JP2002034598A - Method for detecting base polymorphism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To readily detect the kind of a base polymorphism contained in a specimen. SOLUTION: In this method for detecting a base polymorphism by treating a base polymorphism sequence part contained in a specific nucleic acid sequence existing in a specimen with two or more probes specific to the base polymorphism sequence part and obtaining the ratio of detection signals obtained from each probe, the method for detecting the base polymorphism is characterized in that a part to be hybridized with at least polymorphism sequences of the probes specific to the base polymorphism part is an RNA.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for analyzing a nucleic acid sequence. More specifically, the present invention relates to a nucleic acid sequence analysis method capable of detecting a nucleotide polymorphism contained in a specific nucleic acid sequence. The nucleic acid sequence analysis method of the present invention is useful in genetic engineering, molecular biology and related industrial fields.

[0002]

BACKGROUND OF THE INVENTION In the present invention, a nucleotide polymorphism is a genotype having a sequence different from that of a wild type, and the polymorphism gene is a cause of inter-individual variation in drug metabolism and in the occurrence of side effects and treatment failures. Plays an important role. It is also known as a cause of individual differences such as basal metabolism known as a constitution. Moreover, they also serve as genetic markers for a number of diseases. Therefore, the elucidation of these mutations is clinically important, and routine phenotyping is particularly recommended for psychiatric patients and volunteers in clinical studies (Gram and Brsen, Europe
n Consensus Conference on Pharmacogenetics.Commis
sion of the European Communities, Luxembourg, 199
0, pp87-96; Balant et al., Eur. J. Clin. Pharmacol. 36,
551-554, 1989). Therefore, nucleic acid sequence analysis for the detection of each genotype following the identification of the causative polymorphic gene is desired.

[0003] As a conventional nucleic acid sequence analysis technique, there is, for example, 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 labor to perform preparation of template nucleic acid, polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequence, etc. Time is needed. In addition, although labor saving can be achieved by using a recent automatic sequencer, there is a problem that an expensive apparatus is required.

[0004] As another method, there is the Southern hybridization method (Laboratory Manual Engineering Enhancement Edition, edited by Masami Muramatsu, Maruzen Co., Ltd., pp. 70-75). According to this method, a region of DNA having a base sequence complementary to the labeled DNA probe can be identified.
That is, in the Southern hybridization method, a nucleic acid fragment is electrophoresed on a plate of an agarose gel or polyacrylamide gel, separated according to the size (length) of the fragment, denatured into a single strand, and the plate is treated with nitro. After attaching a membrane such as cellulose or nylon, transferring the nucleic acid fragment as it is in the electrophoresis pattern, fixing it, and forming a hybrid with a nucleotide polymorphism-specific DNA probe labeled with RI (radioactive isotope), complements the probe. Nucleic acid fragments on a typical membrane are detected by autoradiography or the like.

[0005] According to the Southern hybridization method, the electrophoresis position and molecular weight of the target DNA fragment can be determined. However, the operation such as electrophoresis and autoradiography requires a long time, and the analysis is performed quickly. If the probe is not strictly specific, a similar sequence for detecting the presence or absence of a nucleotide polymorphism is identified because the detection cannot be performed only by hybridization or not with the nucleotide polymorphism-specific DNA probe. There was a problem that it was difficult.

[0006]

SUMMARY OF THE INVENTION An object of the present invention is to provide a nucleic acid sequence analysis method which can easily detect the type of nucleotide polymorphism in a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample. is there.

[0007]

Means for Solving the Problems The present inventors focused on the interaction in the formation of complementary nucleic acid hybrids, and used a nucleic acid having a base sequence complementary to a target nucleic acid fragment as a probe, It has been found that the sequence of the target nucleic acid fragment can be analyzed by utilizing the specific interaction shown in (1).

Here, it is generally known that even if the sequence and temperature are strictly defined, only perfectly complementary probes react, and it is not possible to prevent a probe containing a mismatch from reacting. Therefore, when the genotype is determined using a mismatch probe, it is difficult to clearly distinguish the wild type, the mutant type, and the mixed type depending on the presence or absence of a detection signal even if the temperature and sequence are defined.

Therefore, the present inventors, in the course of developing and developing nucleic acid sequence analysis methods, use a probe specific to the polymorphic sequence portion where the portion that hybridizes to the polymorphic sequence is RNA, and Alternatively, it has been found that by performing RNA selective cleavage endonuclease treatment at the same time, it is possible to reduce non-specific binding that occurs in a combination where the polymorphic sequence portions are not complementary. Thus, the present invention has been completed.

That is, the present invention has the following configuration.

(1) Two or more probes specific to a nucleotide polymorphism sequence portion contained in a specific nucleic acid sequence present in a sample are allowed to act, and the ratio of detection signals obtained by each probe is determined. A method for detecting a nucleotide polymorphism, wherein at least a portion that hybridizes to a polymorphic sequence of a probe specific to the polymorphic sequence portion is RNA.

(2) A method for detecting a nucleotide polymorphism by allowing two or more probes specific to a polymorphic sequence portion contained in the nucleic acid sequence to detect RNA polymorphism, wherein RNA selective cleavage endonuclease treatment is performed. The method for detecting a nucleotide polymorphism according to claim 1, wherein

(3) The method according to (2), wherein the RNA-selective cleavage endonuclease is ribonuclease A or a ribonuclease A-like enzyme.

(4) In the method for detecting a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, the method of (1), wherein the nucleic acid sequence is amplified in advance.

(5) PCR, NASBA, LCR, S
The method according to (4), wherein the nucleic acid sequence is amplified by any method selected from the group consisting of DA, RCR and TMA.

(6) The method according to any one of (1) to (5), wherein the nucleotide polymorphism includes one of a single nucleotide polymorphism, an insertion and a deletion polymorphism.

(7) A method for identifying a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, comprising:
(1) amplifying a nucleic acid fragment containing the nucleotide polymorphism of the specific nucleic acid sequence, (2) hybridizing the amplified nucleic acid with two or more polymorphism-specific probes, (3) RNA selective cleavage endonuclease (4) measuring the detection signal of each probe, and (5) identifying the polymorphic sequence based on the ratio of each detection signal.

(8) PCR, NASBA, LCR, S
The method of (7), wherein the nucleic acid sequence is amplified by any method selected from the group consisting of DA, RCR and TMA.

(9) The method according to (7) or (8), wherein the nucleotide polymorphism includes one of a single nucleotide polymorphism, an insertion and a deletion polymorphism.

(10) A method for identifying a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, comprising:
(1) Amplify using a labeled primer a nucleic acid sequence containing a nucleotide polymorphism of the specific nucleic acid sequence, (2) hybridized with a different polymorphism specific probe from the amplified nucleic acid, (3) RNA
Treating with a selective cleavage endonuclease, (4) detecting whether or not hybridized with each probe by labeling the primer, and (5) identifying a polymorphic sequence by a ratio of each detected signal. And how.

(11) PCR, NASBA, LCR,
The method according to (10), wherein the nucleic acid sequence is amplified by any method selected from the group consisting of SDA, RCR and TMA.

(12) The nucleotide polymorphism is a single nucleotide polymorphism, insertion,
The method according to (11) or (12), which comprises any of the deletion polymorphisms.

(13) The primer is labeled with biotin,
The method according to any one of (10) to (12), which is any one selected from the group consisting of dicoxigenin, a fluorescent substance, a luminophore, and an enzyme.

(14) A method for identifying a single nucleotide polymorphism of a specific nucleic acid sequence containing a single nucleotide polymorphism contained in a sample, comprising: (1) a nucleic acid fragment containing the single nucleotide polymorphism of the specific nucleic acid sequence (2) amplified nucleic acid and 2
Hybridized with a wild-type probe and a mutant probe immobilized on each of the at least one solid phase, (3) treated with RNA-selective cleavage endonuclease, and (4) determined whether or not each probe was hybridized. (5) The single nucleotide polymorphism is determined to be wild-type by the ratio of the detection signal of the wild-type probe to the detection signal of the mutant-type probe.
A method characterized by identifying either a mutant type or a mixed type.

(15) PCR, NASBA, LCR,
Amplifying the nucleic acid sequence by any method selected from the group consisting of SDA, RCR and TMA (14)
the method of.

(16) The primer is labeled with biotin,
(14) or (15) selected from the group consisting of dicoxigenin, a fluorescent substance, a luminophore, and an enzyme
The method described in.

(17) A probe for wild type and a probe for at least one mutant, wherein the nucleotide capable of hybridizing to the nucleotide polymorphism is RNA, and RNA
A kit for detecting a base polymorphism containing a selective cleavage endonuclease (18) further comprising a set of a forward primer and a reverse primer for amplifying a nucleic acid sequence containing at least one of which is labeled and having a base polymorphism site; The kit according to 17).

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION The method of the present invention comprises reacting two or more probes corresponding to wild-type and mutant types specific to a nucleotide polymorphism sequence portion contained in a specific nucleic acid sequence present in a sample. In the method of determining the ratio of the detection signal obtained by each probe, at least a site that hybridizes with the polymorphic sequence in each probe as RNA, preferably by performing RNA selective cleavage endonuclease treatment, the polymorphic sequence site The RNA that does not hybridize with is removed, and whether or not the RNA has been removed is detected, so that the polymorphic sequence site can be any of wild-type (homo), mixed (wild-type / mutant), and mutant (homo). Is detected.

Here, examples of the RNA selective cleavage endonuclease include ribonuclease A and ribonuclease A-like enzyme. Examples of the nucleotide polymorphism include single nucleotide polymorphism, insertion, and deletion polymorphism.

The specific nucleic acid sequence present in the sample is not particularly limited as long as it is a nucleic acid sequence containing a nucleotide polymorphism that carries information on the target gene. Examples of the target nucleic acid sequence include:
Examples include Alu sequences, exons, introns, and promoters of genes encoding proteins. More specifically, examples include genes related to various diseases including genetic diseases, drug metabolism, and lifestyle-related diseases (hypertension, diabetes, etc.). For example, ACE (Angiotensin I Converting En
zyme) gene.

In the present invention, a nucleic acid sequence may be simply referred to as a nucleic acid. A mutant nucleic acid sequence is a sequence in which at least one, preferably one nucleotide, of the wild-type nucleic acid sequence is point-mutated and replaced with another nucleotide, or inserted or deleted into a part of the wild-type nucleic acid. A nucleic acid containing a sequence or the like, in which nucleotide at which site is mutated has been elucidated. It has been elucidated that the constitution and the like are different 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.

Each of the wild-type probe and at least one mutant probe used in the present invention contains a nucleotide polymorphism site consisting of RNA, more preferably one or more bases before and after the nucleotide polymorphism site are also RNA. Something is good. The probe capable of hybridizing to the wild-type or mutant nucleic acid of the present invention is preferably at least 15 contiguous bases,
More preferably, it is a nucleotide sequence consisting of at least 18 consecutive nucleotides. Examples of the probe include those having 15 to 35 bases, particularly 18 to 30 bases. As long as the probe forms a complementary strand with a specific nucleic acid sequence except that the portion that hybridizes to the polymorphic sequence is RNA, RN can be used for DNA.
It may be A or PNA and can be prepared by chemical synthesis or biological methods. For example, it can be synthesized by a phosphoramidite method using a DNA synthesizer 391 manufactured by Perkin Elmer. Purification is MON by FPLC
It may be carried out using an OQ column or a reversed phase column. Other synthesis methods include the phosphoric acid triester method, the H-phosphonate method, and the thiophosphite method.

The position of the polymorphic sequence in the probe is not particularly limited, but is preferably a position other than the 5 'end or 3' end, more preferably 2 bases or more, preferably 3 bases or more from the 5 'end or 3' end. Are preferably at least 5 bases apart.

In addition, a nucleotide or oligo dG having biotin, a linker arm, a fluorescent substance and the like during synthesis.
TP, oligo dATP, oligo dTTP, oligo dCT
A modifying group may be introduced by adding P or the like to the 5 ′ end or 3 ′ end. Alternatively, a nucleotide having biotin, a linker arm, a fluorescent substance, or the like may be substituted for the nucleotide in the oligonucleotide sequence and synthesized to introduce a modifying group. Alternatively, ³²
Radioactive substances such as P, ³⁵ S, enzymes such as ALP and POD, FITC,
A known oligonucleotide label may be introduced into a fluorescent substance such as HEX, 6-FAM, or TET. These labels can be similarly introduced not only into the probe but also into the forward primer and the reverse primer for amplifying the target nucleic acid. In the present invention, a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample may be amplified in advance before detection with a probe.

The method for amplifying the nucleic acid sequence is not particularly limited, but may be PCR (Polymerase chain reactio).
n; JP-B-4-67960, JP-B-4-6795
No. 7), NASBA (Nucleic acid sequence-base
d amplification method; Nature Volume 350,
91 (1991)), LCR (WO089 / 12696, JP-A-2-293)
No. 4), SDA (Strand Displacment Amplificatio)
n; Nucleic Acids Res. Vol. 20, p. 1691 (199
2)), RCR (WO90 / 01069), TMA (Transcriptio
n Mediated amplification Method; J. Clin. Microbiol.
Vol. 31, p. 3270 (1993)), and any of them can be applied. Specifically, for example, after amplifying a specific nucleic acid sequence using primers (forward primer and reverse primer) labeled with biotin or the like, the amplified nucleic acid is converted into a single strand, for example, on a solid phase such as a microtiter plate. Hybridized with the bound wild-type and mutant polymorphism-specific probes, washing out the unhybridized nucleic acids,
A method of detecting whether or not hybridization has occurred by labeling a primer of a nucleic acid hybridized with a probe, and identifying whether the base polymorphism is a wild type, a mutant type or a mixed type based on a ratio of detection signals of each probe. Can be

The specific probe of the wild type and the specific probe of the mutant type may be any as long as they contain a polymorphic sequence portion, and are preferably 5 bases or more, more preferably 10 bases or more.
It has a sequence that hybridizes to the target nucleic acid in common with at least bases, more preferably at least 15 bases.

Kits In the present invention, the kits include a wild type probe, one or two types of mutant type probes, and a reagent kit for detecting a nucleotide polymorphism including RNA endonuclease. The predicted nucleotide at the nucleotide polymorphism site consists of RNA. The kit optionally includes a forward primer and a reverse primer for amplifying the target nucleic acid sequence, and these primers are preferably labeled.

[0038]

The present invention will be described more specifically with reference to the following examples. The present invention is not particularly limited by the examples. Example 1 Human β2 adrenergic receptor (ADR
B2) Degradation of Specific Probe for Genetic Detection by RNaseA (1) Synthesis of Oligonucleotide Having Linker Arm Using PerkinElmer DNA Synthesizer Model 392, the nucleotide sequence shown in SEQ ID NO: 3 was obtained by the phosphoramidite method. An oligonucleotide (A probe) and an oligonucleotide (G probe) having the base sequence shown in SEQ ID NO: 4 were synthesized. The A probe (SEQ ID NO: 3) and the G probe (SEQ ID NO: 4) are shown below. A probe (SEQ ID NO: 3; 24 bases): TTTTTCACCCA AT
A GAAGCCA TGC (underlined 13th " A " from the 5 'end (single nucleotide polymorphism site) and T and G before and after RN
A and all others are DNA. G probe (SEQ ID NO: 4; 23 bases): TTTTTCACCCA AT
G GAAGCCA TG (14th (1
" G " in the base polymorphism site) and T and G before and after " G " are RNA
And all others are DNA.

At this time, uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine was introduced into the above-mentioned oligonucleotide by the synthesis method disclosed in JP-T-60-500717. The synthesized linker oligonucleotide is made up of 50
Deprotection treatment overnight at ℃ C
Performed on a PC column. (2) Cleavage of polymorphic sequence-specific probe by RNase A A specific probe for ADRB2 detection was added to 500 pmol of ribonuclease I “A” (manufactured by Pharmacia), followed by
The reaction was performed at 20 ° C. for 20 minutes to decompose the RNA site.

Next, the treated sample was applied to a 10% polyacrylamide gel, and subjected to electrophoresis at 20 mM and 120 V for 30 minutes. After the electrophoresis, the cells were stained with Cyber Green II (Amersham) for 30 minutes, and photographed with Polaroid (registered trademark) film.

As a control, a gel obtained by directly applying 500 pmol of the same probe and an alkali-treated gel were added. (3) R of specific probe for detecting human ADRB2 gene
Results of Degradation Examination by NaseA FIG. 1 shows the results of degradation examination by RNaseA of the specific probe for detection of the human ADRB2 gene detected in (2) above.

As can be seen from FIG. 1, the probe in which the portion that hybridizes to the polymorphic sequence was RNA was degraded by RNase A in the RNA portion and detected as a short expected fragment. Example 2 Human β2 adrenergic receptor (ADR
B2) Detection of nucleotide polymorphism in gene (1) Synthesis of primer for amplifying ADRB2 gene fragment Oligonucleotide (primer) having the nucleotide sequence shown in SEQ ID NO: 1 by a phosphoramidite method using DNA synthesizer 392 manufactured by PerkinElmer 1)
An oligonucleotide (primer 2) having the base sequence shown in SEQ ID NO: 2 was synthesized. Primer 1
Has a sequence homologous to the human ADRB2 gene sequence, primer 2 has a sequence complementary to the human ADRB2 gene sequence, and has biotin ligated to the 5 ′ side. The synthesis was performed according to a manual, and deprotection of various oligonucleotides was performed overnight at 55 ° C. with aqueous ammonia. Oligonucleotide purification was carried out using a Perkin Elmer OPC column. (2) Synthesis of oligonucleotide having linker arm Oligonucleotide (A probe) having the nucleotide sequence shown in SEQ ID NO: 3 and the nucleotide sequence shown in SEQ ID NO: 4 by the phosphoramidite method using DNA synthesizer 392 manufactured by PerkinElmer. An oligonucleotide (G probe) having a base sequence to be synthesized was synthesized.

At this time, uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine was introduced into the above-mentioned oligonucleotide by the synthesis method disclosed in Japanese Patent Publication No. 60-500717. The synthesized linker oligonucleotide is made up of 50
Deprotection treatment overnight at ℃ C
Performed on a PC column. (3) Binding of probe oligonucleotide to microtiter plate The probe oligonucleotide synthesized in (2) above was bound to the inner surface of the microtiter plate via its linker arm. 50m oligonucleotide
M borate buffer (pH 10), diluted to a concentration of 0.05 pmol / μl in a solution of 100 mM MgCl ₂ , dispensed into a microtiter plate (MicroFLUOR B, Dynatech) 100 μl each, and left at room temperature for about 15 hours. The linker oligonucleotide was allowed to bind to the inner surface of the microtiter plate by allowing to stand. Then, 0.1p
The mixture was replaced with mol dNTP, 0.5% PVP (polyvinylpyrrolidone), and 5 × SSC, and blocking for suppressing nonspecific reaction was performed at room temperature for about 2 hours. Finally one
Washed with XSSC and dried. (4) Amplification of human ADRB2 gene fragment by PCR method Using a DNA solution extracted from human leukocytes as a sample, adding the following reagents, and adding human AD under the following conditions
The RB2 gene fragment was amplified. Reagent: A 25 μl solution containing the following reagents was prepared.

Primer 1 5 pmol Primer 2 5 pmol × 10 buffer 2.5 μl 2 mM dNTP 2.5 μl KODplus DNA polymerase 0.2 U extracted DNA solution 100 ng Amplification conditions 94 ° C. / 5 minutes 94 ° C./15 seconds, 60 ° C./30 seconds, 68 ° C, 30 seconds (3
(5 cycles) 68 ° C., 2 minutes (5) Hybridization in microtiter plate (4) Dilute the amplification reaction solution 10-fold to 0.3 N Na
The amplified nucleic acid in the amplification reaction solution was denatured in OH, and for each sample, 20 μl of the amplification reaction solution was subjected to 200 mM citrate-phosphate buffer (pH 6.0), 2% SDS (sodium dodecyl sulfate), 750 mM NaCl , 0.1%
The solution was added to a microtiter plate to which the capture probe of the above (3) was bound in addition to 100 μl of the NaN ₃ solution. Liquid paraffin was overlaid to prevent evaporation and shaken at 37 ° C for 30 minutes. Thereby, the amplified human A
The DRB2 gene fragment is specifically captured on the microtiter plate by the immobilized probe.

Next, after replacing with 2 × SSC (pH 7.0), RNase A (25 or 50 μl) was added to each well.
g) was added and allowed to warm at 37 ° C. for 20 minutes. Then 2
XSSC (pH 7.0), replaced with 1% SDS, 25% formamide, and similarly in order to prevent evaporation, liquid paraffin was overlaid and shaken at 37 ° C for 20 minutes. Furthermore, after that, streptavidin labeled with alkaline phosphatase (D0396, manufactured by DAKO) was replaced with 100 μl of a 50 mM Tris-HCl buffer (pH 7.5), which was diluted 2000-fold with a 1% BSA solution, and incubated at 37 ° C. for 15 minutes. Shake for minutes. As a result, streptavidin labeled with alkaline phosphatase was specifically bound to biotin of the captured DNA. After washing three times with 250 μl of a 50 mM Tris-HCl buffer (pH 7.5) and 0.025% Tween20 solution, a dioxetane compound (trade name: Lumiphos480; Lumigen; luminescent substrate of alkaline phosphatase)
Was injected at 50 ° C. for 15 minutes, and the luminescence was measured with a photon counter (Hamamatsu Photonics) in a dark room. (6) Detection of nucleotide polymorphism in human ADRB2 gene Human AD amplified in (4) above and detected in (5)
Table 1 shows the results of detecting nucleotide polymorphisms in the RB2 gene. The numerical value is displayed by the light emission amount (cps: count / second). Asignal
Indicates a detection signal of the amplified nucleic acid fragment reacted with the A probe, and Gsignal indicates a detection signal of the nucleic acid fragment reacted with the G probe. As can be seen from Table 1, the single nucleotide polymorphism of the ADRB2 gene can be identified by taking the logarithm of the ratio of the signal obtained with each probe. That is, as is apparent from FIG. 2, when the logarithm of the signal ratio is 1.0 or more, A
A homogenous genotype of the base type, -1.0 or less can be identified as a homogenous genotype of the G base type, and -1.0 to 1.0 can be identified as a heterogenous type of the A base type and the G base type. A decrease in the noise value caused by the non-specific reaction in the case of acting was observed, and it was a result that the noise value could be expected to be improved with a probe in which the non-specific reaction was likely to appear.

[0046]

[Table 1]

Example 3 Detection of Nucleotide Polymorphism in Human β3 Adrenergic Receptor (3AR) Gene (1) Synthesis of 3AR Gene Fragment Amplification Primer Using a PerkinElmer DNA synthesizer 392, the phosphoramidite method was used. Oligonucleotide having the nucleotide sequence of SEQ ID NO: 5 (primer 1)
An oligonucleotide (primer 2) having the base sequence shown in SEQ ID NO: 6 was synthesized. Primer 2
Has biotin linked to the 5 'side. The synthesis was performed according to a manual, and deprotection of various oligonucleotides was performed overnight at 55 ° C. with aqueous ammonia. Oligonucleotide purification was carried out using a Perkin Elmer OPC column. (2) Synthesis of oligonucleotide having linker arm Oligonucleotide (T probe) having the base sequence shown in SEQ ID NO: 7 and the nucleotide sequence shown in SEQ ID NO: 8 by the phosphoramidite method using DNA synthesizer 392 manufactured by PerkinElmer. An oligonucleotide having a base sequence (C probe) was synthesized.

At this time, uridine having a linker arm at the 5-position prepared by chemical synthesis from deoxyuridine was introduced into the above-mentioned oligonucleotide by the synthesis method disclosed in JP-T-60-500717. The synthesized linker oligonucleotide is made up of 50
Deprotection treatment overnight at ℃ C
Performed on a PC column. (3) Binding of probe oligonucleotide to microtiter plate The probe oligonucleotide synthesized in (2) above was bound to the inner surface of the microtiter plate via its linker arm. 50m oligonucleotide
M borate buffer (pH 10), diluted to a concentration of 0.05 pmol / μl in a solution of 100 mM MgCl ₂ , dispensed into a microtiter plate (MicroFLUOR B, Dynatech) 100 μl each, and left at room temperature for about 15 hours. The linker oligonucleotide was allowed to bind to the inner surface of the microtiter plate by allowing to stand. Then, 0.1p
The mixture was replaced with mol dNTP, 0.5% PVP (polyvinylpyrrolidone), and 5 × SSC, and blocking for suppressing nonspecific reaction was performed at room temperature for about 2 hours. Finally one
Washed with XSSC and dried. (4) Amplification of human 3AR gene fragment by PCR method Using a DNA solution extracted from human leukocytes as a sample, the following reagents were added, and human 3A was prepared under the following conditions.
The R gene fragment was amplified. Reagents: 25 including the following reagents
A μl solution was prepared.

Primer 1 5 pmol Primer 2 5 pmol × 10 buffer 2.5 μl 2 mM dNTP 2.5 μl KODplus DNA polymerase 0.2 U extracted DNA solution 100 ng Amplification conditions 94 ° C./5 minutes 94 ° C./15 seconds, 60 ° C./30 seconds, 68 ° C, 30 seconds (3
(5 cycles) 68 ° C., 2 minutes (5) Hybridization in microtiter plate (4) Dilute the amplification reaction solution 10-fold to 0.3 N Na
Denature the amplified DNA in the amplification reaction solution in OH,
For each sample, add 20 μl of the amplification reaction solution to 200 mM citrate-phosphate buffer (pH 6.0), 2% SDS (sodium dodecyl sulfate), 750 mM NaCl, 0.1%
% In addition to NaN _3, 35% formamide solution 100 [mu] l, it was placed into microtiterplates capture probe in the above (3) is bonded. The plate was covered with a plate seal to prevent evaporation, and heated at 37 ° C. for 30 minutes. Thereby, the amplified human 3AR gene fragment is specifically captured on the microtiter plate by the immobilized probe.

Next, after replacing with 2 × SSC (pH 7.0), RNase A (50 μg) was added to each well.
Warmed at 37 ° C. for 20 minutes. Then 2 × SSC (pH
7.0) In order to prevent evaporation by substituting with 1% SDS and 35% formamide, liquid paraffin is overlaid at 37 ° C.
For 20 minutes. After that, streptavidin labeled with alkaline phosphatase (DAKO: D
0396) in 50 mM Tris-HCl buffer (pH 7.5),
100 μl solution diluted 2000-fold with a 1% BSA solution
and shaken at 37 ° C. for 15 minutes. As a result, streptavidin labeled with alkaline phosphatase was specifically bound to biotin of the captured DNA. 250 μl of 50 mM Tris-HCl buffer (pH
7.5), after washing three times with 0.025% Tween20 solution,
50 μl of a dioxetane compound (trade name: Lumiphos480; Lumigen) which is a luminescent substrate of alkaline phosphatase
Was injected and kept at 37 ° C. for 15 minutes, and the luminescence was measured with a photon counter (Hamamatsu Photonics) in a dark room. (6) Detection of nucleotide polymorphism in human 3AR gene Human 3A amplified in (4) above and detected in (5)
Table 2 shows the results of detecting nucleotide polymorphisms in the R gene. The numerical value is displayed by the light emission amount (cps: count / second). Tsignal is T
The detection signal of the amplified nucleic acid fragment reacted with the probe is converted to C
signal indicates a detection signal of the nucleic acid fragment reacted with the C probe. As can be seen from Table 2, the single nucleotide polymorphism of the 3AR gene can be identified in a 37 ° C isothermal reaction by taking the logarithm of the ratio of the signals obtained with each probe. That is, as is clear from Table 2, when RNaseA treatment is performed, three types of T homo, C homo, and hetero can be identified.

[0051]

[Table 2]

[0052]

As described above, the method of the present invention uses two or more types of probes specific to the polymorphic sequence to obtain a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample. This makes it possible to easily detect and identify the type of nucleotide polymorphism.

[0053]

[Sequence List] SEQUENCE LISTING <110> TOYO BOSEKI KABUSHIKI KAISHA <120> A METHOD FOR DETECTING SNPs <130> 20C00JP <140> <141> <160> 8 <170> PatentIn Ver. 2.1 <210> 1 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 1 ACGGCAGCGC CTTCTTG 17 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: primer <400> 2 TTGCTGCGTG ACGTCGTG 18 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 3 TTTTTCACCCA ATAGAAGCCA TGC 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 4 TTTTTCACCCA ATGGAAGCCA TG 22 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 5 CGCCCAATAC CGCCAACAC 19 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 6 ACGAACACGT TGGTCATGGT CT 22 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 7 TTTTTGGCCA TCGCCTGGAC TCCGAG 22 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer <400> 8 TTTTTGCCAT CGCCCGGACT CCGAG 25

[Brief description of the drawings]

FIG. 1 shows that the method of the present invention in Example 1
It is a figure which shows the decomposition | disassembly examination result of RNaseA of the specific probe for DRB2 gene detection.

FIG. 2 is a diagram showing the results of nucleotide polymorphism detection of the ADRB2 gene when hybridization was performed at 37 ° C. and RNaseA treatment was performed by the method of the present invention in Example 2.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Toshiya Aono 2-1-1 Katata, Otsu-shi, Shiga Prefecture Toyobo Co., Ltd. Research Institute (72) Inventor Masaya Segawa 2-1-1 Katata, Otsu-shi, Shiga Prefecture F-term (reference) in Toyobo Co., Ltd. Research Laboratory 4B024 AA11 AA20 CA01 CA09 HA12 HA19 4B050 LL03 4B063 QA12 QA17 QQ42 QR08 QR14 QR42 QR55 QR62 QS25 QS34 QX02

Claims (18)

[Claims] 1. A method comprising the steps of: allowing two or more types of probes specific to a nucleotide polymorphism sequence portion contained in a specific nucleic acid sequence present in a sample to act, and determining a ratio of detection signals obtained by each probe; A method for detecting a polymorphism, wherein at least a portion that hybridizes to a polymorphic sequence of a probe specific to the polymorphic sequence portion is RNA. 2. A method for detecting a base polymorphism by allowing two or more probes specific to a polymorphism sequence portion contained in the nucleic acid sequence to detect a nucleotide polymorphism, comprising performing RNA selective cleavage endonuclease treatment. The method for detecting a nucleotide polymorphism according to claim 1. 3. The method of claim 1, wherein the RNA selective cleavage endonuclease comprises:
3. The method according to claim 2, which is ribonuclease A or a ribonuclease A-like enzyme. 4. The method according to claim 1, wherein in the method for detecting a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, the nucleic acid sequence is amplified in advance. 5. PCR, NASBA, LCR, SDA,
The method according to claim 4, wherein the nucleic acid sequence is amplified by any method selected from the group consisting of RCR and TMA. 6. The method according to claim 1, wherein the nucleotide polymorphism includes one of a single nucleotide polymorphism, an insertion and a deletion polymorphism. 7. A method for identifying a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, comprising: (1) amplifying a nucleic acid fragment containing the nucleotide polymorphism of the specific nucleic acid sequence; (2) hybridized with the amplified nucleic acid and two or more polymorphism specific probes, (3) treated with RNA selective cleavage endonuclease, (4) measuring the detection signal of each probe, (5 A) identifying a polymorphic sequence based on the ratio of each detection signal; 8. The PCR, NASBA, LCR, SDA,
The method according to claim 7, wherein the nucleic acid sequence is amplified by any method selected from the group consisting of RCR and TMA. 9. The method according to claim 7, wherein the nucleotide polymorphism includes one of a single nucleotide polymorphism, an insertion and a deletion polymorphism. 10. A method for identifying a nucleotide polymorphism of a specific nucleic acid sequence containing a nucleotide polymorphism contained in a sample, comprising: (1) a primer labeled with a nucleic acid sequence containing the nucleotide polymorphism of the specific nucleic acid sequence; (2) hybridized with a different polymorphism-specific probe each amplified nucleic acid, (3) treated with RNA selective cleavage endonuclease, (4) whether or not hybridized with each probe (5) A method comprising identifying the polymorphic sequence based on the ratio of each detection signal, wherein the detection is performed by labeling the primer. 11. PCR, NASBA, LCR, SD
The method according to claim 10, wherein the nucleic acid sequence is amplified by any method selected from the group consisting of A, RCR and TMA. 12. The method according to claim 11, wherein the nucleotide polymorphism includes one of a single nucleotide polymorphism, an insertion, and a deletion polymorphism. 13. The method according to claim 10, wherein the label of the primer is any one selected from the group consisting of biotin, dicoxigenin, a fluorescent substance, a luminophore, and an enzyme. 14. A method for identifying a single nucleotide polymorphism of a specific nucleic acid sequence containing a single nucleotide polymorphism contained in a sample,
(1) a nucleic acid fragment containing the single nucleotide polymorphism of the specific nucleic acid sequence is amplified using a labeled primer, and (2) a wild-type probe and a mutant probe immobilized on each of the amplified nucleic acid and two or more solid phases. And (3) treated with RNA selective cleavage endonuclease, (4) whether or not hybridized with each probe is detected by the label of the primer, (5) detection signal of the wild-type probe A method characterized in that the single nucleotide polymorphism identifies any one of a wild type, a mutant type, and a mixed type based on a ratio of a detection signal of the mutant type probe. 15. PCR, NASBA, LCR, SD
The method according to claim 14, wherein the nucleic acid sequence is amplified by any method selected from the group consisting of A, RCR and TMA. 16. The method according to claim 14, wherein the label of the primer is any one selected from the group consisting of biotin, dicoxigenin, a fluorescent substance, a luminophore, and an enzyme. 17. A kit for detecting a nucleotide polymorphism comprising a wild-type probe and a probe for at least one mutant in which the nucleotide capable of hybridizing to the nucleotide polymorphism portion is RNA, and an RNA-selective cleavage endonuclease 18. The kit according to claim 17, further comprising a set of a forward primer and a reverse primer for amplifying a nucleic acid sequence containing a nucleotide polymorphism site, at least one of which is labeled.