucleotide consisting of the 5' side portion of the P6-2 region of the sixth oligonucleotide generated by the cutting is used as the seventh oligonucleotide.

[0241] The sixth oligonucleotide may be the substrate described in the first embodiment. As the sixth oligonucleotide, for example, an oligonucleotide in which a fluorescent molecule that functions as a fluorescent substance is labeled in the P6-3 region (the portion that becomes the seventh oligonucleotide) and a fluorescent molecule that functions as a quenching substance (Q) is labeled in the P6-2 region may be used.

[0242] That is, in the sixth oligonucleotide, the fluorescent substance is located near the space of the quenching substance, and the fluorescence emission from the fluorescent substance is suppressed by the quenching substance. The peak wavelength of the absorption spectrum of the quenching substance is located on the short wavelength side relative to the peak wavelength of the emission spectrum of the fluorescent substance.

[0243] The sixth oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.

[0244] When the sixth oligonucleotide is labeled with a fluorescent substance and a quenching substance, the number of bases between the nucleic acid bound to the fluorescent substance and the nucleic acid bound to the quenching substance is preferably more than 11 bases and less than 30 bases, more preferably more than 4 bases and less than 10 bases, and further preferably more than 1 base and less than 3 bases.

[0245] By making the fluorescent substance of the sixth oligonucleotide bind to the part other than the base, a three-layer chain structure can be efficiently formed, which can improve the reactivity of ICA. The fluorescent substance is bound to the part other than the base, which means that the fluorescent substance is bound to the sugar residue, phosphate group, sugar residue or phosphate group in the base of the oligonucleotide forming the fluorescent substrate. Among them, it is preferred that the fluorescent substance is bound to the phosphate group of the fluorescent substrate.

[0246] The sixth oligonucleotide (which may be referred to as a fluorescent substrate) preferably has a phosphate group added to the 5' end and a fluorescent substance is bound to the phosphate group added to the 5' end.

[0247] As described later in the Examples, a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bound to the phosphate group added to the 5' end has higher ICA reactivity than a fluorescent substrate having a fluorescent substance bound to a base, and can form a triple-stranded structure more efficiently.

[0248] The above formula (A1) is a chemical formula representing an example of a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bonded to the phosphate group added to the 5' end. As shown in the above formula (A1), the fluorescent substance can be bonded to the phosphate group added to the 5' end via a linking group composed of a hydrocarbon group having about 1 to 100 carbon atoms.

[0249] The number of carbon atoms of the linking group is preferably about 1 to 30, and more preferably about 1 to 10. By making the number of carbon atoms of the linking group above the lower limit, the distance between the fluorescent substance and the cutting site of the cleavage agent having 5' nuclease activity can be appropriately maintained. As a result, the cleavage reaction performed by the cleavage agent is not easily hindered by the fluorescent substance, and the efficiency of the reaction performed by the cleavage agent is improved. By making the number of carbon atoms of the linking group below the upper limit, the distance between the fluorescent substance and the quenching substance becomes shorter. As a result, the quenching effect of the quenching substance is improved.

[0250] By the cleavage reaction in step (g), the fluorescent substance and the quencher substance (Q) in the seventh oligonucleotide are separated, and fluorescence is emitted from the fluorescent substance.

[0251] 《Process (h)》

[0252] In step (h), fluorescence emitted from the fluorescent substance of the seventh oligonucleotide produced in step (g) is detected. Fluorescence can be observed using a fluorescence microscope.

[0253] There are no particular restrictions on the temperature, pH, salt concentration, buffer, etc. of the reaction solution for forming the first cleavage structure, the second α cleavage structure, and the second β cleavage structure described above, and those skilled in the art can set them appropriately. The pH of the reaction solution is preferably 6.5 to 10.0, and more preferably 7.5 to 9.0. The concentration of the salt with a buffering effect is preferably 5 to 250 mM, and more preferably 10 to 100 mM. There are no particular restrictions on the salt with a buffering effect, and examples thereof include cacodylates, phosphates, Tris salts, and the like. The reaction solution preferably contains salts of alkali metals and / or alkaline earth metals, and more preferably contains sodium chloride and / or magnesium chloride.

[0254] <Implementation Method B>

[0255] [Single-stranded oligonucleotide]

[0256] In one embodiment, the present invention provides a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light by the flap nuclease that recognizes the triple-chain structure.

[0257] The single-stranded oligonucleotide of this embodiment is a fluorescent substrate for ICA, and can also be said to be a substrate for flap endonuclease. Examples of flap endonucleases include flap endonuclease 1 (NCBI accession number: WP_011012561.1, Holliday junction 5'flap endonuclease (GEN1)) (NCBI accession number: NP_001123481.3), excision repair protein (NCBI accession number: AAC37533.1), and the like.

[0258] Here, the single-stranded oligonucleotide forms a hairpin structure at the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-stranded structure formed at the 5' terminal portion corresponds to Figure 1 The second triple strand structure 160 is shown. That is, the single-stranded oligonucleotide having a base sequence complementary to the 3′ side corresponds to the nucleic acid fragment 140 derived from the flap site 140 .

[0259] As described later in the examples, by making the fluorescent substance of the fluorescent substrate be combined with the part other than the base, a three-layer chain structure can be formed efficiently, which can improve the reactivity of ICA. The fluorescent substance being combined with the part other than the base means that the fluorescent substance is combined with the sugar residue, phosphate group, sugar residue or phosphate group in the base of the oligonucleotide forming the fluorescent substrate. Among them, it is preferred that the fluorescent substance is combined with the phosphate group of the fluorescent substrate.

[0260] In this specification, the reactivity of ICA can be improved by the fluorescence signal detected by the result of ICA reaching a specified value in a shorter time. Alternatively, the fluorescence signal intensity detected by the result of ICA can reach a higher value. Alternatively, the fluorescence signal of the background detected by the result of ICA can be maintained at a lower value.

[0261] The single-stranded oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.

[0262] (Fluorescent substance)

[0263] The fluorescent substance is not particularly limited, and examples thereof include fluorescein (molecular weight: 332.3), ATTO425 (molecular weight: 401.45), Alexa488 (molecular weight: 534.47), ATTO542 (molecular weight: 914), Yakima Y (molecular weight: 718.33), Redmond R (molecular weight: 445.3), ATTO643 (molecular weight: 836), Alexa647 (molecular weight: 860), Alexa680 (molecular weight: 1050), Alexa568 (molecular weight: 694.7), FAM (molecular weight: 332.3), ATTO633 (molecular weight: 652.2), Cy5 (molecular weight: 483.7), HiLyte Fluor 647 (molecular weight: 1205.6), ATTO665 (molecular weight: 723), Alexa594 (molecular weight: 722.8), Cy3 (molecular weight: 457.6), and ROX (molecular weight: 534.6). The molecular weight of the fluorescent substance is preferably 350 to 1100, more preferably 445 to 1100, and further preferably 445 to 914. As described later in Examples, when the molecular weight of the fluorescent substance is within the above range, a high signal-to-noise ratio (S / N ratio) tends to be obtained, which is preferred.

[0264] The single-stranded oligonucleotide of the fluorescent substrate preferably has a phosphate group added to the 5' end and the fluorescent substance is bound to the phosphate group added to the 5' end.

[0265] As described later in the Examples, a fluorescent substrate having a phosphate group added to the 5' end and a fluorescent substance bound to the phosphate group added to the 5' end has higher ICA reactivity than a fluorescent substrate having a fluorescent substance bound to a base, and can form a triple-stranded structure more efficiently.

[0266] The following formula (B1) is a chemical formula representing an example of a fluorescent substrate in which a phosphate group is added to the 5' end and a fluorescent substance is bound to the phosphate group added to the 5' end. As shown in the following formula (B1), the fluorescent substance can be bound to the phosphate group added to the 5' end via a linking group composed of a hydrocarbon group having a carbon number of about 1 to 100. The number of carbon atoms of the linking group is preferably about 1 to 30, and more preferably about 1 to 10. By making the number of carbon atoms of the linking group above the lower limit, the distance between the fluorescent substance and the cutting site of the flap endonuclease can be appropriately maintained. As a result, the cutting reaction performed by the flap endonuclease is not easily hindered by the fluorescent substance, and the cutting efficiency performed by the flap endonuclease is improved. In addition, by making the number of carbon atoms of the linking group below the upper limit, the distance between the fluorescent substance and the quenching substance becomes shorter. As a result, the quenching effect of the quenching substance is improved.

[0267] [Chemical formula 3]

[0268]

[0269] The following formula (B2) is a chemical formula representing an example of a fluorescent substrate in which a fluorescent substance is bound to a base.

[0270] [Chemical formula 4]

[0271]

[0272] (Quenching substance)

[0273] The quenching substance is not particularly limited as long as it is a substance that can quench the fluorescence of the fluorescent substance used, and examples thereof include Black Hole Quencher (BHQ) (registered trademark)-1, BHQ (registered trademark)-2, BHQ (registered trademark)-3, Fluorescence Quencher 1 (Tide Quencher 1, TQ1), Fluorescence Quencher 2 (Tide Quencher 2, TQ2), Fluorescence Quencher 2WS (Tide Quencher 2WS, TQ2WS), Fluorescence Quencher 3 (Tide Quencher 3, TQ3), Fluorescence Quencher 3WS (Tide Quencher 3WS, TQ3WS), Fluorescence Quencher 4 (Tide Quencher 4, TQ4), Fluorescence Quencher 4WS (Tide Quencher 4WS, TQ4WS), Fluorescence Quencher 5 (Tide Quencher 5, TQ5), Fluorescence Quencher 5WS (Tide Quencher 5WS, TQ5WS), fluorescence quencher 6WS (Tide Quencher 6WS, TQ6WS), fluorescence quencher 7WS (Tide Quencher 7WS, TQ7WS), QSY35, QSY7, QSY9, QSY21, Iowa Black FQ, Iowa Black RQ, etc. As the quenching substance, a substance that can quench the fluorescence of the fluorescent substance used is selected.

[0274] In the case where the fluorescent substance is located near the space of the quenching substance, the fluorescence from the fluorescent substance is quenched by the quenching substance. "Quenched fluorescence" means the following. In the absence of a quenching substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as A. In addition, in the case where the quenching substance is located near the space of the fluorescent substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as B. Here, "quenched fluorescence" means that the value of the above B / A is less than 40%.

[0275] The distance between the fluorescent substance and the quenching substance when the fluorescent substance is located near the space of the quenching substance is not particularly limited as long as the fluorescence emission from the fluorescent substance can be suppressed by the quenching substance, but is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 2 nm or less.

[0276] In the fluorescent substrate of the present embodiment, the quencher substance is preferably bound to the base of the nucleotide residue located at the 3′ side of the fluorescent substance.

[0277] As described later in the Examples, a fluorescent substrate in which the quencher is located on the 3' side of the fluorescent substance tends to be more reactive in ICA than a fluorescent substrate in which the quencher is located on the 5' side of the fluorescent substance. That is, when the quencher is labeled on the 3' side of the fluorescent substance and is cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released, which tends to be more reactive in ICA than the quencher is released from the fluorescent substrate.

[0278] The quenching substance may also be incorporated into the sugar-phosphate backbone. The following formula (B3) is a chemical formula representing an example of a quenching substance incorporated into the sugar-phosphate backbone of a nucleotide. The quenching substance in the following formula (B3) is BHQ (registered trademark)-1.

[0279] [Chemical formula 5]

[0280]

[0281] However, as described later in Examples, a fluorescent substrate in which a quencher is bound to a base tends to have a higher reactivity in ICA than a fluorescent substrate in which a quencher is incorporated into a sugar-phosphate backbone.

[0282] The following formula (B4) is a chemical formula representing an example of a fluorescent substrate in which a quenching substance is bound to a base. The quenching substance in the following formula (B4) is BHQ (registered trademark)-1.

[0283] [Chemical formula 6]

[0284]

[0285] (Oligonucleotide)

[0286] The single-stranded oligonucleotide constituting the fluorescent substrate of the present embodiment is not particularly limited as long as it can exert the effects of the present invention, and may be a natural nucleic acid or a synthetic nucleic acid.

[0287] As natural nucleic acid, for example, genomic DNA, mRNA, rRNA, hnRNA, miRNA, tRNA, etc. can be mentioned. Natural nucleic acid can be nucleic acid recovered from an organism, or nucleic acid recovered from water, organic matter, etc. that have been in contact with an organism. As a method for recovering natural nucleic acid, known methods such as the phenol / chloroform method can be mentioned.

[0288] Examples of synthetic nucleic acids include synthetic DNA, synthetic RNA, cDNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and the like.

[0289] The method for synthesizing nucleic acid is not particularly limited, and examples thereof include known chemical synthesis methods such as solid phase DNA synthesis using the phosphoramidite method, known nucleic acid amplification methods, reverse transcription reaction, etc. Examples of nucleic acid amplification methods include PCR, LAMP, SMAP, NASBA, and RCA.

[0290] In the fluorescent substrate of this embodiment, it is preferred that the fluorescent substance is arranged near the nucleotide residue at the 5' end, and the base of the nucleotide residue at the 5' end is a pyrimidine base. Purine bases tend to quench the fluorescence of the fluorescent substance. Therefore, if the base of the nucleotide residue at the 5' end is a pyrimidine base, the quenching of the fluorescence of the fluorescent substance is suppressed, so it is preferred.

[0291] In the fluorescent substrate of this embodiment, it is preferred that both the fluorescent substance and the quencher substance exist on one strand of the double-stranded oligonucleotide portion of the hairpin structure.

[0292] As described later in the Examples, a fluorescent substrate having both a fluorescent substance and a quenching substance on one strand of a double-stranded oligonucleotide portion of a hairpin structure tends to have better reactivity in ICA than a fluorescent substrate having a fluorescent substance on one strand of a double-stranded oligonucleotide portion of a hairpin structure and a quenching substance on the other strand. More specifically, in a fluorescent substrate having a fluorescent substance on one strand of a double-stranded oligonucleotide portion of a hairpin structure and a quenching substance on the other strand, the background fluorescence signal may increase.

[0293] [Method for improving the formation efficiency of the triple-chain structure in the fluorescent substrate of ICA]

[0294] In one embodiment, the present invention provides a method for improving the formation efficiency of a triple-stranded structure in a fluorescent substrate of ICA, comprising the step of adding a fluorescent substrate in which a fluorescent substance is bound to a portion other than a base to a reaction solution of ICA.

[0295] The fluorescent substrate of ICA is a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance. The single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization. When a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion. The fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light by the flap nuclease that recognizes the triple-chain structure.

[0296] The method of this embodiment is a method for improving the formation efficiency of a triple-stranded structure in a fluorescent substrate of a flap nuclease, which comprises the steps of preparing a first single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance and a second single-stranded oligonucleotide having a base sequence complementary to the 3' side of the first single-stranded oligonucleotide; and a step of bringing the first single-stranded oligonucleotide into contact with the second single-stranded oligonucleotide; wherein the first single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when the second single-stranded oligonucleotide hybridizes, a triple-stranded structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease, and emit fluorescence under the irradiation of excitation light, and the fluorescent substance is bound to the portion of the first single-stranded oligonucleotide other than the base.

[0297] In the method of this embodiment, ICA, fluorescent substance, quenching substance, oligonucleotide, etc. are the same as described above.

[0298] In the method of this embodiment, the fluorescent substrate preferably has a phosphate group added to the 5' end, and the fluorescent substance is bound to the phosphate group added to the 5' end.

[0299] In the method of this embodiment, the fluorescent substrate preferably has a quenching substance bound to a base of a nucleotide residue located at the 3' side of the fluorescent substance.

[0300] In the method of this embodiment, the base of the nucleotide residue at the 5' end of the fluorescent substrate is preferably a pyrimidine base.

[0301] In the method of this embodiment, the fluorescent substrate is preferably labeled with a quencher substance at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0302] In the method of this embodiment, the molecular weight of the fluorescent substrate is preferably 350 to 1100, more preferably 445 to 1100, and further preferably 445 to 914. As described later in the Examples, the molecular weight of the fluorescent substance within the above range tends to provide a high signal-to-noise ratio (S / N ratio), which is preferred.

[0303] In the method of this embodiment, the fluorescent substrate preferably contains both a fluorescent substance and a quenching substance on one strand of the double-stranded oligonucleotide portion of the hairpin structure.

[0304] In the method of this embodiment, ICA is preferably performed in a microscopic space. Specifically, ICA is preferably performed using a device having a hole array having a plurality of microscopic holes.

[0305] The wells may be used without treatment, or may be pre-treated in advance such as immobilizing extraction reagents, detection reagents such as antibodies, etc. on the inner wall of the wells, or covering the well openings with a lipid bilayer membrane, depending on the purpose.

[0306] The device may have a flow channel, and the ICA reaction solution may be supplied to the wells of the well array via the flow channel.

[0307] The diameter of the hole may be, for example, about 3 μm, and the depth of the hole may be, for example, about 4.5 μm. The holes may be arranged in a triangular lattice or a square lattice on the substrate to form a hole array.

[0308] Examples of the material of the substrate include cycloolefin polymers, cycloolefin copolymers, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins.

[0309] The number of wells in the well array is preferably about 100,000 to 6,000,000 per device, and the volume per well is preferably 1 fL to 6 pL.

[0310] <Implementation Method C>

[0311] [Single-stranded oligonucleotide]

[0312] In one embodiment, the present invention provides a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed and fluoresce under the irradiation of excitation light by the flap nuclease that recognizes the triple-chain structure. The single-stranded oligonucleotide contains non-complementary base pairs inside the hairpin structure.

[0313] The single-stranded oligonucleotide of this embodiment is a fluorescent substrate for ICA, and can also be said to be a substrate for flap endonuclease. Examples of flap endonucleases include flap endonuclease 1 (NCBI accession number: WP_011012561.1, Holliday junction 5'flap endonuclease (GEN1)) (NCBI accession number: NP_001123481.3), excision repair protein (NCBI accession number: AAC37533.1), and the like.

[0314] Here, the single-stranded oligonucleotide forms a hairpin structure at the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-stranded structure formed at the 5' terminal portion corresponds to Figure 1 The second three-stranded structure 160 is shown. That is, the single-stranded oligonucleotide having a base sequence complementary to the 3′ side corresponds to the nucleic acid fragment 140 derived from the flap site 140 .

[0315] As described later in Examples, by making the single-stranded oligonucleotide contain a non-complementary base pair inside the hairpin structure, an increase in background in ICA can be suppressed.

[0316] In the single-stranded oligonucleotide of the present embodiment, the number of non-complementary base pairs may be 1 to 3, 1 or 2, or even 1.

[0317] In this specification, the background in ICA refers to, for example, a fluorescent signal detected when ICA is performed in the absence of a target nucleic acid. Suppressing the increase in background in ICA means that the fluorescent signal of the background detected as a result of ICA is maintained at a lower value.

[0318] The single-stranded oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.

[0319] (Fluorescent substance)

[0320] The fluorescent substance is not particularly limited, and examples thereof include fluorescein, ATTO425, Alexa488, ATTO542, Yakima Y, Redmod R, ATTO643, Alexa647, Alexa680, Alexa568, FAM, ATTO633, Cy5, HiLyteFluor 647, and ATTO663.

[0321] The single-stranded oligonucleotide of the present embodiment preferably has a fluorescent substance labeled on the nucleotide residue at the 5' end, and a quencher substance labeled on the nucleotide residue located further to the 5' side among the nucleotide residues constituting the non-complementary base pair.

[0322] As described later in Examples, by performing ICA using such a fluorescent substrate, the background can be significantly suppressed.

[0323] (Quenching substance)

[0324] The quenching substance is not particularly limited as long as it is a substance that can quench the fluorescence of the fluorescent substance used, and examples thereof include Black Hole Quencher (BHQ) (registered trademark)-1, BHQ (registered trademark)-2, BHQ (registered trademark)-3, Fluorescence Quencher 1 (Tide Quencher 1, TQ1), Fluorescence Quencher 2 (Tide Quencher 2, TQ2), Fluorescence Quencher 2WS (Tide Quencher 2WS, TQ2WS), Fluorescence Quencher 3 (Tide Quencher 3, TQ3), Fluorescence Quencher 3WS (Tide Quencher 3WS, TQ3WS), Fluorescence Quencher 4 (Tide Quencher 4, TQ4), Fluorescence Quencher 4WS (Tide Quencher 4WS, TQ4WS), Fluorescence Quencher 5 (Tide Quencher 5, TQ5), Fluorescence Quencher 5WS (Tide Quencher 5WS, TQ5WS), fluorescence quencher 6WS (Tide Quencher 6WS, TQ6WS), fluorescence quencher 7WS (Tide Quencher 7WS, TQ7WS), QSY35, QSY7, QSY9, QSY21, Iowa Black FQ, Iowa Black RQ, etc. As the quenching substance, a substance that can quench the fluorescence of the fluorescent substance used is selected.

[0325] In the case where the fluorescent substance is located near the space of the quenching substance, the fluorescence from the fluorescent substance is quenched by the quenching substance. "Quenched fluorescence" means the following. In the absence of a quenching substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as A. In addition, in the case where the quenching substance is located near the space of the fluorescent substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as B. Here, "quenched fluorescence" means that the value of the above B / A is less than 40%.

[0326] The distance between the fluorescent substance and the quenching substance when the fluorescent substance is located near the space of the quenching substance is not particularly limited as long as the fluorescence emission from the fluorescent substance can be suppressed by the quenching substance, but is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 2 nm or less.

[0327] (Oligonucleotide)

[0328] The single-stranded oligonucleotide forming the fluorescent substrate of the present embodiment is not particularly limited as long as it can exert the effects of the present invention, and may be a natural nucleic acid or a synthetic nucleic acid.

[0329] As natural nucleic acid, for example, genomic DNA, mRNA, rRNA, hnRNA, miRNA, tRNA, etc. can be mentioned. Natural nucleic acid can be nucleic acid recovered from an organism, or nucleic acid recovered from water, organic matter, etc. that have been in contact with an organism. As a method for recovering natural nucleic acid, known methods such as the phenol / chloroform method can be mentioned.

[0330] Examples of synthetic nucleic acids include synthetic DNA, synthetic RNA, cDNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and the like.

[0331] The method for synthesizing nucleic acid is not particularly limited, and examples thereof include known chemical synthesis methods such as solid phase DNA synthesis using the phosphoramidite method, known nucleic acid amplification methods, reverse transcription reaction, etc. Examples of nucleic acid amplification methods include PCR, LAMP, SMAP, NASBA, and RCA.

[0332] In the fluorescent substrate of this embodiment, it is preferred that the fluorescent substance is arranged near the nucleotide residue at the 5' end, and the base of the nucleotide residue at the 5' end is a pyrimidine base. Purine bases tend to quench the fluorescence of the fluorescent substance. Therefore, if the base of the nucleotide residue at the 5' end is a pyrimidine base, the quenching of the fluorescence of the fluorescent substance is suppressed, so it is preferred.

[0333] [Method of suppressing background rise in ICA]

[0334] In one embodiment, the present invention provides a method for suppressing background increase in ICA, comprising the step of adding a fluorescent substrate containing a non-complementary base pair inside a hairpin structure to a reaction solution of ICA.

[0335] The fluorescent substrate of ICA is a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance. The single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization. When a single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion. The triple-chain structure is cut by the flap nuclease that recognizes the flap nuclease, and the fluorescent substance and the quenching substance are freed to emit fluorescence under the irradiation of excitation light.

[0336] In the method of this embodiment, ICA, fluorescent substance, quenching substance, oligonucleotide, etc. are the same as described above.

[0337] In the method of this embodiment, the fluorescent substrate is preferably labeled with a fluorescent substance on the nucleotide residue at the 5' end, and the quencher substance is labeled with a nucleotide residue located further to the 5' side among the nucleotide residues constituting the non-complementary base pair.

[0338] In the method of this embodiment, the fluorescent substrate preferably has a fluorescent substance disposed in the vicinity of the nucleotide residue at the 5' terminal, and the base of the nucleotide residue at the 5' terminal is a pyrimidine base.

[0339] In the method of this embodiment, the fluorescent substrate is preferably labeled with a quencher substance at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

[0340] In the method of this embodiment, ICA is preferably performed in a microscopic space. Specifically, ICA is preferably performed using a device having a hole array having a plurality of microscopic holes.

[0341] The wells may be used without treatment, or may be pre-treated in advance such as immobilizing extraction reagents, detection reagents such as antibodies, etc. on the inner wall of the wells, or covering the well openings with a lipid bilayer membrane, depending on the purpose.

[0342] The device may have a flow channel, and the ICA reaction solution may be supplied to the wells of the well array via the flow channel.

[0343] The diameter of the hole may be, for example, about 3 μm, and the depth of the hole may be, for example, about 4.5 μm. The holes may be arranged in a triangular lattice or a square lattice on the substrate to form a hole array.

[0344] Examples of the material of the substrate include cycloolefin polymers, cycloolefin copolymers, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins.

[0345] The number of wells in the well array is preferably about 100,000 to 6,000,000 per device, and the volume per well is preferably 1 fL to 6 pL.

[0346] <Implementation Method D>

[0347] [Single-stranded oligonucleotide]

[0348] In one embodiment, the present invention provides a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, which is the following single-stranded oligonucleotide: a hairpin structure is formed on the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease that recognizes the triple-chain structure, and emit fluorescence under the irradiation of excitation light; wherein the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by more than 4 residues.

[0349] The single-stranded oligonucleotide of this embodiment is a fluorescent substrate for ICA, and can also be said to be a substrate for flap endonuclease. Examples of flap endonucleases include flap endonuclease 1 (NCBI accession number: WP_011012561.1, Holliday junction 5'flap endonuclease (GEN1)) (NCBI accession number: NP_001123481.3), excision repair protein (NCBI accession number: AAC37533.1), and the like.

[0350] Here, the single-stranded oligonucleotide forms a hairpin structure at the 5' side by self-hybridization, and when a single-stranded oligonucleotide having a base sequence complementary to the 3' side is hybridized, a triple-stranded structure formed at the 5' terminal portion corresponds to Figure 1 The second triple strand structure 160 is shown. That is, the single-stranded oligonucleotide having a base sequence complementary to the 3′ side corresponds to the nucleic acid fragment 140 derived from the flap site 140 .

[0351] As described later in the examples, in the fluorescent substrate, by separating the nucleotide residues labeled with the fluorescent substance and the nucleotide residues labeled with the quenching substance by more than 4 residues, the cleavage efficiency by flap endonuclease in ICA can be improved. As a result, the fluorescent signal detected during ICA can be increased.

[0352] Improved cleavage efficiency by flap endonuclease in ICA means that a higher fluorescent signal is detected when performing ICA compared to when performing ICA using a fluorescent substrate in which the distance between a nucleotide residue labeled with a fluorescent substance and a nucleotide residue labeled with a quencher substance is less than 4 residues.

[0353] The single-stranded oligonucleotide of the present embodiment can be said to be a single-stranded oligonucleotide with improved cleavage efficiency by flap endonuclease.

[0354] The nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by 4 residues or more means that the distance between the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance is 4 nucleotide residues or more.

[0355] For example, when the fluorescent substance is labeled on the first nucleotide residue and the quenching substance is labeled on the fifth nucleotide residue, the distance between the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance is 4 residues.

[0356] In the present specification, "the distance is 4 residues" can be used interchangeably with "the distance is 4 bases in length", "the distance is 4 bases", "the number of bases is 4", etc.

[0357] As described in the Examples, the distance between the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quencher substance is preferably 4 or 5 residues, more preferably 4 residues.

[0358] The single-stranded oligonucleotide of this embodiment preferably has a length of about 20 to 150 bases. In addition, the number of base pairs forming the hairpin structure is preferably about 5 to 50.

[0359] The single-stranded oligonucleotide of this embodiment preferably contains a non-complementary base pair inside the hairpin structure, and the quenching substance is labeled on the nucleotide residue that is more located on the 5' side of the nucleotide residues constituting the non-complementary base pair. As described later in the examples, the single-stranded oligonucleotide having such a structure tends to have a high cutting efficiency using flap endonuclease.

[0360] In the single-stranded oligonucleotide of the present embodiment, the number of non-complementary base pairs may be 1 to 3, 1 or 2, or even 1.

[0361] (Fluorescent substance)

[0362] The fluorescent substance is not particularly limited, and examples thereof include fluorescein, ATTO425, Alexa488, ATTO542, Yakima Y, Redmod R, ATTO643, Alexa647, Alexa680, Alexa568, FAM, ATTO633, Cy5, HiLyteFluor 647, and ATTO663.

[0363] The single-stranded oligonucleotide of the present embodiment is preferably labeled with fluorescent substance on the nucleotide residue at the 5' end, and when cut by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is free. In addition, at this moment, the length of the free oligonucleotide fragment is preferably more than 2 residues. The length of the free oligonucleotide fragment is preferably 2 or 3 residues, more preferably 2 residues. As described later in the embodiment, such single-stranded oligonucleotide has the high tendency of the cutting efficiency that utilizes flap endonuclease to carry out.

[0364] (Quenching substance)

[0365] The quenching substance is not particularly limited as long as it is a substance that can quench the fluorescence of the fluorescent substance used, and examples thereof include Black Hole Quencher (BHQ) (registered trademark)-1, BHQ (registered trademark)-2, BHQ (registered trademark)-3, Fluorescence Quencher 1 (Tide Quencher 1, TQ1), Fluorescence Quencher 2 (Tide Quencher 2, TQ2), Fluorescence Quencher 2WS (Tide Quencher 2WS, TQ2WS), Fluorescence Quencher 3 (Tide Quencher 3, TQ3), Fluorescence Quencher 3WS (Tide Quencher 3WS, TQ3WS), Fluorescence Quencher 4 (Tide Quencher 4, TQ4), Fluorescence Quencher 4WS (Tide Quencher 4WS, TQ4WS), Fluorescence Quencher 5 (Tide Quencher 5, TQ5), Fluorescence Quencher 5WS (Tide Quencher 5WS, TQ5WS), fluorescence quencher 6WS (Tide Quencher 6WS, TQ6WS), fluorescence quencher 7WS (Tide Quencher 7WS, TQ7WS), QSY35, QSY7, QSY9, QSY21, Iowa Black FQ, Iowa Black RQ, etc. As the quenching substance, a substance that can quench the fluorescence of the fluorescent substance used is selected.

[0366] In the case where the fluorescent substance is located near the space of the quenching substance, the fluorescence from the fluorescent substance is quenched by the quenching substance. "Quenched fluorescence" means the following. In the absence of a quenching substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as A. In addition, in the case where the quenching substance is located near the space of the fluorescent substance, the intensity of the fluorescence emitted from the fluorescent substance when the fluorescent substance is irradiated with excitation light is recorded as B. Here, "quenched fluorescence" means that the value of the above B / A is less than 40%.

[0367] The distance between the fluorescent substance and the quenching substance when the fluorescent substance is located near the space of the quenching substance is not particularly limited as long as the fluorescence emission from the fluorescent substance can be suppressed by the quenching substance, but is preferably 10 nm or less, more preferably 5 nm or less, and further preferably 2 nm or less.

[0368] (Oligonucleotide)

[0369] The single-stranded oligonucleotide forming the fluorescent substrate of the present embodiment is not particularly limited as long as it can exert the effects of the present invention, and may be a natural nucleic acid or a synthetic nucleic acid.

[0370] As natural nucleic acid, for example, genomic DNA, mRNA, rRNA, hnRNA, miRNA, tRNA, etc. can be mentioned. Natural nucleic acid can be nucleic acid recovered from an organism, or nucleic acid recovered from water, organic matter, etc. that have been in contact with an organism. As a method for recovering natural nucleic acid, known methods such as the phenol / chloroform method can be mentioned.

[0371] Examples of synthetic nucleic acids include synthetic DNA, synthetic RNA, cDNA, bridged nucleic acid (BNA), locked nucleic acid (LNA), and the like.

[0372] The method for synthesizing nucleic acid is not particularly limited, and examples thereof include known chemical synthesis methods such as solid phase DNA synthesis using the phosphoramidite method, known nucleic acid amplification methods, reverse transcription reaction, etc. Examples of nucleic acid amplification methods include PCR, LAMP, SMAP, NASBA, and RCA.

[0373] In the fluorescent substrate of this embodiment, it is preferred that the fluorescent substance is arranged near the nucleotide residue at the 5' end, and the base of the nucleotide residue at the 5' end is a pyrimidine base. Purine bases tend to quench the fluorescence of the fluorescent substance. Therefore, if the base of the nucleotide residue at the 5' end is a pyrimidine base, the quenching of the fluorescence of the fluorescent substance is suppressed, so it is preferred.

[0374] [Method for improving the cleavage efficiency when cleaving a triple-stranded structure of nucleic acid using flap endonuclease]

[0375] In one embodiment, the present invention provides a method for improving the cutting efficiency when a nucleic acid triple-chain structure is cut with a flap nuclease, which comprises a step of contacting the nucleic acid triple-chain structure with the flap nuclease, wherein the nucleic acid triple-chain structure comprises a single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance and a single-stranded oligonucleotide having a base sequence complementary to the 3' side, the single-stranded oligonucleotide forms a hairpin structure on the 5' side by self-hybridization, and when the single-stranded oligonucleotide having a base sequence complementary to the 3' side hybridizes, a triple-chain structure is formed at the 5' end portion, and the fluorescent substance and the quenching substance are freed by being cut by the flap nuclease, and emit fluorescence under the irradiation of excitation light; wherein the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by more than 4 residues.

[0376] In the method of this embodiment, ICA, fluorescent substance, quenching substance, oligonucleotide, etc. are the same as those described above. By using a single-stranded oligonucleotide in which a nucleotide residue labeled with a fluorescent substance and a nucleotide residue labeled with a quenching substance are separated by 4 or more residues as a fluorescent substrate, the cleavage efficiency when using flap endonuclease can be improved.

[0377] In the method for the present embodiment, the single-stranded oligonucleotide is preferably labeled with a fluorescent substance on the nucleotide residue at the 5' end, and when cut by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is free. In addition, at this moment, the length of the free oligonucleotide fragment is preferably more than 2 residues. The length of the free oligonucleotide fragment is preferably 2 or 3 residues, more preferably 2 residues. As described later in the embodiment, such single-stranded oligonucleotide has a high tendency of cutting efficiency by utilizing flap endonuclease.

[0378] In the method of this embodiment, the fluorescent substance is preferably arranged near the nucleotide residue at the 5' end of the single-stranded oligonucleotide, and the base of the nucleotide residue at the 5' end is a pyrimidine base. As described above, if the base of the nucleotide residue at the 5' end is a pyrimidine base, the quenching of the fluorescence of the fluorescent substance is suppressed, so it is preferred.

[0379] In the method of this embodiment, the single-stranded oligonucleotide preferably contains a non-complementary base pair inside the hairpin structure, and the quenching substance is labeled on the nucleotide residue located more on the 5' side of the nucleotide residues constituting the non-complementary base pair. As described later in the examples, the single-stranded oligonucleotide having such a structure tends to have a high cleavage efficiency by flap endonuclease.

[0380] In the method of this embodiment, ICA is preferably performed in a microscopic space. Specifically, it is preferable to use a device having a hole array having a plurality of microscopic holes.

[0381] The wells may be used without treatment, or may be pre-treated in advance such as immobilizing extraction reagents, detection reagents such as antibodies, etc. on the inner wall of the wells, or covering the well openings with a lipid bilayer membrane, depending on the purpose.

[0382] The device may have a flow channel, and the ICA reaction solution may be supplied to the wells of the well array via the flow channel.

[0383] The diameter of the hole may be, for example, about 3 μm, and the depth of the hole may be, for example, about 4.5 μm. The holes may be arranged in a triangular lattice or a square lattice on the substrate to form a hole array.

[0384] Examples of the material of the substrate include cycloolefin polymers, cycloolefin copolymers, silicone, polypropylene, polycarbonate, polystyrene, polyethylene, polyvinyl acetate, fluororesins, and amorphous fluororesins.

[0385] The number of wells in the well array is preferably about 100,000 to 6,000,000 per device, and the volume per well is preferably 1 fL to 6 pL.

[0386] Example

[0387] Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

[0388] [Experimental Example A1]

[0389] A reaction solution containing multiple target nucleic acids, allele probes, invader oligonucleotides, and FRET boxes was prepared for ICA, and red fluorescence was detected.

[0390] The target nucleic acid, allele probe, invader oligonucleotide, and FRET box shown in Table 1 below were prepared.

[0391] [Table 1]

[0392]

[0393] Target nucleic acid 1 and target nucleic acid 2 are target nucleic acids consisting of the same base sequence except for one base in the underlined portion. Target nucleic acid 3 and target nucleic acid 4 are target nucleic acids consisting of the same base sequence except for one base in the underlined portion.

[0394] The target nucleic acid 1 is detected using the allele probe 1, the invader oligonucleotide 1, and the FRET cassette 1 that can hybridize with the target nucleic acid 1. The bases in lowercase letters in the target nucleic acid 1 and the allele probe 1 are base pairs that can hybridize with each other.

[0395] The target nucleic acid 2 is detected using the allele probe 2 that can hybridize with the target nucleic acid 2, the invader oligonucleotide 1, and the FRET cassette 3. The bases in lowercase letters in the target nucleic acid 2 and the allele probe 2 are base pairs that can hybridize with each other.

[0396] The target nucleic acid 3 is detected using the allele probe 3 that can hybridize with the target nucleic acid 3, the invader oligonucleotide 2, and the FRET cassette 2. The bases in lowercase letters in the target nucleic acid 3 and the allele probe 3 are base pairs that can hybridize with each other.

[0397] The target nucleic acid 4 is detected using the allele probe 4, the invader oligonucleotide 2, and the FRET cassette 4-1, 4-2, or 4-3 that can hybridize with the target nucleic acid 4. The lowercase bases in the target nucleic acid 4 and the allele probe 4 are base pairs that can hybridize with each other.

[0398] The fluorescent substance and quenching substance in each FRET box are as follows.

[0399] FRET Box 1: Alexa Fluor 488, BHQ-1

[0400] FRET box 2: ATTO542, BHQ-1

[0401] FRET Box 3: Alexa Fluor 568, BHQ-2

[0402] FRET box 4-1: ATTO643, BHQ-2

[0403] FRET box 4-2: ATTO643, BHQ-1

[0404] FRET box 4-3: ATTO643, BHQ-3

[0405] As shown in Table 2 below, the reagents were mixed to prepare reaction solutions 1 to 3.

[0406] [Table 2]

[0407] Reagents Final concentration Allele probe 1 2μM Allele Probe 2 1μM Invader Oligonucleotide 1 1μM Allele probe 3 2μM Allele Probe 4 2μM Invader Oligonucleotide 2 1μM FRET Box 1 4μM FRET Box 2 4μM FRET Box 3 4μM FRET box 4-1 or 4-2 or 4-3 4μM FEN-1 0.4mg / mL <![CDATA[MgCl 2 ]]> 25mM Tris pH 8.5 50mM Tween 20 0.05％ Target nucleic acid mixture 15fM each DW Up to 10μL

[0408] Reaction solutions 1 to 3 each contain one selected from FRET cassette 1, FRET cassette 2, and FRET cassette 3, and FRET cassette 4-1, FRET cassette 4-2, and FRET cassette 4-3.

[0409] Reaction solution 1 includes a FRET box 4 - 1 , reaction solution 2 includes a FRET box 4 - 2 , and reaction solution 3 includes a FRET box 4 - 3 .

[0410] (Measurement equipment)

[0411] A substrate with many microscopic holes was made of COP (cycloolefin polymer) and a cover made of COP was attached to produce a measuring device. 2 The total volume of the wells was 0.93 μL, and the total number of wells used for measurement was 1,000,000.

[0412] (Transportation of reaction mixture solution)

[0413] 8 μL of reaction solution 1 was delivered to the device and introduced into each well. Then, 200 μL of FC-40 (Sigma) was delivered as a sealing liquid to seal each well. Thus, less than 1 molecule of target nucleic acid per well was sealed in the well. That is, each well contained or did not contain the target nucleic acid.

[0414] (Nucleic acid detection reaction)

[0415] The above device after the reaction mixture solution was transferred was placed on a hot plate and reacted at 66°C for 25 minutes. Thus, the target nucleic acid was identified by the allele probe and the invader oligonucleotide, the allele probe was cut by FEN-1, the released allele probe fragments were bound to the FRET box, and the FRET box was cut by FEN-1, and a fluorescent signal was emitted. The schematic diagram of the reaction process is shown in FIG. Figure 6 shown.

[0416] (Fluorescence intensity measurement)

[0417] After heating the device 1 containing the reaction solution 1 at 66°C for 25 minutes, the fluorescence signal images of each well in the device were captured using a fluorescence microscope (BZ-700, KEYENCE) with a 4x objective lens and a fluorescence filter (excitation: 607nm / 70nm, dichroic mirror: 665nm, fluorescence: 700nm / 75nm) and an exposure time of 2 seconds. The results are shown in Figure 7 In addition, the fluorescent signal images obtained in the same manner except that the reaction solution 2 and the reaction solution 3 were used are shown in FIG. Figure 8 , Fig. 9 .

[0418] The results of measuring the fluorescence intensity of each reaction solution are shown in Figures 10-12 . Fig.10 is the measurement result of the fluorescence intensity of the reaction solution 1, Fig.11 is the measurement result of the fluorescence intensity of the reaction solution 2, Fig.12 This is the measurement result of the fluorescence intensity of the reaction solution 3.

[0419] Fig.10 In the above analysis, the fluorescence intensity of 3000 to 12000 is determined as noise (N), and the fluorescence intensity of 15000 to 45000 is determined as fluorescence signal (S). Fig.11 In the above analysis, the fluorescence intensity of 1500 to 9000 is determined as noise (N), and the fluorescence intensity of 12000 to 42000 is determined as fluorescence signal (S). Fig.12 In the above analysis, the fluorescence intensity of 1000 to 3000 is judged as noise (N), and the fluorescence intensity of 30000 to 12000 is judged as fluorescence signal (S).

[0420] According to the above-mentioned judgment criteria, the average value and standard deviation of noise (N) and fluorescent signal (S) were calculated. In addition, the S / N value (i.e., the value obtained by dividing the average value of S by the average value of N) and the SN value (i.e., the difference between the average value of S and the average value of N) were calculated. The results are shown in Table 3. Fig.13 A graph showing the S / N value, Fig.14 The SN value is shown.

[0421] [Table 3]

[0422]

[0423] When 4-1 was used among the FRET cassettes 4-1, 4-2, and 4-3, both the S / N value and the SN value were the highest.

[0424] BHQ-3 is recommended as a quenching substance for ATTO643. However, as described above, the S / N value and SN value of BHQ-2 are unexpectedly higher than those of BHQ-3. Therefore, it is shown that from the viewpoint of more accurately distinguishing noise and signal, when ATTO643 is used as a fluorescent substance, it is more preferable to use BHQ-2 as a quenching substance.

[0425] [Experimental Example A2]

[0426] (Study on fluorescent substrates)

[0427] ICA was performed using a fluorescent substrate in which a fluorescent substance was bound to a base and a fluorescent substrate in which a fluorescent substance was bound to a portion other than a base, and the reactivity was compared.

[0428] ICA was performed using the nucleic acid fragments shown in Tables 4 and 5 below. The target nucleic acid (sequence number 17) in Table 4 is the nucleic acid to be detected. In the ICA of this experimental example, the presence of g (guanine) represented by lowercase letters in the target nucleic acid was detected. The flap probe (sequence number 18) and the invasion probe (sequence number 19) each have a base sequence complementary to the target nucleic acid (sequence number 17).

[0429] When the flap probe (SEQ ID NO. 18) and the invader probe (SEQ ID NO. 19) are hybridized with the target nucleic acid (SEQ ID NO. 17), a three-layered structure is formed. Here, the lowercase letter portion "5'-cgcgccgaggc-3'" (SEQ ID NO. 20) of the flap probe (SEQ ID NO. 18) does not form a base pair but forms a flap site.

[0430] Flap endonuclease recognizes the above-mentioned three-layer chain structure, cuts the flap probe (SEQ ID NO: 18), and cuts out the nucleic acid fragment (SEQ ID NO: 20).

[0431] [Table 4]

[0432]

[0433] [Table 5]

[0434] name Serial Number Base sequence (5'→3') Fluorescent substrate 1 21 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG Fluorescent Substrate 2 22 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0435] The fluorescent substrate corresponds to the above-mentioned FRET box. The fluorescent substrate 1 (SEQ ID NO. 21) shown in Table 5 above is a fluorescent substrate in which a fluorescent substance is bound to a base, and specifically has a structure as shown in the following formula (A5). F in the fluorescent substrate 1 represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0436] [Chemical formula 7]

[0437]

[0438] Fluorescent substrate 2 (SEQ ID NO: 22) shown in Table 5 is a fluorescent substrate in which a fluorescent substance is bound to a part other than a base, and specifically has a structure as shown in the following formula (A6): F in fluorescent substrate 2 represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0439] [Chemical formula 8]

[0440]

[0441] When the excised nucleic acid fragment (SEQ ID NO: 20) hybridizes with fluorescent substrate 1 or fluorescent substrate 2, the T (thymine) residues indicated in bold in the above Table 5 form a triple-chain structure, which becomes a substrate for flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0442] First, a reaction solution was prepared with the composition shown in Table 6 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66°C for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0443] [Table 6]

[0444] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent Substrate 1 or Fluorescent Substrate 2 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0445] Fig.15 : is a graph showing the results of measuring the change in fluorescence intensity when using the fluorescent substrate 1. Fig.16 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate 2 was used. Fig.15 and Fig.16 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0446] As a result, it was found that the reactivity of ICA was higher when the fluorescent substrate 2 in which the fluorescent substance was bound to a part other than the base was used.

[0447] [Experimental Example B1]

[0448] (Study on fluorescent substrates 1)

[0449] ICA was performed using a fluorescent substrate in which a fluorescent substance was bound to a base and a fluorescent substrate in which a fluorescent substance was bound to a portion other than a base, and the reactivity was compared.

[0450] ICA was performed using the nucleic acid fragments shown in Tables 7 and 8 below. The target nucleic acid (sequence number 23) in Table 7 is the nucleic acid to be detected. In the ICA of this experimental example, the presence of g (guanine) represented by lowercase letters in the target nucleic acid was detected. The flap probe (sequence number 24) and the invasion probe (sequence number 25) each have a base sequence complementary to the target nucleic acid (sequence number 23).

[0451] When the flap probe (SEQ ID NO. 24) and the invader probe (SEQ ID NO. 25) are hybridized with the target nucleic acid (SEQ ID NO. 23), a three-layered structure is formed. Here, the lowercase letter portion "5'-cgcgccgaggc-3" (SEQ ID NO. 26) of the flap probe (SEQ ID NO. 24) does not form a base pair but forms a flap site.

[0452] Flap endonuclease recognizes the above-mentioned three-layer chain structure, cuts the flap probe (SEQ ID NO: 24), and cuts out the nucleic acid fragment (SEQ ID NO: 26).

[0453] [Table 7]

[0454]

[0455] [Table 8]

[0456] name Serial Number Base sequence (5'→3') Fluorescent substrate B1 27 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG Fluorescent substrate B2 28 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0457] The fluorescent substrate B1 (SEQ ID NO: 27) shown in Table 8 is a fluorescent substrate in which a fluorescent substance is bound to a base, and specifically has a structure as shown in the following formula (B5). In the fluorescent substrate B1, F represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0458] [Chemical formula 9]

[0459]

[0460] The fluorescent substrate B2 (SEQ ID NO: 28) shown in Table 8 is a fluorescent substrate in which a fluorescent substance is bound to a part other than a base, and specifically has a structure as shown in the following formula (B6). F in the fluorescent substrate B2 represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0461] [Chemical formula 10]

[0462]

[0463] When the excised nucleic acid fragment (SEQ ID NO. 4) hybridizes with the fluorescent substrate B1 or the fluorescent substrate B2, the T (thymine) residues indicated in bold in the fluorescent substrate B1 or the fluorescent substrate B2 in the above Table 8 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0464] First, a reaction solution was prepared with the composition shown in Table 9 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66° C. for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0465] [Table 9]

[0466] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent substrate B1 or fluorescent substrate B2 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0467] Fig.17 : is a graph showing the results of measuring the change in fluorescence intensity when the fluorescent substrate B1 is used. Fig.18 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate B2 was used. Fig.17 and Fig.18 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0468] As a result, it was found that the reactivity of ICA was higher when the fluorescent substrate B2 in which the fluorescent substance was bound to a part other than the base was used.

[0469] [Experimental Example B2]

[0470] (Study on fluorescent substrates 2)

[0471] ICA was performed using a fluorescent substrate in which the quencher was incorporated into the sugar-phosphate backbone and a fluorescent substrate in which the quencher was bound to the base, and the reactivity was compared.

[0472] The target nucleic acid (SEQ ID NO: 23), flap probe (SEQ ID NO: 24), and invader probe (SEQ ID NO: 25) were the same as those in Experimental Example B1, and ICA was performed using the fluorescent substrates B3 and B4 shown in Table 10 below.

[0473] [Table 10]

[0474] name Serial Number Base sequence (5'→3') Fluorescent substrate B3 29 F-TCT-Q-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG Fluorescent substrate B4 30 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0475] The fluorescent substrate B3 (SEQ ID NO: 29) shown in Table 10 is a fluorescent substrate in which a quenching substance is incorporated into a sugar-phosphate backbone, and the quenching substance has a structure as shown in the following formula (B7). In the fluorescent substrate B3, F represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0476] [Chemical formula 11]

[0477]

[0478] The fluorescent substrate B4 (SEQ ID NO: 30) shown in Table 10 is a fluorescent substrate in which a quenching substance is bound to a base, and the quenching substance has a structure as shown in the following formula (B8): F in the fluorescent substrate B4 represents fluorescein, and Q represents BHQ (registered trademark)-1.

[0479] [Chemical formula 12]

[0480]

[0481] When the nucleic acid fragment (SEQ ID NO. 26) cut out from the flap probe (SEQ ID NO. 24) hybridizes with the fluorescent substrate B3 or the fluorescent substrate B4, the T (thymine) residues indicated in bold on the fluorescent substrate B3 or the fluorescent substrate B4 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0482] First, a reaction solution was prepared with the composition shown in Table 11 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66°C for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0483] [Table 11]

[0484] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent substrate B3 or fluorescent substrate B4 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0485] Fig.19 : is a graph showing the results of measuring the change in fluorescence intensity when the fluorescent substrate B3 is used. Fig. 20 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate B4 was used. Fig.19 and Fig. 20 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0486] The results showed that the reactivity of ICA was higher when the fluorescent substrate B4 in which a quenching substance was bound to a base was used.

[0487] [Experimental Example B3]

[0488] (Study on fluorescent substrates 3)

[0489] ICA was performed using a fluorescent substrate in which the quencher substance was located on the 5' side of the fluorescent substance and a fluorescent substrate in which the quencher substance was located on the 3' side of the fluorescent substance, and the reactivity was compared.

[0490] The target nucleic acid (SEQ ID NO: 23), flap probe (SEQ ID NO: 24), and invader probe (SEQ ID NO: 25) were the same as those in Experimental Example B1, and ICA was performed using the fluorescent substrates B5 and B6 shown in Table 12 below.

[0491] [Table 12]

[0492] name Serial Number Base sequence (5'→3') Fluorescent substrate B5 31 T(Q)-TCT-T(F)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG Fluorescent substrate B6 32 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0493] The fluorescent substrate B5 (sequence number 31) shown in Table 12 above is a fluorescent substrate in which the quenching substance is located closer to the 5' side than the fluorescent substance, and specifically has a structure as shown in the following formula (B9). F in the fluorescent substrate B5 represents fluorescein, and Q represents BHQ (registered trademark)-1. In the following formula (B9), Q is modified on the base of the thymine residue at the 5' end, and F is modified on the base of the thymine residue in the chain.

[0494] [Chemical formula 13]

[0495]

[0496] The fluorescent substrate B6 (sequence number 32) shown in Table 12 is a fluorescent substrate in which the quenching substance is located on the 3' side of the fluorescent substance, and specifically has a structure as shown in the following formula (B10). F in the fluorescent substrate B6 represents fluorescein, and Q represents BHQ (registered trademark)-1. In the following formula (B10), F is modified on the base of the thymine residue at the 5' end, and Q is modified on the base of the thymine residue in the chain.

[0497] [Chemical formula 14]

[0498]

[0499] When the nucleic acid fragment (SEQ ID NO. 26) cut out from the flap probe (SEQ ID NO. 24) hybridizes with the fluorescent substrate B5 or the fluorescent substrate B6, the T (thymine) residues indicated in bold in the fluorescent substrate B5 or the fluorescent substrate B6 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0500] First, a reaction solution was prepared with the composition shown in Table 13 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66° C. for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0501] [Table 13]

[0502] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent substrate B5 or fluorescent substrate B6 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0503] Fig.21 : is a graph showing the results of measuring the change in fluorescence intensity when the fluorescent substrate B5 is used. Fig. 22 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate B6 was used. Fig.21 and Fig. 22 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0504] The results showed that the reactivity of ICA was higher when the fluorescent substrate B6 in which the quenching substance was located at the 3' side relative to the fluorescent substance was used.

[0505] [Experimental Example B4]

[0506] (Study on fluorescent substrates 4)

[0507] ICA reaction was performed using a fluorescent substrate having a fluorescent substance on one strand of a double-stranded oligonucleotide having a hairpin structure and a quenching substance on the other strand and a fluorescent substrate having both a fluorescent substance and a quenching substance on one strand of a double-stranded oligonucleotide having a hairpin structure, and the reactivity was compared.

[0508] The target nucleic acid (SEQ ID NO: 23), flap probe (SEQ ID NO: 24), and invader probe (SEQ ID NO: 25) were the same as those in Experimental Example B1, and ICA was performed using the fluorescent substrates shown in Table 14 below.

[0509] [Table 14]

[0510] name Serial Number Base sequence (5'→3') Fluorescent substrate B7 33 F-TCTGAGCCGGTTTTCCGGCT-T(Q)-AGACCTCGGCGCG Fluorescent substrate B8 34 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0511] The fluorescent substrate B7 (sequence number 33) shown in Table 14 above is a fluorescent substrate in which a fluorescent substance exists on one strand of the double-stranded oligonucleotide part of the hairpin structure and a quenching substance exists on the other strand, and specifically has a structure as shown in the following formula (B11). F in the fluorescent substrate B7 represents fluorescein, and Q represents BHQ (registered trademark)-1. In the following formula (B11), F is modified on the base of the thymine residue at the 5' end, and Q is modified on the base of the thymine residue in the chain.

[0512] [Chemical formula 15]

[0513]

[0514] The fluorescent substrate B8 (sequence number 34) shown in Table 14 is a fluorescent substrate in which both a fluorescent substance and a quenching substance exist on one strand of the double-stranded oligonucleotide part of the hairpin structure, and specifically has a structure as shown in the following formula (B12). F in the fluorescent substrate B8 represents fluorescein, and Q represents BHQ (registered trademark)-1. In the following formula (B12), F is modified on the base of the thymine residue at the 5' end, and Q is modified on the base of the thymine residue in the chain.

[0515] [Chemical formula 16]

[0516]

[0517] When the nucleic acid fragment (SEQ ID NO. 26) cut out from the flap probe (SEQ ID NO. 24) hybridizes with the fluorescent substrate B7 or the fluorescent substrate B8, the T (thymine) residues indicated in bold of the fluorescent substrate 7 or the fluorescent substrate B8 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0518] First, a reaction solution was prepared with the composition shown in Table 15 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66° C. for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0519] [Table 15]

[0520] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent substrate B7 or fluorescent substrate B8 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0521] Fig.23 : is a graph showing the results of measuring the change in fluorescence intensity when the fluorescent substrate B7 is used. Fig.24This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate B8 was used. Fig.23 and Fig.24 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0522] The results showed that the reactivity of ICA was higher when the fluorescent substrate B8 in which both a fluorescent substance and a quenching substance were present on one strand of the double-stranded oligonucleotide portion of the hairpin structure was used.

[0523] [Experimental Example B5]

[0524] (Study on fluorescent substrates 5)

[0525] ICA reaction was performed using a plurality of fluorescent substrates modified with fluorescent substances having different molecular weights, and the S / N ratio was determined. The correlation between the molecular weight of the fluorescent substance and the S / N ratio was studied.

[0526] The target nucleic acid (SEQ ID NO: 23), flap probe (SEQ ID NO: 24), and invader probe (SEQ ID NO: 25) were the same as those in Experimental Example B1, and ICA was performed using the fluorescent substrates shown in Table 16 below.

[0527] [Table 16]

[0528]

[0529] The fluorescent substrates B9 to B16 (SEQ ID NO: 35) shown in Table 16 above are fluorescent substrates in which the quenching substance is located on the 3' side of the fluorescent substance, and specifically have a structure as shown in the following formula (B13). In the following formula (B13), the fluorescent substance F is modified on the base of the thymine residue at the 5' end, and Q is modified on the base of the thymine residue in the chain.

[0530] [Chemical formula 17]

[0531]

[0532] The combinations of the fluorescent substance F and the quenching substance Q in the fluorescent substrates B9 to B16 are shown in the following Table 17. The following Table 17 also shows the molecular weight of the fluorescent substance and the S / N ratio measured by the method described below.

[0533] [Table 17]

[0534] name Fluorescent substances Molecular weight of fluorescent substance Quenching substance S / N Ratio Fluorescent substrate B9 ATTO 425 401.45 BHQ(Registered Trademark)-0 1.5 Fluorescent substrate B10 Alexa 488 534.47 BHQ(Registered Trademark)-1 3.5 Fluorescent substrate B11 ATTO 542 914 BHQ(Registered Trademark)-1 3.6 Fluorescent substrate B12 Yakima Y 718.33 BHQ(Registered Trademark)-1 3.7 Fluorescent substrate B13 Redmond R 445.3 BHQ(Registered Trademark)-2 3.3 Fluorescent substrate B14 ATTO 643 836 BHQ(Registered Trademark)-2 5.2 Fluorescent substrate B15 Alexa 647 860 BHQ(Registered Trademark)-2 5.5 Fluorescent substrate B16 Alexa 680 1,050 BHQ(Registered Trademark)-2 2.5

[0535] When the nucleic acid fragment (SEQ ID NO. 26) cut out from the flap probe (SEQ ID NO. 24) hybridizes with the fluorescent substrates B9 to B16, the T (thymine) residues in bold of the fluorescent substrates B9 to B16 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0536] First, reaction solutions were prepared with the compositions shown in Table 18 and introduced into the wells of a fluidic device having a well array. The well array had approximately 930,000 wells, and the volume of each well was approximately 1683 fL.

[0537] Next, the fluid device was placed in an aluminum block thermostatic bath (model "DTU-Mini", TAITE), heated at 66°C for 25 minutes, and then observed using a microscope (product name "All-in-One Fluorescence Microscope", model "BZ-X810", KEYENCE).

[0538] Next, based on the microscope observation image, the luminous holes (S) and non-luminous holes (N) are identified, and the brightness of the luminous holes and non-luminous holes is calculated. Next, the ratio of the brightness of the luminous holes to the brightness of the non-luminous holes, that is, the S / N ratio, is calculated.

[0539] [Table 18]

[0540] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Any one of fluorescent substrates B9 to B16 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0541] The calculated S / N ratio is shown in the above Table 17. Fig.25 This is a graph summarizing the relationship between the molecular weight of the fluorescent substance and the S / N ratio. The results show that when the molecular weight of the fluorescent substance is 350 to 1100, the S / N ratio is 1.5 or more. It is also known that when the molecular weight of the fluorescent substance is 445 to 1100, the S / N ratio is 2 or more. It is also known that when the molecular weight of the fluorescent substance is 445 to 914, the S / N ratio is 3 or more.

[0542] [Experimental Example C1]

[0543] (Study on fluorescent substrates)

[0544] ICA was performed using a fluorescent substrate that does not contain a non-complementary base pair inside the hairpin structure and a fluorescent substrate that contains a non-complementary base pair inside the hairpin structure, and the reactivity was compared.

[0545] ICA was performed using the nucleic acid fragments shown in Tables 19 and 20 below. The target nucleic acid (sequence number 1) in Table 19 is the nucleic acid to be detected. In the ICA of this experimental example, the presence of g (guanine) represented by lowercase letters in the target nucleic acid was detected. The flap probe (sequence number 2) and the invasion probe (sequence number 3) each have a base sequence complementary to the target nucleic acid (sequence number 1).

[0546] When the flap probe (SEQ ID NO. 37) and the invader probe (SEQ ID NO. 38) are hybridized with the target nucleic acid (SEQ ID NO. 36), a three-layered structure is formed. Here, the lowercase letter portion "5'-cgcgccgaggc-3'" (SEQ ID NO. 39) of the flap probe (SEQ ID NO. 37) does not form a base pair but forms a flap site.

[0547] Flap endonuclease recognizes the above-mentioned three-layer chain structure, cuts the flap probe (SEQ ID NO: 37), and cuts out the nucleic acid fragment (SEQ ID NO: 39).

[0548] [Table 19]

[0549]

[0550] [Table 20]

[0551] name Serial Number Base sequence (5'-3') Fluorescent substrate C1 40 F-TCT-T(Q)-AGCCGGTTTTCCGGCTAAGACCTCGGCGCG Fluorescent substrate C2 41 F-TCT-T(Q)-AGCCGGTTTTCCGGCTGAGACCTCGGCGCG

[0552] The fluorescent substrate C1 (SEQ ID NO: 40) shown in Table 20 above is a fluorescent substrate that does not contain non-complementary base pairs inside the hairpin structure, and specifically has a structure as shown in the following formula (C1). F in the fluorescent substrate C1 represents fluorescein, and Q represents BHQ (registered trademark)-1. As shown in the following formula (C1), the fluorescent substrate C1 does not contain non-complementary base pairs inside the hairpin structure.

[0553] [Chemical formula 18]

[0554]

[0555] The fluorescent substrate C2 (SEQ ID NO: 41) shown in Table 20 above is a fluorescent substrate containing non-complementary base pairs inside the hairpin structure, and specifically has a structure as shown in the following formula (C2). F in the fluorescent substrate C2 represents fluorescein, and Q represents BHQ (registered trademark)-1. As shown in the following formula (C2), the fluorescent substrate C2 contains non-complementary base pairs inside the hairpin structure. In the following formula (C2), the T (thymine) residue and the G (guanine) residue surrounded by the quadrilateral are not complementary to each other.

[0556] [Chemical formula 19]

[0557]

[0558] When the excised nucleic acid fragment (SEQ ID NO: 39) hybridizes with the fluorescent substrate C1 or the fluorescent substrate C2, the T (thymine) residues indicated in bold in the fluorescent substrate C1 or the fluorescent substrate C2 in the above Table 20 form a triple-chain structure, which becomes a substrate for the flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light.

[0559] First, a reaction solution was prepared with the composition shown in Table 21 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66° C. for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0560] [Table 21]

[0561] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Fluorescent substrate C1 or fluorescent substrate C2 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0562] Fig.26 : is a graph showing the results of measuring the change in fluorescence intensity when the fluorescent substrate C1 is used. Fig. 27 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate C2 was used. Fig.26 and Fig. 27 In the graph, the horizontal axis represents the elapsed time (seconds) from the start of the reaction, and the vertical axis represents the fluorescence intensity (relative value).

[0563] The results showed that when the fluorescent substrate C2 containing a non-complementary base pair inside the hairpin structure was used, the background increase in the ICA method was more significantly suppressed than when the fluorescent substrate C1 was used.

[0564] [Experimental Example D1]

[0565] (Study on fluorescent substrates)

[0566] ICA was performed using various fluorescent substrates in which the distance between the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance was changed, and the reactivity was compared.

[0567] ICA was performed using the nucleic acid fragments shown in Tables 22 and 23 below. The target nucleic acid (sequence number 42) in Table 22 is the nucleic acid to be detected. In the ICA of this experimental example, the presence of g (guanine) represented by lowercase letters in the target nucleic acid was detected. The flap probe (sequence number 43) and the invasion probe (sequence number 44) each have a base sequence complementary to the target nucleic acid (sequence number 42).

[0568] When the flap probe (SEQ ID NO. 43) and the invader probe (SEQ ID NO. 44) are hybridized with the target nucleic acid (SEQ ID NO. 42), a three-layered structure is formed. Here, the lowercase letter portion "5'-cgcgccgaggc-3'" (SEQ ID NO. 45) of the flap probe (SEQ ID NO. 43) does not form a base pair but forms a flap site.

[0569] Flap endonuclease recognizes the above-mentioned three-layer chain structure, cuts the flap probe (SEQ ID NO: 43), and cuts out the nucleic acid fragment (SEQ ID NO: 45).

[0570] [Table 22]

[0571]

[0572] [Table 23]

[0573]

[0574] The fluorescent substrate D1 (SEQ ID NO: 46) shown in Table 23 above is a fluorescent substrate in which the distance between the nucleotide residue labeled with a fluorescent substance and the nucleotide residue labeled with a quenching substance is 2 residues, and specifically has a structure as shown in the following formula (D1). F in the fluorescent substrate D1 represents fluorescein, and Q represents BHQ (registered trademark)-1. In addition, as shown in the following formula (D1), the fluorescent substrate 1 contains non-complementary base pairs inside the hairpin structure.

[0575] [Chemical formula 20]

[0576]

[0577] The fluorescent substrate D2 (SEQ ID NO: 47) shown in Table 23 above is a fluorescent substrate in which the distance between the nucleotide residue labeled with a fluorescent substance and the nucleotide residue labeled with a quenching substance is 3 residues, and specifically has a structure as shown in the following formula (D2). F in the fluorescent substrate D2 represents fluorescein, and Q represents BHQ (registered trademark)-1. In addition, as shown in the following formula (D2), the fluorescent substrate D2 contains non-complementary base pairs inside the hairpin structure.

[0578] [Chemical formula 21]

[0579]

[0580] The fluorescent substrate D3 (SEQ ID NO: 48) shown in Table 23 above is a fluorescent substrate in which the distance between the nucleotide residues labeled with a fluorescent substance and the nucleotide residues labeled with a quenching substance is 4 residues, and specifically has a structure as shown in the following formula (D3). F in the fluorescent substrate D3 represents fluorescein, and Q represents BHQ (registered trademark)-1. In addition, as shown in the following formula (D3), the fluorescent substrate D3 contains non-complementary base pairs inside the hairpin structure.

[0581] [Chemical formula 22]

[0582]

[0583] The fluorescent substrate D4 (SEQ ID NO: 49) shown in Table 23 above is a fluorescent substrate in which the distance between the nucleotide residue labeled with a fluorescent substance and the nucleotide residue labeled with a quenching substance is 5 residues, and specifically has a structure as shown in the following formula (D4). F in the fluorescent substrate D4 represents fluorescein, and Q represents BHQ (registered trademark)-1. In addition, as shown in the following formula (D4), the fluorescent substrate D4 contains non-complementary base pairs inside the hairpin structure.

[0584] [Chemical formula 23]

[0585]

[0586] When the excised nucleic acid fragment (SEQ ID NO: 45) is hybridized with the fluorescent substrate D1, the C (cytosine) residue indicated in bold in the above Table 23 is cut off by the fluctuation of the substrate recognition ability of the flap endonuclease. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light. In the fluorescent substrate D1, when cut by the flap endonuclease, the length of the free oligonucleotide fragment is 1 residue.

[0587] When the excised nucleic acid fragment (SEQ ID NO: 45) hybridizes with the fluorescent substrates D2, D3, and D4, the T (thymine) residues indicated in bold in the fluorescent substrates D2, D3, and D4 in Table 23 above form a three-layer chain structure, which becomes a substrate for flap endonuclease and is cut. As a result, the fluorescent substance is separated from the quenching substance, and the fluorescent signal can be detected by irradiation with excitation light. In the case of the fluorescent substrates D2, D3, and D4 being cut by flap endonuclease, the lengths of the free oligonucleotide fragments are 1, 2, and 3 residues, respectively.

[0588] First, a reaction solution was prepared with the composition shown in Table 24 below and added to a micro test tube. Next, the tube was placed in a real-time PCR device, and the change in fluorescence intensity (excitation wavelength of 490 nm, fluorescence wavelength of 520 nm) when heated at 66° C. for 60 minutes was measured over time. In addition, for comparison, a reaction solution to which sterilized water was added instead of the target nucleic acid was prepared, and the same measurement was performed.

[0589] [Table 24]

[0590] Reagents Final concentration Target nucleic acid 1μM Flap Probe 1μM Intrusion Probe 1μM Any one of fluorescent substrates D1 to D4 2μM 3-Morpholinepropanesulfonic acid (pH 7.9) 10mM <![CDATA[MgCl 2 ]]> 10mM Tween 20 0.05(v / v)% Flap endonuclease 1,000U Distilled water Moderate total 10μL

[0591] Fig.28 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate D1 was used. Fig.29 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate D2 was used. Fig.30 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate D3 was used. Fig.31 This is a graph showing the results of measuring changes in fluorescence intensity when the fluorescent substrate D4 was used. Figure 28 to Figure 31 In the embodiment, the distances between the nucleotide residues labeled with the fluorescent substance of each fluorescent substrate and the nucleotide residues labeled with the quenching substance are 2, 3, 4, and 5 residues, respectively.

[0592] The results showed that when the distance between the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quencher substance in the fluorescent substrate is 4 residues or more, the efficiency of cleavage of the fluorescent substrate by flap endonuclease is high, and particularly high when the distance is 4 residues.

[0593] Industrial availability

[0594] According to the present invention, it is possible to provide a technique for improving the reaction efficiency in a detection reaction of a trace amount of an analyte molecule.

[0595] Description of Reference Numerals

[0596] 100 Target nucleic acid

[0597] 101 bases

[0598] 110 flap probe

[0599] 120 Intrusion Probe

[0600] 130 The first three-layer chain structure

[0601] 140, 170 flap part (nucleic acid fragment)

[0602] 141 Area

[0604] 150 Nucleic Acid Fragments

[0605] 151 Mismatch site

[0606] 160 The second third-layer chain structure

[0607] F Fluorescent material

[0608] Q Quenching substance

Claims

1. A single-stranded oligonucleotide labeled with a fluorescent substance and a quenching substance, wherein the single-stranded oligonucleotide forms a hairpin structure at the 5' side by self-hybridization, forms a triple-stranded structure at the 5' end portion when hybridized with a single-stranded oligonucleotide having a base sequence complementary to the 3' side, and is cut by a flap endonuclease that recognizes the triple-stranded structure, so that the fluorescent substance and the quenching substance are freed and emit fluorescence under the irradiation of excitation light; in, The single-stranded oligonucleotide satisfies any one of the following (A) to (C): (A) a single-stranded oligonucleotide comprising a non-complementary base pair in the interior of the hairpin structure; (B) a single-stranded oligonucleotide in which the nucleotide residue labeled with the fluorescent substance and the nucleotide residue labeled with the quenching substance are separated by 4 residues or more; (C) A single-stranded oligonucleotide in which the fluorescent substance is bound to a portion other than the base.

2. The single-stranded oligonucleotide according to claim 1, wherein The above (A) is satisfied, wherein the fluorescent substance is labeled on the nucleotide residue at the 5' end, and the quenching substance is labeled on the nucleotide residue located further to the 5' side among the nucleotide residues constituting the non-complementary base pair.

3. The single-stranded oligonucleotide according to claim 1, wherein The base of the nucleotide residue at the 5' end is a pyrimidine base.

4. The single-stranded oligonucleotide according to claim 1, wherein The quencher substance is labeled at a position closer to the 3' side than the fluorescent substance, and when cleaved by flap endonuclease, the oligonucleotide fragment containing the fluorescent substance is released.

5. The single-stranded oligonucleotide according to claim 1, wherein The fluorescent substance is labeled on the nucleotide residue at the 5' end, and when cleaved by flap endonuclease, an oligonucleotide fragment containing the fluorescent substance is released, and the length of the oligonucleotide fragment is more than 2 residues.

6. The single-stranded oligonucleotide according to claim 1, wherein A phosphate group is added to the 5' end and the fluorescent substance is bound to the phosphate group.

7. The single-stranded oligonucleotide according to claim 1, wherein The quenching substance is bound to the base of the nucleotide residue located at the 3' side of the fluorescent substance.

8. The single-stranded oligonucleotide according to claim 1, wherein The molecular weight of the fluorescent substance is 350-1100.

9. The single-stranded oligonucleotide according to claim 1, wherein Both the fluorescent substance and the quenching substance exist on one strand of the double-stranded oligonucleotide portion of the hairpin structure.

10. A method for detecting fluorescence from a matrix having a fluorescent substance, a quenching substance and a matrix body, The method comprises the step of separating the fluorescent substance and the quenching substance in the matrix so that the fluorescent substance emits fluorescence. in, When the fluorescent substance is located in the spatial vicinity of the quenching substance, the fluorescent emission from the fluorescent substance is suppressed by the quenching substance. The peak wavelength of the absorption spectrum of the quenching substance is located on the shorter wavelength side with respect to the peak wavelength of the emission spectrum of the fluorescent substance.

11. The method according to claim 10, wherein: The step of causing the fluorescent substance to emit fluorescence is performed in the presence of a target substance.

12. The method according to claim 10 or 11, wherein: The peak wavelength of the absorption spectrum of the quenching substance is shorter than the peak wavelength of the emission spectrum of the fluorescent substance by 50 to 100 nm.

13. The method according to claim 10 or 11, wherein: The matrix is ​​mainly nucleic acid.

14. The method according to claim 10 or 11, wherein: The peak wavelength of the luminescence spectrum of the fluorescent substance is 600-700 nm, and the peak wavelength of the absorption spectrum of the quenching substance is 550-640 nm.

15. The method according to claim 14, wherein: In the step of causing the fluorescent substance to emit fluorescence, the fluorescent substance and the quenching substance in the matrix are separated by the action of the nuclease.

16. The method according to claim 10 or 11, wherein: The process of causing the fluorescent substance to emit fluorescence is performed in a microscopic space.

17. The method according to claim 11, wherein: The target substance is nucleic acid.

18. The method according to claim 10 or 11, wherein: The fluorescence was further observed using a fluorescence microscope.

Images