JP2008017752A - Method for identifying base polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for clearly detecting a polymorphism in a nucleic acid sequence in excellent repeatability and to obtain a reagent therefor. <P>SOLUTION: The method for identifying a base polymorphism comprises detecting whether a first ligand and a third ligand coexist in a complex (Pa) containing the extension product of at least one kind of an oligonucleotide primer (A) for base judgment, to which the first ligand is bonded and which contains a base site (X) desired to be judged and at least one kind of an oligonucleotide probe (D) for detection, to which the third ligand is bonded and which is complementary to a part of the extension product and detecting whether a second ligand and a third ligand coexist in a complex (Pb) containing the extension product of at least one kind of an oligonucleotide primer (B) for base judgment, to which the second ligand is bonded and which contains a base sequence (X) desired to be judged and at least one kind of an oligonucleotide probe (D) for detection, to which the third ligand is bonded and which is complementary to a part of the extension product. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for identifying a nucleotide polymorphism. The present invention is particularly useful for diagnosis of genetic diseases, nucleotide polymorphism analysis, and the like.

  In the present invention, the nucleotide polymorphism means having a nucleotide sequence different from the wild type. The nucleotide polymorphism of a gene plays an important role as a cause of inter-individual variation in the occurrence of side effects and treatment failures in drug metabolism, and is also known as a cause of individual differences such as basic metabolism known as constitution. In addition, they also serve as genetic markers for a number of diseases. Therefore, elucidation of these mutations is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical studies (Non-Patent Document 1, Non-Patent Document 2). In addition, a nucleic acid sequence analysis method for detecting each genotype following identification of the causative mutant gene is desired.

  As a conventional nucleic acid sequence analysis technique, for example, there is a nucleic acid sequencing method (sequencing method). Nucleic acid sequencing can detect and identify nucleotide polymorphisms contained in nucleic acid sequences, but requires a great deal of effort to prepare template nucleic acids, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequences, etc. And time is needed. Further, labor can be saved by using a recent automatic sequencer, but there is a problem that an expensive apparatus is required.

  On the other hand, there are various known genetic diseases caused by point mutations in genes, and some of them know which part of the gene and how point mutations cause genetic diseases. Not a few.

  As a method for detecting such a predicted point mutation, detection of a point mutation of a gene using a gene amplification method such as a PCR (polymerase chain reaction) method (see, for example, Patent Documents 1 and 2) has been conventionally performed. The method is known. A method (PCR-RFLP) is used in which amplified gene fragments are treated with a restriction enzyme that cleaves a specific nucleic acid sequence, and the size of the resulting fragments is judged. Similarly, as a detection method using the PCR method, the Alelle specific amplification method using the specificity of the primer to be used is used. In this method, as one of the pair of oligonucleotides used in the gene amplification method, a wild-type oligonucleotide that is completely complementary to the end region of the wild-type gene amplification region and the mutant-type gene amplification A mutant oligonucleotide that is completely complementary to the end region of the region is used. Mutant oligonucleotides have nucleotides complementary to the predicted point mutation at the 3 'end. Such wild-type and mutant-type oligonucleotides are separately used to subject the sample gene to the gene amplification method.

  If the sample gene is wild-type, nucleic acid amplification occurs when a wild-type oligonucleotide is used, but if a mutant-type oligonucleotide is used, the 3 ′ end of the oligonucleotide corresponds to the sample gene. Since it is not complementary to the nucleotide (mismatch), no extension reaction occurs, and no nucleic acid amplification occurs. On the other hand, if the sample gene is a mutant type, conversely, amplification does not occur when the wild-type oligonucleotide is used, and amplification occurs when the mutant-type oligonucleotide is used. Therefore, by examining whether amplification occurs when using each oligonucleotide, it is possible to determine whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. Can do. As a method to check whether amplification has occurred at this time, the amplified product is subjected to agarose gel electrophoresis, stained with a nucleic acid-specific binding fluorescent reagent such as ethidium bromide, and then irradiated with UV to detect the presence of amplified nucleic acid. it can. Other methods include Southern blotting, in which amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe, sandwich hybridization that is detected by using a detection probe after capturing with a capture probe immobilized on an individual carrier. Laws have been developed.

  Although it seems that the amplified nucleic acid can be detected by the method as described above and the polymorphism can be easily identified, in practice, the operation is complicated, and in order to analyze a large amount of samples quickly, Requires labor or needs to build a large lab automation system.

For example, according to the electrophoresis method, it is necessary to separately detect the wild type and the mutant type, and it is difficult to accurately quantify the amount of nucleic acid from the electrophoretic image. Further, Southern blotting and sandwich hybridization require a hybridization reaction with a probe, and it is necessary to strictly adjust the conditions, and the operation is very complicated.
Japanese Examined Patent Publication No. 4-67960 Japanese Patent Publication No. 4-67957 Gram and Brsen, European Consensus Conference on Pharmacogenetics. Commission of the European Communities, Luxembourg, 87-96 (1990) Balant et al., Eur. J. Clin. Pharmacol. 36, 551-554, (1989)

An object of the present invention is to provide a method and a reagent therefor that can solve the above-mentioned problems and detect a polymorphism in a nucleic acid sequence clearly and reproducibly.

In view of the above circumstances, the present inventors have completed the present invention as a result of intensive studies.

That is, the present invention has the following configuration.
1. A method for determining a base site on a target nucleic acid sequence contained in a sample solution, comprising at least the following steps (1) to (4):
(1) At least one base determination oligonucleotide primer (A) containing the base site (X) to which the first ligand is bound and the base ligand to be judged, and the base to be judged and the second ligand are bound At least one base determination oligonucleotide primer (B) containing the site (X) and at least one oligonucleotide primer complementary to a part of the extension product of the primer (A) and / or (B) ( C), and a first ligand (A) and / or (B) extension product obtained in the first step (2) in which an amplification reaction is carried out from the target nucleic acid sequence, and the third ligand binds to each other. And at least one detection oligonucleotide probe (D) complementary to a part of the extension product of the primer (A) and / or (B). Soybean is formed to form a complex (Pa) containing the extension product of the primer (A) and the probe (D), and a complex (Pb) containing the extension product of the primer (B) and the probe (D) The second step (3) for detecting whether the first ligand and the third ligand coexist in the complex (Pa) obtained in the second step (4) the second step 4. The fourth step of detecting whether or not the second ligand and the third ligand coexist in the complex (Pb) obtained in step 2. The base determination method according to 1, wherein an oligonucleotide probe (D) is present in the first step.
3. 1 or 2 base judging method characterized by performing a 3rd process and a 4th process simultaneously.
4). The base determination method according to any one of 1 to 3, wherein the first ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
5. The base determination method according to any one of 1 to 3, wherein the second ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
6). The base determination method according to any one of 1 to 3, wherein the third ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
7). The base determination method according to any one of 1 to 6, wherein the third step includes the following step (i) or (ii):
(I) a step of detecting the first ligand after capturing the complex (Pa) on a solid phase to which a capture agent for the third ligand is bound; and (ii) capable of binding to and labeling the first ligand. At least one type of physiologically active substance is captured on the solid phase to which the third ligand capture agent is bound via the complex (Pa), and then captured on the solid phase via the complex (Pa). 7. detecting a label of the physiologically active substance thus produced; The fourth step includes any one of the following steps (i) and (ii): The base determination method according to any one of 1 to 7, wherein
(I) a step of detecting the first ligand after capturing the complex (Pb) on a solid phase to which a capture agent for the third ligand is bound; and (ii) being capable of binding to and labeling the second ligand. At least one kind of physiologically active substance is captured on the solid phase to which the capture agent of the third ligand is bound via the complex (Pb), and then captured on the solid phase via the complex (Pb). 8. detecting a label of the physiologically active substance thus produced; 7. The base determination method according to 7 or 8, wherein the capture agent for the third ligand is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.
10. The base determination method according to any one of 7 to 9, wherein the physiologically active substance is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.
11. The label bonded to the physiologically active substance is at least one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance and a conductive substance. The base determination method in any one of -10.
12 The base determination according to any one of 1 to 11, wherein the second base from the 3 ′ end of the primer (A) is the same or complementary base as the predicted base of the base site (X) to be determined Method.
13. 13. The primer according to any one of 1 to 12, wherein at least one of the 3rd to 5th bases from the 3 ′ end of the primer (A) is substituted with a base that is not complementary or homologous to the nucleic acid sequence used as a template. Base determination method.
14 A kit for base determination, comprising at least the following (1) to (4).
(1) At least one type of oligonucleotide primer for base determination (A) to which the first ligand is bound and which includes the base site (X) to be determined
(2) At least one type of oligonucleotide primer for base determination (B) to which the second ligand is bound and containing the base site (X) to be determined
(3) At least one kind of oligonucleotide primer (C) complementary to a part of the extension product of the primer (A) and / or (B)
(4) At least one type of oligonucleotide probe for detection (D) to which a third ligand is bound and which is complementary to a part of the extension product of the primer (A) and / or (B)
15. The kit for base determination according to claim 14, further comprising the following (5) and (6).
(5) at least one physiologically active substance capable of binding to and labeled with the first ligand and / or at least one physiologically active substance capable of binding to and labeled with the second ligand (6) third Solid phase to which ligand capture agent is bound 16. The 14 or 15 base determination kit, wherein the first ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
17. The 14 or 15 base determination kit, wherein the second ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
18. The 14 or 15 base determination kit, wherein the third ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
19. 15. A 15-base determination kit, wherein the third ligand capture agent is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.
20. 15. The kit for determining bases according to 15, wherein the physiologically active substance is at least one selected from the group consisting of antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances and avidin.
21. The label bonded to the physiologically active substance is at least one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. Kit for base determination.

  The present invention provides a method capable of clearly and easily detecting a base polymorphism in a sample nucleic acid. Since the method of the present invention does not require a complicated operation as in the conventional methods, results with good reproducibility can be obtained quickly and easily.

Hereinafter, the present invention will be described in detail.
In the present invention, the “sample solution” refers to a solution containing a nucleic acid having a sequence complementary to a target nucleic acid sequence, that is, a chromosome containing a nucleotide polymorphism site to be analyzed or a fragment thereof, for example, bacteria, animals or plants. It can be prepared from any material such as tissue, lysates from individual cells. The method for preparing the sample solution is not particularly limited. For example, it may be prepared from a patient's blood or tissue by a known method. Typical examples include phenol / chloroform extraction method (Biochimica et Biophysica acta 72, 619-629, 1963), alkaline SDS method (Nucleic Acid Research 7th, 1513-1523, 1979). There is a method of performing in the liquid phase. Further, as a system using a nucleic acid binding carrier for nucleic acid isolation, a method using glass particles and a sodium iodide solution (Proc. Natl. Acad. USA, Vol. 76-2, 615-619, 1979). ) And a method using hydroxyapatite (Japanese Patent Laid-Open No. 63-263093). Examples of other methods include a method using silica particles and chaotropic ions (J. Clinical Microbiology Vol. 28-3, 495-503, 1990, JP-A-2-289596).

In the present invention, the “target nucleic acid sequence” refers to a target nucleic acid, that is, a nucleic acid sequence containing a nucleotide polymorphism site to be analyzed. Examples of the target nucleic acid include an Alu sequence, a ribosome gene, an exon of a gene encoding a protein, an intron, a promoter, and the like. More specifically, genes related to various diseases including genetic diseases, drug metabolism, lifestyle-related diseases (hypertension, diabetes, etc.) can be mentioned. For example, the CYP2C19 (Cytochrome P450 2C19) gene can be mentioned as a gene related to drug metabolism.

In the present invention, the “base polymorphism” means that the nucleic acid has a base sequence different from that of the wild type. At least one of wild-type nucleic acids having a wild-type base sequence, preferably one nucleotide is point-mutated and replaced with another nucleotide, or inserted or deleted in a part of the wild-type nucleic acid It has been elucidated which part of the nucleotide is mutated in a nucleic acid containing a sequence, that is, a mutant nucleic acid. In addition, it has been elucidated that the constitution is different depending on such base polymorphism, and the method of the present invention examines whether or not the nucleic acid in the sample has such an expected mutation. This is a method, and “base part (X)” indicates a base part to be determined as a test target in the base polymorphism. There may be a plurality of “base sites (X)”, and for each “base site (X)”, at least one kind of base including the base site (X) to which the first ligand is bound and to be determined. An oligonucleotide primer for determination (A) and at least one base determination oligonucleotide primer (B) containing a base site (X) to which the second ligand is bound and to be determined may be set.

  In the present invention, at least one base determination oligonucleotide primer (A) containing the base site (X) to which the first ligand is bound and the second ligand is bound, and the second ligand is bound. The at least one base determination oligonucleotide primer (B) containing the desired base site (X) is an oligonucleotide having a sequence complementary to the chromosome containing the target nucleotide polymorphism site or a fragment thereof, There is no particular limitation as long as it can be used for known amplification methods such as PCR, NASBA, LCR, SDA, RCA, TMA, LAMP, ICAN and UCAN.

  In the present invention, when it is desired to distinguish whether the “base site (X)” is a wild type or a mutant type, for example, at least one type of base determination including the base site (X) to which the first ligand is bound and to be determined Oligonucleotide primer (A) is designed for wild-type amplification, and at least one base determination oligonucleotide primer (B) containing the base site (X) to which the second ligand is bound and to be determined is mutated It may be designed for mold amplification. When a plurality of mutations occur, a plurality of base determination oligonucleotide primers (B) may be set according to the mutations. In this case, by setting a second ligand having substantially different properties for each of a plurality of oligonucleotide primers for base determination (B), when the base site (X) is mutated, the base site The type of mutation produced in (X) can be identified.

  In the present invention, when there are a plurality of “base sites (X)”, a plurality of base determination oligonucleotide primers (A) and base determination oligonucleotide primers (B) may be set. In this case, a first ligand having a substantially different property is set for each of the plurality of base determination oligonucleotide primers (A), and a first property having a substantially different property for each of the plurality of base determination oligonucleotide primers (B). Two ligands may be set. The first ligand and the second ligand are preferably different.

At least one base determination oligonucleotide primer (A) to which the first ligand is bound and containing the base site (X) to be judged, and the base site (to which the second ligand is bound and to be judged The at least one base determination oligonucleotide primer (B) containing X) is preferably designed so that the first or second base from the 3 ′ end corresponds to the position of the base to be determined. Preferably, the second base is designed so as to correspond to the position of the base to be determined. In order not to impair the effects of the present invention, the third or third base from the 3 'end may be designed to correspond to the position of the base to be determined. Furthermore, by substituting at least one base from the 3rd to 5th from the 3 ′ end with a base that is not complementary to the template nucleic acid sequence, a primer that can be amplified more selectively when the base to be determined is present. Also good. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases.

As long as at least one oligonucleotide primer (C) complementary to a part of the extension product of primer (A) and / or (B) has characteristics generally used for nucleic acid amplification, It may be modified as necessary. Usually, when primers (A) and (B) are sense primers (also referred to as forward primers), primer (C) may be set as an antisense primer (also referred to as reverse primer). Conversely, when primers (A) and (B) are antisense primers, primer (C) may be set as a sense primer. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases. The amplification method using these primers is not particularly limited. A known amplification method may be used. Known amplification methods include, for example, PCR, NASBA, LCR, SDA, RCA, TMA, LAMP, ICAN, and UCAN methods.

  In the present invention, at least one base determination oligonucleotide primer (A) containing the base site (X) to which the first ligand is bound and the second ligand is bound, and the second ligand is bound. The extension method of at least one base determination oligonucleotide primer (B) containing the desired base site (X) can basically be performed using a conventional method. Usually, a target nucleic acid is used as a template by allowing four types of deoxynucleoside triphosphates (dNTP) and DNA polymerase to act on a chromosome or fragment thereof containing a specific nucleotide polymorphism site denatured into a single strand. Oligonucleotide primers (A) and (B) are extended.

  The extension reaction can be performed according to the method described in Molecular Cloning, A Laboratory Manual, Third Edition (Sambrook et al., Chapter 8, 8.1-8.126, 2001). In the method for detecting a base polymorphism based on whether or not the oligonucleotide primers (A) and (B) are extended, if the target nucleic acid does not contain an amount sufficient for detection, the polymorphism It is also possible to amplify a nucleic acid fragment containing the sequence by the amplification reaction shown below.

  In the present invention, a method for amplifying a chromosome or fragment containing a specific nucleotide polymorphism site can also be basically performed using a conventional method, and usually a specific nucleotide polymorphism denatured into a single strand. By using 4 types of deoxynucleoside triphosphate (dNTP), DNA polymerase and oligonucleotide primers (A), (B) and (C) on the chromosome containing the site or its fragment, the target nucleic acid can be used as a template. The sequence between the oligonucleotide primers was amplified.

  Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic acid research, Volume 16, 1691 (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Volume 31, 3270 (1993)), LAMP (loop-mediated isothermal amplification method: J Clin Microbiol. 42: 1,956 (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 78, 533 (2003)).

  In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of oligonucleotides, and a heat-resistant DNA polymerase, and thereby the above paired pair. In this method, the region of the sample nucleic acid sandwiched between the oligonucleotide primers is exponentially amplified. That is, the nucleic acid of the sample is denatured in the denaturation step, each oligonucleotide primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each in the subsequent annealing step, and each oligonucleotide primer is then synthesized in the subsequent extension step. The DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of DNA polymerase from the starting point to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of oligonucleotide primers is theoretically amplified to 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

  “At least one detection oligonucleotide probe (D) to which a third ligand is bound and complementary to a part of the extension product of the primer (A) and / or (B)” used in the present invention Is preferably one in which a ligand is bound to an oligonucleotide complementary to a part of the extension product of the primer (A) and / or (B), more preferably the oligonucleotide primer (A), (B ) And (C) can be used which has a ligand bound to an oligonucleotide complementary to a part of the extension product of the primer (A) and / or (B). The site where the ligand binds to the oligonucleotide is not particularly limited. The oligonucleotide probe (D) is preferably designed so as not to inhibit the amplification reaction using the oligonucleotide primers (A), (B) and (C). As an example, the Tm value of the oligonucleotide probe (D) may be designed to be lower than the Tm value of the primer (A, B, C).

The Tm value of the oligonucleotide primer is a temperature at which 50% of the double-stranded DNA molecules forming the hybrid are separated from each other, and the calculation method is not particularly limited as long as it is a known method, but the Neighbor Neighbor method, It is obtained by either the Wallace method or the GC% method and needs to satisfy the characteristics of the present invention. Note that a particularly preferable Tm value is calculated by the Nearest neighbor method. The difference in Tm value between the oligonucleotide probe (D) and the primer (A, B, C) is not particularly limited as long as it is 0.5 ° C or higher, but is preferably 1 ° C or higher.

  In the present invention, at least one base determination oligonucleotide primer (A) containing the base site (X) to which the first ligand is bound and the second ligand is bound, A part of the extension product of the primer (A) and / or (B) in which at least one base determination oligonucleotide primer (B) containing the base site (X) to be determined is bound to the third ligand. And at least one kind of oligonucleotide probe for detection (D) can be allowed to act separately or simultaneously.

When acting separately, as an example, after performing an amplification reaction using the oligonucleotide primers (A), (B), and (C), the oligonucleotide probe (D) is added, followed by 98 ° C. for 3 minutes. The temperature can be gradually lowered to near room temperature after the thermal denaturation of is carried out.

When acting simultaneously, as an example, an amplification reaction is performed in a state where the oligonucleotide primers (A), (B), (C) and the oligonucleotide probe (D) are mixed. After performing heat denaturation for 3 minutes, the temperature can be gradually lowered to near room temperature.

  As an example of the method using the nucleic acid amplification method as described above, an oligonucleotide primer (A) capable of specifically amplifying a wild type sequence and an oligonucleotide primer (B) capable of specifically amplifying a mutant type sequence are used. If the template sequence contained in the sample is a wild type sequence after the reaction, a complex (Pa) containing a probe (D) in which the nucleic acid fragment amplified by the oligonucleotide primer (A) and the third ligand are bound. When the template sequence contained in the sample is a mutant sequence, a complex (Pb) in which the nucleic acid fragment amplified by the oligonucleotide primer (B) and the third ligand are bound is formed.

  The first ligand, the second ligand, and the third ligand to be used are not particularly limited as long as they do not interfere with the detection of the nucleic acid sequence, but are preferably antigens, antibodies, fluorescent substances, luminescence The group can be selected from the group consisting of enzymes, avidin, biotin, digokeshigenin. Antigens, fluorescent substances, biotin, digoxigenin, and the like are preferable, and antigens, fluorescent substances, biotin, and digoxigenin are particularly preferable. In practicing the present invention, the first ligand, the second ligand, and the third ligand are preferably different. For example, FITC may be used as the first ligand, digoxigenin as the second ligand, and biotin as the third ligand.

  In the present invention, whether the first ligand and the third ligand coexist in the formed complex (Pa), and the second ligand and the third ligand in the formed complex (Pb). What is necessary is just to be able to confirm whether the ligand coexists. For example, the first ligand and the second ligand may be detected after capturing the complexes (Pa) and (Pb) on a solid phase to which a capture agent for the third ligand is bound. At this time, the ligand may be directly detected, or at least one physiologically active substance that can be bound to the ligand and is labeled is captured on a solid phase via the complex, and the physiologically active substance is captured. May be detected. Further, the first ligand and the second ligand may be detected simultaneously or separately.

As an example of the above, as shown in FIG. 1, the complexes (Pa) and (Pb) are captured on a solid phase to which a capture agent of a third ligand is bound, can bind to the first ligand, and are labeled. At least one type of physiologically active substance is captured on the solid phase to which the third ligand capture agent is bound via the complex (Pa), and then captured on the solid phase via the complex (Pa). The label of the bioactive substance thus detected is detected, and the capture agent for the third ligand is bound via the complex (Pb) to at least one bioactive substance that can bind to the second ligand and is labeled. After capturing on the solid phase, the label of the physiologically active substance captured on the solid phase via the complex (Pb) can be detected. As another example, as shown in FIG. 2, after the complexes (Pa) and (Pb) are captured on the solid phase to which the capture agent of the third ligand is bound, the first ligand is directly detected. After capturing at least one physiologically active substance capable of binding to and labeled with the second ligand on the solid phase to which the capture agent of the third ligand is bound via the complex (Pb), The label of the physiologically active substance captured on the solid phase via the body (Pb) can be detected.

As the solid phase, a metal plate, a piece of wood, a plastic plate, a glass plate, a rubber plate, a polystyrene foam, a film, a membrane, a liquid-permeable filter, a gel, an assay plate, or the like may be used. An assay plate is preferable.

  The physiologically active substance to be used is not particularly limited as long as it has an affinity for the first or second ligand, but is preferably an antibody, oligonucleotide, receptor, nucleic acid-specific binding substance, avidin, It can be selected from the group consisting of biotin and digokesigenin. An antibody and avidin are preferable, and an antibody is particularly preferable. For example, when the ligand is FITC, the physiologically active substance may be an anti-FITC antibody, and when the ligand is digoxigenin, the physiologically active substance may be an anti-digoxigenin antibody.

  When selecting a first ligand, a second ligand, a third ligand, a third ligand capture agent and a bioactive substance that binds to the first or second ligand, the first ligand and the first ligand The bioactive substance that binds to the first ligand and the second ligand may be selected so that the bioactive substance that binds to the first ligand and the second ligand and the capture agent for the third ligand cannot bind. Select so that the second ligand and the second ligand capture agent can bind, and the second and first biologically active substances that bind to the first and third ligands cannot bind. It is good to do. Furthermore, it is preferable that the physiologically active substance that binds to the third ligand and the first ligand and the physiologically active substance that binds to the second ligand are selected so that they cannot bind.

  The label bonded to the physiologically active substance can be selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. Preferred are fluorescent chemical substances, fluorescent proteins, luminophores, enzymes and conductive substances, more preferred are fluorescent chemical substances, fluorescent proteins and enzymes, and particularly preferred are enzymes. This label can be any of a first ligand, a second ligand, a third ligand, a third ligand capture agent, a physiologically active substance that binds to the first ligand, and a physiologically active substance that binds to the second ligand. Substances that do not bind to each other are preferred.

  In particular, humans and other higher organisms have one gene each derived from a father and one from a mother, but according to these methods, whether a sample gene is a wild-type homolog or not. It is also possible to distinguish between mutant homozygous or both heterozygous. That is, in the case of heterogeneity, both wild type and mutant types are detected because both wild type genes and mutant genes exist.

Kit In this invention, it is a kit for base determination characterized by including at least the following (1)-(4).
(1) at least one base determination oligonucleotide primer (A) to which the first ligand is bound and which includes the base site (X) to be determined;
(2) at least one base determination oligonucleotide primer (B) to which the second ligand is bound and containing the base site (X) to be determined;
(3) at least one oligonucleotide primer (C) complementary to a part of the extension product of the primer (A) and / or (B),
(4) At least one type of oligonucleotide probe for detection (D) to which a third ligand is bound and which is complementary to a part of the extension product of the primer (A) and / or (B).

Furthermore, it is a kit for base determination characterized by including the following (5) and (6).
(5) at least one physiologically active substance capable of binding to and labeled with the first ligand and / or at least one physiologically active substance capable of binding to and labeled with the second ligand (6) third A solid phase to which a ligand capture agent is bound.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
Detection of nucleotide polymorphism (636G → A) of Cytochrome P450 2C19 (CYP2C19) gene (1) Synthesis of oligonucleotide for detecting 636th polymorphism of CYP2C19 gene By the method, oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 4 (hereinafter referred to as oligos 1 to 4) were synthesized. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C. with ammonia water. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, DNA commissioned companies (Nippon Bioservice, Sigma-Aldrich Japan, Operon Biotechnology, etc.) were requested.
Oligo 1 is the sense strand and oligonucleotide primer (C), and is used as an oligonucleotide for the amplification reaction in combination with oligo 2 or oligo 3 of the antisense strand. Oligo 2 is a wild-type amplification oligonucleotide primer (A), and oligo 3 is a mutation-type amplification oligonucleotide primer (B). Oligo 4 is a detection oligonucleotide probe (D). Oligo 2 is labeled with FITC at the 5 ′ end, oligo 3 is labeled with digoxigenin at the 5 ′ end, and oligo 4 is labeled with biotin at the 5 ′ end.

(2) Analysis of CYP2C19 gene polymorphism by PCR method Amplification reaction by PCR method Using a DNA solution extracted from human leukocytes by phenol-chloroform method as a sample, the following reagents were added, and human CYP2C19 gene was The nucleotide polymorphism (636G → A) was analyzed.

Reagents A 25 μl solution containing the following reagents was prepared.
Taq DNA polymerase reaction solution Oligo 1 5 pmol,
Oligo 2 (5 ′ end labeled with FITC) 5 pmol,
Oligo 3 (5 ′ end labeled with digoxigenin) 5 pmol,
Oligo 4 (5 ′ end labeled with biotin) 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1.5 μl of 25 mM MgCl ₂ ,
Taq DNA polymerase 1.3U,
Extracted DNA solution 100ng

Amplification conditions 95 ° C, 5 minutes 95 ° C, 30 seconds,
60 ° C for 30 seconds,
72 ° C, 30 seconds (35 cycles)
98 ℃, 3 minutes,
65 ° C for 1 minute,
55 ° C for 1 minute,
45 ° C for 1 minute,
35 ° C for 1 minute,
25 ° C for 15 minutes.

(3) Detection using an assay plate 15 μl of an amplification reaction solution was added to 30 μl of a POD-labeled anti-digoxigenin antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the POD-labeled anti-digoxigenin antibody binds to the amplified CYP2C19 gene fragment. When this reaction solution is added to an avidin-bound assay plate (BD Biosciences), oligo 4 which is a biotin-labeled oligonucleotide bound to the amplified CYP2C19 gene fragment is captured in the well of the plate. After washing the plate with the washing solution, the washing solution was added to the well and irradiated with excitation light, and the fluorescence by FITC, which is the oligo 2 label, was confirmed. Next, the washing solution was discarded, and after adding the POD substrate solution, the color development of the solution by the POD-labeled anti-digoxigenin antibody bound to digoxigenin, which is the oligo 3 labeled product, was visually confirmed. In Table 1, Sample No. Regarding the samples shown in 1 to 7 and the case without the sample, “+” indicates that the fluorescence was confirmed by FITC and color development of the solution by the POD-labeled anti-digoxigenin antibody was “+”; As shown.

  As described above, the genotype could be clearly determined easily and quickly.

According to the present invention, it is possible to clearly and easily detect a base polymorphism in a sample nucleic acid, and it is possible to quickly and easily obtain a reproducible result without requiring a complicated operation as in the conventional method. Therefore, it is expected to greatly contribute to the industry.

Complex (Pa) is detected using at least one type of physiologically active substance that can bind to and labeled with the first ligand, and at least one type of physiologically active substance that can bind to and label the second ligand Example of detecting complex (Pb) An example of detecting the complex (Pb) using at least one type of physiologically active substance that can be bound to the second ligand and labeled after directly detecting the first ligand in the complex (Pa)

Claims (21)

A method for determining a base site on a target nucleic acid sequence contained in a sample solution, comprising at least the following steps (1) to (4):
(1) At least one base determination oligonucleotide primer (A) containing the base site (X) to which the first ligand is bound and the base ligand to be judged, and the base to be judged and the second ligand are bound At least one base determination oligonucleotide primer (B) containing the site (X) and at least one oligonucleotide primer complementary to a part of the extension product of the primer (A) and / or (B) ( C), and a first ligand (A) and / or (B) extension product obtained in the first step (2) in which an amplification reaction is carried out from the target nucleic acid sequence, and the third ligand binds to each other. And at least one detection oligonucleotide probe (D) complementary to a part of the extension product of the primer (A) and / or (B). Soybean is formed to form a complex (Pa) containing the extension product of the primer (A) and the probe (D), and a complex (Pb) containing the extension product of the primer (B) and the probe (D) The second step (3) for detecting whether the first ligand and the third ligand coexist in the complex (Pa) obtained in the second step (4) the second step A fourth step of detecting whether or not the second ligand and the third ligand coexist in the complex (Pb) obtainable by The base determination method according to claim 1, wherein an oligonucleotide probe (D) is present in the first step. The base determination method according to claim 1 or 2, wherein the third step and the fourth step are performed simultaneously. The base determination method according to claim 1, wherein the first ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination method according to claim 1, wherein the second ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination method according to any one of claims 1 to 3, wherein the third ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination method according to any one of claims 1 to 6, wherein the third step includes the following step (i) or (ii).
(I) a step of detecting the first ligand after capturing the complex (Pa) on a solid phase to which a capture agent for the third ligand is bound; and (ii) capable of binding to and labeling the first ligand. At least one type of physiologically active substance is captured on the solid phase to which the third ligand capture agent is bound via the complex (Pa), and then captured on the solid phase via the complex (Pa). Detecting a label of the bioactive substance that has been produced The base determination method according to any one of claims 1 to 7, wherein the fourth step includes the following step (i) or (ii).
(I) a step of detecting the second ligand after capturing the complex (Pb) on a solid phase to which a capture agent for the third ligand is bound (ii) capable of binding to and labeling the second ligand At least one kind of physiologically active substance is captured on the solid phase to which the capture agent of the third ligand is bound via the complex (Pb), and then captured on the solid phase via the complex (Pb). Detecting a label of the bioactive substance that has been produced The base determination according to claim 7 or 8, wherein the capture agent for the third ligand is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin. Method. The base determination method according to claim 7, wherein the physiologically active substance is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin. . The label bound to the physiologically active substance is at least one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. The base determination method according to any one of Items 7 to 10. 12. The base according to claim 1, wherein the second base from the 3 ′ end of the primer (A) is the same or complementary base as the expected base of the base site (X) to be determined. The base determination method as described. The at least one base from 3 to 5 from the 3 'end of the primer (A) is substituted with a base that is not complementary or homologous to the nucleic acid sequence used as a template. The base determination method according to. A kit for base determination, comprising at least the following (1) to (4).
(1) At least one type of oligonucleotide primer for base determination (A) to which the first ligand is bound and which includes the base site (X) to be determined
(2) At least one type of oligonucleotide primer for base determination (B) to which the second ligand is bound and containing the base site (X) to be determined
(3) At least one kind of oligonucleotide primer (C) complementary to a part of the extension product of the primer (A) and / or (B)
(4) At least one type of oligonucleotide probe for detection (D) to which a third ligand is bound and which is complementary to a part of the extension product of the primer (A) and / or (B) The kit for base determination according to claim 14, further comprising the following (5) and (6).
(5) at least one physiologically active substance capable of binding to and labeled with the first ligand and / or at least one physiologically active substance capable of binding to and labeled with the second ligand (6) third Solid phase with ligand capture agent bound The base determination kit according to claim 14 or 15, wherein the first ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination kit according to claim 14 or 15, wherein the second ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination kit according to claim 14 or 15, wherein the third ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme. The base determination kit according to claim 15, wherein the capture agent for the third ligand is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin. . The base determination kit according to claim 15, wherein the physiologically active substance is at least one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin. The label bound to the physiologically active substance is at least one selected from the group consisting of a fluorescent chemical substance, a luminophore, an enzyme, a fluorescent protein, a photoprotein, a magnetic substance, and a conductive substance. Item 16. The base determination kit according to Item 15.

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