JP2004121044A - Method for analyzing nucleic acid sequence, substrate for immobilizing probe, assay kit and probe set - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for analyzing a nucleic acid sequence for detecting the nucleic acid sequence with high specificity and to provide a substrate for immobilizing a probe, an assay kit and a probe set. <P>SOLUTION: The method for analyzing the nucleic acid sequence comprises reacting a nucleic acid in a sample with a first probe containing a first target site and a first sequence and a second probe containing a sequence shorter than the first sequence by one base and homologous except the one base of a second target site corresponding to the position of the first target site under conditions for initiating suitable hybridization. <P>COPYRIGHT: (C)2004,JPO

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]
TECHNICAL FIELD 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]
At present, 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 gene expression analysis, a denatured PCR product is immobilized on a substrate as a probe. When high sensitivity is required as in expression analysis, a relatively long probe consisting of several hundred bases is often used because a longer chain is more sensitive.
[0003]
On the other hand, when very high specificity is required such as detection of single nucleotide polymorphism (hereinafter referred to as SNP or SNPs) or point mutation, a probe comprising a relatively short synthetic oligonucleotide of about 15 to 20 bases is required. Is used (see Non-Patent Documents 1 and 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, a single base mismatch does 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 of combining enzyme reactions such as nucleases and ligases, and methods using PNA probes have been reported. However, (1) the detection system becomes complicated, (2) the analysis cost increases, (3) It was pointed out that it takes time.
[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]
An object of the present invention is to solve the above problems and to provide a nucleic acid sequence analysis method, a probe-immobilized substrate, an assay kit, and a probe set for detecting a nucleic acid sequence with high specificity.
[0008]
[Means for Solving the Problems]
The present invention provides a means for solving the above problems. That is, the present invention
(1) a first probe containing a first target site and a first sequence, and one base of a second target site shorter than the first sequence by one base and corresponding 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 for the conditions under conditions under which appropriate hybridization occurs;
(2) Homologous to the first nucleic acid probe containing the first sequence site except for one base shorter than the first sequence by one base and one base of the second target site corresponding to the position of the first target site. A probe-immobilized substrate comprising a second nucleic acid probe containing a unique sequence;
(3) The probe-immobilized substrate according to (2) and 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. (4) an assay kit for analyzing a nucleic acid sequence, comprising at least a component selected from the group consisting of a probe set with a second nucleic acid probe containing a homologous sequence except for one base of the second target site; A) a first nucleic acid probe comprising a first sequence, a sequence which is shorter by one base than the first sequence and is homologous except for one base in a second target site corresponding to the position of the first target site; And a probe set with a second nucleic acid probe.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
1. Analysis Methods Generally, mismatches in which the base of the target site in the target and the probe is GA, GT, or GU are said to be difficult to distinguish from the case of a full match of AT or AU. Has been done. The present inventors have found that in the case of a target in which GT or GA or GU mismatch is considered as a possibility, specific detection becomes possible 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 including a plurality of probes 2 to 9 immobilized on a 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 shown 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 counted 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 counted 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 a further example case, the first probe 7 includes a first target site whose base is "C" and a first sequence indicated by 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 counted 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 counted 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 strand has a G / T-type single nucleotide polymorphism (ie, SNP), a G-type detection probe (the base at the target site is C), a T-type detection probe (the base at the target site is A) is used. In this case, CG, CT, AG, AT can be considered as a combination of the probe and the base at the target site of the target. In this combination, CG and CT are easy to distinguish, but it is very difficult to distinguish AG from AT. Therefore, the probe is set as short as possible so that the AG mismatch does not occur. However, considering the detection sensitivity, the longer probe length is preferable. Therefore, as a result, the T-type detection probe (the base at the target site is A) is shorter than the G-type detection probe. That is, the second probe is shorter than the first probe. Then, it is possible to specifically detect SNPs and point mutations without lowering the S / N only by the hybridization reaction. Such a method of controlling the specificity by changing the length of the probe by discriminating SNPs or detecting point mutations 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 arrange the first and second probes at positions farthest from the substrate.
[0017]
As used herein, the term "nucleic acid" refers to any nucleic acid analog, such as DNA and RNA, S-oligos, methyl phosphonate oligos, any nucleic acid analog such as PNA (i.e., peptide nucleic acids) and LNA (i.e., locked nucleic acids). The term is a general term for a substance whose partial structure can be represented by a base sequence. Such a nucleic acid may be a naturally occurring one, an artificially synthesized one, or a mixture thereof. The probe according to the embodiment of the present invention may be any of the above-described nucleic acids, and may further have a labeling substance, a tag, and / or other sequences as long as they do not prevent the method of the present invention from being performed. Good.
[0018]
As used herein, the term "under appropriate hybridization conditions" 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 an 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 reacted under appropriate hybridization-producing conditions. By detecting, the nucleic acid sequence of the nucleic acid contained in the sample can be analyzed.
[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 a nucleic acid in a sample under conditions under which appropriate hybridization occurs, and after the reaction, the resulting double strand is detected to be included in the sample. The nucleic acid sequence can be analyzed for the nucleic acid to be used.
[0021]
Regardless of whether a released probe or a probe-immobilized substrate is used, a double-stranded detecting means used in the present invention may be a means known per se. For example, the nucleic acid in the sample may be labeled in advance with a fluorescent substance or the like, or may be detected using an antigen-antibody reaction. Alternatively, double-stranded nucleic acids may be detected using an intercalator.
[0022]
2. Probe-immobilized Substrate The probe-immobilized substrate used in the embodiment of the present invention may be a commonly used substrate, for example, various substrates such as a silicon substrate, a glass substrate and a resin, a carrier such as beads, a microtiter plate. And a device in which a probe is immobilized on a container such as a multiwell plate by a method known per se. The shape of the substrate is not particularly limited. Further, the type of the probe immobilized on one substrate is such that the first probe and the second probe can be identified by detecting which probe the nucleic acid in the sample binds to, and can be detected. It is desirable to fix. For example, they may be immobilized on different immobilized regions, or may be immobilized on the same immobilized region and competitively hybridized.
[0023]
Further, a plurality of combinations of the first probe and the second probe may be immobilized on one substrate, and further, probes other than the set of the first probe and the second probe according to the embodiment of the present invention are simultaneously immobilized. It may be.
[0024]
An example of the probe-immobilized substrate according to the embodiment of the present invention will be described below.
[0025]
(A) First Example FIG. 2 schematically shows a first example of a probe-immobilized substrate that can be used according to an embodiment of the present invention. A probe-immobilized substrate as a first example includes a substrate 16 and one or more probes 11 to 14 immobilized on one or more immobilized regions 15 existing on the surface thereof (FIG. 2). According to the aspect of the present invention, at least, for example, a first probe may be immobilized as the probe 11 and a second probe may be immobilized as the probe 12.
[0026]
Such a probe-immobilized substrate can be manufactured, for example, by immobilizing a probe on a substrate such as a silicon substrate by a means known per se.
[0027]
According to the embodiment of the present invention, the number of the immobilization regions 15 arranged on one substrate and the number of the probes immobilized thereon are not limited thereto, and may be changed as desired. Further, a plurality of types of base sequences may be arranged on one substrate as probes. The solid phase pattern of a plurality and / or a plurality of types of probes on a substrate can be appropriately changed by a person skilled in the art as needed. Such probe-immobilized substrates are within the scope of the present invention.
[0028]
(B) Second Example A second example of the probe-immobilized substrate that can be used in the embodiment of the present invention will be described with reference to FIG. The probe-immobilized substrate according to the second example includes one or more probes 17 to 20 immobilized on at least one electrode 23 provided on the substrate 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 arranging electrodes on a substrate such as a silicon substrate by means known per se, and fixing the probe to the electrode surface. According to the aspect of the present invention, at least, for example, the first probe may be immobilized as the probe 17 and the second probe may be immobilized as the probe 18.
[0030]
In the present embodiment, the number of electrodes is five, but the number of electrodes arranged on one base is not limited to this. Further, the arrangement pattern of the electrodes is not limited to the pattern shown in FIG. 3, and a person skilled in the art can appropriately change the design as needed. A reference electrode and a counter electrode may be provided as necessary. Such probe-immobilized substrates are 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 substrates. In the case of a probe-immobilized substrate for performing electrochemical detection as described in the first example, electrodes are arranged on any of the substrates so that electrochemical detection is possible. Then, the probe may be immobilized on the electrode.
[0032]
Electrodes that can be used in the present invention are not particularly limited, for example, graphite, glassy carbon, pyrolytic graphite, carbon paste, carbon electrode such as carbon fiber, platinum, platinum black, gold, palladium, Examples include a noble metal electrode such as rhodium, an oxide electrode such as titanium oxide, tin oxide, manganese oxide, and lead oxide; a semiconductor electrode such as Si, Ge, ZnO, CdS, TiO ₂ , and GaAs; and titanium. These electrodes may be coated with a conductive polymer or a monomolecular film, and may be treated with another surface treating agent as desired.
[0033]
Further, the probes having different base sequences may be respectively immobilized on different electrodes, or a plurality of types of probes having different base sequences may be immobilized on one electrode in a mixed state. Good.
[0034]
(C) Detection When a probe-immobilized substrate according to an embodiment of the present invention is used, 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 It is possible to use an electrochemical method and a fluorescence detection method.
[0035]
(A) Electrochemical detection The double-stranded nucleic acid can be detected electrochemically using, for example, a double-stranded recognition substance known per se.
[0036]
The double-stranded recognizer used here is not particularly limited, but for example, bisintercalators such as Hoechst 33258, acridine orange, quinacrine, dounomycin, metallointercalators, and bisacridines, tris intercalators and polyintercalators Etc. can be used. Furthermore, these intercalators can be modified with an electrochemically active metal complex such as ferrocene, viologen, or the like. Further, 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 immobilized on the electrode. Detection of a double-stranded nucleic acid using such an electrode may further use a counter electrode or a reference electrode, as in other general electrochemical detection methods. When a reference electrode is provided, 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 may 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 their state. It can be appropriately selected. Although not limited thereto, for example, the reaction may be performed under the following conditions.
[0039]
For example, the hybridization reaction solution is performed in a buffer having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The sample nucleic acid obtained here is added and denatured at 90 ° C. or higher. The insertion of the probe-immobilized substrate into the heat-denatured sample nucleic acid may be performed immediately after denaturation or after quenching to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a 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 about 1 night. After the hybridization reaction, the electrodes are 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. If 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, double-stranded nucleic acid generated by the following procedure is detected by electrochemical means. Generally, after the hybridization reaction, the substrate is washed, a double-stranded recognizer is allowed to act on the double-stranded portion formed on the electrode surface, and a signal generated thereby is electrochemically measured.
[0042]
The concentration of the double-stranded recognizer varies depending on its type, but is generally used in the range of 1 ng / mL to 1 mg / mL. In this case, 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, in the electrochemical measurement, a potential equal to or higher than the potential at which the double-stranded recognizer reacts electrochemically may be applied, and the reaction current value derived from the double-stranded recognizer may be measured. At this time, the potential may be swept at a constant speed, applied as a pulse, or a constant potential may be applied. At the time of measurement, for example, the current and voltage may be controlled 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 also be preferably used in the method of the present invention. In that article, Hashimoto et al. Reported sequence-specific gene detection using a gold electrode modified with a DNA probe and an electrochemically active dye. The anodic current from the dye correlates with the concentration of the target DNA. Wang et al. Also reported indicator-free electrochemical DNA hybridization. The configuration of this biosensor involves immobilization of an inosine-substituted probe (without guanine) on 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 using a fluorescent labeling substance, the target nucleic acid may be labeled with a substance capable of obtaining a fluorescent activity such as a fluorescent dye such as FITC, Cy3, Cy5 or rhodamine. Good. Alternatively, a second probe labeled with the aforementioned substance may be used instead of such a label. Further, 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 having an ionic strength of 0.01 to 5 and a pH of 5 to 10. To this solution, dextran sulfate as a hybridization accelerator, salmon sperm DNA, bovine thymus DNA, EDTA, a surfactant and the like may be added. The extracted nucleic acid component is added thereto, and denatured at 90 ° C. or higher. Insertion of the probe-immobilized chip may be performed immediately after denaturation or after rapid cooling to 0 ° C. Further, it is also possible to carry out a hybridization reaction by dropping a solution on a 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 about 1 night. After the hybridization reaction, washing is performed. For 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 detection of the hybridization reaction is performed by detecting the labeled base sequence in the sample or the label in the secondary probe using an appropriate detection device according to the type of the label. When the label is a fluorescent substance, the label may be detected using, for example, a fluorescence detector.
[0048]
3. Probe Set The first probe and the second probe according to the above-described embodiment 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 embodiment of the present invention, the above-described probe-immobilized substrate and / or probe set may be provided as an assay kit. For example, they may be selected and combined according to the purpose of use, and provided as an appropriate assay kit. Further, in addition to them, salts for buffers, other necessary reagents, labeling substances, and / or reaction vessels may be combined and provided as an assay kit. In addition, a probe and a substrate that are not immobilized on the substrate may be provided in the assay kit.
[0050]
For example, the first and second probes and / or probe-immobilized substrates according to the present invention are used as an assay kit in combination with a reaction vessel, salts for buffer solution and / or other necessary reagents such as a labeling substance. May be provided.
[0051]
【Example】
Example 1
SNP (position -123) in the promoter region of the human MxA gene was detected. As a target gene, synthetic oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 whose 5 'end was labeled with Cy5 were used as a model. There are two types of SNPs, in which the -123 position is "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 for immobilizing the probe, and a probe-immobilized substrate was manufactured as follows.
[0052]
First, the slide glass was treated with a solution of sulfuric acid: aqueous hydrogen peroxide = 2: 1 to create a normal surface. Next, after treating with 1% of γ-aminopropyltriethoxysilane for 1 hour, the mixture was 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 the fluorescence intensity was measured. As a result, when a 15-mer probe was used, the signal intensity was considerably high even with 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, according to the method of the present invention, it was possible to detect SNP with high specificity. The use of the nucleic acid sequence analysis method, probe-immobilized substrate, assay kit and probe set according to the present invention makes it possible to analyze a target nucleic acid sequence simply and in a short time.
[0055]
Example 2
SNP (-88 position) in the promoter region of the human MxA gene was detected. As a target gene, synthetic oligonucleotides of SEQ ID NO: 7 and SEQ ID NO: 8 were used as models. The type of SNP is two patterns of “G” or “T” at position −88. Therefore, it was considered that a GA mismatch that was difficult to detect was formed. Therefore, a thiol group was introduced at the 5 ′ end of the G-type detection probe as a 14mer (SEQ ID NO: 9) and the A-type detection probe as a 15mer (SEQ ID NO: 11). For the G-type detection probe, a 15-mer was also synthesized as a control (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 formed by forming a titanium / gold thin film on a glass substrate and patterning the thin film 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 the 15-mer probes were used, the GA mismatch signal became large, and the S / N was about 1.5 (FIG. 6). On the other hand, in the chip in which the G-type detection probe was changed to 14 mer, GA mismatch was also successfully suppressed, indicating that SNP type determination was possible (FIG. 7).
[0057]
As is clear from the above results, according to the method of the present invention, it was possible to detect SNP with high specificity. 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 easily and quickly analyzed.
[0058]
【The invention's effect】
The use of the nucleic acid sequence analysis method, probe-immobilized substrate, assay kit, and probe set according to the present invention makes it possible to detect SNPs and point mutations with high specificity.
[0059]
[Sequence list]

[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 view showing an example of a probe-immobilized substrate according to an embodiment of the present invention.
FIG. 3 is a schematic view showing an example of a probe-immobilized substrate according to an embodiment of the present invention.
FIG. 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.
[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. Fixed area 16. Substrate 22. Substrate 23. Electrode 24. Pat

Claims (10)

A first probe comprising a first target site and a first sequence, and one base shorter than the first sequence, except for one base of a second target site corresponding 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 under conditions that cause appropriate hybridization. The combination of bases in 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, and cytosine and uracil. The method for analyzing a nucleic acid sequence according to claim 1, wherein 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, wherein the method 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 3. The method for analyzing a nucleic acid sequence according to any one of Items 1 and 2. A first nucleic acid probe containing a first sequence site and a sequence that is one base shorter than the first sequence and is homologous except for one base of a second target site corresponding to the position of the first target site; A probe-immobilized substrate comprising a second nucleic acid probe. 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, and cytosine and uracil. The probe-immobilized substrate according to claim 5, wherein: The probe-immobilized substrate according to claim 5, and a first nucleic acid probe including the first sequence site, and a second nucleic acid probe that is shorter than the first sequence by one base and corresponds 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 except for one base at the target site. The probe-immobilized substrate according to claim 6, a first nucleic acid probe including the first target site and the first sequence, and one base shorter than the first sequence, at a position of the first target site. A probe set with a second nucleic acid probe containing a homologous sequence except for one base of a corresponding second target site, wherein a combination of bases of the first target site and the second target site is thymine and guanine. Adenine and guanine, cytosine and adenine, cytosine and thymine, uracil and guanine, nucleic acid sequence analysis comprising at least a component selected from the group consisting of a probe set characterized by being selected from the group consisting of cytosine and uracil Assay kit for A first nucleic acid probe containing the first sequence, a sequence shorter by one base than the first sequence and containing a sequence homologous except for 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. 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, and cytosine and uracil. The probe set according to claim 9.

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