JP3697436B2 - Nucleic acid sequence analysis method, probe-immobilized substrate, assay kit, and probe set - Google Patents

Description

【図面の簡単な説明】
【図１】本発明の態様に従うプローブの例を示す模式図。
【図２】本発明の態様に従うプローブ固定化基体の例を示す模式図。
【図３】本発明の態様に従うプローブ固定化基体の例を示す模式図。
【図４】実施例１の結果を示すグラフ。
【図５】実施例１の結果を示すグラフ。
【図６】実施例２の結果を示すグラフ。
【図７】実施例２の結果を示すグラフ。
【符号の説明】
１．基体 ２，４，６，８．第２のプローブ ３，５，７，９．第１のプローブ １１，１２，１３，１４，１７，１８，１９，２０．プローブ １５．固定化領域 １６．基体 ２２．基体 ２３．電極 ２４．パット[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for specifically analyzing a nucleic acid sequence, a nucleic acid chain having a specific sequence, particularly a nucleic acid having a single nucleotide polymorphism or a point mutation, a probe-immobilized substrate, an assay kit, and a probe set.
[0002]
[Prior art]
Currently, in order to detect a nucleic acid chain having a specific nucleic acid sequence, a DNA chip in which a nucleic acid probe complementary to a sequence to be detected is immobilized on a substrate such as glass or silicon is used. For example, in a gene expression analysis or the like, a denatured PCR product is immobilized on a substrate as a probe. When high sensitivity is required as in expression analysis, the long chain is more advantageous in terms of sensitivity, and thus a relatively long chain probe consisting of several hundred bases is often used.
[0003]
On the other hand, a probe comprising a relatively short synthetic oligonucleotide of about 15 to 20 bases when extremely high specificity is required, such as detection of single nucleotide polymorphism (hereinafter referred to as SNP or SNPs) or point mutation Is used (see Non-Patent Document 1 and Non-Patent Document 2).
[0004]
. Usually, in the type determination of SNPs, a probe having a base sequence corresponding to the type of SNPs is immobilized on a substrate and a hybridization reaction with a target is performed. Under certain conditions, even a single base mismatch will not cause hybridization. Therefore, the type of SNPs can be determined by detecting the presence or absence of hybridization using fluorescence or the like as an index. However, in practice, it is difficult to distinguish a single base difference only by a method based on a hybridization reaction. In particular, a mismatch consisting of a bond of GA, GT, or GU is very difficult to distinguish from an AT full match. In order to solve this, methods such as combining nucleases and ligases and using PNA probes have been reported, but (1) the detection system is complicated, (2) the analysis cost is high, (3) Problems such as taking time were pointed out.
[0005]
[Non-Patent Document 1]
Nature Biotechnology 18, 438 (2000)
[0006]
[Non-Patent Document 2]
Science 274, 610 (1996)
[0007]
[Problems to be solved by the invention]
The present invention solves the above-described problems, and an object thereof is to provide a method for analyzing a nucleic acid sequence, a probe-immobilized substrate, an assay kit, and a probe set for detecting the nucleic acid sequence with high specificity.
[0008]
[Means for Solving the Problems]
The present invention provides means for solving the above problems. That is, the present invention
(1) a first probe including a first target site and a first sequence, and one base of a second target site that is shorter by one base than the first sequence and corresponds to the position of the first target site A method for analyzing a nucleic acid sequence, comprising reacting a nucleic acid in a sample with a second probe containing a homologous sequence except under the conditions under which appropriate hybridization occurs;
(2) Homologous to the first nucleic acid probe including the first sequence site, except for one base of the second target site that is shorter by one base than the first sequence and corresponding to the position of the first target site A probe-immobilized substrate comprising a second nucleic acid probe containing a specific sequence;
(3) The probe-immobilized substrate according to (2) above, the first nucleic acid probe including the first sequence site, and one base shorter than the first sequence, corresponding to the position of the first target site An assay kit for nucleic acid sequence analysis comprising at least a component selected from the group consisting of a probe set with a second nucleic acid probe containing a homologous sequence excluding one base of the second target site
(4) The first nucleic acid probe containing the first sequence is homologous to the first sequence by one base, except for one base of the second target site corresponding to the position of the first target site. Probe set with second nucleic acid probe containing sequence
It is.
[0009]
DETAILED DESCRIPTION OF THE INVENTION
1. analysis method
In general, mismatches in which the base of the target site in the target and the probe is GA or GT or GU are said to be indistinguishable from the case of a full match of AT or AU. Yes. The present inventors have found that in the case of a target in which GT, GA, or GU mismatch is considered as a possibility, specific detection can be performed by changing the length of the probe. . According to an embodiment of the present invention, there are provided a nucleic acid sequence analysis method, a probe-immobilized substrate, an assay kit, and a probe set utilizing such a principle.
[0010]
The present invention will be described with reference to FIG. For example, FIG. 1 shows a probe-immobilized substrate having a plurality of probes 2 to 9 fixed to the substrate 1.
[0011]
The first probe 3 shown second from the left includes a first target site whose base is “T” and a first sequence indicated by a straight line. The second probe 2 includes a second target site whose base is “G” and a sequence indicated by a straight line. Here, the second probe 2 is shorter than the first probe 3 by one base. The first target site and the second target site are arranged at the same position counting from the end of the probe fixed to the substrate. The sequences of the first probe 3 and the second probe 2 are homologous to each other except for the target site.
[0012]
Further examples of the first probe and the second probe are as follows. That is, the first probe 5 includes a first target site whose base is “A” and a first sequence indicated by a straight line. The second probe 4 includes a second target site whose base is “G” and a sequence indicated by a straight line. Here, the second probe 4 is shorter than the first probe 5 by one base. The first target site and the second target site are arranged at the same position counting from the end of the probe fixed to the substrate. The sequences of the first probe 5 and the second probe 4 are homologous to each other except for the target site.
[0013]
Similarly, in the case of a further example, the first probe 7 includes a first target site whose base is “C” and a first sequence shown in a straight line. The second probe 6 includes a second target site whose base is “A” and a sequence indicated by a straight line. Here, the second probe 6 is shorter than the first probe 7 by one base. The first target site and the second target site are arranged at the same position counting from the end of the probe fixed to the substrate. The sequences of the first probe 7 and the second probe 6 are homologous to each other except for the target site.
[0014]
The same applies to further examples. That is, the first probe 9 includes a first target site whose base is “C” and a first sequence indicated by a straight line. The second probe 8 includes a second target site whose base is “A” and a sequence indicated by a straight line. Here, the second probe 8 is shorter than the first probe 9 by one base. The first target site and the second target site are arranged at the same position counting from the end of the probe fixed to the substrate. The sequences of the first probe 9 and the second probe 8 are homologous to each other except for the target site.
[0015]
When the target nucleic acid chain has a single nucleotide polymorphism (ie, SNP) consisting of G / T type, a G type detection probe (the target site base is C), a T type detection probe (the target site base is A) is used. In that case, CG, CT, AG, AT can be considered as a combination of the base of the target site | part of a probe and a target. In this combination, it is easy to distinguish CG and CT, but it becomes very difficult to distinguish AG and AT. Therefore, the probe is set as short as possible so that no A-G mismatch occurs. However, considering the detection sensitivity, a longer probe length is preferable. Therefore, as a result, the probe for T-type detection (the base of the target site is A) is made shorter than the probe for G-type detection. That is, the second probe is made shorter than the first probe. Then, it becomes possible to specifically detect SNPs and point mutations without reducing the S / N only by the hybridization reaction. A method of controlling the specificity by changing the length of the probe by such SNP discrimination or point mutation detection is proposed for the first time by the present invention.
[0016]
The position of the target site in the first and second probes may be any position of the probe. However, it is not desirable to dispose the first and second probes farthest from the substrate.
[0017]
As used herein, the term “nucleic acid” refers to any nucleic acid analog such as DNA and RNA, S-oligo, methylphosphonate oligo, PNA (ie, peptide nucleic acid) and LNA (ie, lock nucleic acid), etc. In addition, it is a term that collectively indicates substances whose partial structure can be represented by a base sequence. Such nucleic acids may be naturally occurring or artificially synthesized and may be a mixture thereof. The probe according to the embodiment of the present invention may be any nucleic acid as described above, and may further have a labeling substance, a tag, and / or other sequences as long as it does not interfere with the implementation of the method of the present invention. Good.
[0018]
As used herein, “under conditions that cause appropriate hybridization” refers to conditions under which hybridization occurs when a target and a probe react in an appropriate combination that is complementary to each other. Any condition is acceptable.
[0019]
According to the embodiment of the present invention, for example, a sample nucleic acid is added to the probe-immobilized substrate according to the above-described embodiment of the present invention, and the reaction is performed under conditions that cause appropriate hybridization. By detecting this, it is possible to analyze the nucleic acid sequence of the nucleic acid contained in the sample.
[0020]
Alternatively, a probe that is not immobilized on the substrate may be used. That is, the first and second probes according to the embodiment of the present invention are reacted with the nucleic acid in the sample under conditions that cause appropriate hybridization, and the resulting double strand is detected after the reaction, thereby being contained in the sample. It is possible to analyze the nucleic acid sequence of the nucleic acid.
[0021]
Regardless of whether a free probe or a probe-immobilized substrate is used, a means known per se may be used as the double-stranded detection means used in the present invention. For example, the nucleic acid in the sample may be labeled with a fluorescent substance in advance, or may be detected using an antigen-antibody reaction. Moreover, you may detect a double stranded nucleic acid using an intercalator.
[0022]
2. Probe immobilization substrate
The probe-immobilized substrate used in the embodiment of the present invention is a commonly used substrate, for example, various substrates such as silicon substrate, glass substrate and resin, carrier such as beads, microtiter plate and multiwell plate. This is a device in which a probe is immobilized on a container such as the above by a method known per se. The shape of the substrate is not particularly limited. In addition, the type of probe immobilized on one substrate is such that the first probe and the second probe can be identified and detected to which probe the nucleic acid in the sample is bound. It is desirable to fix it. For example, they may be immobilized on different immobilized regions, or may be immobilized on the same immobilized region and competitively hybridized.
[0023]
In addition, a plurality of combinations of the first probe and the second probe may be fixed to one base, and probes other than the first probe and the second probe set according to the embodiment of the present invention are fixed simultaneously. May be used.
[0024]
Examples of probe-immobilized substrates according to embodiments of the present invention are described below.
[0025]
(A) First example
FIG. 2 schematically shows a first example of a probe-immobilized substrate that can be used in accordance with an embodiment of the present invention. The probe-immobilized substrate as a first example includes the substrate 11 and probes 11 to 14 immobilized at one or more on one or more immobilization regions 15 existing on the surface of the substrate 16 (FIG. 2). According to the aspect of the present invention, at least, for example, the first probe may be fixed as the probe 11 and the second probe may be fixed as the probe 12.
[0026]
Such a probe-immobilized substrate can be produced, for example, by immobilizing the probe to a substrate such as a silicon substrate by means known per se.
[0027]
According to the aspect of the present invention, the number of the immobilization regions 15 arranged on one substrate and the number of probes immobilized thereon are not limited to this, and may be changed as desired. A plurality of types of base sequences may be arranged as a probe on one substrate. A person skilled in the art can appropriately change the design of the solid phase pattern of a plurality of and / or a plurality of types of probes on the substrate. Such a probe-immobilized substrate is within the scope of the present invention.
[0028]
(B) Second example
A second example of a probe-immobilized substrate that can be used in the embodiment of the present invention will be described with reference to FIG. The probe-immobilized base as a second example includes probes 17 to 20 fixed at one or more to one or more electrodes 23 provided on the base 22 (FIG. 3). The electrode 23 is connected to a pad 24 for extracting electrical information.
[0029]
Such a probe-immobilized substrate can be manufactured, for example, by disposing an electrode on a substrate such as a silicon substrate by means known per se and immobilizing the probe on the electrode surface. According to the aspect of the present invention, at least, for example, the first probe may be fixed as the probe 17 and the second probe may be fixed as the probe 18.
[0030]
In this embodiment, the number of electrodes is five, but the number of electrodes arranged on one substrate is not limited to this. Also, the arrangement pattern of the electrodes is not limited to that shown in FIG. 3, and those skilled in the art can appropriately change the design as necessary. You may provide a reference electrode and a counter electrode as needed. Such a probe-immobilized substrate is also within the scope of the present invention.
[0031]
In the case of a probe-immobilized substrate for performing fluorescence detection as described in the above example, the probe may be immobilized on any of the above-described substrates. In the case of a probe-immobilized substrate for performing electrochemical detection as described in the first example above, electrodes are arranged on any of the above substrates so that electrochemical detection is possible. And what is necessary is just to fix | immobilize a probe on the electrode.
[0032]
The electrode that can be used in the present invention is not particularly limited. For example, carbon electrodes such as graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon fiber, platinum, platinum black, gold, palladium, Noble metal electrodes such as rhodium, oxide electrodes such as titanium oxide, tin oxide, manganese oxide, lead oxide, Si, Ge, ZnO, CdS, TiO ₂ And semiconductor electrodes such as GaAs, titanium and the like. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with other surface treatment agents as desired.
[0033]
In addition, probes having different base sequences may be immobilized on different electrodes, respectively, or may be immobilized on one electrode in a state where a plurality of types of probes having different base sequences are mixed. Good.
[0034]
(C) Detection
When a probe-immobilized substrate according to an embodiment of the present invention is used, as a means for detecting the presence of a double strand resulting from a hybridization reaction between a probe immobilized on the substrate and a target nucleic acid, electrochemical Manual methods and fluorescence detection methods can be used.
[0035]
(A) Electrochemical detection
Electrochemical detection of double-stranded nucleic acid may be performed using, for example, a publicly known double-stranded recognition substance.
[0036]
The double-stranded recognizer used here is not particularly limited. For example, Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalator, bisintercalator such as bisacridine, trisintercalator and polyintercalator Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene or viologen. In addition, any other known double-stranded recognition substance is preferably used in the present invention.
[0037]
In the probe-immobilized substrate according to the embodiment of the present invention, the probe is fixed to the electrode. In the detection of double-stranded nucleic acid using such an electrode, a counter electrode or a reference electrode may be used in the same manner as in other general electrochemical detection methods. When arranging the reference electrode, for example, a general reference electrode such as a silver / silver chloride electrode or a mercury / mercury chloride electrode may be used.
[0038]
Subsequently, the reaction is performed under conditions that allow appropriate hybridization. Such appropriate conditions can be determined by those skilled in the art depending on various conditions such as the type of base contained in the target sequence, the type of probe provided on the probe-immobilized substrate, the type of sample nucleic acid, and the state thereof. It is possible to select appropriately. Although not limited to this, for example, the reaction may be performed under the following conditions.
[0039]
For example, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The sample nucleic acid obtained here is added and heat denatured at 90 ° C. or higher. The probe-immobilized substrate may be inserted into the heat-denatured sample nucleic acid immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate.
[0040]
During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, the electrode is washed. For washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 may be used. When the target nucleic acid containing the target sequence is present in the sample nucleic acid, it hybridizes with the probe, thereby generating a double-stranded nucleic acid.
[0041]
Subsequently, the double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. In general, after the hybridization reaction, the substrate is washed, and a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and the resulting signal is electrochemically measured.
[0042]
The concentration of the double-stranded recognizer varies depending on the type, but is generally used in the range of 1 ng / mL to 1 mg / mL. At this time, a buffer solution having an ionic strength of 0.001 to 5 and a pH of 5 to 10 may be used.
[0043]
For example, the electrochemical measurement may be performed by applying a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically and measuring the reaction current value derived from the double-stranded recognizer. At this time, the potential may be swept at a constant speed, applied with a pulse, or a constant potential may be applied. In the measurement, for example, the current and voltage may be controlled by using a device such as a potentiostat, a digital multimeter, and a function generator. For example, the concentration of the target nucleic acid may be calculated from a calibration curve based on the obtained current value.
[0044]
In addition, electrochemical detection means known per se, for example, means disclosed in the following documents (Hashimoto et al. 1994, Wang et al. 1998) can be preferably used in the method of the present invention. In this document, Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anode current derived from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. This biosensor configuration includes immobilization of an inosine displacement probe (without guanine) to a carbon paste electrode and chronopotentiometric detection of duplex formation due to the presence of the guanine oxidation peak of the label. The detection means described in these documents may preferably be used in the present invention.
[0045]
(B) Fluorescence detection method
In the case of a method using a fluorescent labeling substance, the target nucleic acid may be labeled with a substance capable of obtaining fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Alternatively, a second probe labeled with the aforementioned substance may be used instead of such a label. A plurality of labeling substances may be used simultaneously.
[0046]
In some embodiments, a hybridization reaction between a nucleic acid component extracted from a sample and a probe immobilized on a probe-immobilized chip is performed, for example, as follows. For example, the hybridization reaction solution is performed in a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10. In this solution, dextran sulfate, which is a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, and a surfactant may be added. The nucleic acid component extracted here is added and heat-denatured at 90 ° C. or higher. The probe-immobilized chip may be inserted immediately after denaturation or after rapid cooling to 0 ° C. It is also possible to perform a hybridization reaction by dropping a solution on the substrate. During the reaction, the reaction rate may be increased by an operation such as stirring or shaking. The reaction temperature may be, for example, in the range of 10 ° C. to 90 ° C., and the reaction time may be about 1 minute to overnight. After the hybridization reaction, washing is performed. For the washing, for example, a buffer solution having an ionic strength of 0.01 to 5 and a pH of 5 to 10 is used.
[0047]
In the case of fluorescence detection, the hybridization reaction is detected by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device corresponding to the type of label. When the label is a fluorescent substance, for example, the label may be detected using a fluorescence detector.
[0048]
3. Probe set
The first probe and the second probe according to the above-described aspect of the present invention may be provided as a probe set corresponding to a target to be detected.
[0049]
4). Assay kit
According to the above-described aspect of the present invention, the above-described probe-immobilized substrate and / or probe set may be provided as an assay kit. For example, it may be selected according to the purpose of use, combined, and provided as an appropriate assay kit. Furthermore, in addition to them, buffer salts, other necessary reagents, labeling substances and / or reaction containers may be combined and provided as an assay kit. In addition, the assay kit may include a probe and a substrate that are not immobilized on the substrate.
[0050]
For example, the first and second probes and / or probe-immobilized substrates according to the present invention can be combined with reaction vessels, buffer salts and / or other necessary reagents such as labeling substances as assay kits. May be provided.
[0051]
【Example】
Example 1
SNP (position -123) in the promoter region of human MxA gene was detected. As a target gene, synthetic oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 having 5 ′ ends labeled with Cy5 were used as models. There are two types of SNP patterns, -123 at position "C" or "A". Therefore, 15-mer (ie, SEQ ID NO: 3 and SEQ ID NO: 4) and 14-mer (ie, SEQ ID NO: 5 and SEQ ID NO: 6) probes were synthesized, respectively. A thiol group was introduced at the 3 ′ end of the probe. A slide glass was used as a substrate on which the probe was immobilized, and a probe-immobilized substrate was produced as follows.
[0052]
First, the slide glass was treated with a solution of sulfuric acid: hydrogen peroxide = 2: 1 to create a normal surface. Next, it was treated with 1% γ-aminopropyltriethoxysilane for 1 hour, washed with water, and reacted with 0.3 mg / ml EMCS (Dojin Laboratories) for 2 hours. After washing, the substrate was dried, and a probe solution having a concentration of 50 μg / mL was spot-fixed.
[0053]
A hybridization reaction was performed on a substrate on which two types of probes were immobilized, and fluorescence intensity was measured. As a result, when the 15-mer probe was used, the signal intensity was considerably high even with a mismatch, and the S / N was about 1.5 (FIG. 4). N was improved to about 5 (FIG. 5).
[0054]
As is clear from the above results, it was possible to detect SNPs with high specificity according to the method of the present invention. By using the nucleic acid sequence analysis method, probe-immobilized substrate, assay kit and probe set according to the present invention, the target nucleic acid sequence can be analyzed easily and in a short time.
[0055]
Example 2
SNP (position -88) in the promoter region of human MxA gene was detected. As target genes, synthetic oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 were used as models. There are two types of SNP types: -G at position -88, "G" or "T". Therefore, it was considered that a GA mismatch that was difficult to detect was formed. Therefore, the G-type detection probe was 14mer (SEQ ID NO: 9) and the A-type detection probe was 15mer (SEQ ID NO: 11), and a thiol group was introduced at the 5 'end. As a control for the G-type detection probe, a 15mer was also synthesized (SEQ ID NO: 10).
[0056]
A thiol group was introduced at the 5 ′ end of all probes and immobilized on a gold electrode. The gold electrode was prepared by forming a titanium / gold thin film on a glass substrate and patterning it by photolithography. A hybridization reaction was performed on the probe-immobilized electrode, and after washing, the oxidation current value of Hoechst 33258 was measured. As a result, when all 15-mer probes were used, the signal of GA mismatch became large and the S / N was about 1.5 (FIG. 6). On the other hand, in the chip where the G-type detection probe was changed to 14mer, GA mismatch was well suppressed, indicating that SNP type determination was possible (FIG. 7).
[0057]
As is clear from the above results, it was possible to detect SNPs with high specificity according to the method of the present invention. By using the nucleic acid sequence analysis method, probe-immobilized substrate, assay kit and probe set according to the present invention, the target nucleic acid sequence can be analyzed easily and in a short time.
[0058]
【The invention's effect】
By using the nucleic acid sequence analysis method, probe-immobilized substrate, assay kit and probe set according to the present invention, SNPs and point mutations can be detected with high specificity.
[0059]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram showing an example of a probe according to an embodiment of the present invention.
FIG. 2 is a schematic diagram showing an example of a probe-immobilized substrate according to an embodiment of the present invention.
FIG. 3 is a schematic diagram showing an example of a probe-immobilized substrate according to an embodiment of the present invention.
4 is a graph showing the results of Example 1. FIG.
5 is a graph showing the results of Example 1. FIG.
6 is a graph showing the results of Example 2. FIG.
7 is a graph showing the results of Example 2. FIG.
[Explanation of symbols]
1. Substrate 2,4,6,8. Second probe 3,5,7,9. First probe 11, 12, 13, 14, 17, 18, 19, 20,. Probe 15. Immobilization region 16. Substrate 22. Substrate 23. Electrode 24. Pat

Claims (6)

A first probe comprising a first target site and a first sequence, and a base shorter than the first sequence by one base, except for one base of the second target site corresponding to the position of the first target site Reacting a nucleic acid in a sample with a second probe containing a homologous sequence under conditions that cause appropriate hybridization;
The base combination of the first target site and the second target site is selected from the group consisting of thymine and guanine, adenine and guanine, cytosine and adenine, cytosine and thymine, uracil and guanine, cytosine and uracil. A method for analyzing a characteristic nucleic acid sequence.   After the reaction, the method further comprises determining the genotype of the single nucleotide polymorphism of the nucleic acid by detecting the presence of the binding of the nucleic acid in the sample to the first nucleic acid probe and the second nucleic acid probe. The method for analyzing a nucleic acid sequence according to claim 1.   The method further comprises detecting a point mutation of the nucleic acid by detecting the presence of binding of the nucleic acid in the sample to the first nucleic acid probe or the second nucleic acid probe after the reaction. Item 2. A method for analyzing a nucleic acid sequence according to Item 1. A first nucleic acid probe comprising a first target site and a first sequence, and a base shorter than the first sequence by one base and excluding one base of the second target site corresponding to the position of the first target site A second nucleic acid probe comprising a homologous sequence,
The combination of the base of the first target site and the second target site is selected from the group consisting of thymine and guanine, adenine and guanine, cytosine and adenine, cytosine and thymine, uracil and guanine, cytosine and uracil. A probe-immobilized substrate . The probe-immobilized substrate according to claim 4 , the first nucleic acid probe including the first target site and the first sequence, and one base shorter than the first sequence, at the position of the first target site A probe set with a second nucleic acid probe containing a homologous sequence except for one base of the corresponding second target site, wherein the combination of the bases of the first target site and the second target site is thymine and guanine A nucleic acid sequence analysis comprising at least a component selected from the group consisting of a probe set, selected from the group consisting of adenine and guanine, cytosine and adenine, cytosine and thymine, uracil and guanine, cytosine and uracil Assay kit for use. A first nucleic acid probe comprising a first target site and a first sequence, and a base shorter than the first sequence by one base and excluding one base of the second target site corresponding to the position of the first target site A probe set with a second nucleic acid probe comprising a homologous sequence,
The combination of the bases of the first target site and the second target site is selected from the group consisting of thymine and guanine, adenine and guanine, cytosine and adenine, cytosine and thymine, uracil and guanine, cytosine and uracil, and Probe set .

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