JP2003144163A - Method for identifying base polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting variation or polymorphism of a nucleic acid sequence which is especially useful in diagnosis of genetic diseases and analysis of a base polymorphism and to provide an oligonucleotide used therefor. SOLUTION: Wild type oligonucleotides in which at least one is labeled and one or two kinds of variant oligonucleotides are simultaneously or separately reacted with a nucleic acid sequence containing a specific base polymorphism site contained in a sample and the base polymorphism is identified by detecting whether the oligonucleotides react or not. In the method, a specifically labeled nucleic acid acts on the reaction system during reaction or the reaction product and the base polymorphism is identified by mutual action between the label and the nucleic acid-specific label.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for identifying a mutation or polymorphism in a nucleic acid sequence. The present invention is particularly useful for diagnosis of genetic diseases, nucleotide polymorphism analysis and the like.

[0002]

2. Description of the Related Art In the present invention, nucleotide polymorphism means having a nucleotide sequence different from that of 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 failure in drug metabolism, and is also known as a cause of individual differences such as basal metabolism known as constitution. Moreover, they serve as genetic markers for many diseases. Therefore, elucidation of these mutations is clinically important, and routine phenotyping is especially recommended for psychiatric patients and volunteers in clinical research (Gram and Brsen, European Consensus Co.
nference on Pharmacogenetics. Commission of the Eu
ropean Communities, Luxembourg, 1990, 87-96
P .; Balant et al., Eur. J. Clin. Pharmacol. Vol. 36,
551-554, (1989)). Also desired is a nucleic acid sequence analysis method for detection of each genotype following identification of the causative mutant gene.

A conventional nucleic acid sequence analysis technique is, for example, a nucleic acid sequence determination method (sequencing method). The nucleic acid sequencing method can detect and identify nucleotide polymorphisms contained in a nucleic acid sequence, but it requires a great deal of labor because it involves preparation of template nucleic acid, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequence, etc. And time is needed. Further, although labor saving can be achieved by using an automatic sequencer in recent years, there is a problem that an expensive device is required.

[0004] On the other hand, various genetic diseases caused by point mutations of genes are known, and among them, it has been known that which part of the gene and how the point mutation causes the genetic disease. There are not a few

As a method for detecting such an expected point mutation, PCR (polymerase chain r
eaction) method (Japanese Patent Publication No. 4-67960, Japanese Patent Publication No. 4-
There is known a method for detecting a point mutation of a gene using a gene amplification method (eg, Japanese Patent Publication No. 67957). In this method, as one of the pair of oligonucleotides used in the gene amplification method, a wild-type oligonucleotide completely complementary to the end region of the amplification region of the wild-type gene and amplification of the mutant gene are used. A mutant oligonucleotide completely complementary to the end region of the region is used. The mutant oligonucleotide has a nucleotide complementary to the expected point mutation at the 3'end thereof. A sample gene is subjected to a gene amplification method by separately using such wild-type and mutant-type oligonucleotides.

When the sample gene is wild type, amplification of nucleic acid occurs when the wild type oligonucleotide is used, but when the mutant type oligonucleotide is used, the 3'end of the oligonucleotide is the sample. Since it is not complementary (mismatch) to the corresponding nucleotide of the gene, extension reaction does not occur and nucleic acid amplification does not occur. On the other hand, when 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 or not amplification occurs when each oligonucleotide is used, it is possible to distinguish whether the sample gene is a wild type or a mutant type, thereby identifying a point mutation in the sample gene. You can 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 or absence of amplified nucleic acid. it can. In addition, other methods include Southern blotting in which amplified nucleic acid is immobilized on a nylon membrane and detected using a labeled probe, sandwich hybridization in which detection is performed by capturing with a complementary probe immobilized on a solid carrier, and then detecting. Laws have been developed.

Further, the recently developed detection method does not directly detect the fluorescence intensity in detecting the hybridization of the probe, but rather the fluorescence resonance energy transfer (Fluorescence Resonance Energy Transfer).
nance Energy Transfer; FRET) method is used. Fluorescence resonance energy transfer occurs between the donor fluorophore and the quencher dye (which may or may not be a fluorophore), and the absorption spectrum of one (quencher) and the other (donor)
It occurs when the two dyes are close to each other and overlaps the emission spectrum of. Dyes with these properties are called donor / quencher dye pairs or energy transfer dye pairs. The excited state energy of the donor fluorophore is due to the dipole interaction caused by the resonant dipole,
Moved to a nearby quencher. As a result, quenching of the donor phosphor occurs. In some cases, when the quenching agent is also a fluorescent substance, its fluorescence intensity may be enhanced. Energy transfer efficiency is highly dependent on the distance between the donor and the quencher, and the formula for predicting these relationships is Forster (1948. Ann. P.
hys.2, 55-75). The distance between the donor and the quencher dye, which has an energy transfer efficiency of 50%, is Forster.
It is called the distance (Ro). Other mechanisms of fluorescence quenching include, for example, charge transfer quenching and collision quenching.

Energy transfer and other mechanisms, which are based on the interaction of two dyes in close proximity to cause quenching, are an attractive means of detecting or identifying nucleotide sequences because they can be performed in a homogeneous manner. The homogeneous assay format is simpler than the conventional probe hybridization assay method, which is based on the detection of the fluorescence of one fluorophore label. This is because heterogeneous analysis generally requires the additional step of separating the hybridized label from the unhybridized free label.

[0009] Typically, FRET and related methods are based on monitoring changes in the fluorescent properties of one or both dye labels when two complementary oligonucleotides are joined by hybridization. In this manner, changes in fluorescence properties are measured as changes in energy transfer or changes in fluorescence quenching, and are typically shown as an increase in fluorescence intensity of one dye.
In this method, it is possible to detect the nucleotide sequence of interest without separating the oligonucleotide that has not hybridized and the oligonucleotide that has hybridized. Hybridization can occur between two separate complementary oligonucleotides, one labeled with the donor fluorophore and the other labeled with the quencher. At this time, when one of the oligonucleotides has a nucleotide polymorphism-specific sequence, the nucleotide polymorphism can be identified by obtaining a FRET signal in a polymorphism-specific manner.

Several approaches to FRET hybridization analysis have been described by Nonisotopic DNA Probe Techniques (199).
2. Academic Press, Inc., pags. 311-352). Alternatively, the donor and quencher can be combined into a single oligonucleotide so that there is a detectable difference in the fluorescence properties of one or both of the oligonucleotides when unhybridized and when hybridized to complementary sequences. Can be combined.

In this approach, donor fluorescence typically increases and energy transfer / quenching decreases when the oligonucleotides hybridize. For example, a self-complementary oligonucleotide labeled at both ends forms a hairpin structure, which results in two fluorophores (i.e., 5 ').
Energy transfer and quenching occur due to the proximity of the end and the 3'end. When the self-complementary oligonucleotide and the complementary sequence in the second oligonucleotide hybridize,
The hairpin structure is destroyed and the distance between the two dyes is increased, resulting in reduced quenching. The drawbacks of hairpin structures are that they are very stable, their conversion to unquenched hybrids is often slow, with only a slight bias, and generally poor performance.

Tyagi and Kramer (1996. Nature Biotech.
14, 303-308) report a hairpin structure labeled as described above which contains a detection sequence in the loop between the self-complementary arms of the hairpin structure forming the stem. The base-paired stem must melt in order for the detection sequence to hybridize to the target and cause reduced quenching. A "double hairpin" probe and method of using it is described in B. Bagwell, et al. (1994. Nucl. Acids Res. 22,
2424-2425; U.S. Pat. No. 5,607,834).
These structures contain the target binding sequence within the hairpin structure, thus involving competitive hybridization between the target and the self-complementary sequence of the hairpin structure. Bagwel
l solves the problem of unfavorable hybridization kinetics by destabilizing the hairpin structure by a mismatch.

By selecting a nucleotide polymorphism-specific sequence for the target binding sequence, it is possible to identify the nucleotide polymorphism using this method. Generally, the difference of 1 nucleotide is discriminated by the oligonucleotide hybridization method. Therefore, it is necessary to set strict hybridization conditions.

Homogeneous methods that utilize energy transfer or other fluorescence quenching mechanisms to detect nucleic acid amplification have also been reported. LG Lee, et al. (1993. Nuc. Acids
Res. 21, 3761-3766) discloses a real-time detection method in which a double-labeled detection probe is cleaved specifically during PCR during target amplification. When the detector probe hybridizes downstream of the amplification primer, the 5'-3 'of Taq polymerase
Exonuclease activity digests the detection probe and separates the two fluorochromes that form an energy transfer pair. Also in this method, the nucleotide polymorphism can be identified by using the double-labeled detection probe as a nucleotide polymorphism-specific probe.

[0015]

It seems that the amplified nucleic acid can be detected by the method as described above and the polymorphism can be easily identified. However, in practice, the conventional method is complicated in operation and the detection is difficult. It was difficult to quantify the value, which made it difficult to analyze the ratio of the wild type signal to the polymorphic signal, and required a lot of work to identify the correct polymorphism. .

For example, according to the electrophoresis method, it is necessary to detect the wild type and the polymorphism separately, and it is difficult to accurately quantify the amount of nucleic acid from the electrophoretic image. Further, in the Southern blotting method and the sandwich hybridization method, a hybridization reaction with a probe is necessary, and the conditions must be strictly adjusted. Furthermore, a step of removing excess probe is required, and the operation is very complicated.

Further, the method using FRET allows uniform analysis and does not require removal of an excessive probe or the like, which facilitates detection, but two polymorphism-specific two labels are provided. Oligonucleotide probes are required and doubly labeled probes are required.

An object of the present invention is to solve the above problems and provide a method and a reagent therefor capable of clearly and reproducibly detecting a polymorphism in a nucleic acid sequence.

[0019]

[Means for Solving the Problems] In view of the above circumstances, the inventors of the present invention have conducted intensive studies and found that at least one nucleic acid sequence containing a specific nucleotide polymorphism site is labeled with respect to the above conventional method. After reacting the wild-type oligonucleotide and one or two mutant-type oligonucleotides simultaneously or separately, a nucleic acid-specific label is allowed to act, and the interaction between the label and the nucleic acid-specific label is measured. By doing so, it was possible to obtain data that was easily quantified without requiring a large number of complicated detection operations and expensive labeled probes, and therefore a method that enables clear polymorphism identification, and completed the present invention. .

That is, the present invention has the following configuration. [1] A nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample is reacted with at least one labeled wild type oligonucleotide and one or two mutant type oligonucleotides simultaneously or separately. Then, a nucleic acid-specific label is allowed to act, and the nucleotide polymorphism is identified by the interaction between the label and the nucleic acid-specific label. [2] The method according to [1], wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. [3] The method according to [1] or [2], wherein the nucleic acid-specific label is a fluorescent dye. [4] The method according to [1] to [3], wherein the oligonucleotide label is a fluorescent dye. [5] The method according to [1] to [4], wherein the interaction is fluorescence resonance energy transfer. [6] Performing a polymorphism-specific amplification reaction using at least one labeled wild-type oligonucleotide and one or two mutant-type oligonucleotides, and measuring during and / or after the amplification reaction. Features [1]
~ The method described in [5]. [7] A wild type oligonucleotide in which at least one is labeled in a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample and one or two kinds of mutant oligonucleotides are used as primers at the same time or separately. To perform a polymorphism-specific extension reaction, and then a nucleic acid-specific label is allowed to act on the extension product, and the nucleotide polymorphism is identified by the interaction between the oligonucleotide label and the nucleic acid-specific label. Method. [8] The method according to [7], wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. [9] The method according to [7] or 8, wherein the nucleic acid-specific label is a fluorescent dye. [10] The method according to [7] to [9], wherein the oligonucleotide label is a fluorescent dye. [11] The method according to [7] to [10], wherein the interaction is fluorescence resonance energy transfer. [12] The method according to [7] to [11], wherein the specific nucleic acid sequence contained in the sample is previously amplified. [13] Performing a polymorphism-specific amplification reaction using at least one labeled wild-type oligonucleotide and one or two mutant-type oligonucleotides, and measuring during and / or after the amplification reaction. Features [7]
~ The method described in [12]. [14] In the method of identifying a nucleotide polymorphism present in a specific nucleic acid sequence contained in a sample, the following steps are performed: 1) A wild type oligonucleotide in which at least one is fluorescently labeled, and one or two mutations An extension reaction is performed by using the template oligonucleotide as a primer. 2) After the extension product is denatured into a single strand, an oligonucleotide complementary to the extension product is allowed to act to perform an extension reaction to produce a double-stranded nucleic acid. 3) A double-stranded nucleic acid-specific fluorescent dye is allowed to act, and the interaction between the fluorescent dye and the label of the oligonucleotide and the fluorescence signal of the nucleic acid-specific label are measured. A method comprising: [15] The method according to [14], wherein the interaction is fluorescence resonance energy transfer. [16] The method according to [14] to [15], wherein the specific nucleic acid sequence contained in the sample is previously amplified. [17] The method according to [14] to [16], wherein steps 1 and 2 are repeated to carry out an amplification reaction, and measurement is performed during and / or after the amplification reaction. [18] A nucleotide polymorphism present in a specific nucleic acid sequence contained in a sample containing at least one fluorescent-labeled wild-type oligonucleotide and one or two mutant-type oligonucleotides, a polymerase, and a nucleic acid-specific label A kit to identify.

[0021]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.
A chromosome or a fragment thereof containing a specific nucleotide polymorphism site contained in a sample is not particularly limited as long as it is a target nucleic acid containing a nucleotide polymorphism site carrying information of a target gene. Examples of the target nucleic acid include Alu sequences, exons and introns of genes encoding proteins, promoters 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, as hypertension, ACE (Angiotens
in IConverting Enzyme) gene.

In the present invention, the nucleic acid sequence may be simply referred to as nucleic acid. The mutant nucleic acid refers to a wild-type nucleic acid in which at least one nucleotide, preferably one nucleotide is point-mutated to be replaced with another nucleotide, or an insertion or deletion sequence in a part of the wild-type nucleic acid. It is a nucleic acid containing, and it has been elucidated at which site the nucleotide is mutated. It has been elucidated that constitutions and the like differ depending on such nucleotide polymorphisms, and the method of the present invention is a method for examining whether or not a nucleic acid in a sample has such an expected mutation. is there.

In the present invention, the reaction of reacting a wild-type oligonucleotide with a mutant-type oligonucleotide generally means that a target nucleic acid denatured into a single strand has an oligonucleotide and four kinds of deoxynucleoside triphosphates (dNT).
When P) and DNA polymerase are allowed to act on each other, an oligonucleotide extension reaction using the target nucleic acid as a template occurs and a complementary strand of the nucleic acid sequence is synthesized.

In the present invention, a wild-type oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome containing a nucleotide polymorphic site having a normal phenotype or a fragment thereof, and for a mutant type. The oligonucleotide is an oligonucleotide having a sequence complementary to a chromosome containing a nucleotide polymorphism site having a sequence different from the wild type or a fragment thereof.

The above-mentioned oligonucleotide used in the present invention was designed so that the 3'end or the second nucleotide from the 3'end of the wild type and mutant oligonucleotides correspond to the nucleotide of the mutant sequence. When designed in this way, in the combination of wild-type oligonucleotide / wild-type nucleic acid and mutant-type oligonucleotide / mutant-type nucleic acid, each oligonucleotide matches at least at the 3 ′ end or the second sequence from the 3 ′ end. Therefore, extension reaction occurs. On the other hand, wild type oligonucleotide / mutant nucleic acid and mutant oligonucleotide /
In the combination of wild-type nucleic acids, extension reaction does not occur because of an artificial mismatch between the 3'end of the oligonucleotide or the second base from the 3'end, and further one base.

The oligonucleotide of the present invention has a length of 13 to 35 bases, preferably 16 to 30 bases, and at least one mismatch site is present in the oligonucleotide. Also, its position is
It is not particularly limited as long as it is from the 3rd end of the 3'end to the 5'end, but it is preferably at a position close to the 3rd end of the 3'end, more preferably at the 3rd end of the 3'end.

Third, using an artificial mismatch,
When a nucleic acid sequence that does not expect an extension reaction is used as a template, a mismatch of 2 bases is generated together with the nucleotide polymorphism site, and the extension reaction is strongly inhibited.

In the present invention, the above-mentioned wild type oligonucleotide and one or two mutant type oligonucleotides are allowed to act on the sample separately or simultaneously.

In the present invention, a method for extending an oligonucleotide can be basically performed by using a conventional method. Usually, a chromosome containing a specific nucleotide polymorphism site denatured into a single strand or a fragment thereof is used together with four types of deoxynucleoside triphosphates (dNTP) and a DNA polymerase together with a wild type oligonucleotide, one type or two types. The oligonucleotides are extended using the target nucleic acid as a template by allowing the mutant oligonucleotides of different species to act simultaneously or separately.

The elongation reaction is performed by Molecular Cloning, A Lab.
Oratory Manual (Sambrook et al., 1989). Further, in the method of detecting a nucleotide polymorphism depending on whether or not the oligonucleotide has been extended, when the target nucleic acid does not contain a sufficient amount for detection, a nucleic acid fragment containing the polymorphic sequence is previously prepared as follows. It is also possible to perform amplification by the amplification reaction shown.

In the present invention, a method for amplifying a chromosome or a fragment containing a specific nucleotide polymorphism site can also be basically performed by a conventional method, and usually a specific single-stranded modified Four types of deoxynucleoside triphosphates (dNTP) and D are added to a chromosome containing a nucleotide polymorphism site or a fragment thereof.
A target nucleic acid is used as a template by allowing a wild-type oligonucleotide and one or two mutant-type oligonucleotides (corresponding to a forward primer) to act simultaneously or separately with NA polymerase and a reverse primer. Amplified between the forward and reverse oligonucleotides.

As a nucleic acid amplification method, PCR, NASB
A (Nucleic acid sequence-basedamplification metho
d; Nature, Volume 350, page 91 (1991)), L
CR (International Publication 89/12696, Japanese Patent Laid-Open No. 2-2
934), SDA (Strand Displacement Amplif)
ication: Nucleic acid research Volume 20, 169
1 page (1992)), RCR (International Publication 90/1069), TMA (Transcription mediated amplification)
method; J. Clin. Microbiol. Vol. 31, p. 3270
(1993)) and the like.

Among them, the PCR method involves denaturation in the presence of a sample nucleic acid, four kinds of deoxynucleoside triphosphates, a pair of oligonucleotides and a thermostable DNA polymerase,
This is a method of exponentially amplifying the region of the sample nucleic acid sandwiched by the pair of oligonucleotides by repeating a cycle consisting of three steps of annealing and extension. That is, the nucleic acid of the sample is denatured in the denaturation step, each oligonucleotide is hybridized with the region on the single-stranded sample nucleic acid complementary thereto in the subsequent annealing step, and each oligonucleotide is started in the subsequent extension step. As a result, the DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of the DNA polymerase to form a double-stranded DNA. By this one cycle, one double-stranded D
NA 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 oligonucleotides is theoretically amplified 2 ⁿ times. Since there is a large amount of amplified DNA region,
It can be easily detected by a method such as electrophoresis. Therefore, using the gene amplification method, it could not be detected in the past.
It is possible to detect a very small amount (even one molecule) of sample nucleic acid, and this is a technique that has been very widely used recently.

In the method utilizing the nucleic acid amplification method as described above, gene amplification is carried out by separately or simultaneously using a wild type oligonucleotide capable of amplifying a wild type nucleic acid and a mutant type oligonucleotide capable of amplifying a mutant type nucleic acid. Do the law.

When the sample nucleic acid is extended or amplified using the wild type oligonucleotide, the reaction occurs if the sample nucleic acid is the wild type, but the reaction does not occur in the mutant type. On the contrary, when the sample nucleic acid is extended or amplified using the mutant oligonucleotide, the reaction occurs if the sample nucleic acid is mutant, but the reaction does not occur if the sample nucleic acid is wild type. Therefore, by dividing one sample into two, one using the wild type oligonucleotide, the other using the mutant type oligonucleotide, by checking whether the reaction has occurred , It is possible to clearly know whether the sample nucleic acid is a wild type or a mutant type. In particular, humans and other higher organisms have one gene for each of one father-derived gene and one mother-derived gene.
According to this method, it is possible to distinguish whether the sample gene is a wild type homo, a mutant homo, or both hetero. That is, in the case of hetero, both the wild-type gene and the mutant-type gene are present, so that the reaction occurs regardless of whether the wild-type oligonucleotide or the mutant-type oligonucleotide is used.

The present invention can also be used in a hybridization method using a labeled oligonucleotide.
That is, for the target nucleic acid, polymorphism-specific and wild-type specific oligonucleotides are prepared, and each oligonucleotide is allowed to act simultaneously and / or separately, and the interaction between each oligonucleotide label and the nucleic acid-specific label The nucleotide polymorphism can be identified by detecting Further, the nucleotide sequence polymorphism can be identified by using the above-mentioned polymorphism-specific probe to act while performing a nucleic acid amplification reaction such as PCR by using an outer sequence as a primer for hybridization, or after performing the nucleic acid amplification reaction such as PCR. Is.

In the present invention, the oligonucleotide label may be any label as long as it interacts with the nucleic acid specific label, such as FITC, 6-FAM, HEX, TET, TA.
MRA, Texas Red, Cy3, Cy5, Rhodamine, etc., but not limited to these.

In the present invention, the nucleic acid-specific labeling substance specifically binds to the nucleic acid reacted with the labeled oligonucleotide,
It may be anything as long as it interacts with the label of the oligonucleotide, preferably a double-stranded nucleic acid-specific label.
The double-stranded specific label includes, but is not limited to, Cybergreen I intercalating into a double-stranded chain, ethidium bromide, acridine orange, thiazole orange, oxazole yellow, rhodamine and the like.

Examples of the enzyme include alkaline phosphatase and peroxidase. As the fluorescent substance, FITC, 6-FAM, HEX, TET, TA
Examples include MRA, Texas Red, Cy3, Cy5 and the like. Examples of the hapten include biotin and digoxigenin. Examples of radioactive materials include ³² P and ³⁵ S. Examples of the luminophore include ruthenium.

The label may be attached to any position of the oligonucleotide as long as it does not affect the hybridization and extension reaction of the oligonucleotide. The 5'site is preferred. When different labels are used for the wild type oligonucleotide and the mutant type oligonucleotide, detection can be performed once.

Kit In the present invention, the kit includes a nucleotide polymorphism detection containing a wild-type oligonucleotide and one or two mutant-type oligonucleotides, a DNA polymerase, and four types of deoxynucleoside triphosphates (dNTP). It includes a reagent kit for use. When detecting by amplification,
Further, it may contain a reverse oligonucleotide. Further, each of the oligonucleotides may be labeled in advance with an enzyme, biotin, a fluorescent substance, a hapten, an antigen, an antibody, a radioactive substance, a luminophore or the like as described above.

[0042]

EXAMPLES The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

Example 1 Detection of nucleotide polymorphism (Trp64Arg) of β3 adrenergic receptor gene (1) 190th polymorphism of β3 adrenergic receptor gene
Synthesis of oligonucleotides for detecting Perkin Elmer DNA Synthesizer 392 type, in the phosphoamidite method, oligonucleotide having a nucleotide sequence shown in SEQ ID NO: 1-3 (hereinafter, oligo 1
Designated as ~ 3) was synthesized. The synthesis follows the manual, and various oligonucleotides are deprotected with ammonia water at 55 ° C.
It was carried out overnight. Purification of the oligonucleotide was carried out on a Perkin Elmer OPC column. Or a DNA synthesis outsourcing company (Nippon Bioservices, Sawasdee,
We requested GENSET KK, Amersham Pharmacia Biotech Co., Ltd., etc. Oligo 1 has a wild type (T) nucleotide sequence 2nd from the 3'end and an artificial mismatch (C → A) at the 3rd end, and oligo 2 has a mutant type (C) 2nd from the 3'end. And an artificial mismatch (C → A) at the third position, oligo 3 is an antisense strand, and is used as an oligonucleotide in an amplification reaction in combination with oligos 1 and 2. In addition, oligos 1, 2, and 3 are used by labeling if necessary.

(2) β3 adrenergic recept by PCR method
or analysis of gene polymorphism 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 β3 adrenergic receptor under the following conditions
The nucleotide polymorphism (Trp64Arg) of the gene was analyzed.

[0045] was prepared 25μl solution containing the following reagents reagent. Taq DNA polymerase reaction solution Oligo 1 and / or 2 (oligo 1 labeled 5'with Texas Red, oligo 2 unlabeled) 5 pmol oligo 3 (unlabeled) 5 pmol x 10 buffer 2.5 μl 2 mM dNTP 2.5 μl 25 mM MgCl ₂ 1.5 μl Taq DNA polymerase 1.3 U Extracted DNA solution 100 ng

Amplification conditions 95 ° C. for 5 minutes 95 ° C. for 30 seconds, 62.5 ° C. for 30 seconds, 72 ° C. for 30 seconds (35 cycles) 72 ° C. for 2 minutes. 5 μl of amplification reaction solution for detection by FRET was added to Syber Green I (Molecular probes).
100 μl of the solution of the company) and reacted at room temperature for 10 minutes. This amplified human β3 adrenergic rec
Syber Green I is incorporated into the eptor gene fragment.
Next, in a dark room, fluorescent plate reader (Dainippon Pharmaceutical Co., Ltd.)
The amount of fluorescence intensity was measured with. It took a few minutes.

From the obtained fluorescence intensity, the fluorescence intensity amount of the sample was calculated using the following formula. [Formula 1] FL1 (fluorescence intensity of sample) = FLb1-FLs1 FLb1: Negative Control (no sample added) excitation wavelength: 355 / fluorescence intensity at fluorescence wavelength 612 FLs1: excitation wavelength of each sample: 355 / fluorescence wavelength 612 [Equation 2] FL2 (fluorescence intensity of sample) = FLb2-FLs2 FLb2: Negative Control (no sample added) excitation wavelength: 355 / fluorescence intensity at fluorescence wavelength 538 FLs2: excitation wavelength of each sample: 355 / Fluorescence intensity at fluorescence wavelength 538

[0048]

Industrial Applicability As described above, the present invention provides a method capable of clearly and simply detecting a nucleotide polymorphism in a sample nucleic acid. Since the method of the present invention does not require a complicated operation as in the conventional methods, quick and easy reproducible results were obtained.

[0049]

[Sequence list]                                SEQUENCE LISTING <110> TOYO BOSEKI KABUSHIKI KAISHA <120> A METHOD FOR DETECTING SNPs <130> 01-0938 <141> 2001-11-07 <160> 2 <170> PatentIn Ver. 2.1 <210> 1 <211> 20 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide described in example 1 <400> 1 gtcatcgtgg ccatcgcatg 20

[0050] <210> 2 <211> 20 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide described in example 1 <400> 2 gtcatcgtgg ccatcgcacg 20

[0051] <210> 3 <211> 22 <212> DNA <213> Artificial sequence <220> <223> the sequence of designed polynucleotide described in example 1 <400> 3 acgaacacgt tggtcatggt ct 22

[Brief description of drawings]

[Fig. 1] Fluorescence intensity of each sample in detection of nucleotide polymorphism of β3 adrenergic receptor gene

   ─────────────────────────────────────────────────── ─── Continued front page    F term (reference) 2G045 DA12 DA13 DA14 FB02 FB07                       FB12 GC15                 4B024 AA11 BA80 CA01 CA11 HA12                 4B063 QA01 QA17 QA19 QQ42 QQ52                       QR08 QR42 QR55 QR62 QS25                       QS34 QX10

Claims (18)

[Claims] 1. A wild-type oligonucleotide and at least one kind of mutant-type oligonucleotide in which at least one is labeled in a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample, simultaneously or separately. A method of identifying a nucleotide polymorphism by allowing a nucleic acid-specific label to act on the nucleic acid and reacting the label with the nucleic acid-specific label. 2. The method according to claim 1, wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. 3. The method according to claim 1, wherein the nucleic acid-specific label is a fluorescent dye. 4. The method according to claim 1, wherein the label of the oligonucleotide is a fluorescent dye. 5. The method according to claim 1, wherein the interaction is fluorescence resonance energy transfer. 6. A polymorphism-specific amplification reaction is performed using at least one labeled wild-type oligonucleotide and one or two mutant-type oligonucleotides, and is measured during and / or after the amplification reaction. The method according to claim 1, wherein 7. A wild-type oligonucleotide in which at least one is labeled in a nucleic acid sequence containing a specific nucleotide polymorphism site contained in a sample and one or two mutant-type oligonucleotides are simultaneously used as primers. Alternatively, the polymorphism-specific extension reaction is performed separately, and then the extension product is allowed to act on the nucleic acid-specific label, and the nucleotide polymorphism is detected by the interaction between the oligonucleotide label and the nucleic acid-specific label. How to identify. 8. The method according to claim 7, wherein the nucleic acid-specific label is double-stranded nucleic acid-specific. 9. The method according to claim 7, wherein the nucleic acid-specific label is a fluorescent dye. 10. The method according to claim 7, wherein the label of the oligonucleotide is a fluorescent dye. 11. The method according to claim 7, wherein the interaction is fluorescence resonance energy transfer. 12. The method according to claim 7, wherein the specific nucleic acid sequence contained in the sample is previously amplified. 13. A polymorphism-specific amplification reaction is carried out using at least one labeled wild-type oligonucleotide and one or two mutant-type oligonucleotides, and is measured during and / or after the amplification reaction. 13. The method according to claims 7 to 12, characterized in that 14. A method for identifying a nucleotide polymorphism present in a specific nucleic acid sequence contained in a sample, comprising the steps of: 1) at least one fluorescently labeled wild type oligonucleotide and one or two species. The oligonucleotide for mutation type of is used as a primer to perform extension reaction. 2) After the extension product is denatured into a single strand, an oligonucleotide complementary to the extension product is allowed to act to perform an extension reaction to produce a double-stranded nucleic acid. 3) A double-stranded nucleic acid-specific fluorescent dye is allowed to act, and the interaction between the fluorescent dye and the label of the oligonucleotide and the fluorescence signal of the nucleic acid-specific label are measured. A method comprising: 15. The method of claim 14, wherein the interaction is fluorescence resonance energy transfer. 16. The method according to claim 14, wherein the specific nucleic acid sequence contained in the sample is previously amplified. 17. The method according to claim 14, wherein steps 1 and 2 are repeated to carry out an amplification reaction, and the measurement is carried out during and / or after the amplification reaction. 18. A base present in a specific nucleic acid sequence contained in a sample containing a wild-type oligonucleotide at least one of which is fluorescently labeled and one or two mutant-type oligonucleotides, a polymerase and a nucleic acid-specific label. A kit for identifying polymorphisms.