KR20120124033A - Gene mutation detection probe, method of detecting gene mutation and gene mutation detection reagent kit - Google Patents

Abstract

[Problem] Even if the nucleotide sequence encoding the gene contains a plurality of gene mutations, the gene enables specific detection of specific gene mutations to be detected without being affected by gene mutations other than the detection target. Provided is a probe for detecting a mutation.
[Remedy] (P1) A base having a base complementary to any of the wild type base and the mutant base of the target gene mutation at a base position corresponding to the target gene mutation, and at a base position corresponding to the nontarget gene mutation. Oligos having non-complementary bases in any of the wild-type bases and variant bases of the non-target gene variant, having at least 80% identity to the base sequence complementary to some or all of the base sequences encoding the gene of interest A gene mutation detection probe for detecting a target gene variation in a nucleotide sequence that is a nucleotide or the like and encodes a target gene including target gene variation and non-target gene variation.

Description

Gene Mutation Detection Probes, Gene Mutation Detection Methods, and Gene Mutation Detection Reagent Kits {GEN MUTATION DETECTION PROBE, METHOD OF DETECTING GENE MUTATION AND GENE MUTATION DETECTION REAGENT KIT}

The present invention relates to a probe for detecting a genetic variation.

A "gene variant" means that the base in the base sequence encoding the gene is a base different from the majority of the traits of a certain population. For example, the gene structure (base sequence) such as single nucleotide substitution, deletion, insertion, gene fusion, etc. ) Is a difference.

On the other hand, "gene polymorphism" is a part of "gene mutation", and the most frequent variation or allel (allele) in a group is 99% or less, in other words, the total frequency of low frequency variation or allel Indicates that there is more than 1%. This 1% value is convenient, but meaningful. Since most of the new mutations that occur every generation are removed from the population by natural selection, the equilibrium frequency is theoretically calculated. The value higher than this equilibrium frequency and 1% is made into the criterion of a "polymorphism." In other words, the appearance of a polymorphism means that the frequency of their variation in a group is increased by some mechanism and has been maintained for generations.

It has been pointed out that the association between the genetic variation and the morbidity of various diseases and the frequency of adverse reactions caused by the administration of the drug, and by detecting the gene polymorphism, it is possible to predict the frequency of various diseases, drug resistance and the like. Therefore, there has been a demand for a method for measuring gene mutations accurately and in a short time and at low cost.

The types of gene polymorphisms and gene variants include single base substitution resulting from substitution of one base in a base sequence, deletion type mutation in which one or more bases are deleted, insertional mutation in which one or more bases are inserted, gene fusion, and the like. Hereinafter, the gene polymorphism and gene mutations are collectively called genetic mutations.

Currently, as a method for measuring genetic variation, after amplifying a region containing the mutation by PCR, a nucleic acid probe labeled with a fluorescent dye is hybridized to a target nucleic acid to measure the amount of decrease in luminescence of the fluorescent dye, The method of analyzing the variation of a nucleotide sequence based on the result of a melting curve analysis is known (refer patent document 1).

Japanese Patent Laid-Open No. 2002-119291

However, in the method using the nucleic acid probe, it is necessary to use a nucleic acid probe having an appropriate sequence for each mutation in order to detect genetic variation. In addition, when performing a fusion curve analysis using a nucleic acid probe, if a mutation other than the gene mutation to be detected exists in the nucleic acid probe, it is affected by the gene mutation other than the gene mutation detection target, thereby affecting the melting temperature (Tm value). Since nonuniformity arises, it becomes difficult to specifically detect the genetic variation to be detected.

In view of these phenomena, even if the nucleotide sequence encoding a gene contains a plurality of gene mutations, it is possible to precisely detect specific gene mutations to be detected without being affected by gene mutations other than the gene mutation detection target. New technology development was desired.

The present invention makes it possible to specifically detect only a specific gene variant to be detected from a plurality of gene variants included in a base sequence encoding a gene, without being affected by the base type of the non-target gene variant. An object of the present invention is to provide a gene mutation detection probe and a gene mutation detection method using the same. It is another object of the present invention to provide a reagent kit for detecting a genetic mutation using the gene mutation detecting probe.

The present invention is as follows.

<1> at least one oligonucleotide selected from the group consisting of the following P1, P1-1, P1 ', and P1'-1, and a nucleotide sequence encoding a target gene including a target gene mutation and an untarget gene mutation. Gene mutation detection probe for detecting the target gene mutation in:

(P1) the non-target gene at a base position corresponding to the target gene mutation having a base complementary to either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having bases which are non-complementary to any of the wild type and variant bases of the variant and having at least 80% identity to the base sequence complementary to some or all of the base sequence encoding the gene of interest;

(P1-1) a base having a base complementary to either the wild-type base or the mutant base of the target gene mutation at a base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligos that hybridize under oligonucleotide conditions to oligonucleotides having non-complementary bases and having the same base sequence as the base sequence encoding the gene of interest. Nucleotides;

(P1 ′) the non-target gene at a base position corresponding to the target gene mutation having the same base for either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having other bases in any of the wild-type and variant bases of the variant and having at least 80% or more identity to some or all of the base sequences encoding the genes of interest; And

(P1′-1) has a base identical to any of the wild-type base and the mutant base of the target gene mutation at the base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligonucleotides that hybridize under oligonucleotide conditions to oligonucleotides having a base sequence complementary to the base sequence encoding the gene of interest and having a base different from any of the wild type base and the variant base of the desired gene variant. .

<2> Gene mutation detection probe according to <1>, which satisfies any one of the following (a) to (f):

(a) when the wild-type base at the base position of the non-target gene variant is either A or T, and the variant base of the non-target gene variant is another one of A or T, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases, or which are either wild-type bases or variant bases of said non-target gene variants, are C or G.

(b) when the wild-type base at the base position of the non-target gene variant is either C or G and the variant base of the non-target gene variant is another one of C or G, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases or which are either wild-type bases or variant bases of said non-target gene variants are A or T.

(c) when the wild-type base at the base position of the non-target gene variant is either A or C, and the variant base of the non-target gene variant is another one of A or C, the wild-type base of the non-target gene variant And A or C, which is non-complementary to any of the variant bases, is A or C, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or G.

(d) when the wild-type base at the base position of the non-target gene variant is either A or G, and the variant base of the non-target gene variant is another one of A or G, the wild-type base of the non-target gene variant And A or G, which is non-complementary to any of the variant bases, is A or G, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or C.

(e) when the wild-type base at the base position of the non-target gene variant is either T or C, and the variant base of the non-target gene variant is another one of T or C, the wild-type base of the non-target gene variant And bases that are non-complementary to any of the variant bases are T or C, and bases other than any of the wild-type bases and variant bases of the non-target gene variant are A or G.

(f) when the wild-type base at the base position of the non-target gene variant is either T or G, and the variant base of the non-target gene variant is another one of T or G, the wild-type base of the non-target gene variant And a base non-complementary to any of the variant bases is T or G, and a base other than either the wild-type base or the variant base of the non-target gene variant is A or C.

<3> The oligonucleotide is a gene mutation detection probe according to <1> or <2>, which is a fluorescently labeled oligonucleotide.

<4> The gene-detection probe according to <3>, wherein the fluorescently labeled oligonucleotide is a cytosine having a base at the 3 'end or the 5' end, and the cytosine is labeled with a fluorescent dye.

<5> The probe for gene mutation detection according to <3> or <4>, wherein the fluorescently labeled oligonucleotide is located at any one of the first to third bases by counting from the 3 'end or the 5' end.

<6>? 1 which is a probe for melting curve analysis? The gene mutation detection probe according to any one of <5>.

<7> Does the fluorescent label oligonucleotide decrease or increase the fluorescence intensity when hybridizing to the target sequence compared to the fluorescence intensity when not hybridizing to the target sequence? The probe for gene mutation detection in any one of <6>.

In <3>? <7>, the <8> above-mentioned fluorescently labeled oligonucleotide decreases the fluorescence intensity at the time of hybridizing to a target sequence compared with the fluorescence intensity at the time of not hybridizing to a target sequence. Gene mutation detection probe according to any one.

<9> <1>? The gene mutation detection method which detects the target gene mutation used as a detection object by using the gene mutation detection probe in any one of <8>.

<10> (Ⅰ) <3>? Contacting the single stranded nucleic acid in the gene mutation detection probe and the sample according to any one of <8> to obtain a hybrid, and (II) changing the temperature of the sample containing the hybrid to dissociate the hybrid, Measuring the fluctuation of the fluorescence signal based on the dissociation of the hybrid, (III) obtaining the Tm value, which is the dissociation temperature of the hybrid, based on the fluctuation of the fluorescence signal, and (IV) on the basis of the Tm value A target gene mutation detection method comprising detecting the presence of a target gene mutation in a single-stranded nucleic acid.

<11> The target gene mutation detection method according to <10>, further comprising amplifying the nucleic acid before obtaining the hybrid of (I) or simultaneously with obtaining the hybrid of (I).

<12> <1>? Reagent kit for detecting a genetic mutation comprising the probe for detecting the genetic mutation according to any one of <8>.

<13> <1>? The reagent kit for detecting a gene mutation according to <12>, further comprising a primer capable of amplifying a nucleotide sequence having a region to which the gene mutation detection probe according to any one of <8> hybridizes.

According to the present invention, even if the nucleotide sequence encoding a gene includes a plurality of gene mutations, it is possible to specifically detect specific gene mutations to be detected without being affected by gene mutations other than the detection target. A genetic mutation detection probe can be provided, and a genetic mutation detection method using the same can be provided. Moreover, according to this invention, the reagent kit for gene mutation detection using the said gene mutation detection probe can be provided.

(A) is an example of the melting curve of a nucleic acid mixture, (B) is an example of a differential melting curve.
(A)-(P) is the differential melting curve of the sample by Example 1 of this invention.
(A)-(H) is the differential melting curve of the sample by Example 2 of this invention.
(A)-(P) is the differential melting curve of the sample by the comparative example 1 of this invention.

The gene mutation detection probe according to the present invention is at least one oligonucleotide selected from the group consisting of P1, P1-1, P1 ', and P1'-1, and includes a target gene mutation and an untarget gene mutation. A gene mutation detection probe for detecting a target gene mutation in a nucleotide sequence encoding a gene.

Accordingly, the gene mutation detection probe according to the present invention is capable of detecting a specific gene mutation to be detected without being affected by gene mutations other than the detection target, even if the nucleotide sequence encoding the gene includes a plurality of gene mutations. Specifically detect.

The gene mutation detection method according to the present invention uses at least one gene mutation detection probe for detecting the gene mutation, and the gene mutation detection target is performed even if the base sequence encoding the gene contains a plurality of gene mutations. It is a method including detecting the target gene mutation to be detected, without being influenced by other gene mutations.

The reagent set for detecting a genetic mutation according to the present invention includes the above-described genetic mutation detecting probe for detecting a target gene mutation.

In the present invention, regarding the individual sequences of the sample nucleic acid, the gene mutation detection probe or the primer in the sample to be detected, the matters described based on the complementary relationship with each other, unless otherwise stated, The same applies to the base sequence and the base sequence complementary to each base sequence. When applying the matter of this invention with respect to the said base sequence complementary to each base sequence, the base sequence which the said complementary base sequence recognizes is described in the corresponding specification within the range of technical common sense by those skilled in the art. The sequence complementary to the sequence shall be read interchangeably.

In the present invention, the "Tm value" is a temperature at which the double-stranded nucleic acid dissociates (dissociation temperature: Tm), and is generally defined as the temperature when the absorbance at 260 nm reaches 50% of the total absorbance increase. . That is, when the solution containing a double stranded nucleic acid, for example, double stranded DNA, is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both chains in double-stranded DNA are released by heating, and dissociate into single-stranded DNA (fusion of DNA). When all the double-stranded DNA is dissociated into single-stranded DNA, the absorbance represents about 1.5 times the absorbance at the start of heating (absorbance only of the double-stranded DNA), whereby it can be determined that the fusion is completed. The Tm value is set based on this phenomenon.

In the present invention, when referring to the "first to third bases counted from the 3 'end" with respect to the oligonucleotide sequence, the 3' terminal base of the oligonucleotide chain is counted as the first. Similarly, in the case of the "first to third base counting from the 5 'end," the 5' terminal base of the oligonucleotide chain is counted as the first base.

In this specification, the term "process" is included in this term as long as the desired action of this process is achieved, even if it is not only an independent process but also a case where it cannot be clearly distinguished from other processes.

The numerical range shown using "?" In this specification shows the range which includes the numerical value described before and after "?" As minimum value and the maximum value, respectively.

In addition, in this invention, when there exists a plurality of substances corresponding to each component in a composition, unless there is particular notice, the quantity of each component in a composition means the total amount of the said some substance which exists in a composition.

EMBODIMENT OF THE INVENTION Hereinafter, this invention is demonstrated.

<Gene mutation detection probe>

The genetic variation detection probe according to the present invention is at least one oligonucleotide selected from the group consisting of the following P1, P1-1, P1 'and P1'-1, and includes a target gene mutation and an untarget gene mutation. A gene mutation detection probe for detecting the target gene mutation in the nucleotide sequence encoding the gene of interest.

Accordingly, the gene mutation detection probe according to the present invention is capable of detecting a specific gene mutation to be detected without being affected by gene mutations other than the detection target, even if the nucleotide sequence encoding the gene includes a plurality of gene mutations. Specifically detect.

(P1) the non-target gene at a base position corresponding to the target gene mutation having a base complementary to either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having bases which are non-complementary to any of the wild type and variant bases of the variant and having at least 80% identity to the base sequence complementary to some or all of the base sequence encoding the gene of interest;

(P1-1) a base having a base complementary to either the wild-type base or the mutant base of the target gene mutation at a base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligos that hybridize under oligonucleotide conditions to oligonucleotides having non-complementary bases and having the same base sequence as the base sequence encoding the gene of interest. Nucleotides;

(P1 ′) the non-target gene at a base position corresponding to the target gene mutation having the same base for either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having other bases in any of the wild-type and variant bases of the variant and having at least 80% or more identity to some or all of the base sequences encoding the genes of interest; And

(P1′-1) has a base identical to any of the wild-type base and the mutant base of the target gene mutation at the base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligonucleotides that hybridize under oligonucleotide conditions to oligonucleotides having a base sequence complementary to the base sequence encoding the gene of interest and having a base different from any of the wild type base and the variant base of the desired gene variant. .

In the present invention, the "base position corresponding to the target gene mutation" means the target gene in the sample base sequence including the target gene mutation and the non-target gene mutation when the base sequence of the oligonucleotide and the sample base sequence are best aligned with each other. It refers to the base position in the oligonucleotide corresponding to the base position of the mutation. Similarly, in the present invention, the "base position corresponding to the non-target gene mutation" means a sample base sequence containing the target gene mutation and the non-target gene mutation when the base sequence of the oligonucleotide and the sample base sequence are best aligned with each other. Refers to the base position in the oligonucleotide corresponding to the base position of the nontarget gene mutation. The wild-type base of the target gene variant refers to the wild-type base of the base position of the target gene variant (ie, the base position where the mutation of the target gene variant may occur to the variant base).

Adenine (A) and thymine (T) and guanine (G) and cytosine (C) each form a hydrogen bond. Whereas the AT pair forms two hydrogen bonds, the GC pair forms three hydrogen bonds. That is, a hydrogen bond is formed in "between A-T" and "between G-C", and a double helix structure is maintained. Combinations of bases other than these cannot form sufficient hydrogen bonds to maintain the double helix structure.

For example, when the combination of the wild type base and the mutant base at the base position of the nontarget gene mutation contained in the sample nucleic acid is a combination of A and T, the nontarget gene mutation in the probe for detecting the genetic mutation If the base that is non-complementary to any of the wild-type and mutant bases of C is G or G, the wild-type base and the mutant base (base selected from A and T) at the base position of the non-target gene mutation, and the non-target Non-complementary bases (C or G) are not hydrogen-bonded to any of the wild type base and the variant base of the genetic variation, and the Tm value of the genetic variation detection probe is not affected by non-target genetic variation. On the other hand, if the non-complementary base is any of the wild-type base and the mutant base of the non-target gene mutation in the gene mutation detection probe, the base at the base position of the non-target gene mutation contained in the sample nucleic acid is A. Is not hydrogen-bonded, and is hydrogen-bonded when the base at the base position of the non-target gene mutation is T. Under such a situation, the Tm value of the gene mutation detection probe is influenced not only by the target gene mutation but also by the non-target gene mutation, and the target gene is appropriately used when the gene mutation detection probe is used. The state of the mutation cannot be detected.

As described above, in the case of a plurality of gene variants included in the base sequence encoding the gene of interest, when the target gene mutation is detected using a gene mutation detection probe having homology to the base sequence complementary to the base sequence. The base on the probe at the base position corresponding to the non-target gene mutation is a base (non-complementary base) which does not bind to either the normal base or the mutant base at the base position of the non-target gene mutation. Gene mutation detection probes composed of the above (P1) or (P1-1) oligonucleotides according to the present invention are capable of specifically detecting target gene mutations without being affected by the base type of the base position of the non-target gene mutation. It becomes possible.

Similarly, in a plurality of gene mutations included in the nucleotide sequence encoding the gene of interest, when the target gene mutation is detected by using a gene mutation detection probe having homology to the same nucleotide sequence as that of the nucleotide sequence, The base (P1) according to the present invention is obtained by making the base on the probe at the base position corresponding to the target gene mutation different from any of the normal base and the variant base at the base position of the non-target gene mutation (other bases). Gene mutation detection probes consisting of ') or (P1'-1) oligonucleotides can be specifically detected for target gene mutations without being affected by the base type of the base position of the non-target gene mutation.

The gene mutation detection probe in the present invention can recognize any of the mutations consisting of a single base substitution type, a deletion type and an insertion type. Among these, the gene mutation detection probe in the present invention is particularly useful as a gene mutation detection probe that recognizes a single base substitution type mutation (single base variation) from the viewpoint of detection sensitivity.

The oligonucleotide of (P1) or (P1-1) according to the present invention has a base sequence complementary to the base sequence encoding the gene containing the target gene mutation, except for the base position corresponding to the non-target gene mutation. , Homology.

On the other hand, the oligonucleotide of (P1 ') or (P1'-1) has the same base sequence as that of the base sequence encoding the gene containing the target gene mutation, except for the base position corresponding to the non-target gene mutation, It has homology.

All known genes are applicable to the gene of interest in the present invention. In addition, the gene of interest in the present invention includes any of a structural gene and a transcriptional regulatory region.

In the present specification, the transcriptional control region is a sequence that exists in many 5 'upstream regions of the gene signal sequence (may be present in the first intron), and when RNA polymerase transcribes RNA from the gene, Sequence.

Moreover, in the structural gene, a gene mutation may exist in the site | part of an exon or a site of intron, and in a more specific embodiment, a genetic mutation may exist in the site | part of an exon.

In the present invention, the term "having homology" refers to a base sequence encoding the gene mutation detection probe of the present invention and a base sequence complementary to a part or all of the base sequence encoding the gene of interest. And oligonucleotides having a homology of 80% to 100%. In terms of detection sensitivity, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

When the homology is 80% or more, the detection sensitivity of the sample nucleic acid containing the target gene including the target gene mutation to be detected becomes good.

In one embodiment, the (P1) or (P1-1) oligonucleotide of the present invention corresponds to a portion of the nucleotide sequence encoding a gene of interest comprising a target gene variation and a nontarget gene mutation, corresponding to the nontarget gene mutation. It is complementary (completely complementary) except for the base position and, optionally, the base position corresponding to the desired gene mutation. In another embodiment, a (P1) or (P1-1) oligonucleotide of the invention corresponds to an untargeted genetic variation, relative to all of the base sequences encoding the gene of interest, including the desired and untargeted genetic variation. Except for the base position and, optionally, the base position corresponding to the gene mutation of interest, it is completely complementary.

In another embodiment, a (P1 ') or (P1'-1) oligonucleotide of the present invention corresponds to a portion of a nucleotide sequence encoding a gene of interest comprising a target gene mutation and a nontarget gene mutation and corresponding to the nontarget gene mutation. Except that the base position and, optionally, the base position corresponding to the desired gene mutation. In another embodiment, the (P1 ') or (P1'-1) oligonucleotide of the present invention corresponds to the entirety of the base sequence encoding the target gene, including the desired and untargeted genetic variation, and corresponding to the untargeted genetic variation. Except that the base position and, optionally, the base position corresponding to the desired gene mutation.

In the present invention, "hybridize under stringent conditions" will be described below.

Hybridization can be performed according to a known method or a method similar thereto, for example, a method described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is incorporated herein by reference.

Stringent conditions refer to conditions under which specific hybrids are formed and nonspecific hybrids are not formed. Typical stringent conditions include, for example, conditions for performing hybridization in a potassium concentration of about 25 mM to about 50 mM, and a magnesium concentration of about 1.0 mM to about 5.0 mM. Examples of the conditions of the present invention include, but are not limited to, conditions for hybridization in Tris-HCl (pH8.6), 25 mM KCl, and 1.5 mM MgCl ₂ . Other stringent conditions are described in Molecular Cloning 3rd (J. Sambrook et al., Cold Spring Harbor Lab. Press, 2001). This document is incorporated herein by reference. Those skilled in the art can easily select such conditions by changing the hybridization reaction, the salt concentration of the hybridization reaction solution, and the like.

Hereinafter, oligonucleotides of (P1), (P1-1), (P1 ') and (P1'-1) according to the present invention will be described. If necessary, the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 is described as an example, but the oligonucleotides of (P1), (P1-1), (P1 ') and (P1'-1) according to the present invention are It is not limited to this.

The nucleotide sequence shown in SEQ ID NO: 1 is a nucleotide sequence encoding CYP3A4 gene mutation (CYP3A4 * 1B rs2740574) which is a drug metabolizing enzyme. The CYP3A4 gene encoding CYP3A4 is located on the seventh chromosome of human. CYP3A4 gene mutations have been reported, for example, in association with drug metabolism such as immunosuppressant tacrolimus (Clin. Pharmacol. Ther., 2006, Vol. 80, p. 179-191). Therefore, the detection result of the mutation in the CYP3A4 gene is extremely important information when selecting a more effective treatment method for the disease.

Here, rs2740574, which is a CYP3A4 gene mutation, is the 501st base of SEQ ID NO: 1. This rs number shows the registration number of the dbSNP database (//www.ncbi.nlm.nih.gov/projects/SNP/) of National Center for Biotechnology Information (it is the same below). In the wild type of the CYP3A4 gene, the 501st base of the nucleotide sequence shown in SEQ ID NO: 1 is A (adenine), but in the mutant type, it is mutated to G (guanine).

The nucleotide sequence shown in SEQ ID NO: 2 shows an example of a gene mutation detection probe according to the present invention for detecting the 501st target gene mutation among the nucleotide sequences shown in SEQ ID NO: 1.

In the present invention, the nucleotide sequence encoding the gene of interest includes a plurality of gene variants. Specifically, the nucleotide sequence encoding the target gene includes two or more gene variants, and for example, may include three gene variants and four gene variants.

Of the gene mutations included in the base sequence encoding the gene of interest, the target gene mutation can be appropriately selected from base sequences encoding the gene of interest, as necessary. For example, in the nucleotide sequence encoding the CYP3A4 gene of SEQ ID NO: 1, the 501st gene mutation is selected as the target gene mutation to be detected. Therefore, using SEQ ID NO: 1 as an example, A (adenine) or G (guanine) corresponds to the "mutant base of the base position of the target gene mutation" according to the present invention, and the "base of the target gene mutation" according to the present invention. Wild-type base at the position &quot; corresponds to either A (adenine) or G (guanine).

In the (P1) or (P1-1) oligonucleotide, for example, the nucleotide sequence shown in SEQ ID NO: 1 corresponds to the target gene mutation, which is complementary to either the wild type base or the variant base of the target gene mutation. The base at the base position "corresponds to T (thymine) or C (cytosine).

In (P1) or (P1-1) oligonucleotide, taking the nucleotide sequence shown in SEQ ID NO: 1 as an example, "the base of the base position of the non-target gene mutation" is the 507th position of the nucleotide sequence shown in SEQ ID NO: 1; R corresponds. R in position 507 is A (adenine) in the wild type, but mutated to G (guanine) in the mutant type.

In the (P1) or (P1-1) oligonucleotide, the nucleotide sequence shown in SEQ ID NO: 1 is exemplified by the following. A (adenine) or G (guanine) corresponds to "base at the corresponding base position".

In the (P1) oligonucleotide, taking the nucleotide sequence shown in SEQ ID NO: 1 as an example, the "nucleotide sequence complementary to the nucleotide sequence encoding the target gene" is the 496th to 516th nucleotide sequences encoding the CYP3A4 gene. The base sequence complementary to the base sequence formed from the base of this corresponds.

In the (P1-1) oligonucleotide, taking the nucleotide sequence shown in SEQ ID NO: 1 as an example, "the oligonucleotide having the same nucleotide sequence as the nucleotide sequence encoding the gene of interest '' is, among the nucleotide sequences encoding the CYP3A4 gene, The oligonucleotide which has the same base as the base sequence formed by the 496th-516th base corresponds.

In the (P1 ') or (P1'-1) oligonucleotide, the nucleotide sequence shown in SEQ ID NO: 1 is exemplified as "the base identical to either the wild-type base or the variant base of the target gene mutation". Adenine) or G (guanine).

In the (P1 ') or (P1'-1) oligonucleotide, for example, the nucleotide sequence shown in SEQ ID NO: 1 is &quot; a base different from any of the wild type base and the variant base of non-target gene mutation &quot; Thymine) or C (cytosine).

About (P1 ') or (P1'-1) oligonucleotide, about (P1) or (P1-1) oligonucleotide about the point of that excepting the above in common with (P1) or (P1-1) oligonucleotide All of the information provided for is applicable.

Also, when the base at the base position of the desired gene mutation is one wild type base and one variant type base, one wild type base and two variant bases, and one wild type base and 3 In any case of the base of the variant of the dogs, the "target gene mutation" in the present invention is included.

The oligonucleotide of (P1), (P1-1), (P1 ') or (P1'-1) in this invention satisfy | fills the relationship in any one of following (a)-(f). have.

(a) when the wild-type base at the base position of the non-target gene variant is either A or T, and the variant base of the non-target gene variant is another one of A or T, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases, or which are either wild-type bases or variant bases of said non-target gene variants, are C or G.

(b) when the wild-type base at the base position of the non-target gene variant is one of C and G, and the variant base of the non-target gene variant is the other of C and G, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases or which are either wild-type bases or variant bases of said non-target gene variants are A or T.

(c) when the wild-type base at the base position of the non-target gene mutation is any one of A and C, and the mutant base of the non-target gene mutation is another one of A and C, the wild-type base of the non-target gene mutation And A or C, which is non-complementary to any of the variant bases, is A or C, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or G.

(d) when the wild-type base at the base position of the non-target gene mutation is any one of A and G, and the mutant base of the non-target gene mutation is the other of A and G, the wild-type base of the non-target gene mutation And A or G, which is non-complementary to any of the variant bases, is A or G, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or C.

(e) when the wild type base at the base position of the non-target gene variant is one of T and C, and the variant base of the non-target gene variant is the other of T and C, the wild type base of the non-target gene variant And bases that are non-complementary to any of the variant bases are T or C, and bases other than any of the wild-type bases and variant bases of the non-target gene variant are A or G.

(f) when the wild-type base at the base position of the non-target gene mutation is one of T and G, and the mutant base of the non-target gene mutation is the other of T and G, the wild-type base of the non-target gene mutation And a base non-complementary to any of the variant bases is T or G, and a base other than either the wild-type base or the variant base of the non-target gene variant is A or C.

Further, in the oligonucleotide of P1, P1-1, P1 'or P1'-1 in the present invention, a base is inserted, deleted or substituted into the oligonucleotide of P1, P1-1, P1' or P1'-1. One oligonucleotide is also included in the oligonucleotide in the present invention.

The oligonucleotide to which the base is inserted, deleted or substituted may have an effect equivalent to that of the oligonucleotide of P1, P1-1, P1 'or P1'-1. When the base is inserted, deleted or substituted, the position of the insertion, deletion or substitution is not particularly limited. Examples of the number of bases inserted, deleted or substituted include one or two or more bases, and depending on the length of the oligonucleotide as a whole, for example, there may be mentioned one or ten bases or one or five bases.

As a length of the oligonucleotide of said P1, P1-1, P1 ', or P1'-1 of this invention, the case of 11mer-60mer is mentioned. In the said range, the detection sensitivity of gene mutation will become favorable.

The length of the oligonucleotide P1, P1-1, P1 'or P1'-1 of the present invention can be 12mer-55mer, 15mer-45mer, or 18mer-40mer. It can be in the range of 12mer-55mer. This tends to increase the detection sensitivity, for example.

In addition, by changing the base length of the oligonucleotide of P1, P1-1, P1 'or P1'-1, for example, the oligonucleotide of P1, P1-1, P1' or P1'-1 is the complementary chain (target Tm value which is the dissociation temperature of the hybrid to form) can be adjusted to a desired value.

Hereinafter, the method for designing the genetic variation detection probe of this invention is demonstrated, giving a specific example. However, the present invention is not limited to these.

The nucleotide sequence shown in SEQ ID NO: 3 is a sequence containing rs20542. The base sequence shown in SEQ ID NO: 3 includes a target gene mutation at the 251 nucleotide position and an untarget gene mutation at the 256 nucleotide position in the vicinity thereof.

In this case, the gene mutation detection probe corresponding to the oligonucleotide of (P1) or (P1-1) has a corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 251st base. As a base, a base complementary to either a wild-type base or a mutant base is selected, and as the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 256th base, Non-complementary bases are selected for any of the wild type and variant bases of the genetic variant.

Specifically, in Table 1, the gene mutation detection probe shown in SEQ ID NO: 4 or SEQ ID NO: 5 is mentioned.

In addition, the gene mutation detection probe corresponding to the oligonucleotide of (P1 ') or (P1'-1), in the oligonucleotide of (P1') or (P1'-1) with respect to the 251st base, As the base of the base position, the same base is selected for either the wild-type base or the variant base, and for the 256th base, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) As a base, another base is selected for either wild-type base or variant base of non-target gene mutation.

Specifically, in Table 1, the gene mutation detection probe shown in SEQ ID NO: 6 or SEQ ID NO: 7 may be mentioned.

By using one of the obtained gene mutation detection probes shown in SEQ ID NO: 4 to SEQ ID NO: 7, the presence or absence of the target gene mutation in the 251 th sequence in the nucleotide sequence shown in SEQ ID NO: 3 is 251 th. Even when a nontarget gene mutation exists in the vicinity of the base, it is possible to specifically detect the presence or absence of the 251nd target gene mutation.

In Table 1, the underlined uppercase letters indicate the target gene variants to be detected, and the underlined uppercase letters indicate the nontarget gene variants. The same shall apply hereinafter.

[Table 1]

The nucleotide sequence shown in SEQ ID NO: 8 is a sequence containing rs11881222. In the nucleotide sequence shown in SEQ ID NO: 8, the target gene mutation at the 355th and the nontarget gene mutation at the 364th of the vicinity are included.

In this case, the gene mutation detection probe corresponding to the oligonucleotide of (P1) or (P1-1) has a corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 355th base. As a base, a base complementary to either the wild-type base or the mutant base is selected, and as the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 364th base, Non-complementary bases are selected for any of the wild type and variant bases of the gene variant of interest. Specifically, in Table 2, the gene mutation detection probe shown in SEQ ID NO: 9 or SEQ ID NO: 10 is mentioned.

Further, the gene mutation detection probe corresponding to the oligonucleotide of (P1 ') or (P1'-1) has a corresponding position in the oligonucleotide of (P1') or (P1'-1) with respect to the 355th base. As the base of the base position, the same base is selected for either the wild-type base or the mutant base, and for the 364th base, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) As the base, other bases are selected for either wild-type base or variant base of non-target gene variation. Specifically, in Table 2, the gene mutation detection probe shown in SEQ ID NO: 11 or SEQ ID NO: 12 is mentioned.

By using any of the obtained gene mutation detection probes shown in SEQ ID NO: 9 to SEQ ID NO: 12, the presence or absence of the target gene mutation in the 355th sequence in the nucleotide sequence shown in SEQ ID NO: 8 is 355 Even when a nontarget gene mutation exists in the vicinity of the first base, it is possible to specifically detect the presence or absence of the target gene mutation in the 355th base.

[Table 2]

The nucleotide sequence shown in SEQ ID NO: 13 is a sequence containing rs80230660. The base sequence shown in SEQ ID NO: 13 includes a target gene mutation in 201st and an untargeted genetic mutation in 187th of the vicinity.

In this case, the gene mutation detection probe corresponding to the oligonucleotide of (P1) or (P1-1) has a base position corresponding to the oligonucleotide of (P1) or (P1-1) with respect to the 201st base. As a base, a base complementary to either the wild-type base or the mutant base is selected, and as the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 187th base, Non-complementary bases are selected for any of the wild type and variant bases of the gene variant of interest. Specifically, in Table 3, a gene mutation detection probe shown in SEQ ID NO: 14 or SEQ ID NO: 15 may be mentioned.

In addition, the gene mutation detection probe corresponding to the oligonucleotide of (P1 ') or (P1'-1), in the oligonucleotide of (P1') or (P1'-1) with respect to the 201st base, As the base of the base position, the same base is selected for either the wild-type base or the mutant base, and for the 187th base, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) As the base, another base is selected for either wild-type base or variant base of non-target gene variation. Specifically, in Table 3, the gene mutation detection probe shown in SEQ ID NO: 16 or SEQ ID NO: 17 may be mentioned.

The nucleotide sequence shown in SEQ ID NO: 13 is used to detect the presence or absence of the target gene mutation in the 201st sequence by using any one of the obtained gene mutation detection probes shown in SEQ ID NO: 14 to SEQ ID NO: 17. Even when a non-target gene mutation exists in the vicinity of the base, it is possible to specifically detect the presence or absence of the target gene mutation in the 201st.

[Table 3]

The base sequence shown in SEQ ID NO: 13 includes a target gene mutation at 187th and a nontarget gene mutation at 201 in the vicinity.

In this case, the gene mutation detection probe corresponding to the oligonucleotide of (P1) or (P1-1) has a corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 187th base. As a base, a base complementary to either the wild-type base or the mutant base is selected, and as the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 201st base, Non-complementary bases are selected for any of the wild type and variant bases of the gene variant of interest. Specifically, in Table 4, the gene mutation detection probe shown in SEQ ID NO: 18 is mentioned.

In addition, the gene mutation detection probe corresponding to the oligonucleotide of (P1 ') or (P1'-1) corresponds to the oligonucleotide of (P1') or (P1'-1) with respect to the 187th base. As the base of the base position, the same base is selected for either the wild-type base or the mutant base, and for the 201st base, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) As the base, another base is selected for either wild-type base or variant base of non-target gene variation. Specifically, in Table 4, the gene mutation detection probe shown in SEQ ID NO: 19 may be mentioned.

By using any of the obtained gene mutation detection probes shown in SEQ ID NO: 18 or SEQ ID NO: 19, the presence or absence of the target gene mutation in the 187th sequence in the nucleotide sequence shown in SEQ ID NO: 13 is 187. Even when a non-target gene mutation exists in the vicinity of the first base, it becomes possible to specifically detect the presence or absence of the target gene mutation in the 187th base.

[Table 4]

The nucleotide sequence shown in SEQ ID NO: 20 is a sequence including rs74182491. The base sequence shown in SEQ ID NO: 20 includes a target gene mutation at 247th and a non-target gene mutation at 251th.

In this case, the gene mutation detection probe corresponding to the oligonucleotide of (P1) or (P1-1) has a corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 247th base. As a base, a base complementary to either the wild-type base or the mutant base is selected, and as the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) with respect to the 251st base, Non-complementary bases are selected for any of the wild type and variant bases of the gene variant of interest. Specifically, in Table 5, the gene mutation detection probe shown in SEQ ID NO: 21 or SEQ ID NO: 22 may be mentioned.

Further, the gene mutation detection probe corresponding to the oligonucleotide of (P1 ') or (P1'-1), in the oligonucleotide of (P1') or (P1'-1) with respect to the 247th base, As the base of the base position, the same base is selected for either the wild-type base or the mutant base, and for the 251st base, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) As the base, other bases are selected for either wild-type base or variant base of non-target gene variation. Specifically, in Table 5, the gene mutation detection probe shown in SEQ ID NO: 23 or SEQ ID NO: 24 may be mentioned.

By using any of the gene mutation detection probes shown in SEQ ID NO: 21 to SEQ ID NO: 24, the presence or absence of the target gene mutation in the 247th sequence from the nucleotide sequence shown in SEQ ID NO: 20 is determined. Even when there is a non-target gene mutation in the vicinity of the base, it becomes possible to specifically detect the presence or absence of the target gene mutation at the 247th.

[Table 5]

Further, according to the present invention, not only when the non-target gene mutation present in the vicinity of the target gene mutation is a single base substitution mutation as described above, but also when a deletion mutation or an insertion mutation is present, the detection sensitivity is high. Probe for detecting genetic variation can be provided.

If the non-target gene mutation is a deletion mutation, the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) (ie, (P1) or (located at the base position corresponding to the deleted base) ( As the base of P1-1), a gene corresponding to the oligonucleotide of (P1) or (P1-1) according to the present invention is selected by selecting the deleted base and the non-complementary base, and otherwise following the method described above. The probe for detecting a mutation can be obtained. Specifically, when A is a deletion, the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) (that is, (P1) located at the base position corresponding to the deleted base A). Or (P1-1) base) may be at least one base selected from A, C, and G which are bases not complementary to A.

Similarly, if the non-target gene mutation is a deletion mutation, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) (ie, located at the base position corresponding to the deleted base ( As the base of P1 ') or (P1'-1)), a base different from the deleted base is selected, and the others are followed by the above-described method, so that of (P1') or (P1'-1) A gene mutation detection probe corresponding to an oligonucleotide can be obtained. Specifically, when A is a deletion, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) (i.e., located at the base position corresponding to the deleted base A) What is necessary is just to make P1 ') or the base of (P1'-1)) into at least 1 base chosen from G, T, and C which are bases different from A.

If the non-target gene mutation is an insert mutation, the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) (ie, (P1) or (P1 located at the base position corresponding to the inserted base) -1) base non-complementary to the inserted base, and the other method is followed by the above-described method, to detect the genetic mutation corresponding to the oligonucleotide of (P1) or (P1-1) according to the present invention Probe for this can be obtained. Specifically, in the case where the G is inserted, the base of the corresponding base position in the oligonucleotide of (P1) or (P1-1) (that is, (P1) located at the base position corresponding to the inserted base G). Or (P1-1) base) may be at least one base selected from A, T, and G which are bases complementary to G.

Similarly, if the non-target gene mutation is an insert mutation, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) (ie, the base position corresponding to the inserted base (P1) As the base of ') or (P1'-1)), an oligonucleotide of (P1') or (P1'-1) according to the present invention is selected by selecting a base different from the inserted base and following the above-described method. A gene mutation detection probe corresponding to a nucleotide can be obtained. Specifically, in the case where the G-inserted mutation, the base of the corresponding base position in the oligonucleotide of (P1 ') or (P1'-1) (that is, located at the base position corresponding to the inserted base G) ( What is necessary is just to make P1 ') or the base of (P1'-1)) into at least 1 base chosen from A, T, and C which are bases different from G.

The (P1), (P1-1), (P1 ') or (P1'-1) oligonucleotide according to the present invention may be a fluorescently labeled oligonucleotide. The fluorescently labeled oligonucleotide may be a fluorescently labeled oligonucleotide in which the fluorescence intensity when hybridizing to the target sequence is reduced (quenched) or increased as compared to the fluorescence intensity when the hybridized to the target sequence is not hybridized. . In particular, a fluorescently labeled oligonucleotide may be used in which the fluorescence intensity when hybridizing to the target sequence is reduced compared to the fluorescence intensity when the hybridization is not performed to the target sequence. A probe using such a fluorescence quenching phenomenon is generally called a guanine quenching probe and is known as QProbe (registered trademark). In particular, the oligonucleotide may be designed such that the 3 'end or 5' end is C, and the C at the end thereof is an oligonucleotide labeled with a fluorescent dye so that light emission becomes weak when it approaches G.

 In addition to the detection method using QProbe, a known detection mode may be applied. Examples of such a detection mode include a Taq-man Probe method, a Hybridization Probe method, a Molecular Beacon method, an MGB Probe Method, and the like.

Although it does not restrict | limit especially as said fluorescent dye, For example, a fluorescein, a phosphor, rhodamine, a polymethine pigment derivative, etc. are mentioned. As a commercial item of these fluorescent dyes, Pacific Blue, Bodipy FL, FluorePrime, Fluoredite, FAM, Cy3, Cy5, TAMRA, etc. are mentioned, for example.

The detection conditions of the fluorescently labeled oligonucleotide are not particularly limited and can be appropriately determined depending on the fluorescent dye to be used. For example, Pacific Blue can detect 445 nm-480 nm, TAMRA can detect 585 nm-700 nm, and Bodipy FL can detect 520 nm-555 nm.

By using a probe having such a fluorescent dye, hybridization and dissociation can be easily confirmed by variation of each fluorescent signal. The binding of the fluorescent dye to the oligonucleotide can be performed according to a conventional method, for example, the method described in JP-A-2002-119291.

In the fluorescently labeled oligonucleotide, for example, a phosphate group may be added to the 3 'end. By adding a phosphate group to the 3 'end of the fluorescently labeled oligonucleotide, it is possible to sufficiently suppress that the probe itself is elongated by a gene amplification reaction. As mentioned later, DNA (target DNA) which detects the presence or absence of a mutation can be prepared by gene amplification methods, such as PCR. At that time, by using a fluorescently labeled oligonucleotide having a phosphoric acid group added to the 3 'end, it can coexist in the reaction solution of the amplification reaction.

Similar effects can also be obtained by adding the above-mentioned labeling substance (fluorescent dye) to the 3 'end.

The oligonucleotide of P1, P1-1, P1 'or P1'-1 is a target for detection without being affected by gene mutations other than the detection target, even if the nucleotide sequence encoding the gene contains a plurality of gene mutations. It can be used as a gene mutation detection probe for specifically detecting a specific gene mutation.

Moreover, the said gene mutation detection probe can be used as a probe for a melting curve analysis.

<Primer>

In the gene mutation detection method of the present invention described later, a primer can be used when amplifying a sequence containing a target gene mutation to be detected by PCR.

The primer used in the present invention is a nucleic acid containing a base corresponding to the 501st base and the 507th base in the base sequence shown in SEQ ID NO: 1, for example, a site of gene mutation of a target gene to be detected. If possible, the amplification is not particularly limited.

The primer to be applied to the PCR method is not particularly limited as long as it can amplify a region capable of hybridizing the gene mutation detection probe of the present invention. For example, those skilled in the art can appropriately use the nucleotide sequence shown in SEQ ID NO: 1. Can be designed. The length and the Tm value of the primer can be usually 12mer to 40mer and 40 ° C to 70 ° C, or 16mer to 30mer and 55 ° C to 60 ° C.

In addition, although the length of each primer of a primer set does not need to be the same, the Tm value of both primers may be substantially the same (or the difference in both primers of Tm value may be less than 5 degreeC).

The method for detecting the genetic variation is not particularly limited as long as the method uses the fluorescently labeled nucleotides as a probe. As an example of a gene mutation detection method using the oligonucleotide of P1, P1-1, P1 'or P1'-1 as a probe, a gene mutation detection method using Tm analysis will be described below.

<Gene mutation detection method>

Gene mutation detection method according to the present invention is a gene mutation detection probe containing an oligonucleotide of the fluorescently labeled P1, P1-1, P1 'or P1'-1 (hereinafter referred to as "fluorescent labeled gene mutation detection probe Gene mutation detection method using at least one).

According to the method for detecting genetic variation according to the present invention, by including at least one fluorescently labeled probe for detecting genetic variation, the genetic variation can be detected easily and with high sensitivity.

In addition, the gene mutation detection method of the present invention is a method for detecting gene mutation in various human genes, and may include the following steps (I) to (IV), and may include the following step (V). The gene mutation detection method of the present invention is characterized by using the above gene mutation detection probe, and other configurations, conditions, and the like are not limited to the following description.

(I) A hybrid is obtained by contacting the fluorescently labeled gene mutation detection probe and a single-stranded nucleic acid in a sample.

(II) Dissociating the hybrid by changing the temperature of the sample containing the hybrid, and measuring the fluctuation of the fluorescence signal based on the dissociation of the hybrid.

(III) Detecting the Tm value which is the dissociation temperature of a hybrid based on the fluctuation of the said fluorescence signal.

(IV) Detecting the presence of the target gene mutation in the single-stranded nucleic acid in the sample based on the Tm value.

(V) Determining the abundance of single-stranded nucleic acid having a genetic variation in the total single-stranded nucleic acid in the sample based on the presence of the genetic variation.

Moreover, in this invention, in addition to the said process (I)-(IV) or the said process (I)-(V), before obtaining the hybrid of a process (I), or simultaneously with obtaining a hybrid of a process (I), a nucleic acid is carried out. It may further include amplifying.

In addition, measuring the Tm value in (III) includes not only measuring the dissociation temperature of the hybrid, but also measuring the magnitude of the derivative value of the fluorescent signal that varies with temperature at the time of melting of the hybrid forming body. do.

In the present invention, the nucleic acid in the sample may be a single stranded nucleic acid or a double stranded nucleic acid. In the case where the nucleic acid is a double-stranded nucleic acid, for example, prior to hybridizing with the fluorescently labeled gene mutation detection probe, the double-stranded nucleic acid in the sample is heated to dissociate (dissociate) into a single-stranded nucleic acid. It may contain. By dissociating the double-stranded nucleic acid into single-stranded nucleic acids, hybridization with the fluorescently labeled gene mutation detection probes becomes possible.

In the present invention, the nucleic acid contained in the sample to be detected may be, for example, a nucleic acid originally contained in a biological sample. However, since the detection accuracy can be improved, the nucleic acid originally contained in the biological sample is used as a template. Or an amplification product obtained by amplifying a region including a region of mutated gene of interest. Although the length of an amplification product is not specifically limited, For example, it can be 50mer-1000mer, or 80mer-200mer. In addition, the nucleic acid in a sample may be cDNA synthesize | combined by RT-PCR (Reverse Transcription PCR) from RNA (total RNA, mRNA, etc.) derived from a biological sample, for example.

In the present invention, the addition ratio (molar ratio) of the gene mutation detection probe of the present invention to the nucleic acid in the sample is not particularly limited. The DNA in the sample can be, for example, 1 times or less. In addition, from the viewpoint of ensuring a sufficient detection signal, the addition ratio (molar ratio) of the gene mutation detection probe of the present invention to the nucleic acid in the sample can be 0.1 times or less.

Here, the nucleic acid in the sample may be, for example, the sum of the detection target nucleic acid in which the gene mutation for detection has occurred and the non-detection nucleic acid in which the gene mutation has not occurred, or the gene mutation for detection purpose is generated. The sum of the amplification product containing the sequence to be detected and the amplification product containing the non-detection sequence in which the gene mutation does not occur may be sufficient. In addition, although the ratio of the said detection target nucleic acid in the nucleic acid in a sample is not known previously, As a result, the addition ratio (molar ratio) of the said genetic mutation detection probe is a detection target nucleic acid (containing a detection target sequence) 10 times or less with respect to the amplification product). In addition, the addition ratio (molar ratio) of the said polymorphism detection probe can be 5 times or less, or 3 times or less with respect to the nucleic acid to be detected (amplification product containing a sequence to be detected). The lower limit thereof is not particularly limited, but may be, for example, 0.001 times or more, 0.01 times or more, or 0.1 times or more.

The addition ratio of the fluorescently labeled gene mutation detection probe in the present invention to the DNA may be, for example, a molar ratio with respect to the double-stranded nucleic acid or a molar ratio with respect to the single-stranded nucleic acid.

In the present invention, the measurement of the signal variation due to the temperature change for determining the Tm value may be performed by absorbance measurement at 260 nm from the principle described above, but the label added to the fluorescently labeled gene mutation detection probe. As a signal based on the signal of, the signal which fluctuates according to the state of hybrid formation between a single-stranded DNA and the said gene mutation detection probe is measured. To this end, the fluorescently labeled oligonucleotide described above may be used as the fluorescently labeled gene mutation detection probe. As an oligonucleotide of fluorescently labeled P1, P1-1, P1 'or P1'-1 (hereinafter also referred to as "fluorescently labeled oligonucleotide" at once), for example, fluorescence intensity when not hybridized to a target sequence Compared to the fluorescent label oligonucleotide which decreases (quenches) the fluorescence intensity when hybridizing to the target sequence, or the fluorescence intensity when not hybridizing to the target sequence, And a fluorescently labeled oligonucleotide which increases in fluorescence intensity when present.

In the case of a fluorescently labeled oligonucleotide such as the former, the fluorescent signal does not show when the hybrid (double-stranded DNA) is formed with the sequence to be detected, or the fluorescent signal is weak. Or the fluorescence signal is increased.

In the latter fluorescently labeled oligonucleotide, a fluorescent signal is generated by forming a hybrid (double-stranded DNA) with a detection target sequence, and when the fluorescent labeled oligonucleotide dissociates by heating, the fluorescent signal decreases (dissipates). Therefore, by detecting the change in the fluorescence signal based on the fluorescent label under conditions specific to the fluorescent label (fluorescence wavelength or the like), the advancing of the melting and the Tm value can be determined similarly to the absorbance measurement at 260 nm.

Next, the method for detecting the change in the signal based on the fluorescent dye will be described with reference to the gene mutation detection method of the present invention with specific examples. The gene mutation detection method of the present invention is characterized by the use of the fluorescently labeled probe for detecting gene mutation, and is not limited to other processes and conditions at all.

The sample containing the nucleic acid as a template at the time of nucleic acid amplification only needs to contain a nucleic acid, in particular, a gene of interest, and is not particularly limited. For example, tissues such as colon or lung, blood cells such as white blood cells, whole blood, plasma, sputum, oral mucosa suspension, somatic cells such as nails and hair, germ cells, milk, ascites fluid, paraffin embedded tissue, gastric juice, gastric lavage fluid, And those derived from, or derived from, any biological origin such as urine, peritoneal fluid, amniotic fluid, cell culture, and the like. In addition, the sample collection method, the preparation method of the sample containing a nucleic acid, etc. are not restrict | limited, Any conventionally well-known method can be used. In addition, the nucleic acid to be used as a template can be used either directly or after pretreatment in order to modify the characteristics of the sample.

For example, when whole blood is used as a sample, isolation of genomic DNA from whole blood can be performed by a conventionally well-known method. For example, a commercial genomic DNA isolation kit (trade name GFX Genomic Blood DNA Purification kit; manufactured by GE Healthcare Bioscience Co., Ltd.) may be used.

Next, a fluorescently labeled gene mutation detection probe containing the fluorescently labeled oligonucleotide is added to the sample containing the isolated genomic DNA.

The fluorescently labeled gene mutation detection probe may be added to a liquid sample containing isolated genomic DNA or mixed with genomic DNA in a suitable solvent. As the solvent, not particularly limited, and for example may be mentioned such as Tris-HCl buffer, KCl, MgCl _2, MgSO _4, glycerol, and the like, such as solvent, PCR reaction solution containing a conventionally known of.

The timing of addition of the fluorescently labeled gene mutation detection probe is not particularly limited. For example, when amplification processing such as PCR described below is performed, the amplification processing may be added to the PCR amplification product after the amplification processing. You may add before.

As described above, when the fluorescently labeled gene mutation detection probe is added before the amplification process by PCR or the like, for example, as described above, a fluorescent dye or a phosphate group can be added to the 3 'end thereof. .

As a method of nucleic acid amplification, the method of using a polymerase, etc. are mentioned, for example. Examples thereof include PCR method, ICAN method, LAMP method, NASBA method and the like. As amplification by a method using a polymerase, amplification is performed in the presence of a fluorescently labeled gene mutation detection probe in the present invention. It is easy for a person skilled in the art to adjust the reaction conditions and the like of amplification according to the fluorescently labeled gene mutation detection probe and polymerase to be used. As a result, the gene mutation can be detected only by analyzing the Tm value of the fluorescently labeled gene mutation detection probe after amplification of the nucleic acid. Therefore, it is not necessary to separate the amplification product after the reaction is completed. Therefore, there is no worry of contamination by the amplification product. In addition, since it can be detected by a device such as a device required for amplification, there is no need to move the container, and automation is easy.

As the DNA polymerase to be used in the PCR method, a DNA polymerase which is usually used can be used without particular limitation. For example, GeneTaq (made by Nippon Jinjin), Prime STAR Max DNA Polymerase (made by Takara Bio Co., Ltd.), Taq polymerase, etc. are mentioned.

The amount of polymerase to be used is not particularly limited as long as it is a concentration normally used. For example, when using Taq polymerase, it can be made into the density | concentration of 0.01U-100U with respect to 50 microliters of reaction solutions, for example. This tends to increase the detection sensitivity of the target gene mutation.

In addition, a PCR method can be performed by selecting suitably the conditions used normally.

In addition, when amplifying, amplification can be monitored by real-time PCR to examine the copy number of the DNA (sequence to be detected) contained in the sample. That is, the proportion of probes that form hybrids increases with amplification of DNA (detection sequence) by PCR, so that the fluorescence intensity fluctuates. By monitoring this, the copy number and abundance ratio of the detection target sequence (normal DNA or mutated DNA) contained in a sample can be detected.

In the genetic mutation detection method of the present invention, the fluorescently labeled oligonucleotide and a single-stranded nucleic acid in a sample are contacted to hybridize both. The single-stranded nucleic acid in the sample can be prepared, for example, by dissociating the PCR amplification product obtained as described above.

The heating temperature in the dissociation (dissociation step) of the PCR amplification product is not particularly limited as long as it is a temperature at which the amplification product can be dissociated. For example, it is 85 degreeC-95 degreeC. The heating time is not particularly limited either. Heating time can be made into 1 second-10 minutes, or 1 second-5 minutes, for example.

In addition, hybridization of the dissociated single-stranded DNA with the fluorescently labeled oligonucleotide can be performed by, for example, lowering the heating temperature in the dissociation step after the dissociation step. As temperature conditions, they are 40 degreeC-50 degreeC, for example.

The volume and the concentration of each composition in the reaction solution of the hybridization step are not particularly limited. As a specific example, the density | concentration of DNA in the said reaction liquid can be 0.01 micrometer-1 micrometer, or 0.1 micrometer-0.5 micrometer, for example. The concentration of the fluorescently labeled oligonucleotide is, for example, a range that satisfies the addition ratio to the DNA, and may be, for example, 0.001 μM to 10 μM, or 0.001 μM to 1 μM.

Then, the hybrid of the obtained single-stranded DNA and the fluorescently labeled oligonucleotide is gradually heated, and the fluctuation of the fluorescence signal accompanying the temperature rise is measured. For example, when QProbe is used, in the state of hybridization with single-stranded DNA, the fluorescence intensity is reduced (or quenched) as compared with the dissociated state. Therefore, for example, what is necessary is just to gradually heat the hybrid whose fluorescence is decreasing (or quenching), and to measure the increase in fluorescence intensity accompanying a temperature rise.

 Although the temperature range at the time of measuring the fluctuation | variation of fluorescence intensity | strength is not restrict | limited, For example, start temperature can be room temperature -85 degreeC or 25 degreeC-70 degreeC. End temperature can be 40 degreeC-105 degreeC, for example. In addition, the rate of temperature rise is not particularly limited, but may be, for example, 0.1 ° C./second to 20 ° C./second or 0.3 ° C./second to 5 ° C./second.

Next, the variation of the signal is analyzed and determined as a Tm value. Specifically, the derivative value (-d fluorescence intensity / dT) at each temperature can be calculated from the obtained fluorescence intensity, and the temperature showing the lowest value can be determined as the Tm value. Further, the point where the fluorescence intensity increase amount (fluorescence intensity increase amount / t) per unit time is the highest can be determined as the Tm value. As a fluorescently labeled probe for detecting a genetic variation, instead of a quenching probe, when a probe whose signal intensity increases due to hybrid formation is used, the amount of decrease in fluorescence intensity may be measured on the contrary.

In the present invention, the hybrid is heated as described above, and the fluorescence signal fluctuation (preferably an increase in fluorescence intensity) is measured in accordance with the temperature rise, but instead of this method, for example, at the time of hybrid formation, Signal fluctuations may be measured. That is, you may measure the fluorescence signal fluctuation accompanying the said temperature fall at the time of forming a hybrid by reducing the temperature of the sample which added the said probe.

As a specific example, when QProbe is used, when the probe is added to a sample, since the probe is in a dissociated state, the fluorescence intensity is large, but when the hybrid is formed by a drop in temperature, the fluorescence is reduced (or quenched). ) do. Therefore, for example, the temperature of the heated sample is gradually lowered, and the decrease in fluorescence intensity accompanying the temperature drop may be measured.

On the other hand, when a probe having a signal increasing due to hybrid formation is used, when the probe is added to a sample, since the probe is in a dissociated state, the fluorescence intensity is small (or quenched). , The fluorescence intensity is increased. Therefore, for example, the temperature of the sample may be gradually lowered, and an increase in fluorescence intensity accompanying the temperature drop may be measured.

The method for detecting a plurality of gene mutations in the same system is not particularly limited. For example, each probe which can detect each genetic variation may be mixed beforehand, and may be added to a sample, and each probe which can detect each genetic variation may be continuously added to the sample containing single-stranded nucleic acid.

Here, "system" means one independent reaction system formed from a sample containing hybrid hybridized from fluorescently labeled oligonucleotide and single-stranded nucleic acid.

<Reagent Kit for Gene Mutation Detection>

The reagent kit for detecting a genetic mutation of the present invention includes the above-described probe for detecting a genetic mutation.

In the reagent kit for detecting a genetic variation, even if the nucleotide sequence encoding the gene contains a plurality of genetic variations, the specific genetic variation to be detected is specifically detected without being affected by the genetic variation other than the detection target. By including at least one kind of gene mutation detection probe of a technology that can be used, there is a tendency that detection of gene mutation can be performed more easily.

When two or more types of oligonucleotides are included as a gene mutation detection probe, each oligonucleotide may be included in a mixed state or separately.

Two or more kinds of the fluorescent label oligonucleotides may be labeled with fluorescent dyes having different emission wavelengths.

By changing the kinds of fluorescent dyes as described above, detection of each fluorescently labeled oligonucleotide can be performed simultaneously even in the same reaction system.

In addition, the reagent kit for detecting genetic variation in the present invention may further include a primer for amplifying a nucleotide sequence having a region hybridized by the gene mutation detecting probe.

In addition, the above-mentioned matters can be applied as it is about the probe and primer which can be included in the reagent kit for detecting the genetic variation.

In addition, the reagent kit for detecting a gene mutation in the present invention may further include reagents necessary for performing nucleic acid amplification in the detection method of the present invention, in addition to the gene mutation detection probe and the primer. In addition, gene mutation detection probes, primers and other reagents may be accommodated separately, or a part of them may be a mixture.

In addition, "accommodating separately" should just be divided | segmented so that each reagent can maintain a non-contact state, and does not necessarily need to be contained in the individual container which can be handled independently.

By including a primer set for amplifying a base sequence (a region hybridized by a probe for detecting a gene mutation) including a base showing a gene mutation, the gene mutation can be detected with a higher sensitivity, for example.

In addition, the reagent kit for detecting a genetic variation of the present invention, using the gene mutation detection probe, generates a differential melting curve for a sample containing a nucleic acid to be detected, analyzes its Tm value, and encodes a gene. The instruction manual describing detecting the genetic variation in the sequence, or the instruction manual described with respect to various reagents that may be included or additionally included in the reagent kit for detecting the genetic variation may be included.

[Example]

Hereinafter, the present invention will be described in detail in the Examples. However, the present invention is not limited to them at all.

 Example 1

Tm analysis was performed using the fully automatic SNPs inspection apparatus (brand name i-densy (trademark), the product made by AKray Co., Ltd.), and the test reagent of the prescription shown in Table 6 below.

(Reaction amount: 50 µl) 1 × Gene Taq Universal buffer ※
Probe: 3PB-CYP3A4 * 1B-R3
Mold (Complementary Offset)
0.2 μM
0.2 μM

※ Nippon Jinsha

The details of the probe (SEQ ID NO: 2) used in Table 6 are shown in Table 7 below, and the details of the template (complementary chain) (ID-1 to ID-16) are shown in Tables 8 and 9 below. In addition, the parenthesis of the 3 'terminal in a probe shows the kind of fluorescent dye.

The Tm value was calculated under Meltcalc 99 free (http://www.meldcalc.com/) under conditions of setting conditions: Oligoconc [μM] 0.2 and Na eq. [MM] 50.

In Table 7, Tm (WT) represents the Tm value when the template is wild type with respect to the genetic variation of detection object, and Tm (mt) represents the Tm value when the template is mutant with respect to the genetic variation of detection object. Indicates. Δ represents the difference between the Tm values of mt (variant type) and WT (wild type).

 [Table 7]

[Table 8]

TABLE 9

The Tm analysis was performed at 95 ° C. for 1 second and 40 ° C. for 60 seconds, and then the temperature was increased from 40 ° C. to 80 ° C. at a rate of temperature increase of 1 ° C./3 sec. Was measured.

The excitation wavelength of the fluorescent dye Pacific Blue was 365 nm to 415 nm, and the detection wavelength was 445 nm to 480 nm. The change in fluorescence intensity derived from the fluorescent labeling probe was measured for each sample according to each wavelength.

By Tm analysis, FIG. 2 (A)-(P) which showed the amount of change of the fluorescence value of a probe were obtained. In the figure, the vertical axis represents the amount of change in fluorescence intensity (d fluorescent intensity increase / t) per unit time, and the horizontal axis represents temperature (° C.), respectively. In Fig. 2, (A) is a mold ID-1, (B) is a mold ID-2, (C) is a mold ID-3, (D) is a mold ID-4, (E) is a mold ID-5, (F) as template ID-6, (G) as template ID-7, (H) as template ID-8, (I) as template ID-9, (J) as template ID-10, (K) as template ID-11, (L) as template ID-12, (M) as template ID-13, (N) as template ID-14, (O) as template ID-15 and (P) are figures which show the amount of change in fluorescence value when ID-16 is used as a template, respectively.

When the probe shown in SEQ ID NO: 2 is used from the results of Figs. 2 (A) to (P), the 501th subject to be detected is the 501st sequence of the base sequence shown in SEQ ID NO: 1 regardless of the presence or absence of nontarget gene mutation. With respect to the base, it has been found that the presence or absence of a genetic mutation can be detected.

[Example 2]

The Tm analysis was performed using the fully automatic SNPs tester IS-5310 (trade name i-densy (trademark), manufactured by Akray Co., Ltd.) and the reagents for testing the prescriptions shown in Table 10 below. In addition, even if the prescription is changed to the probe used in Example 2 and the same experiment is performed, equivalent results are obtained.

[Table 10]

 The Tm analysis was performed at 95 ° C. for 1 second and 40 ° C. for 60 seconds, and then the temperature was increased from 40 ° C. to 75 ° C. at a rate of temperature increase of 1 ° C./3 sec. Was measured. The excitation wavelength was set at 420 nm to 485 nm and the measurement wavelength was set at 520 nm to 555 nm, and the change in fluorescence intensity derived from the fluorescent labeling probe was measured, respectively.

 TABLE 11

 The detail of the probe and single-stranded nucleic acid of the said description is shown below.

[Table 12]

As a result, by using the gene mutation detection probe according to the present invention, wild type (the 234th base of SEQ ID NO: 29 is G) 1 and 2, and variant (the 234th base of the SEQ ID NO: 29 is C) 1 and It was confirmed that the peaks at both sides could be detected. In addition, even when the mixed type 1-4 which mixed 1: 1 wild-type nucleic acid of a human genome type and single-stranded nucleic acid of a human genome type | mold was used, the peak was obtained by using the genetic mutation detection probe by this invention. It was confirmed that it could be detected.

The Tm values of wild type 1 and wild type 2 were 50 ° C, and the Tm values of variant 1 and variant 2 were 57 ° C.

In addition, the Tm values of mixed type 1 are 50 ° C and 56 ° C, the Tm values of mixed type 2 are 50 ° C and 56 ° C, the Tm values of mixed type 3 are 48 ° C and 56 ° C, and the Tm values of mixed type 4 are 49 ° C and 57 ° C. ° C.

3 shows wild type 1 (A), wild type 2 (C), variant 1 (B), variant 2 (D), mixed type 1 (E), mixed type 2 (F), and mixed type 3 (G) obtained by Tm analysis. ) And mixed type 4 (H) are shown, respectively. The vertical axis represents the temperature differential value of the fluorescence intensity, and the horizontal axis represents the temperature, respectively.

Comparative Example 1

Tm analysis was performed using the fully automatic SNPs test | inspection apparatus (brand name i-densy (trademark), the product made by AKRAY Co., Ltd.), and the test reagent of the prescription shown in following Table 13.

(Reaction amount: 50 µl) 1 × Gene Taq Universal buffer ※
Probe: 3PB-CYP3A4 * 1B-R3TK
Mold (Complementary Offset)
0.2 μM
0.2 μM

※ Nippon Jinsha

The details of the probe (SEQ ID NO: 35) used in Table 13 are shown in Table 14 below, and the details of the template (complementary chain) (ID-17 to ID-32) are shown in Table 15 below. In addition, the parenthesis of the 3 'terminal in a probe shows the kind of fluorescent dye.

[Table 14]

TABLE 15

The Tm analysis was performed at 95 ° C. for 1 second and 40 ° C. for 60 seconds, and then the temperature was increased from 40 ° C. to 80 ° C. at a rate of temperature increase of 1 ° C./3 sec. Was measured.

By Tm analysis, FIG. 4 which showed the change amount of the fluorescence value of a probe was obtained. In the figure, the vertical axis represents the amount of change in fluorescence intensity (d fluorescent intensity increase / t) per unit time, and the horizontal axis represents the temperature (° C), respectively. In Fig. 4, (A) is a mold ID-17, (B) is a mold ID-18, (C) is a mold ID-19, (D) is a mold ID-20, (E) is a mold ID-21, (F) as template ID-22, (G) as template ID-23, (H) as template ID-24, (I) as template ID-25, (J) as template ID-26, (K) as template ID-27, (L) as template ID-28, (M) as template ID-29, (N) as template ID-30, (O) as template ID-31, (P) is a figure which shows the change amount of the fluorescence value when ID-32 is used as a template, respectively.

As a result, when the probe used in Comparative Example 1 was used, the presence or absence of non-target gene mutations was affected. Thus, among the nucleotide sequences shown in SEQ ID NO. It has been found to be difficult to detect the presence or absence.

As described above, according to the present invention, from a plurality of gene mutations included in a base sequence encoding a gene, only a specific gene mutation to be detected is specifically affected without being affected by the base type of the non-target gene mutation. It was found that it can be detected.

As for the indication of Japanese patent application 2011-102987 (application date May 2, 2011) and Japanese patent application 2011-218114 (application date September 30, 2011), the whole is integrated in this specification by reference.

All documents, patent applications, and technical specifications described herein are incorporated by reference to the same extent as if the individual documents, patent applications, and technical specifications were specifically and individually described by reference.

                         SEQUENCE LISTING <110> ARKRAY, INC.   <120> GENE MUTATION DETECTION PROBE, METHOD OF DETECTING GENE MUTATION AND GENE MUTATION DETECTION REAGENT KIT <130> P1433A <150> JP2011-218114 <151> 2011-09-30 <150> JP2011-102987 <151> 2011-05-02 <150> JP2012-082391 <151> 2012-03-30 <160> 35 <170> PatentIn version 3.4 <210> 1 <211> 701 <212> DNA <213> Homo sapiens <220> <221> misc_feature (222) (491) .. (491) R is g or a. <220> <221> misc_feature <222> (501) .. (501) R is g or a. <220> <221> misc_feature (507) (507) .. (507) R is g or a. <400> 1 cagctgaggc acagccaaga gctctggctg tattaatgac ctaagaagtc accagaaagt 60 cagaagggat gacatgcaga ggcccagcaa tctcagctaa gtcaactcca ccagcctttc 120 tagttgccca ctgtgtgtac agcaccctgg tagggaccag agccatgaca gggaataaga 180 ctagactatg cccttgagga gctcacctct gttcagggaa acaggcgtgg aaacacaatg 240 gtggtaaaga ggaaagagga caataggatt gcatgaaggg gatggaaagt gcccagggga 300 ggaaatggtt acatctgtgt gaggagtttg gtgaggaaag actctaagag aaggctctgt 360 ctgtctgggt ttggaaggat gtgtaggagt cttctagggg gcacaggcac actccaggca 420 taggtaaaga tctgtaggtg tggcttgttg ggatgaattt caagtatttt ggaatgagga 480 cagccataga racaagggca rgagagrggc gatttaatag attttatgcc aatggctcca 540 cttgagtttc tgataagaac ccagaaccct tggactcccc agtaacattg attgagttgt 600 ttatgatacc tcatagaata tgaactcaaa ggaggtcagt gagtggtgtg tgtgtgattc 660 tttgccaact tccaaggtgg agaagcctct tccaactgca g 701 <210> 2 <211> 21 <212> DNA <213> Artificial <220> <223> 3PB-CYP3A4 * 1B-R3 <400> 2 taaatcgccg ctctcctgcc c 21 <210> 3 <211> 501 <212> DNA <213> Artificial <220> <223> SEQ3 <220> <221> misc_feature <251> (251) .. (251) R is g or a. <220> <221> misc_feature (222) (256) .. (256) S is g or c. <220> <221> misc_feature <222> (260) .. (260) Y is t or c. <220> <221> misc_feature (273) .. (273) S is g or c. <400> 3 gggctcgagg cgtcccccgg ggagtcgcct cttagcggtg cgtccgggct agcggcgagg 60 ggccgcccca agtcttccca ccgccgccac cttagcagcc cgacttgggg cctggaaagt 120 ggagcacgcg gaggtgggag ggccctgcac gcggcccccg gtggggaagg ggacgggcca 180 gggattcaga ctcgggctct cccctcagga tgcagcaccg aggcttcctc ctcctcaccc 240 tcctcgccct rctggsgcty acctccgcgg tcsccaaaaa gaaaggtgat gggggatgat 300 cgaaggaggg ctggggacgg gcaggcgagg cccctccact tctgggctgg gccgcctggg 360 ttcctagcct ggaaccccag gaaggcggct cccgagggag tctccccgtg ccccagtcct 420 gaactctgtt cctcgcgcgt tgtagataag gtgaagaagg gcggcccggg gagcgagtgc 480 gctgagtggg cctgggggcc c 501 <210> 4 <211> 17 <212> DNA <213> Artificial <220> SEQ 4 <400> 4 ctccagcagg gcgagga 17 <210> 5 <211> 17 <212> DNA <213> Artificial <220> SEQ 5 <400> 5 caccagcagg gcgagga 17 <210> 6 <211> 18 <212> DNA <213> Artificial <220> SEQ 6 <400> 6 cctcgcccta ctggagct 18 <210> 7 <211> 18 <212> DNA <213> Artificial <220> SEQ 223 <400> 7 cctcgcccta ctggtgct 18 <210> 8 <211> 588 <212> DNA <213> Homo sapiens <220> <221> misc_feature <222> (322) .. (322) R is g or a. <220> <221> misc_feature <222> (355) .. (355) R is g or a. <220> <221> misc_feature <222> (364) .. (364) R is g or a. <220> <221> misc_feature <222> (370) .. (370) R is g or a. <400> 8 ctgctcagag ctcacagacc tgggtgcccg ggccctgacg actcacacag gcccggagct 60 gggagaggat atggtgcagg gtgtgaaggg gctggtccaa gacatccccc agggctgggt 120 cagtgtcagc ggtggcctcc agaaccttca gcgtcagggc cagctcagcc tccaaagcca 180 cggggcgctc cctcacctga ggagaggtga gaaagagcag gtgagggggg aggtgagggg 240 aacaggttgg gggaggagga tagagaggaa caagtgaagg tgacaggcac aggggagagg 300 gcacagccag tgtggtcagg trggagcaga gggaaggggt agcaggtgtg gggaraggag 360 agarggacar tggagaagga gaaggtgaag gggccactac agagccaggt gagcagggct 420 gggagggcag gggtgggcct gactccccct ctcacctgca gctgcctcag gtcccaggtc 480 ctggggaaga ggcgggagcg gcacttgcag tccttcagca gaagcgactc ttcctagaca 540 gcaaaggcac aggttagccc cagcaggagg ggtggaggtt agaccact 588 <210> 9 <211> 18 <212> DNA <213> Artificial <220> SEQ 9 <400> 9 tccatctctc ctctcccc 18 <210> 10 <211> 18 <212> DNA <213> Artificial <220> SEQ 223 <400> 10 tccgtctctc ctctcccc 18 <210> 11 <211> 18 <212> DNA <213> Artificial <220> SEQ 223 <400> 11 gggagaggag agatggac 18 <210> 12 <211> 18 <212> DNA <213> Artificial <220> SEQ 12 <400> 12 gggagaggag agacggac 18 <210> 13 <211> 401 <212> DNA <213> Homo sapiens <220> <221> misc_feature <187> (187) .. (187) W is a or t. <220> <221> misc_feature (201). (201) M is a or c. <400> 13 aactatagtt gatacctata aaattgactt cacaatgcac tattgggtca aaatttccag 60 tttgaaaaac agtgtgccta gacagtctgg tgaagttcag tttccaaact aatcatccgc 120 aggtctccta gttcctcaaa tgcacatctt ctgactcctt taggggtttc ctatcctcta 180 gtgttgwtgt cttcctgggg magccctcta ttctgctctt gattgaattt cctctccgat 240 atcacctgct tgcctggcta tgatgatcat tgctgctgta gtgattcata aatgtatatt 300 cctaactgtg gttgcgttcc tgaattctgt atgatgtttc cagcagcatt ccaaacagtt 360 tcatgtagcc ttctgttgta cccttcatct tgaactcagc a 401 <210> 14 <211> 22 <212> DNA <213> Artificial <220> SEQ 14 <400> 14 gctgccccag gaagacacca ac 22 <210> 15 <211> 22 <212> DNA <213> Artificial <220> SEQ 15 <400> 15 gctgccccag gaagacagca ac 22 <210> 16 <211> 24 <212> DNA <213> Artificial <220> SEQ 16 <400> 16 ttgctgtctt cctggggcag cccc 24 <210> 17 <211> 24 <212> DNA <213> Artificial <220> SEQ 17 <400> 17 ttggtgtctt cctggggcag cccc 24 <210> 18 <211> 21 <212> DNA <213> Artificial <220> SEQ 18 <400> 18 ctaccccagg aagacatcaa c 21 <210> 19 <211> 21 <212> DNA <213> Artificial <220> SEQ 19 <400> 19 tgatgtcttc ctgggggagc c 21 <210> 20 <211> 501 <212> DNA <213> Homo sapiens <220> <221> misc_feature (247) (247) .. (247) Y is c or t. <220> <221> misc_feature <251> (251) .. (251) K is g or t. <400> 20 gttttttatg ccatgtatat ttctctatgt gtagcctttg cctaaaacaa cacatgatta 60 atatttgttc attgttcctt ttgctatcac ccctgtctag gatctacaca ttaagaaaca 120 aagacatgaa cgtctccatg gaaagactgg gaaaatggat tgcaggttct agcaggatgt 180 cataataaat ggtgcatatc cagagtgcaa gatgattcag tctcaccaag aacactgaaa 240 gtcacayggc kaccagcatt attgtgataa gaactactat tttgggagat agtttagcaa 300 aggtgccatg tagaaattga ttaagtcaga ggtatcttta acttgccacc acagagaaga 360 gattaatttc atatacttcc attgagaaga gagataagaa tacaaaacca agctgatttg 420 caggagtaaa cttgatattc aaatactatt tcctgaatga cattttctga gacatgctaa 480 ttgtaattac tttcagcttc a 501 <210> 21 <211> 18 <212> DNA <213> Artificial <220> SEQ 21 <400> 21 ggttgccatg tgactttc 18 <210> 22 <211> 18 <212> DNA <213> Artificial <220> SEQ 22 <400> 22 ggtggccatg tgactttc 18 <210> 23 <211> 16 <212> DNA <213> Artificial <220> SEQ 23 <400> 23 aagtcacatg gcaacc 16 <210> 24 <211> 16 <212> DNA <213> Artificial <220> SEQ 223 <400> 24 aagtcacatg gccacc 16 <210> 25 <211> 40 <212> DNA <213> Artificial <220> SEQ 223 <400> 25 agccatagag acaagggcaa gagagaggcg atttaataga 40 <210> 26 <211> 40 <212> DNA <213> Artificial <220> SEQ 223 <400> 26 agccatagag acaagggcag gagagaggcg atttaataga 40 <210> 27 <211> 40 <212> DNA <213> Artificial <220> SEQ 223 <400> 27 agccatagag acaagggcaa gagaggggcg atttaataga 40 <210> 28 <211> 40 <212> DNA <213> Artificial <220> SEQ 223 <400> 28 agccatagag acaagggcag gagaggggcg atttaataga 40 <210> 29 <211> 480 <212> DNA <213> Homo sapiens <220> <221> misc_feature (227) (227) .. (227) Y is t or c. <220> <221> misc_feature (234) .. (234) R is g or a. <220> <221> misc_feature (234) .. (234) S is g or c. <220> <221> misc_feature <245> (245) .. (245) Y is t or c. <400> 29 agtagaattt ggattcaaag tagccatgag atatatagca tgtgttggag ggaaaaaaac 60 cccacaacat atatattctc ttataggtta ttagacccac aacatatatg tcctcttata 120 ggttattaga tgtcttagct gcaaggaaag atccaagtgg attatctgga gatgttctga 180 taaatggagc accgcgacct gccaatttca aatgtaattc aggttaygtg gtasaagtaa 240 gtatyagtgg gtttgcattt tctgtttcct ctgtttctat atgggtaagt gctttsgctg 300 atagttcaat gtgcttccag ttgattatgt gacatggtcc tagaactgac gttctttaca 360 gcagcttttc ttaatttctc atagacactt atgtgaaaag gcagggagaa tctggaatat 420 ggcccttgta aggacagtga taccaattct agtttttgta tcatttctaa aatgatacat 480 <210> 30 <211> 24 <212> DNA <213> Artificial <220> <223> 5FL-ABCG2 Q126X-T-R1 <400> 30 cccacttata cttacttata ccac 24 <210> 31 <211> 50 <212> DNA <213> Artificial <220> <223> ABCG2-Q126X-50-F-WT <400> 31 caaatgtaat tcaggttacg tggtacaagt aagtattagt gggtttgcat 50 <210> 32 <211> 50 <212> DNA <213> Artificial <220> <223> ABCG2-Q126X-50-F-mt <400> 32 caaatgtaat tcaggttacg tggtataagt aagtattagt gggtttgcat 50 <210> 33 <211> 40 <212> DNA <213> Artificial <220> <223> ABCG2-Q126X-40-F-WTC <400> 33 ggttacgtgg tacaagtaag tatcagtggg tttgcatttt 40 <210> 34 <211> 40 <212> DNA <213> Artificial <220> <223> ABCG2-Q126X-40-F-mtC <400> 34 ggttacgtgg tataagtaag tatcagtggg tttgcatttt 40 <210> 35 <211> 21 <212> DNA <213> Artificial <220> <223> 3PB-CYP3A4 1B-R3TK <400> 35 taaatcgcct ctctcctgcc c 21

Claims (13)

At least one oligonucleotide selected from the group consisting of the following P1, P1-1, P1 ', and P1'-1, and the above-described nucleotide sequence encoding a target gene containing a target gene mutation and an untarget gene mutation Gene mutation detection probes for detecting target gene mutations:
(P1) the non-target gene at a base position corresponding to the target gene mutation having a base complementary to either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having bases which are non-complementary to any of the wild type and variant bases of the variant and having at least 80% identity to the base sequence complementary to some or all of the base sequence encoding the gene of interest;
(P1-1) a base having a base complementary to either the wild-type base or the mutant base of the target gene mutation at a base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligos that hybridize under oligonucleotide conditions to oligonucleotides having non-complementary bases and having the same base sequence as the base sequence encoding the gene of interest. Nucleotides;
(P1 ′) the non-target gene at a base position corresponding to the target gene mutation having the same base for either the wild-type base or the mutant base of the target gene mutation, and at the base position corresponding to the non-target gene mutation Oligonucleotides having other bases in any of the wild-type and variant bases of the variant and having at least 80% or more identity to some or all of the base sequences encoding the genes of interest; And
(P1′-1) has a base identical to any of the wild-type base and the mutant base of the target gene mutation at the base position corresponding to the target gene mutation, and the non-base position at the base position corresponding to the non-target gene mutation Oligonucleotides that hybridize under oligonucleotide conditions to oligonucleotides having a base sequence complementary to the base sequence encoding the gene of interest and having a base different from any of the wild type base and the variant base of the desired gene variant. . The method of claim 1,
Gene mutation detection probes satisfying any one of the following relationships (a)? (F):
(a) when the wild-type base at the base position of the non-target gene variant is either A or T, and the variant base of the non-target gene variant is another one of A or T, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases, or which are either wild-type bases or variant bases of said non-target gene variants, are C or G.
(b) when the wild-type base at the base position of the non-target gene variant is either C or G and the variant base of the non-target gene variant is another one of C or G, the wild-type base of the non-target gene variant And bases which are not complementary to any of the variant bases or which are either wild-type bases or variant bases of said non-target gene variants are A or T.
(c) when the wild-type base at the base position of the non-target gene variant is either A or C, and the variant base of the non-target gene variant is another one of A or C, the wild-type base of the non-target gene variant And A or C, which is non-complementary to any of the variant bases, is A or C, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or G.
(d) when the wild-type base at the base position of the non-target gene variant is either A or G, and the variant base of the non-target gene variant is another one of A or G, the wild-type base of the non-target gene variant And A or G, which is non-complementary to any of the variant bases, is A or G, and the base other to any of the wild-type bases and variant bases of the non-target gene variant is T or C.
(e) when the wild-type base at the base position of the non-target gene variant is either T or C, and the variant base of the non-target gene variant is another one of T or C, the wild-type base of the non-target gene variant And bases that are non-complementary to any of the variant bases are T or C, and bases other than any of the wild-type bases and variant bases of the non-target gene variant are A or G.
(f) when the wild-type base at the base position of the non-target gene variant is either T or G, and the variant base of the non-target gene variant is another one of T or G, the wild-type base of the non-target gene variant And a base non-complementary to any of the variant bases is T or G, and a base other than either the wild-type base or the variant base of the non-target gene variant is A or C. The method of claim 1,
The oligonucleotide is a genetic variation detection probe that is a fluorescently labeled oligonucleotide. The method of claim 3,
The fluorescently labeled oligonucleotide is a probe for detecting genetic variation in which the base at the 3 'end or the 5' end is cytosine, and the cytosine is labeled with a fluorescent dye. The method of claim 3,
The fluorescently labeled oligonucleotide is a gene mutation detection probe which is located at any one of the first to third bases by counting from the 3 'end or the 5' end. The method of claim 1,
Probe for detecting genetic variation, which is a probe for melting curve analysis. The method of claim 3,
The fluorescently labeled oligonucleotide is a probe for detecting a genetic variation in which the fluorescence intensity when hybridizing to a target sequence is reduced or increased, compared to the fluorescence intensity when not hybridizing to a target sequence. The method of claim 3,
The fluorescently labeled oligonucleotide is a probe for detecting a genetic variation in which the fluorescence intensity when hybridizing to the target sequence is reduced compared to the fluorescence intensity when not hybridizing to the target sequence. The gene mutation detection method which detects the target gene mutation used as a detection object by using the gene mutation detection probe in any one of Claims 1-8. (I) contacting the single stranded nucleic acid in the gene mutation detection probe according to any one of claims 3 to 8 and a sample to obtain a hybrid,
(II) dissociating the hybrid by changing the temperature of the sample containing the hybrid, and measuring variation in the fluorescence signal based on dissociation of the hybrid;
(III) obtaining a Tm value that is a dissociation temperature of the hybrid based on the fluctuation of the fluorescence signal;
(IV) Detecting the presence of target gene mutation in single-stranded nucleic acid in the sample based on the Tm value.
Gene mutation detection method comprising a. The method of claim 10,
The method for detecting a target gene mutation further comprising amplifying the nucleic acid before obtaining the hybrid of (I) or simultaneously with obtaining the hybrid of (I). Reagent kit for detecting a genetic mutation comprising a probe for detecting the genetic mutation according to any one of claims 1 to 8. A reagent kit for detecting genetic variation, further comprising a primer capable of amplifying a nucleotide sequence having a region hybridized by the gene mutation detecting probe according to any one of claims 1 to 8.

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