Nucleic acid sequence measurement method and nucleic acid sequence measurement kit

Abstract

The present invention uses a detection probe (10) that has a detection part that has a nucleic acid sequence that is complementary to the nucleic acid sequence of a target (50) that has a specific nucleic acid sequence, a label molecule A (20) that has a nucleic acid sequence that is complementary to the nucleic acid sequence of the target (50) but is different from the nucleic acid sequence of the detection part, and a label molecule B (30) that has a nucleic acid sequence that is complementary to the nucleic acid sequence of label molecule A (20). A fluorescent molecule is added to one of label molecule A (20) and label molecule B (30), label molecule A (20) and label molecule B (30) are made to bond at two or more locations, and a branched structure of label molecule A (20) and label molecule B (30) is formed to measure the target (50) included in a sample.

Description

[Technical Field]

[0001] The present invention relates to a nucleic acid sequence measurement method and a nucleic acid sequence measurement kit for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization. [Background technology]

[0002] A widely known method for measuring a target having a specific nucleic acid sequence contained in a sample is to use a DNA microarray (a detection probe having a complementary sequence to a specific nucleic acid sequence is provided on a solid phase surface such as a substrate). This method measures the target by utilizing the property that the target contained in a sample added to the DNA microarray is captured by the detection probe of the DNA microarray through a hybridization reaction. This method can measure not only whether or not the target is contained in the sample, but also the amount of the target contained in the sample. The following Non-Patent Document 1 and Patent Document 1 disclose conventional measurement methods for measuring targets using a DNA microarray. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Koichi Hirayama et al., Development of a DNA chip kit for identifying UGT1A1 gene polymorphisms, Toyo Kohan Vol.38,51-56 [Patent documents]

[0004] [Patent Document 1] Patent No. 4482557 Summary of the Invention [Problem to be solved by the invention]

[0005] However, both of the methods disclosed in Non-Patent Document 1 and Patent Document 1 require a step of washing the DNA microarray before detecting the target, which poses a problem of reducing the reproducibility of the measurement results.

[0006] The present invention has been made in consideration of the above circumstances, and aims to provide a nucleic acid sequence measurement method and a nucleic acid sequence measurement kit that use simple steps and have excellent reproducibility in the measurement results of targets contained in a sample. [Means for solving the problem]

[0007] In order to achieve the above object, the present invention employs the following configuration. [1] A nucleic acid sequence measurement method for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization, comprising: (a) adding a label molecule A to a sample solution containing the target; (b) a step of causing a hybridization reaction between the target and the label molecule A in the sample solution; (c) adding label molecule B to the sample solution; (d) supplying the sample solution to a solid surface of a substrate having detection probes immobilized thereon; (e) a step of hybridizing the detection probe with the target, the label molecule A, and the label molecule B; (f) separating from the solid phase surface the target not bound to the detection probe, the aggregates consisting of the label molecule A and the label molecule B, and the aggregates consisting of the label molecule A and the label molecule B; and (g) irradiating the solid phase surface with excitation light and measuring the amount of fluorescence from the solid phase surface; the detection probe has a detection portion that is a sequence complementary to the target nucleic acid sequence; the label molecule A has a nucleic acid sequence that is complementary to the target nucleic acid sequence and different from the nucleic acid sequence of the detection portion, The label molecule B is The nucleic acid sequence of having a nucleic acid sequence complementary to The label molecule A and the label molecule B are bound to each other at at least two positions, firefly a photomolecule is attached to a predetermined position of at least one of the label molecule A and the label molecule B; A method for measuring a nucleic acid sequence. [2] The label molecule A has an x ​​portion near the 3' end, a y portion near the 5' end, and a z portion which is a nucleic acid sequence complementary to a target nucleic acid sequence and different from the nucleic acid sequence of the detection portion; the label molecule B has an X moiety near the 3' end and a Y moiety near the 5' end, the nucleic acid sequence of the x portion is complementary to the nucleic acid sequence of the X portion, and the nucleic acid sequence of the y portion is complementary to the nucleic acid sequence of the Y portion; The method for measuring a nucleic acid sequence according to [1], characterized in that either one or both of the x portion and the y portion are provided in the label molecule A, or either one or both of the X portion and the Y portion are provided in the label molecule B, or either one or both of the x portion and the y portion are provided in the label molecule A and either one or both of the X portion and the Y portion are provided in the label molecule B. [3] The method for measuring a nucleic acid sequence according to [2], wherein the label molecule A has two or more y moieties. [4] The method for measuring a nucleic acid sequence according to [2] or [3], wherein the label molecule B has two or more Y moieties. [5] The nucleic acid sequence measuring method according to any one of [1] to [4], wherein the predetermined position to which the fluorescent molecule is added is midway along the label molecule A or the label molecule B. [6] The method for measuring a nucleic acid sequence according to any one of [1] to [5], wherein the fluorescent molecule is attached to both the label molecule A and the label molecule B. [7] The method for measuring a nucleic acid sequence according to any one of [1] to [5], wherein a metal fine particle is attached to a predetermined position of either the label molecule A or the label molecule B. [8] The nucleic acid sequence measuring method according to [7], wherein the predetermined position to which the metal microparticle is added is midway along the label molecule A or the label molecule B. [9] The method for measuring a nucleic acid sequence according to any one of [1] to [8], wherein the separation is performed by centrifugation.

[10] The nucleic acid sequence measurement method according to any one of [1] to [9], characterized in that the number of hybridized target molecules is calculated from the change in the amount of fluorescence before and after the hybridization reaction between the detection probe and the target, the label molecule A, and the label molecule B.

[11] A nucleic acid sequence measurement kit for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization, a detection probe having a detection portion having a nucleic acid sequence complementary to the target nucleic acid sequence; a substrate having a solid phase surface on which the detection probes are immobilized; a label molecule A having a nucleic acid sequence complementary to the target nucleic acid sequence and different from the nucleic acid sequence of the detection unit; a label molecule B having a nucleic acid sequence complementary to the nucleic acid sequence of the label molecule A; Including, The label molecule A and the label molecule B are bound to each other at at least two positions, a fluorescent molecule is attached to a predetermined position of at least one of the label molecule A and the label molecule B; A kit for measuring a nucleic acid sequence.

[12] The label molecule A has an x ​​portion near the 3' end, a y portion near the 5' end, and a z portion which is a nucleic acid sequence complementary to a target nucleic acid sequence and different from the nucleic acid sequence of the detection portion; the label molecule B has an X moiety near the 3' end and a Y moiety near the 5' end, the nucleic acid sequence of the x portion is complementary to the nucleic acid sequence of the X portion, and the nucleic acid sequence of the y portion is complementary to the nucleic acid sequence of the Y portion; The kit for measuring nucleic acid sequences according to

[11] , characterized in that either one or both of the x moiety and the y moiety are provided in the label molecule A, or either one or both of the X moiety and the Y moiety are provided in the label molecule B, or either one or both of the x moiety and the y moiety are provided in the label molecule A and either one or both of the X moiety and the Y moiety are provided in the label molecule B.

[13] The nucleic acid sequence measuring kit according to

[12] , wherein the label molecule A has two or more y moieties.

[14] The kit for measuring a nucleic acid sequence according to

[12] or

[13] , wherein the label molecule B has two or more Y moieties.

[15] The kit for measuring nucleic acid sequences according to any one of

[11] to

[14] , wherein the predetermined position to which the fluorescent molecule is added is midway along the label molecule A or the label molecule B.

[16] The kit for measuring a nucleic acid sequence according to any one of

[11] to

[15] , wherein the fluorescent molecule is attached to both the label molecule A and the label molecule B.

[17] The kit for measuring a nucleic acid sequence according to any one of

[11] to

[15] , wherein a metal microparticle is attached to a predetermined position of either the label molecule A or the label molecule B.

[18] The kit for measuring a nucleic acid sequence according to

[17] , wherein the predetermined position to which the metal microparticle is added is midway along the label molecule A or the label molecule B. [Effects of the Invention]

[0008] According to the present invention, the present invention can be easily carried out by a simple process. Target The present invention has the effect of providing a nucleic acid sequence measurement method and a nucleic acid sequence measurement kit that are excellent in reproducibility of measurement results. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 2 is a diagram showing an example of the structure of a label molecule A. [Figure 2] FIG. 10 is a diagram showing an example of the structure of a label molecule B. [Figure 3] FIG. 1 shows a structure having repeated branched structures formed by the binding of label molecules A and B. [Figure 4] FIG. 2 is a diagram schematically illustrating the principle of target detection. [Figure 5] FIG. 1 is a diagram showing the configuration of a fluorescence reading device. [Figure 6] FIG. 1 shows the structure of label molecule A used in the examples. [Figure 7] FIG. 1 shows the structure of label molecule B used in the examples. [Figure 8] FIG. 10 is a diagram showing the spot light intensity when a hybridization reaction is carried out on a sample containing a target and a sample not containing a target in an example. [Figure 9] FIG. 10 is a diagram showing the amount of background light in a solution before and after centrifuging a microarray in an example. DETAILED DESCRIPTION OF THE INVENTION

[0010] Hereinafter, a nucleic acid sequence measurement method and a nucleic acid sequence measurement kit according to an embodiment of the present invention will be described in detail with reference to the drawings. First, an overview of the embodiment of the present invention will be described, followed by a detailed description of the embodiment of the present invention.

[0011] 〔overview〕 The embodiments of the present invention are intended to improve the reproducibility of measurement results of targets contained in a sample through simple steps. Non-Patent Document 1, cited above, discloses a method in which a DNA sample is subjected to PCR using fluorescently modified primers, the resulting fluorescently modified PCR product is added to a DNA microarray, and a hybridization reaction is carried out to detect targets in the sample. Patent Document 1, cited above, also discloses a method for increasing the amount of detected light by labeling with a large number of fluorescent molecules.

[0012] However, the method disclosed in the aforementioned Non-Patent Document 1 requires modification of the target DNA sample with fluorescent molecules, and also requires a washing step of the DNA microarray before detection, making the process complicated. Furthermore, depending on the method of washing the DNA microarray, signal reduction and an increase in background light intensity may occur, and uneven washing may cause uneven signals within the solid phase surface. These factors reduce the reproducibility of measurement results. Furthermore, the method of Patent Document 1 requires washing the solid phase surface before detection, and as with the aforementioned Non-Patent Document 1, there is a problem of reduced reproducibility of measurement results due to washing.

[0013] In the target measurement method of this embodiment, first, label molecule A is added to a sample solution containing the target, and a hybridization reaction between the target and label molecule A occurs. Next, label molecule B is added to the sample solution. Next, the sample solution is supplied to the solid phase surface of a substrate on which a detection probe is immobilized, and a hybridization reaction between the detection probe and the target, label molecule A, and label molecule B occurs. Next, the target not bound to the detection probe, aggregates consisting of label molecule A and label molecule B, and aggregates consisting of label molecule A and label molecule B are separated from the solid phase surface. Then, fluorescent light is irradiated onto the solid phase surface, and the amount of fluorescence from the solid phase surface is measured.

[0014] Here, the detection probe has a detection part that is a sequence complementary to the target nucleic acid sequence, and the label molecule A has a nucleic acid sequence that is complementary to the target nucleic acid sequence but different from the nucleic acid sequence of the detection part, and the label molecule B has a nucleic acid sequence complementary to the target nucleic acid sequence but different from the nucleic acid sequence of the detection part. The nucleic acid sequence of The label molecule A and the label molecule B are bound to each other at at least two positions, and a fluorescent molecule is attached to a predetermined position on at least one of the label molecule A and the label molecule B.

[0015] This allows for a simple process to improve the reproducibility of measurement results for targets contained in samples. Specifically, not only is there no need for a labeling step for the target nucleic acid, but the omission of the washing step prior to the detection step makes it possible to avoid performance degradation, reduced light intensity, increased background light, or variability due to imperfections in the washing step. Conventional methods involve the risk of increased signal and background light and variability due to factors such as the washing method, degree of washing, and uneven washing. However, the present invention avoids these risks. This allows for more uniform results on the array surface and improves detection reproducibility. Furthermore, increasing the number of fluorescent molecules bound to the detection probes hybridized with the target increases the amount of light detected, thereby improving detection sensitivity.

[0016] [Embodiment] <Nucleic acid sequence measurement method> The nucleic acid sequence measurement method of the present invention is a nucleic acid sequence measurement method for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization, comprising: (a) adding a label molecule A to a sample solution containing the target; (b) a step of causing a hybridization reaction between the target and the label molecule A in the sample solution; (c) adding label molecule B to the sample solution; (d) supplying the sample solution to a solid surface of a substrate having detection probes immobilized thereon; (e) a step of hybridizing the detection probe with the target, the label molecule A, and the label molecule B; (f) separating from the solid phase surface the target not bound to the detection probe, the aggregates consisting of the label molecule A and the label molecule B, and the aggregates consisting of the label molecule A and the label molecule B; and (g) irradiating the solid phase surface with excitation light and measuring the amount of fluorescence from the solid phase surface; the detection probe has a detection portion that is a sequence complementary to the target nucleic acid sequence; the label molecule A has a nucleic acid sequence that is complementary to the target nucleic acid sequence and different from the nucleic acid sequence of the detection portion, The label molecule B is The nucleic acid sequence of having a nucleic acid sequence complementary to The label molecule A and the label molecule B are bound to each other at at least two positions, a fluorescent molecule is attached to a predetermined position of at least one of the label molecule A and the label molecule B; It is characterized by:

[0017] Label molecule A has an x ​​portion near the 3' end, a y portion near the 5' end, and a z portion which is a nucleic acid sequence complementary to the target nucleic acid sequence but different from the nucleic acid sequence of the detection portion. Label molecule B has an X portion near the 3' end and a Y portion near the 5' end. The nucleic acid sequence of the x portion is complementary to the nucleic acid sequence of the X portion, and the nucleic acid sequence of the y portion is complementary to the nucleic acid sequence of the Y portion.

[0018] In the present invention, "complementary" means that one nucleic acid sequence has a nucleic acid sequence that can form a double-stranded state with another nucleic acid sequence, and does not necessarily have to be completely complementary and may contain some mismatched base pairs.

[0019] The x portion is located near the 3' end of the label molecule A, and is located several bases from the 3' end of the label molecule A. The y portion is located near the 5' end of the label molecule A, and is located several bases from the 5' end of the label molecule A. The X moiety is located near the 3' end of the label molecule B, and is located several bases from the 3' end of the label molecule B. The Y portion is located near the 5' end of the label molecule B, a few bases away from the 5' end of the label molecule B.

[0020] In label molecule A, the z portion may or may not overlap with all or part of the x portion or the y portion.

[0021] The label molecule A has a z portion which is a nucleic acid sequence complementary to the nucleic acid sequence of the target and different from the nucleic acid sequence of the detection portion, so that the target bound to the z portion of the label molecule A can be attached to the solid phase. surface The detection moiety of the detection probe immobilized thereon can bind to the detection moiety of the detection probe.

[0022] Label molecule A has two or more x moieties or two or more y moieties, label molecule B has two or more X moieties or two or more Y moieties, or label molecule A has two or more x moieties or two or more y moieties and label molecule B has two or more X moieties or two or more Y moieties. For example, label molecule A may have two or more y moieties, label molecule B may have two or more Y moieties, or label molecule A may have two or more y moieties and label molecule B may have two or more Y moieties. The nucleic acid sequence of the x portion is complementary to the X portion, and the nucleic acid sequence of the y portion is complementary to the Y portion. Therefore, the x portion of label molecule A binds to the X portion of label molecule B, and the y portion of label molecule A binds to the Y portion of label molecule B. As a result, a structure in which a plurality of label molecules A and label molecules B have a repeated branched structure is formed.

[0023] A fluorescent molecule is attached to a predetermined position on at least one of label molecule A and label molecule B. The fluorescent molecule does not have to be attached to the tip of label molecule A or label molecule B, but may be located somewhere along label molecule A or label molecule B. Furthermore, multiple types of fluorescent molecules may be attached to multiple positions. By attaching fluorescent molecules to multiple positions, the amount of fluorescence during detection can be increased, enabling more sensitive detection.

[0024] The fluorescent molecule is not particularly limited as long as it can be added to a predetermined position of at least one of the label molecule A and the label molecule B. Examples of the fluorescent molecule include EDANS, Coumarin, FAM, FITC, Cy2 (registered trademark), TF2, TF3, HEX, JOE, TET, Cy3 (registered trademark), Cy5 (registered trademark), Alexa Fluor (registered trademark) 532, Alexa Fluor (registered trademark) 610, Alexa Fluor (registered trademark) 647, ATTO532, ATTO633, Qdot (registered trademark) 565, Qdot (registered trademark) 585, Qdot (registered trademark) 605, Qdot (registered trademark) 705, and iFluor. TM 532, iFluor TM 647 and other known fluorescent molecules.

[0025] FIG. 1 shows an example of the structure of label molecule A. In FIG. Molecule A has one x portion 22 and one y portion 23, and has a fluorescent molecule 21 attached to the 3' end. Fig. 2 is a diagram showing an example of the structure of label molecule B. In Fig. 2, label molecule B has one X moiety 32 and two Y moieties 33, and has metal fine particles 31 attached to the 5' end.

[0026] Figure 3 shows the state in which label molecule A shown in Figure 1 is bound to label molecule B shown in Figure 2. The x portion of label molecule A binds to the X portion of label molecule B, and the y portion of label molecule A binds to the Y portion of label molecule B, resulting in the formation of a structure in which multiple label molecules A and label molecules B have repeated branched structures, as shown in Figure 3.

[0027] The fluorescent molecule may be attached to both label molecule A and label molecule B. By attaching the fluorescent molecule to both label molecule A and label molecule B, the amount of fluorescence during detection can be increased. Metal particles may be attached to a predetermined position on either label molecule A or label molecule B. By attaching metal particles to either label molecule A or label molecule B, when label molecule A and label molecule B bind to form an aggregate having a branched structure, weight can be added to the aggregate, making it easier to separate by centrifugation. Furthermore, magnetism can be imparted to the aggregate, making it possible to separate the aggregate by magnetic force. Metal particles may be attached to the end of label molecule A or label molecule B, or to an intermediate position. Furthermore, multiple types of metal particles may be attached to multiple positions. By attaching metal particles to multiple positions, the weight and magnetism of the aggregate can be increased, making it easier to separate by centrifugation or magnetic force.

[0028] There are no particular limitations on the metal microparticles as long as they can be added to a predetermined position on either label molecule A or label molecule B, and examples include gold nanoparticles, silver nanoparticles, iron oxide, and black silica particles. The particle size of the metal microparticles is preferably such that they are highly dispersible in solution, do not interfere with the hybridization reaction between DNAs, and have a density or mass that allows them to be precipitated by centrifugation. To ensure high dispersibility in solution, iron oxide particles preferably have a particle size of 100 nm or less, and gold nanoparticles preferably have a particle size of 15 nm or less. However, to avoid interfering with the hybridization reaction between DNAs, smaller particle sizes are preferred.

[0029] Each of the above steps will be explained below: Figure 4 is a diagram showing the principle of the nucleic acid sequence measurement method of the present invention. First, a sample solution is prepared containing a target 50 having a specific nucleic acid sequence of interest. In preparing the sample solution, the nucleic acid (target 50) having the specific nucleic acid sequence may be amplified.

[0030] After the gene amplification, a test may be carried out to confirm whether the gene has been amplified, and only if the gene has been amplified may the hybridization reaction described below be carried out.

[0031] The timing for testing the presence or absence of the gene is not limited to after the completion of amplification, but may be during the amplification reaction. Testing methods that can be used include electrophoresis, antigen-antibody reaction, mass spectrometry, real-time PCR, and the like, as appropriate.

[0032] Furthermore, the nucleic acid (target 50) may be bound to a protein, a sugar chain, etc. In this case, the interaction of the protein, sugar chain, etc. with the nucleic acid (target 50) can be confirmed.

[0033] A label molecule A (20) is added to a sample solution containing the target 50 (step (a)). Then, a hybridization reaction between the target 50 and the label molecule A (20) is carried out in the sample solution containing the target 50 (step (b)).

[0034] When the target 50 and the label molecule A(20) are subjected to a hybridization reaction in a sample solution containing the target 50, the target 50 binds to the z portion 24 of the label molecule A(20).

[0035] After the hybridization reaction between the target 50 and the label molecule A (20), the label molecule B (30) is added to the sample solution containing the target 50 and the label molecule A (20) (step (c)). Then, a solution in which the label molecule B (30) is added to the sample solution containing the target 50 and the label molecule A (20) is supplied to the solid phase surface 100 on which the detection probe 10 is immobilized (step (d)).

[0036] Next, the detection probe 10 is subjected to a hybridization reaction with the target 50, label molecule A (20), and label molecule B (30) (step (e)). When the detection probe 10 is subjected to a hybridization reaction with the target 50, label molecule A (20), and label molecule B (30), in step (b), the x portion 22 of the label molecule A (20) bound to the target 50 binds to the X portion 32 of the label molecule B (30), and the y portion 23 of the label molecule A (20) bound to the target 50 binds to the Y portion 33 of the label molecule B (30). As a result, a branched structure in which multiple label molecules A (20) and label molecules B (30) have repeated branched structures is formed, and the target 50 binds to this branched structure, forming a structure consisting of the target 50, label molecule A (20), and label molecule B (30). The target 50 of the structure binds to the detection portion 13 of the detection probe 10 immobilized on the solid-phase surface 100.

[0037] In step (b), the label molecule A (20) that did not bind to the target 50 also binds to the X portion 32 of the label molecule B (30) at the x portion 22 of the label molecule A (20) and to the Y portion 33 of the label molecule B (30). As a result, a plurality of label molecules A (20) and label molecules B (30) form a branched structure having repeated branched structures, and an aggregate consisting of the label molecule A (20) and label molecule B (30) is formed, which exists in a free state in the solution.

[0038] Next, aggregates consisting of the target 50, label molecule A (20), and label molecule B (30) that are not bound to the detection probe 10, and aggregates consisting of label molecule A (20) and label molecule B (30) are separated from the solid surface 100 (step (f)). Centrifugal separation is a preferred method for separating the aggregates from the solid surface 100. The aggregates consisting of the target 50, label molecule A (20), and label molecule B (30) that are not bound to the detection probe 10, and aggregates consisting of label molecule A (20) and label molecule B (30) can be separated from the solid surface 100 by centrifugation. Furthermore, when metal fine particles 31 are attached to either the label molecule A (20) or the label molecule B (30), the aggregates consisting of the target 50, label molecule A (20), and label molecule B (30) that are not bound to the detection probe 10, and aggregates consisting of label molecule A (20) and label molecule B (30) can be separated by magnetic force. In step (f), the target 50 that is not bound to the detection probe 10 and the aggregates consisting of label molecule A (20) and label molecule B (30), as well as the aggregates consisting of label molecule A (20) and label molecule B (30), can be removed without washing, so that the amount of light can be measured without the influence of washing.

[0039] Next, the solid phase surface 100 is irradiated with excitation light, and the fluorescence from the solid phase surface 100 is measured by the fluorescence reader 60 (step (g)). The fluorescence reader 60 can confirm the presence or absence of the target nucleic acid (target 50) in the sample based on whether the detection probe 10 exhibits fluorescence, and can also quantify the hybridized target nucleic acid (target 50). Furthermore, by using the fluorescence reading device 60, it is possible to obtain images of the same coordinates before and after hybridization, and also to obtain fluorescent images separated by wavelength at the same time. By analyzing the fluorescent images, it is also possible to calculate the number of molecules of target 50 that have undergone a hybridization reaction.

[0040] Furthermore, the number of target 50 molecules that have undergone a hybridization reaction can be calculated from the amount of change in fluorescence of the fluorescent molecules 21 before and after the hybridization reaction. For example, a hybridization reaction is performed using a standard solution of target 50 having a known number of molecules, and the amount of change in fluorescence of the fluorescent molecules 21 before and after the reaction is measured, and a calibration curve showing the relationship between the number of molecules and the amount of change in fluorescence is created in advance. The number of target 50 molecules that have undergone a hybridization reaction can be calculated from this calibration curve and the amount of change in fluorescence of the fluorescent molecules 21 before and after the hybridization reaction using a sample.

[0041] The solid phase surface on which the detection probe is immobilized is not limited to a flat surface on the substrate. The detection probe may also be immobilized on the surface of a bead. By immobilizing the detection probe on the surface of a bead, the detection probe will have a shape that spreads out radially from the bead. In this case, the surface area of ​​the solid phase surface on which the probe is immobilized increases, allowing for an increase in the amount of probe per unit area. In addition, by recovering the beads that have captured the target molecules using their size or magnetism, it is possible to selectively recover the target molecules. The recovered molecules can be used for other tests in subsequent processes.

[0042] <Nucleic acid sequence measurement kit> Next, the nucleic acid sequence measurement kit of the present invention will be described. The nucleic acid sequence measurement kit of the present invention can be used in the nucleic acid sequence measurement method of the present invention.

[0043] The nucleic acid sequence measurement kit of this embodiment includes a detection probe 10 having a detection unit 13 having a nucleic acid sequence complementary to that of a target 50, which is a nucleic acid to be detected; a substrate having a solid-phase surface 100 to which the detection probe 10 is fixed; a label molecule A having a nucleic acid sequence complementary to that of the target 50 but different from that of the detection unit 13; and a label molecule B having a nucleic acid sequence complementary to that of the label molecule A. The label molecule A and the label molecule B are bonded at at least two or more sites, and a fluorescent molecule 21 is attached to a predetermined position on at least one of the label molecule A and the label molecule B. Examples of the fluorescent molecule 21 include those described above.

[0044] As shown in Figure 1, label molecule A has an x ​​portion 22 near the 3' end, a y portion 23 near the 5' end, and a z portion 24 which is a nucleic acid sequence complementary to the nucleic acid sequence of target 50 but different from the nucleic acid sequence of detection portion 13. As shown in Figure 2, label molecule B has an X portion 32 near the 3' end and a Y portion 33 near the 5' end. The nucleic acid sequence of x portion 22 is complementary to the nucleic acid sequence of X portion 32, and the nucleic acid sequence of y portion 23 is complementary to the nucleic acid sequence of Y portion 33.

[0045] The x portion 22 is located near the 3' end of the label molecule A, a few bases away from the 3' end of the label molecule A. The y portion 23 is located near the 5' end of the label molecule A, a few bases away from the 5' end of the label molecule A. The X portion 32 is located near the 3' end of the label molecule B, a few bases away from the 3' end of the label molecule B. The Y portion 33 is located near the 5' end of the label molecule B, a few bases away from the 5' end of the label molecule B.

[0046] In label molecule A, z portion 24 may or may not overlap with all or part of x portion 22 or y portion 23.

[0047] Since label molecule A has z portion 24, which is a nucleic acid sequence that is complementary to the nucleic acid sequence of target 50 and different from the nucleic acid sequence of detection portion 13, target 50 bound to z portion 24 of label molecule A can bind to detection portion 13 of detection probe 10 immobilized on solid phase surface 100 of the substrate.

[0048] In label molecule A, two or more of either or both of x moieties 22 and y moieties 23 are provided; in label molecule B, two or more of either or both of X moieties 32 and Y moieties 33 are provided; or in label molecule A, two or more of either or both of x moieties 22 and y moieties 23 are provided and in label molecule B, two or more of either or both of X moieties 32 and Y moieties 33 are provided. For example, label molecule A may have two or more y moieties 23, label molecule B may have two or more Y moieties 33, or label molecule A may have two or more y moieties 23 and label molecule B may have two or more Y moieties 33. The nucleic acid sequence of the x portion 22 is complementary to the X portion 32, and the nucleic acid sequence of the y portion 23 is complementary to the Y portion 33. Therefore, the x portion 22 of the label molecule A binds to the X portion 32 of the label molecule B, and the y portion 23 of the label molecule A binds to the Y portion 33 of the label molecule B. As a result, a structure in which a plurality of label molecules A and label molecules B have a repeated branched structure is formed.

[0049] Fluorescent molecules 21 are attached to predetermined positions on at least one of label molecules A and B. The fluorescent molecules 21 do not have to be attached to the ends of label molecules A or B, but may be located midway through label molecules A or B. Furthermore, multiple types of fluorescent molecules 21 may be attached to multiple positions. By attaching fluorescent molecules 21 to multiple positions, the amount of fluorescence during detection can be increased, enabling more sensitive detection.

[0050] The fluorescent molecule 21 may be attached to both the label molecule A and the label molecule B. By attaching the fluorescent molecule 21 to both the label molecule A and the label molecule B, the amount of fluorescence during detection can be increased. Metal fine particles 31 may be attached to a predetermined position of either the label molecule A or the label molecule B. By attaching metal fine particles 31 to either the label molecule A or the label molecule B, when the label molecule A and the label molecule B bind to form an aggregate having a branched structure, weight can be added to the aggregate, making it easier to separate by centrifugation. Furthermore, magnetism can be imparted to the aggregate, making it possible to separate the aggregate by magnetic force. The metal fine particles 31 may be attached to the end of the label molecule A or the label molecule B, or to an intermediate position. Furthermore, multiple types of metal fine particles 31 may be attached to multiple positions. By attaching metal fine particles 31 to multiple positions, the weight and magnetism of the aggregate can be increased, making it easier to separate by centrifugation or magnetic force. Examples of metal fine particles 31 include those described above.

[0051] The nucleic acid sequence measurement kit of the present invention may further contain standard solutions, necessary buffer solutions, product instructions, and the like required for quantifying the target.

[0052] <How to use the nucleic acid sequence measurement kit> Next, a method for using the nucleic acid sequence measurement kit of the present invention will be described.

[0053] (1) Solution preparation First, a probe solution containing the detection probe 10 is prepared, and the probe concentration is adjusted.

[0054] (2) Fixation to a solid surface Next, the probe solution is spotted onto the solid phase surface 100 of the substrate, and the detection probe 10 is immobilized on the solid phase surface 100 of the substrate.

[0055] (3) Cleaning Next, the solid phase surface 100 is washed to remove any excess probes that have not been immobilized. By the above procedure, a substrate having detection probes immobilized on the solid phase surface 100 is produced.

[0056] (4) Preparation of sample solution Next, a sample solution containing a target 50 having a specific nucleic acid sequence of interest is prepared. In preparing the sample solution, the nucleic acid (target 50) having the specific nucleic acid sequence may be amplified.

[0057] (5) Hybridization reaction between target and label molecule A Next, label molecule A (20) is added to the sample solution containing target 50 prepared in (4). Thereafter, a hybridization reaction between target 50 and label molecule A (20) is caused in the sample solution containing target 50. When a hybridization reaction between target 50 and label molecule A (20) is caused in the sample solution containing target 50, target 50 binds to z portion 24 of label molecule A (20).

[0058] (6) Hybridization reaction between the detection probe, target, label molecule A, and label molecule B After the hybridization reaction between the target 50 and the label molecule A (20), the label molecule B (30) is added to the sample solution containing the target 50 and the label molecule A (20). Then, a solution prepared by adding the label molecule B (30) to the sample solution containing the target 50 and the label molecule A (20) prepared in (4) is supplied to the substrate on which the detection probe 10 is immobilized on the solid phase surface 100, and the hybridization reaction between the detection probe 10 immobilized on the solid phase surface 100 and the target 50, the label molecule A (20), and the label molecule B (30) is caused. When the detection probe 10 immobilized on the solid phase surface 100 undergoes a hybridization reaction with the target 50, label molecule A (20), and label molecule B (30), the x portion 22 of the label molecule A (20) bound to the target 50 binds to the X portion 32 of the label molecule B (30), and the y portion 23 of the label molecule A (20) bound to the target 50 binds to the Y portion 33 of the label molecule B (30). As a result, a branched structure is formed in which multiple label molecules A (20) and label molecules B (30) have repeated branched structures, and the target 50 binds to this branched structure, forming a structure consisting of the target 50, label molecule A (20), and label molecule B (30). The target 50 of this structure binds to the detection portion 13 of the detection probe 10 immobilized on the solid phase surface 100.

[0059] (7) Separation and removal of aggregates Next, aggregates consisting of the target 50 not bound to the detection probe 10, the label molecule A (20), and the label molecule B (30), and aggregates consisting of the label molecule A (20) and the label molecule B (30) are separated from the solid phase surface 100. Centrifugal separation is preferred as a method for separating the aggregates from the solid phase surface 100. When metal fine particles 31 are attached to either the label molecule A (20) or the label molecule B (30), aggregates consisting of the target 50 not bound to the detection probe 10, the label molecule A (20), and the label molecule B (30), and aggregates consisting of the label molecule A (20) and the label molecule B (30) can be separated by magnetic force in addition to centrifugation.

[0060] (8) Fluorescence detection Next, excitation light is irradiated onto the solid phase surface 100 on which the target 50, label molecule A (20), and label molecule B (30) are bound to the detection probe 10, and fluorescence emitted from the solid phase surface 100 is detected by a fluorescence reader 60. The fluorescence reader 60 can confirm the presence or absence of the target nucleic acid (target 50) in the sample based on whether the detection probe 10 exhibits fluorescence, and can also quantify the hybridized target nucleic acid (target 50). Furthermore, by using the fluorescence reading device 60, it is possible to obtain images of the same coordinates before and after hybridization, and also to obtain fluorescent images separated by wavelength at the same time. By analyzing the fluorescent images, it is also possible to calculate the number of molecules of target 50 that have undergone a hybridization reaction.

[0061] Furthermore, the number of target 50 molecules that have undergone a hybridization reaction can be calculated from the amount of change in fluorescence of the fluorescent molecules 21 before and after the hybridization reaction. For example, a hybridization reaction is performed using a standard solution of target 50 having a known number of molecules, and the amount of change in fluorescence of the fluorescent molecules 21 before and after the reaction is measured, and a calibration curve showing the relationship between the number of molecules and the amount of change in fluorescence is created in advance. The number of target 50 molecules that have undergone a hybridization reaction can be calculated from this calibration curve and the amount of change in fluorescence of the fluorescent molecules 21 before and after the hybridization reaction using a sample.

[0062] 5 is a configuration diagram showing the fluorescence reader 60. The fluorescence reader 60 acquires images before and after the target 50 on the DNA chip 40 hybridizes to the detection probe 10. After acquiring the image before hybridization, the temperature of the DNA chip 40 is raised by the temperature control stage 82 to allow the hybridization reaction to proceed, and then the temperature is lowered back to room temperature, after which an image after hybridization is acquired.

[0063] In order to promote hybridization between the target 50 and the detection probe 10, it is preferable that the temperature-controlled stage 82 has a stirring function such as shaking or rotating the DNA chip 40 or using a vortex mixer during the reaction between the target 50 and the detection probe 10.

[0064] In the optical system of the fluorescence reader 60, laser light emitted from a laser light source 61 passes through a mirror 73 and is reflected by a dichroic mirror 74, irradiating the DNA chip 40. The irradiated light serves as excitation light for the fluorescent molecules 21 on the DNA chip 40, and when the wavelength of the laser light source 61 overlaps with the excitation wavelength of the fluorescent molecules 21, the fluorescent molecules 21 enter an excited state.

[0065] The fluorescence emitted from the DNA chip 40 passes through the dichroic mirror 74 and passes through the imaging optical system 81 to be imaged and detected on the detection element of the CCD camera 63. To prevent the excitation light from leaking into the detection light, a bandpass filter matched to the excitation light wavelength may be installed on the excitation light side, or a bandpass filter matched to the fluorescence wavelength to be detected may be installed on the detection light side.

[0066] The fluorescence images obtained by the fluorescence reader 60 can be images obtained before and after the hybridization reaction between the target 50 and the detection probe 10 in the same spot. Therefore, they are not affected by variations in light intensity between solid phases or spots. Furthermore, the amount of change in fluorescence can be calculated from the fluorescence images before and after the hybridization reaction, and the number of molecules that have undergone the binding reaction can be calculated. The amount of change in fluorescence can be calculated using the average light intensity of the entire spot, or the amount of change in fluorescence for each pixel of the spot image.

[0067] The fluorescence reading device 60 may be equipped with a computer that controls the CCD camera 63, an arithmetic unit that calculates the light intensity of the image, and a recording unit that stores the image, the light intensity, and the like.

[0068] The scope of application of the present invention is not limited to the above-described embodiments, and the present invention can be widely applied to nucleic acid sequence measurement methods that measure a target having a specific nucleic acid sequence contained in a sample by hybridization, and nucleic acid sequence measurement kits that use the nucleic acid sequence measurement methods. [Example]

[0069] The present invention will be described in more detail below based on examples, but the present invention is not limited to the following examples.

[0070] Genomic DNA extracted from Staphylococcus aureus (NBRC12732) was used as a template and PCR amplification was performed using primers capable of amplifying the 16S rDNA region. The resulting PCR amplified product was diluted to a DNA concentration of 20 nM, and this was used as the sample solution. Next, label molecule A shown in Fig. 6 was added to the sample and incubated at 65°C for 1 hour to bind the PCR amplification product to label molecule A. As shown in Fig. 6, label molecule A used in this example was modified at its 5' end with the fluorescent molecule Cy3 (registered trademark) and had one x moiety and two y moieties, with the x moiety and the y moiety forming a z moiety.

[0071] Next, label molecule B shown in Figure 7 was added to the sample solution, and then the detection probes were supplied to a microarray in which multiple detection probes were arranged on the solid phase surface of a substrate, and incubated for 3 hours at 60°C. As shown in Figure 7, label molecule B used in this example was modified at its 5' end with Cy3 (registered trademark) and had one X moiety and two Y moieties.

[0072] Next, the microarray was centrifuged at 3000 g in a centrifuge to precipitate aggregates that did not bind to the detection probe. After centrifugation, the spot light intensity was measured using a fluorescence reader. As a negative control, a solution containing genomic DNA extracted from Escherichia coli that did not bind to the primers capable of amplifying the 16S rDNA region (non-target control; hereafter referred to as NTC) was used as the sample solution, and the spot light intensity was measured in the same manner. The results are shown in Figure 8. As shown in Figure 8, a significant increase in fluorescence was observed from the PCR amplification product of the genomic DNA of the Staphylococcus aureus strain compared to the NTC.

[0073] The amount of background light before and after centrifuging the microarray is shown in Figure 9. As shown in Figure 9, centrifuging the microarray reduced the background light to 5% or less. From the above results, it was confirmed that the nucleic acid sequence measurement method of the present invention is capable of detecting targets without requiring a step of modifying the target nucleic acid with fluorescence and a step of washing the solid phase before detection, and that it has excellent detection sensitivity. [Explanation of symbols]

[0074] 10 Detection Probe 13 Detector 20 Label molecule A 21 Fluorescent Molecules 22 x parts 23 y part 24 z section 30 Label molecule B 31 Metal fine particles 32 X section 33 Y section 40 DNA chips 50 targets 60 Fluorescence Reader 61 Laser light source 63 CCD cameras 73 Mirror 74 Dichroic Mirror 81 Imaging optical system 82 Temperature Control Stage 100 Solid surface

Claims

1. A nucleic acid sequence measurement method for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization, comprising: (a) adding a label molecule A to a sample solution containing the target; (b) performing a hybridization reaction between the target and the label molecule A in the sample solution; (c) adding label molecule B to the sample solution in which the hybridization reaction has been carried out; (d) supplying the sample solution to which the label molecule B has been added onto the solid phase surface of the substrate on which the detection probes have been immobilized; (e) a step of hybridizing the detection probe with the target, the label molecule A, and the label molecule B; (f) separating the target not bound to the detection probe, the aggregate consisting of the label molecule A and the label molecule B, and the aggregate consisting of the label molecule A and the label molecule B from the solid phase surface; and (g) irradiating the solid phase surface with excitation light and measuring the amount of fluorescence from the solid phase surface; the detection probe has a detection portion that is a sequence complementary to the target nucleic acid sequence, the label molecule A has a nucleic acid sequence that is complementary to the target nucleic acid sequence and different from the nucleic acid sequence of the detection portion, the label molecule B has a nucleic acid sequence complementary to the nucleic acid sequence of the label molecule A, The label molecule A and the label molecule B are bound to each other at at least two positions, Fluorescent molecules are attached to predetermined positions of both the label molecule A and the label molecule B, the label molecule A has an x ​​portion near the 3' end, a y portion near the 5' end, and a z portion which is a nucleic acid sequence complementary to a target nucleic acid sequence but different from the nucleic acid sequence of the detection portion; The label molecule B has an X moiety near the 3' end and a Y moiety near the 5' end, the nucleic acid sequence of the x portion is complementary to the nucleic acid sequence of the X portion, and the nucleic acid sequence of the y portion is complementary to the nucleic acid sequence of the Y portion; The label molecule A has two or more of either the x moiety or the y moiety, or both of them, and the label molecule B has two or more of either the X moiety or the Y moiety, or both of them. A method for measuring a nucleic acid sequence.

2. 2. The method for measuring a nucleic acid sequence according to claim 1, wherein the label molecule A has two or more y portions.

3. 3. The method for measuring a nucleic acid sequence according to claim 1, wherein the label molecule B has two or more Y moieties.

4. 4. The nucleic acid sequence measuring method according to claim 1, wherein the predetermined position to which the fluorescent molecule is added is midway along the label molecule A or the label molecule B.

5. 5. The nucleic acid sequence measuring method according to claim 1, wherein metal particles are added to a predetermined position of either the label molecule A or the label molecule B.

6. 6. The nucleic acid sequence measuring method according to claim 5, wherein the predetermined position to which the metal fine particle is added is midway along the label molecule A or the label molecule B.

7. 7. The method for measuring a nucleic acid sequence according to claim 1, wherein the separation is performed by centrifugation.

8. The nucleic acid sequence measuring method according to any one of claims 1 to 7, characterized in that the number of hybridized target molecules is calculated from a change in the amount of fluorescence before and after a hybridization reaction between the detection probe and the target, the label molecule A, and the label molecule B.

9. A nucleic acid sequence measurement kit for measuring a target having a specific nucleic acid sequence contained in a sample by hybridization, comprising: a detection probe having a detection portion having a nucleic acid sequence complementary to the target nucleic acid sequence; and a substrate having a solid phase surface on which the detection probe is immobilized; a label molecule A having a nucleic acid sequence complementary to the target nucleic acid sequence and different from the nucleic acid sequence of the detection unit; a label molecule B having a nucleic acid sequence complementary to the nucleic acid sequence of the label molecule A; Including, The label molecule A and the label molecule B are bound to each other at at least two positions, Fluorescent molecules are attached to predetermined positions of both the label molecule A and the label molecule B, the label molecule A has an x ​​portion near the 3' end, a y portion near the 5' end, and a z portion which is a nucleic acid sequence complementary to a target nucleic acid sequence but different from the nucleic acid sequence of the detection portion; The label molecule B has an X moiety near the 3' end and a Y moiety near the 5' end, the nucleic acid sequence of the x portion is complementary to the nucleic acid sequence of the X portion, and the nucleic acid sequence of the y portion is complementary to the nucleic acid sequence of the Y portion; The label molecule A has two or more of either the x moiety or the y moiety, or both of them, and the label molecule B has two or more of either the X moiety or the Y moiety, or both of them. A kit for measuring a nucleic acid sequence.

10. 10. The nucleic acid sequence measuring kit according to claim 9, wherein the label molecule A has two or more y moieties.

11. 11. The nucleic acid sequence measuring kit according to claim 9, wherein the label molecule B has two or more Y moieties.

12. 12. The nucleic acid sequence measuring kit according to claim 9, wherein the predetermined position to which the fluorescent molecule is added is midway along the label molecule A or the label molecule B.

13. 13. The nucleic acid sequence measuring kit according to claim 9, wherein metal particles are attached to a predetermined position of either the label molecule A or the label molecule B.

14. 14. The nucleic acid sequence measuring kit according to claim 13, wherein the predetermined position to which the metal fine particle is added is midway along the label molecule A or the label molecule B.

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