JP2007330137A - Method for identifying base polymorphism - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which a polymorphism 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 comprises modifying a double strand nucleic acid to form a single strand nucleic acid, hybridizing the formed single strand nucleic acid with at least one base judgment oligonucleotide probe (D) complementary to a portion containing a base site (X) to be judged, performing an elongation reaction in the presence of the single strand nucleic acid as a template, forming a complex (P) of the single strand nucleic acid an elongation product of the oligonucleotide probe (D), and detecting the presence of the complex (P). <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for identifying a base polymorphism using an oligonucleotide probe in a form in which a nucleic acid elongation 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.

  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 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. A method for determining a base site in a target nucleic acid sequence in a double-stranded nucleic acid, comprising at least the following steps (1) to (3):
(1) a first step in which a double-stranded nucleic acid containing a base site (X) to be determined is denatured to form a single-stranded nucleic acid;
(2) The single-stranded nucleic acid obtained in the first step contains at least one base determination oligonucleotide probe (D) containing the base site (X) to be determined and complementary to a part of the single-stranded nucleic acid. A second step of hybridizing and performing an extension reaction using the single-stranded nucleic acid as a template to form a complex (P) of the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid,
(3) A third step of detecting the presence of the complex (P).
2. The base determination method according to 1, wherein the oligonucleotide probe (D) has a different sequence depending on the type of base at the base site (X) to be determined.
3. The base determination method according to 1 or 2, wherein 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 (X) to be determined.
4). The base determination method according to 1 or 2, wherein the second base from the 3 ′ end of the oligonucleotide probe (D) is the same or complementary base as the predicted base of the base site (X) to be determined .
5). 3 or 4 base determination characterized in that at least one base from 3 to 5 from the 3 ′ end of the oligonucleotide probe (D) is replaced with a base that is not complementary or homologous to the nucleic acid sequence used as a template Method.
6). The base determination method according to any one of 1 to 5, wherein the oligonucleotide probe (D) is labeled with a ligand.
7). The base determination method according to any one of 1 to 6, wherein at least one nucleotide after the 3 ′ end of the oligonucleotide probe (D) is labeled with a ligand.
8). The base determination method according to any one of 1 to 7, wherein the 5 ′ end of the oligonucleotide probe (D) is labeled with a ligand.
9. The base determination method according to any one of 6 to 8, wherein the ligand is at least one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.
10. The base determination method according to any one of 1 to 9, wherein the oligonucleotide probe (D) is present in the first step.
11. The base determination method according to any one of 1 to 10, wherein the double-stranded nucleic acid is a double-stranded nucleic acid obtained by an amplification reaction.
12 11. The method for determining bases according to 11, wherein the amplification reaction is PCR.
13. The method for determining a base according to 11 or 12, wherein an oligonucleotide probe (D) is present in the amplification reaction.
14 The base determination method according to any one of 11 to 13, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm value of the primer used in the amplification reaction.
15. The base determination method according to any one of 1 to 14, wherein the denaturation in the first step is heat denaturation.

  The present invention provides a method capable of clearly and simply detecting a polymorphism in a sample nucleic acid with good reproducibility.

Hereinafter, the present invention will be described in detail.
In the present invention, the “double-stranded nucleic acid” refers to a nucleic acid having a sequence complementary to a target nucleic acid sequence, that is, a chromosome containing a nucleic acid sequence to be analyzed or a fragment thereof, a sequence complementary to an amplified nucleic acid or a fragment thereof. The nucleic acid etc. which have.

  A nucleic acid having a sequence complementary to the chromosome or a fragment thereof can be prepared from any material such as bacteria, animal or plant tissue, and lysates derived from individual cells. A method for preparing a nucleic acid having a sequence complementary to the chromosome or a fragment thereof is not particularly limited. For example, the nucleic acid 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 7, 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).

  The nucleic acid having a sequence complementary to the amplified nucleic acid or a fragment thereof may be prepared, for example, by a known amplification method. As typical nucleic acid amplification methods, PCR (Molecular Cloning, A Laboratory Manual, Third Edition (Sambrook et al., Chapter 8, 8.1-8.126, 2001)), 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 20, 2091) (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Vol. 31, p. 3270 (1993)), LAMP (loop-mediated isothermal) amplification method: J Clin Microbiol. Volume 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 oligonucleotide primers, and a heat-resistant DNA polymerase. This is a method of exponentially amplifying a sample nucleic acid region sandwiched between a pair of oligonucleotide primers. 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. .

  In the present invention, the “target nucleic acid sequence” refers to a nucleic acid sequence including a target nucleic acid, that is, a nucleic acid sequence 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. This is a method, and “base part (X)” indicates a base part to be determined as a test target in the base polymorphism.

  In the present invention, the “primer” used in the amplification reaction is an 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, There is no particular limitation as long as it can be used for the LCR, SDA, RCA, TMA, LAMP, ICAN and UCAN methods. 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, in order to simultaneously amplify a plurality of types of nucleic acids from a chromosome containing a target nucleic acid sequence or a fragment thereof, primers corresponding to the respective nucleic acid sequences may be mixed and used.

  The “at least one type of oligonucleotide probe for base determination (D) complementary to a part of the single-stranded nucleic acid” obtained by denaturing the double-stranded nucleic acid used in the present invention is contained in the double-stranded nucleic acid. An oligonucleotide complementary to the portion containing the base site (X) to be determined in the target nucleic acid sequence can be used. More preferably, a target nucleic acid sequence in a double-stranded nucleic acid in which a ligand is bound to an oligonucleotide complementary to a portion containing a base site (X) to be determined can be used.

  In the present invention, “denaturation” of a double-stranded nucleic acid is formed in the double-stranded nucleic acid in at least a region of the double-stranded nucleic acid that can hybridize with the oligonucleotide probe for base determination (D). This means that the base pair has been eliminated and is single-stranded. As a method for denaturing the double-stranded nucleic acid, a conventionally known method such as a method by heating (thermal denaturation) or a method of adding formamide can be used, but thermal denaturation is preferable from the viewpoint of simplicity.

  One of the important disclosures of the present invention is that a single-stranded nucleic acid obtained by denaturing a double-stranded nucleic acid has at least a portion complementary to the portion containing the base site (X) to be determined in the single-stranded nucleic acid. When hybridizing one kind of oligonucleotide probe for base determination (D), an extension reaction using the single-stranded nucleic acid as a template is performed to obtain an extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid. By forming the complex (P), the production amount of the complex (P) is greatly improved as compared with the conventionally known hybridization method in which the extension reaction is not performed. Has found a remarkable effect that can be identified.

  This principle is that when the oligonucleotide probe (D) and single-stranded nucleic acid complex is (p ′), the extension product of the oligonucleotide probe (D) and single-stranded nucleic acid complex (P) is It comes from being much more stable than the complex (p ′). In general, double-stranded nucleic acids are much longer than oligonucleotide probes (D), so single-stranded nucleic acids formed by denaturation of the double-stranded nucleic acids hybridize to form the original double-stranded nucleic acid. This reaction is far superior to the reaction in which the oligonucleotide probe (D) hybridizes to one of the single-stranded nucleic acids to form a complex (p ′). The extension product of the oligonucleotide probe (D) formed by hybridizing the oligonucleotide probe (D) to the single-stranded nucleic acid and extending the single-stranded nucleic acid as a template is more suitable for the oligonucleotide Since it is much longer than the probe (D), the complex (P) can be present in a much larger amount 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 the single-stranded nucleic acid as a template. The synthetic reaction is formed by denaturing the double-stranded nucleic acid. Since the single-stranded nucleic acids thus hybridized are far superior to the reaction of forming the original double-stranded nucleic acids, the amount of complex (P) produced is greatly improved by the extension reaction, The complex (P) may be present in much larger amounts than the complex (p ′).

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

  The extension method of the oligonucleotide probe (D) can be basically 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. 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, it is possible to amplify a nucleic acid fragment containing a base site (X) to be determined in advance by the amplification reaction.

  Further, as at least one kind of base determination oligonucleotide probe (D) complementary to a part of the single-stranded nucleic acid obtained by denaturing the double-stranded nucleic acid, among the target nucleic acid sequences in the double-stranded nucleic acid When a ligand is bound to an oligonucleotide complementary to the portion containing the base site (X) to be determined, it is preferable to bind the ligand to a position that does not inhibit the extension reaction. 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 (X) to be determined. Preferably, the second base is designed so as to correspond to the position of the base site (X). In order not to impair the effects of the present invention, the third and third bases from the 3 'end may be designed to correspond to the position of the base site (X). 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 (X) 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 ligand may be changed.

  In the amplification reaction, an oligonucleotide probe (D) may be present. In that case, the oligonucleotide probe (D) is preferably designed so as not to inhibit the amplification reaction using the primer used in the amplification reaction. Furthermore, in order to suppress nonspecific reaction, it is preferable to design the oligonucleotide probe (D) so that the nucleic acid extended from the oligonucleotide probe (D) is not used for the amplification reaction. For example, the oligonucleotide probe (D) may be designed to have a Tm value lower than that of the primer used in the amplification reaction.

  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 used in the amplification reaction 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 type of oligonucleotide probe for detection (D) complementary to a portion containing a base site (X) to be determined among single-stranded nucleic acids obtained by denaturing double-stranded nucleic acids, It is possible to act separately or simultaneously with the denaturation of the double-stranded nucleic acid.

  When acting separately, for example, the oligonucleotide probe (D) is added after the denaturation of the double-stranded nucleic acid, and after heat denaturation at 98 ° C. for 3 minutes, the temperature is gradually increased to near room temperature. Can be lowered.

  When acting simultaneously, as an example, in the mixed state of the above double-stranded nucleic acid and the oligonucleotide probe (D), heat denaturation at 98 ° C. for 3 minutes and then gradually lower the temperature to near room temperature. Can do.

  Until the complex (P) is formed from the nucleic acid amplification method, a homogeneous system may be used. For example, 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 oligonucleotide primers, a base determination oligonucleotide probe (D), and a heat-resistant DNA polymerase To repeat the nucleic acid amplification reaction. Then, after heat denaturation at 98 ° C. for 3 minutes, the temperature is gradually lowered to near room temperature, and the base determination oligonucleotide probe (D) and the single-stranded nucleic acid obtained by denaturing the double-stranded nucleic acid are hybridized. Let it soy. Thereafter, an extension reaction of the oligonucleotide probe for detection (D) is carried out using a single-stranded nucleic acid as a template by a thermostable DNA polymerase present in the reaction system, and the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid A complex (P) is formed. Thereafter, the complex (P) may be detected.

  In the method using the nucleic acid amplification method as described above, after the reaction, a single-stranded nucleic acid obtained by denaturation of the double-stranded nucleic acid, and a detection oligonucleotide probe complementary to a part of the single-stranded nucleic acid ( A complex (P) comprising D) is formed.

  In the present invention, it is only necessary to confirm that the single-stranded nucleic acid and the extension product of the oligonucleotide probe (D) coexist in the formed complex (P). For example, by using the oligonucleotide probe (D) to which a ligand is bound, the complex (P) can be captured and detected on a solid phase to which the ligand capture agent is bound.

That is, the third step may include at least the following steps (i) and (ii).
(I) capturing the complex (P) on the solid phase using the ligand of the oligonucleotide probe (D);
(Ii) A step of detecting the complex (P) captured on the solid phase.
Further, the step (i) may include a step of removing the single-stranded nucleic acid not hybridized with the oligonucleotide probe (D).

  The ligand is not particularly limited as long as it does not interfere with the detection of the nucleic acid sequence, but is preferably selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme, avidin, biotin, and digokesigenin. Can do. Antigens, fluorescent substances, biotin, digoxigenin, and the like are preferable, and antigens, fluorescent substances, biotin, and digoxigenin are particularly preferable.

  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 method for detecting the complex (P) is not particularly limited. As an example, a substance capable of selectively binding to a conventionally known double-stranded nucleic acid represented by ethidium bromide or cyber green is used as the complex (P). Fluorescence can be detected by binding to P) and emitting ultraviolet light.

  As another example, when a ligand is bound to a single-stranded nucleic acid obtained by denaturation of a double-stranded nucleic acid, an oligonucleotide probe (D) is allowed to act to form the complex (P). Thereafter, at least one type of physiologically active substance that can bind to the ligand and is labeled may be bound, and the label of the physiologically active substance may be detected.

  The physiologically active substance to be used is not particularly limited as long as it has an affinity for the ligand bound to the single-stranded nucleic acid, but is preferably an antibody, oligonucleotide, receptor, nucleic acid-specific binding. It can be selected from the group consisting of substance, avidin, 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.

  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.

  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 polymorphism (636G → A) of Cytochrome P450 2C19 (CYP2C19) gene (1) Synthesis of oligonucleotide to detect 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 primer (A), and oligo 2 is the antisense primer (B). Oligo 3 is a wild-type detection oligonucleotide probe (D), and oligo 4 is a mutation-type detection oligonucleotide probe (D). Oligo 1 is labeled with FITC at the 5 ′ end, and oligos 3 and 4 are labeled with biotin at the 5 ′ end. By labeling the 5 ′ ends of oligos 3 and 4 with biotin, an extension reaction from oligos 3 and 4 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 The nucleotide polymorphism (636G → A) was analyzed.

Reagents A 25 μl solution containing the following reagents was prepared.
KOD plus DNA polymerase reaction solution Oligo 1 (5 ′ end labeled with FITC) 5 pmol,
5 pmol of oligo 2,
Oligo 3 or Oligo 4 (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 3 or 4 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 the samples 1 to 7 and the case without the sample, “+” indicates that the color was clearly confirmed, and “−” indicates that the color was not clearly observed.

  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.

Claims (15)

A method for determining a base site in a target nucleic acid sequence in a double-stranded nucleic acid, comprising at least the following steps (1) to (3):
(1) a first step in which a double-stranded nucleic acid containing a base site (X) to be determined is denatured to form a single-stranded nucleic acid;
(2) The single-stranded nucleic acid obtained in the first step contains at least one base determination oligonucleotide probe (D) containing the base site (X) to be determined and complementary to a part of the single-stranded nucleic acid. A second step of hybridizing and performing an extension reaction using the single-stranded nucleic acid as a template to form a complex (P) of the extension product of the oligonucleotide probe (D) and the single-stranded nucleic acid,
(3) A third step of detecting the presence of the complex (P). The base determination method according to claim 1, wherein the sequence of the oligonucleotide probe (D) varies depending on the type of base at the base site (X) to be determined. The base determination according to claim 1 or 2, wherein 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 (X) to be determined. Method. 3. The oligonucleotide according to claim 1, wherein the second base from the 3 ′ end of the oligonucleotide probe (D) is the same or a complementary base as the predicted base of the base site (X) to be determined. Base determination method. 5. The oligonucleotide probe (D) according to claim 3 or 4, wherein at least one of the third to fifth bases 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 oligonucleotide determination method according to any one of claims 1 to 5, wherein the oligonucleotide probe (D) is labeled with a ligand. The base determination method according to any one of claims 1 to 6, wherein at least one nucleotide after the 3 'end of the oligonucleotide probe (D) is labeled with a ligand. The base determination method according to any one of claims 1 to 7, wherein the 5 'end of the oligonucleotide probe (D) is labeled with a ligand. The base determination method according to any one of claims 6 to 8, wherein the 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 an oligonucleotide probe (D) is present in the first step. The base determination method according to any one of claims 1 to 10, wherein the double-stranded nucleic acid is a double-stranded nucleic acid obtained by an amplification reaction. The base determination method according to claim 11, wherein the amplification reaction is PCR. The base determination method according to claim 11 or 12, wherein an oligonucleotide probe (D) is present in the amplification reaction. The base determination method according to claim 11, wherein the Tm value of the oligonucleotide probe (D) is lower than the Tm value of the primer used in the amplification reaction. The base determination method according to claim 1, wherein the denaturation in the first step is heat denaturation.