JP2007330138A - Method for identifying a plurality of base polymorphisms - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which a plurality of polymorphisms in a nucleic acid sequence can clearly be detected in good repeatability, and to provide a reagent therefor. <P>SOLUTION: This method for judging a base site (X) and a base site (Y) on a target nucleic acid sequence comprises the first process for amplifying the target nucleic acid sequence with at least one base judgment oligonucleotide primer (A) containing the base site (X) and an oligonucleotide primer (B), the second process for forming a complex (P) of an elongation product of the primer (A) with an elongation product of a base judgment oligonucleotide probe (D) containing a base site (Y) to be judged, and the third process for detecting the complex (P). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a nucleotide polymorphism identification method using an oligonucleotide primer for base determination and an oligonucleotide probe in a form in which a nucleic acid extension reaction can occur. 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. The amplified gene fragment is treated with a restriction enzyme that cleaves a specific nucleic acid sequence, and a method of judging based on the size of the resulting fragment (PCR-RFLP) is used. 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 Use a mutant oligonucleotide that is completely complementary to the end region of the region. 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.

  Although it seems that the amplified nucleic acid can be detected by the above-mentioned method and polymorphism can be easily identified, in practice, non-specific nucleic acid amplification occurs and it is clear whether the sample gene is wild type or mutant type. Sometimes it cannot be determined. In addition, in the above-mentioned nucleotide polymorphism identification method, it is usually necessary to perform nucleic acid amplification once or twice in order to identify one nucleotide polymorphism. In particular, a point mutation causing a genetic disease is caused by a plurality of gene mutations. When this occurs at a site, the above-described series of operations must be performed a plurality of times, so that a greater effort is required to identify the nucleotide polymorphism of the gene.

  As another typical technique for detecting a specific nucleic acid molecule present in a sample, there is a hybridization method (see, for example, Non-Patent Document 3). Nucleic acid hybridization analysis is a technique for detecting a very small number of target nucleic acids (DNA and RNA) from various types of nucleic acids using probes. In hybridization methods, highly sensitive reporters (enzymes, fluorescent dyes, Even if a radioisotope or the like is used, it is difficult to detect a target nucleic acid molecule having a low copy number (1-1000). Furthermore, since the hybridization method has a big problem of non-specific reaction, operations such as carrier blocking and removal of non-specific signal are indispensable for accurately detecting a specific nucleic acid molecule. Effort is required.

In addition, as a method for solving the problem of non-specific reaction in the past, a competitive hybridization method (for example, for suppressing non-specific reaction by adding similarly prepared non-labeled DNA to labeled DNA prepared from target nucleic acid) Patent Document 3 and Non-Patent Document 4) have been developed. However, the competitive hybridization method is complicated in operation and is not sufficient in terms of detection sensitivity, and therefore cannot be applied particularly to genetic disease diagnosis and nucleotide polymorphism analysis.
Japanese Examined Patent Publication No. 4-67960 Japanese Patent Publication No. 4-67957 Japanese Patent No. 2982304 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) Sambrook et al., Molecular Cloning, A Laboratory Manual, Third Edition, Chapter 10, 10.1-10.52 (2001) Nicolas et al., Anal. Biochem. 205, 193, (1992)

  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. At least one or more types of base sites (X) and at least one or more types of base sites (Y) on a target nucleic acid sequence contained in a sample solution by a method comprising at least the following steps (1) to (3): A base determination method characterized by performing determination.
(1) Complementary to at least one base determination oligonucleotide primer (A) to which the first ligand is bound and containing the base site (X) to be determined, and a part of the extension product of the primer (A) A first step of performing an amplification reaction from the target nucleic acid sequence using at least one kind of oligonucleotide primer (B),
(2) An extension product of the primer (A), which is an oligonucleotide containing the base product (Y) to which the extension product of the primer (A) obtained in the first step is bound and the second ligand is bound. The oligonucleotide probe (D) is hybridized with a part of the product and at least one type of oligonucleotide probe (D) for base determination, and the extension product of the primer (A) is used as a template to perform extension reaction. A second step of forming a complex (P) of the extension product of the primer and the extension product of the primer (A),
(3) A third step of detecting whether or not the first ligand and the second ligand coexist in the complex (P) obtainable in the second step.
2. 2. The base determination method according to 1, wherein at least one nucleotide from the 3 ′ end of the oligonucleotide probe (D) is labeled with a ligand.
3. The method for determining a base according to 1 or 2, wherein the 5 'end of the oligonucleotide probe (D) is labeled with a second ligand.
4). The base determination method according to any one of 1 to 3, wherein the oligonucleotide probe (D) has a different sequence depending on the type of base at the base site (Y) to be determined.
5). The base determination method according to any one of 1 to 4, wherein the base at the 3 ′ end of the oligonucleotide probe (D) is the same or complementary to the expected base of the base site (Y) to be determined .
6). Any one of 1 to 4 characterized in that the second base from the 3 ′ end of the oligonucleotide probe (D) is the same or complementary base as the expected base of the base site (Y) to be determined Base determination method.
7). 5 or 6 base determination characterized in that at least one base from 3 to 5 from the 3 ′ end of the oligonucleotide probe (D) is substituted with a base that is not complementary or homologous to the nucleic acid sequence used as a template Method.
8). The base determination method according to any one of 1 to 7, wherein the primer (A) has a different sequence depending on the type of base at the base site (X) to be determined.
9. The base determination according to any one of 1 to 8, wherein the second base from the 3 ′ end of the primer (A) is the same as or a complementary base to the expected base of the base site (X) to be determined Method.
10. 9. The base determination method according to 9, wherein 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 a nucleic acid sequence that serves as a template.
11. The base determination method according to any one of 1 to 10, wherein the oligonucleotide probe (D) is present in the first step.
12 The base determination method according to any one of 1 to 11, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm values of the oligonucleotide primer (A) and the oligonucleotide primer (B).
13. The base determination method according to any one of 1 to 12, 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.
14 The base determination method according to any one of 1 to 12, 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 present invention provides a method capable of clearly and simply detecting a plurality of nucleotide polymorphisms in a sample nucleic acid with good reproducibility.

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 ribosomal gene, an exon, intron, and promoter of a gene encoding a protein. 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. Is the method.

  “Base site (X)” and “base site (Y)” each indicate a base site to be determined as a test target in the base polymorphism. The base sites (X) and (Y) are preferably different base sites on the same nucleic acid, and more preferably different base sites on the same gene. As an example of the base site to be determined, it is known that polymorphisms of the 636th base (G to A mutation) and 681st base (G to A mutation) affect drug metabolism in the CYP2C19 gene. It has been.

  In the present invention, at least one base determination oligonucleotide primer (A) to which the first ligand is bound and containing the base site (X) to be determined, and a part of the extension product of the primer (A) And at least one type of oligonucleotide primer (B) that is complementary to the oligonucleotide having a sequence complementary to a chromosome containing a target nucleic acid sequence or a fragment thereof, and is a known amplification method, such as PCR, NASBA , LCR, SDA, RCA, TMA, LAMP, ICAN and UCAN method, as long as the amplified nucleic acid fragment can contain the base site (Y) to be determined, it is not particularly limited. May be modified. As an example, when the primer (A) is a sense primer containing a base site (X), the primer (B) is an antisense primer complementary to the downstream region of the base sites (X) and (Y). As another example, when the primer (A) is an antisense primer containing the base site (X), the primer (B) is homologous to the upstream region of the base sites (X) and (Y). A sense primer is preferred. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases. A plurality of the primers may be mixed depending on the nucleic acid sequence to be detected. For example, when there are a plurality of base sites (X) to be determined, a plurality of primers (A) and / or primers (B) may be mixed as desired. An amplification reaction can be performed in one reaction system using a plurality of primers. When a plurality of primers (A) are mixed and used, the type of the first ligand may be changed.

  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 base ligand to be judged and the base to be judged and the second ligand are bound An extension method of at least one type of oligonucleotide probe (D) for base determination, which is an oligonucleotide containing the site (Y) and complementary to a part of the extension product of the primer (A), is basically the conventional method. This method can be used. 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. The oligonucleotide is 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 addition, when the target nucleic acid does not contain a sufficient amount for detection, a nucleic acid fragment containing the base sites (X) and (Y) to be determined may be amplified in advance by the amplification reaction shown below. Is possible.

  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. Oligonucleotide primer using target nucleic acid as template by allowing 4 types of deoxynucleoside triphosphate (dNTP), DNA polymerase and oligonucleotide primers (A) and (B) to act on the chromosome containing the site or fragment thereof The sequence between is 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, whereby the above paired 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. .

  Another important disclosure of the present invention is an oligonucleotide containing the base site (Y) to be determined in the extension product of the oligonucleotide primer (A), and a part of the extension product of the primer (A) When hybridizing at least one complementary oligonucleotide probe (D) for base determination, the extension product of the oligonucleotide probe (D) is subjected to an extension reaction using the extension product of the primer (A) as a template. And the formation of a complex (P) of the extension product of the primer (A), and found a remarkable effect that multiple polymorphisms in the nucleic acid sequence can be clearly and reproducibly detected. It is in.

  “An oligonucleotide containing a base site (Y) to which the second ligand is bound and to be determined and used in the present invention, and is at least one base complementary to a part of the extension product of the primer (A) The “determination oligonucleotide probe (D)” is preferably one in which a ligand is bound to an oligonucleotide complementary to the portion containing the base site (Y) of the extension product of the primer (A), more preferably The oligonucleotide is complementary to the oligonucleotide complementary to the portion containing the base site (Y) in the extension product of the primer (A) of the nucleic acid fragment amplified using the oligonucleotide primers (A) and (B). A bound one can be used, and the ligand is preferably bound to a position that does not inhibit the extension reaction of the probe (D). As the position for binding the ligand, it is preferable to bind the ligand to at least one nucleotide after the 3 ′ end of the oligonucleotide probe (D), and to the 5 ′ end of the oligonucleotide probe (D). More preferably, the ligand is bound.

  The oligonucleotide probe (D) is preferably designed so that the first or second base from the 3 'end corresponds to the position of the base site (Y) to be determined. Preferably, the second base is designed so as to correspond to the position of the base site (Y). 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 site (Y). Further, when at least one base from the 3rd to the 5th from the 3 ′ end is substituted with a base that is not complementary to the template nucleic acid sequence, when the base site (Y) is present, the oligo is more selectively selected. The extension reaction of the nucleotide probe (D) may occur. The chain length of the oligonucleotide probe may be 5 to 25 bases, and preferably 10 to 20 bases.

  A plurality of oligonucleotide probes (D) may be present depending on the base site (Y) to be determined. For example, an oligonucleotide probe (D) for detecting the wild type and an oligonucleotide probe (D) for detecting the mutant type may be mixed and used. When the oligonucleotide probe (D) is mixed and used, the type of the second ligand may be changed.

  The oligonucleotide probe (D) is preferably designed so as not to inhibit the amplification reaction using the oligonucleotide primers (A) and (B). 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).

  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) is not particularly limited as long as it is 1 ° C. or higher, but is preferably 5 ° C. or higher. In addition, the difference of Tm value should just be 50 degrees C or less, Preferably 40 degrees C or less is good. The probe chain length may be 5 to 30 bases, and preferably 8 to 25 bases.

  In the present invention, at least one oligonucleotide primer (A) to which the first ligand is bound and at least one oligonucleotide primer (A) bound to the second ligand and complementary to a part of the extension product of the primer (A). The types of detection oligonucleotide probes (D) can act separately or simultaneously.

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

  When acting simultaneously, as an example, an amplification reaction is performed in a state where the oligonucleotide primers (A), (B) and the oligonucleotide probe (D) are mixed, and the amplification reaction is followed by 98 ° C. for 3 minutes. After heat denaturation, the temperature can be gradually lowered to near room temperature.

  Until the complex (P) is formed from the nucleic acid amplification method, a homogeneous system may be used. For example, three steps of denaturation, annealing, and extension in the presence of sample nucleic acid, four types of deoxynucleoside triphosphates, primer (A), primer (B), detection oligonucleotide probe (D), and heat-resistant DNA polymerase The cycle consisting of is repeated to carry out a nucleic acid amplification reaction. Thereafter, after heat denaturation at 98 ° C. for 3 minutes, the temperature is gradually lowered to near room temperature, and the extension product of the detection oligonucleotide probe (D) and the primer (A) is hybridized. Thereafter, an extension reaction of the oligonucleotide probe for detection (D) is carried out using the extension product of the primer (A) as a template with a heat-resistant DNA polymerase present in the reaction system, and the extension product of the oligonucleotide probe (D) and the primer (A ) Extension product complex (P). Thereafter, the complex (P) may be detected.

  The first and second ligands 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, luminophores, enzymes, and avidin , Biotin, digokesigenin, and dinitrophenyl. Preferably, antigens, fluorescent substances, biotin, digoxigenin, dinitrophenyl and the like are preferable, and antigens, fluorescent substances, biotin, digoxigenin and dinitrophenyl are particularly preferable. In practicing the present invention, the first ligand and the second ligand are preferably different. As an example, FITC may be used as the first ligand and biotin may be used as the second ligand.

  As another example, for the base site (X) to be determined, FITC as the first ligand of the oligonucleotide primer (A) for detecting the wild type, and the oligonucleotide primer (A) for detecting the mutant type Using digoxigenin as the first ligand, for the base site (Y) to be determined, biotin as the second ligand of the oligonucleotide probe (D) for detecting the wild type, oligonucleotide probe for detecting the mutant type (D ), The polymorphisms of the base sites (X) and (Y) can be simultaneously identified from the detected first and / or second ligand combination.

  One of the important disclosures of the present invention is that when the oligonucleotide probe (D) is hybridized to the extension product of the primer (A) that can be obtained in the first step, the extension product of the primer (A) is used as a template. By performing an extension reaction to form a complex (P) of the extension product of the oligonucleotide probe (D) and the extension product of the primer (A), compared with a conventionally known hybridization method that does not perform an extension reaction. The production amount of the complex (P) is greatly improved, and a remarkable effect that the base polymorphism can be clearly and easily identified has been found.

  This principle is based on the assumption that a complex of an oligonucleotide probe (D) and a “primer (A) extension product” is (p ′), an extension product of the oligonucleotide probe (D) and a “primer (A) extension product”. This complex (P) is derived from being much more stable than the complex (p ′). In general, the double-stranded nucleic acid containing “the extension product of the primer (A) that can be obtained in the first step” is much longer than the oligonucleotide probe (D), and thus formed by denaturation of the double-stranded nucleic acid. In the reaction in which single-stranded nucleic acids hybridize to form the original double-stranded nucleic acid, the oligonucleotide probe (D) is attached to one of the single-stranded nucleic acids, ie, the “extension product of primer (A)”. It is far superior to the reaction that hybridizes to form the complex (p ′). The extension product of the oligonucleotide probe (D) formed by hybridizing the oligonucleotide probe (D) to the “product of extension of the primer (A)” and using the extension product as a template is more Since it is much longer than the oligonucleotide probe (D), the complex (P) can be present in much larger amounts than the complex (p ′). In other words, the extension reaction is a reaction in which a synthetic double-stranded nucleic acid is formed by synthesizing a complementary strand using a single-stranded nucleic acid as a template, and the synthetic reaction is formed by denaturation of the double-stranded nucleic acid. Since it is far superior to the reaction in which single-stranded nucleic acids hybridize to form the original double-stranded nucleic acid, the extension reaction of the oligonucleotide probe (D) using the extension product of the primer (A) as a template Greatly improves the production amount of the complex (P), and the complex (P) may be present in a much larger amount than the complex (p ′).

  Therefore, the presence or absence of a base site (Y) on the target nucleic acid sequence is reflected as a detection signal of the complex. If the base site (Y) is present, an extension product of the oligonucleotide probe (D) is obtained, and the amount of complex (P) produced increases, so that a high signal is obtained. On the contrary, if the base site (Y) does not exist, the extension product of the oligonucleotide probe (D) cannot be obtained, so that the complex (P) is not produced and no signal is obtained, or only a few The signal is extremely low due to the complex (p ′). A cut-off value may be set if extremely low signals due to very few complexes (p ') are observed. The cut-off value may be set for each target base site (Y). A person skilled in the art can easily set the cutoff value.

  In the present invention, it is only necessary to confirm that the first ligand and the second ligand coexist in the formed complex (P). For example, one of the first and second ligands is used as a capture ligand for capturing the complex (P) on a solid phase, and the other ligand is captured on the solid phase ( It can be used as a detection ligand for detecting P). As an example, a fluorescent substance may be used as the detection ligand, and fluorescence emitted from the fluorescent substance contained in the complex (P) captured on the solid phase to which the capture agent of the capture ligand is bound may be detected. As another example, at least one physiologically active substance that can be bound to and labeled with a detection ligand is captured via a complex (P) on a solid phase to which a capture agent for the capture ligand is bound. Then, the label of the physiologically active substance captured on the solid phase via the complex (P) may be detected.

That is, one of the first and second ligands is used as a capture ligand for capturing the complex (P) on the solid phase, and the other ligand is captured on the solid phase (P ) May be used as a ligand for detection.
The third step may include at least the following steps (i) and (ii).
(I) a step of capturing at least one physiologically active substance that can be bound to and labeled with a detection ligand on a solid phase to which a capture agent of the capture ligand is bound, via the complex (P);
(Ii) detecting a label of the physiologically active substance captured on the solid phase via the complex (P).
Furthermore, the capture agent for the capture ligand may be at least one selected from the group consisting of antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances, and avidin.
Furthermore, the physiologically active substance may be at least one selected from the group consisting of antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances, and avidin.
Furthermore, the label bound to the physiologically active substance may be 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.

  As the solid phase, any material such as 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, and a gel can be used, but a membrane and a liquid-permeable filter are preferable. If necessary, the surface and / or the inside of the solid phase may be modified to a form capable of capturing the ligand capture agent.

  The ligand capture agent can be appropriately selected from antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances, avidin, biotin, and digokesigenin. An antibody and avidin are preferable, and avidin is particularly preferable. For example, when the ligand is biotin, the ligand capture agent may be avidin.

  The physiologically active substance to be used is not particularly limited as long as it has an affinity for the detection ligand, but preferably from antibodies, oligonucleotides, receptors, nucleic acid-specific binding substances, avidin, biotin, and digokesigenin. You can choose from the group of An antibody and avidin are preferable, and an antibody is particularly preferable. For example, when the detection ligand is FITC, the physiologically active substance may be an anti-FITC antibody.

  When selecting a detection ligand, a capture ligand, a capture agent for the capture ligand, and a physiologically active substance that binds to the detection ligand, the detection ligand and the physiologically active substance that binds to the detection ligand can bind to each other. The capture agent for the capture ligand should be selected so that it cannot bind. The capture ligand and the capture agent for the capture ligand are preferably selected such that they can bind, and the physiologically active substance that binds to the capture ligand and the detection ligand cannot be bound.

  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 is preferably a substance that does not bind to any of the detection ligand, the capture ligand, the capture agent for the capture ligand, and the physiologically active substance that binds to the detection ligand.

  The detection of the label is not particularly limited as long as it is a known method. However, when the label is a fluorescent chemical substance or a fluorescent protein, light of a specific wavelength is irradiated to excite the fluorescent chemical substance or the fluorescent protein to bring it to the ground state. It is possible to measure the amount of fluorescence of a specific wavelength generated when converted. Examples of the fluorescent chemical substance used in these include FITC, FAM, TAMRA, Texas Red, VIC, Cy3, Cy5, HEX, and the like, and examples of the fluorescent protein include GFP, YFP, and RFP. When the label is an enzyme, it can be measured by detecting the reaction product produced by adding the enzyme substrate. The combinations of enzyme and substrate used in these include alkaline phosphatase and paranitrophenyl phosphate, CDP-star, AMPPD, DDAOphosphate, BCIP-NBT, etc., peroxidase and TMB, Lumi-Light (Roche Diagnostics) ), Combinations of SAT-1 (Dojindo), combinations of diaphorase and NTB, various oxidases and substrates, combinations of various dehydrogenases and substrates, etc. It can be used without any problems. When the label is a magnetic substance, measurement can be performed by detecting magnetism. The label can be measured by detecting a conductive substance and a current value.

  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 polymorphisms (636G → A and 681G → A) of Cytochrome P450 2C19 (CYP2C19) gene (1) Synthesis of oligonucleotide for detecting polymorphisms 636 and 681 of CYP2C19 gene DNA synthesizer 392 manufactured by PerkinElmer Using the mold, oligonucleotides having the base sequences shown in SEQ ID NOs: 1 to 5 (hereinafter referred to as oligos 1 to 5) were synthesized by the phosphoramidite method. 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 primer (B). Oligo 2 is a wild-type amplification oligonucleotide primer (A), and oligo 3 is a mutation-type amplification oligonucleotide primer (A). Further, Oligo 4 wild type detection oligonucleotide probe (D), and Oligo 5 is a mutation type detection oligonucleotide probe (D). Oligos 2 and 3 are labeled at the 5 ′ end with FITC and oligos 4 and 5 are labeled at the 5 ′ end with biotin. By labeling the 5 ′ ends of oligos 4 and 5 with biotin, an extension reaction from oligos 4 and 5 is possible.

(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 Nucleotide polymorphisms (636G → A and 681G → A) were analyzed.

Reagents A 25 μl solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 1 5 pmol,
Oligo 2 or Oligo 3 (5 ′ end labeled with FITC) 5 pmol,
Oligo 4 or oligo 5 (5 ′ end labeled with biotin) 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
25 mM MgCl ₂ 1.0 μl,
KOD plus DNA polymerase 0.4U,
Extracted DNA solution 20ng

Amplification conditions 94 ° C, 2 minutes, 94 ° C, 15 seconds,
60 ° C for 30 seconds,
68 ℃, 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 liquid-permeable filter 15 μl of the amplification reaction solution was added to 30 μl of a solution of POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the POD-labeled anti-FITC antibody binds to the amplified CYP2C19 gene fragment. When this reaction solution is added to a liquid-permeable filter to which avidin is bound, oligo 4 or 5 which is a biotin-labeled oligonucleotide bound to the amplified CYP2C19 gene fragment is captured on the filter. Next, after sequentially adding a cleaning solution and a POD substrate solution from the top, color development on the filter surface was visually confirmed. In Table 1, Sample No. In the case of samples 1 to 6 and the case without sample, “+” indicates that the color development was clearly confirmed when oligo 2 or oligo 3 and oligo 4 or oligo 5 were used in combination. The case where no is seen is shown as “−”.

  In Table 1, for example, in sample 1, clear color development can be confirmed only when Oligo 2 indicating that the 681st base is G and Oligo 4 indicating that the 636th base is G are used. Therefore, sample 1 has only the CYP2C19 gene in which the 681st base is G and the 636th base is G. Similarly, in sample 2, when the oligo 2 and oligo 4 and the oligo 2 and oligo 5 are used, clear color development can be confirmed. Therefore, in the sample 2, the 681st base is G and the 636th base is G. And the CYP2C19 gene in which the 681st base is G and the 636th base is A.

  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 plurality of nucleotide polymorphisms 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 contribute greatly to the industrial world.

Claims (14)

At least one or more types of base sites (X) and at least one or more types of base sites (Y) on a target nucleic acid sequence contained in a sample solution by a method comprising at least the following steps (1) to (3): A base determination method characterized by performing determination.
(1) Complementary to at least one base determination oligonucleotide primer (A) to which the first ligand is bound and containing the base site (X) to be determined, and a part of the extension product of the primer (A) A first step of performing an amplification reaction from the target nucleic acid sequence using at least one kind of oligonucleotide primer (B),
(2) An extension product of the primer (A), which is an oligonucleotide containing the base site (Y) to which the extension product of the primer (A) obtained in the first step is bound and the second ligand is bound. The oligonucleotide probe (D) is hybridized with a part of the product and at least one type of oligonucleotide probe (D) for base determination, and the extension product of the primer (A) is used as a template to perform extension reaction. A second step of forming a complex (P) of the extension product of and the extension product of the primer (A),
(3) A third step of detecting whether or not the first ligand and the second ligand coexist in the complex (P) obtainable in the second step. The base determination method according to claim 1, wherein at least one nucleotide from the 3 'end of the oligonucleotide probe (D) is labeled with a ligand. The base determination method according to claim 1 or 2, wherein the 5 'end of the oligonucleotide probe (D) is labeled with a second ligand. The base determination method according to claim 1, wherein the oligonucleotide probe (D) has a different sequence depending on the type of base at the base site (Y) to be determined. The base at the 3 'end of the oligonucleotide probe (D) is the same as or complementary to the expected base of the base site (Y) to be determined. Base determination method. The base according to any one of claims 1 to 4, wherein the second base from the 3 'end of the oligonucleotide probe (D) is the same as or a complementary base to the expected base of the base site (Y) to be determined. The base determination method of crab. 7. The oligonucleotide probe (D) according to claim 5 or 6, wherein at least one base from 3 to 5 from the 3 'end of the oligonucleotide probe (D) is substituted with a base that is not complementary or homologous to a nucleic acid sequence as a template. The base determination method as described. The base determination method according to claim 1, wherein the sequence of the primer (A) is different depending on the type of base at the base site (X) to be determined. The base according to any one of claims 1 to 8, wherein the second base from the 3 'end of the primer (A) is the same as or a complementary base to the expected base of the base site (X) to be determined. The base determination method as described. The base determination according to claim 9, 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. Method. 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, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm values of the oligonucleotide primer (A) and the oligonucleotide primer (B). The base determination method according to any one of claims 1 to 12, 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.