JPWO2006082685A1 - Single nucleotide polymorphism detection method - Google Patents

Abstract

The present invention provides (A) I) a 5 ′ or 3 ′ end at a position capable of complementary hybridization to a nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism. A nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the side, or II) hybridizing complementary to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism A nucleic acid probe having a nucleotide sequence which is possible and has a cytosine residue or a thymine residue added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism on the antisense strand And (B) a compound capable of recognizing a bulge base and (C) the nucleic acid to be evaluated, and the cytosine formed when the nucleic acid probe and the nucleic acid to be evaluated are hybridized. Wherein recognize distearate or Chiminbaruji detects a signal based on the compound of (B), single nucleotide polymorphism detection method characterized by evaluating the single nucleotide polymorphism, as well as the kit.

Description

  The present invention relates to a method for detecting a single nucleotide polymorphism in a target nucleic acid and a kit thereof. More specifically, the present invention relates to a method for detecting a single nucleotide polymorphism in a target nucleic acid with high sensitivity and a kit thereof.

  Single nucleotide polymorphisms (SNPs) are differences between individuals existing on genomic DNA, and can cause various diseases and various phenotypic differences in humans and the like. Therefore, such SNPs are used for analysis of genetic diseases, identification between individuals, and the like.

  The detection of the SNPs is, for example, a detection method by typing based on differences in mobility in electrophoresis, primer extension, hybridization using a probe labeled with a labeling substance such as a fluorescent substance or radioactive substance, amplification by PCR, etc. Etc.

  As the detection method, specifically, for example, the DNA fragment to be analyzed and the reference DNA fragment are subjected to temperature conditions that increase separation of fragments in electrophoresis using an increase in secondary structure difference due to polymorphism as an index. The heterozygous reference DNA fragment and the DNA fragment to be analyzed are electrophoretically separated simultaneously in the same capillary, the peak is corrected by the reference DNA fragment, and the heterozygosity of the DNA fragment to be analyzed is lost. Detection method (Patent Document 1) for examining the presence or absence of a wild-type primer and one or two kinds of mutant-type primers simultaneously or on a chromosome or a fragment thereof containing a specific single nucleotide polymorphism site contained in a sample A detection method (Patent Document 2) that separately acts with a DNA polymerase and examines the presence or absence of extension based on a primer; A detection method for amplifying and typing a plurality of nucleotide sequences including at least one single nucleotide polymorphic site using the primers of the above (patent document 3); for a reference sequence including a single nucleotide polymorphic site and a mutant sequence Detection method (Patent Document 4): Amplification of the target sequence portion using two kinds of specific primers and universal primer, electrophoresis of the obtained reaction solution, and examining the presence or absence of the amplification product (Patent Document 4); A target sample and a plurality of single-stranded peptide nucleic acids that continuously hybridize to the target nucleic acid region when the target nucleic acid region remains unchanged, and then an agent that degrades the mismatch nucleic acid (single-stranded nucleic acid). The detection method (patent document 5) etc. which add and then investigate presence of a decomposition product are mentioned.

However, according to the method described in the above-mentioned patent document, examination of temperature conditions in electrophoresis, electrophoresis, peak correction, examination of DNA polymerase reaction conditions, setting of conditions for the presence or absence of extension, amplification reaction Since it is performed by combining multiple complicated procedures such as examination of conditions, examination of typing conditions, examination of amplification reaction conditions, detection of presence or absence of amplification products, the operation is complicated and takes time. is there.
JP 2002-78500 A International Publication No. 01/042498 Pamphlet JP2002-300894 JP 2003-52372 A Special table 2004-521630

  One aspect of the present invention is to detect a single nucleotide polymorphism in a nucleic acid with high sensitivity, to easily detect a single nucleotide polymorphism in a nucleic acid without performing a labeling step with a labeling substance, and at a low cost. It is an object of the present invention to provide a method for detecting a single nucleotide polymorphism in a nucleic acid, which enables at least one of detecting a single nucleotide polymorphism in a nucleic acid. Another aspect of the present invention is to provide a kit that enables the detection method to be performed efficiently and simply at low cost. In addition, the other subject of this invention can be guide | induced from description etc. of this specification.

That is, the gist of the present invention is as follows.
[1] (A) I) 5 ′ or 3 ′ terminal side of a position capable of complementary hybridization to a nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the nucleic acid, or II) capable of hybridizing complementary to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism on the antisense strand,
When,
(B) a compound capable of recognizing a bulge base;
(C) the nucleic acid to be evaluated;
Mix and
Detecting a signal based on the compound (B) that recognizes cytosine bulge or thymine bulge formed when the nucleic acid probe and the nucleic acid to be evaluated are hybridized;
A method for detecting a single nucleotide polymorphism, characterized by evaluating the single nucleotide polymorphism,
[2] The following 1) to 4):
1) A bulge formed between a wild-type evaluation target nucleic acid containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the wild-type evaluation target nucleic acid,
2) A bulge formed between a mutant nucleic acid to be evaluated containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the nucleic acid to be mutant-type evaluated;
3) a bulge formed between the wild-type evaluation target nucleic acid and a nucleic acid probe capable of hybridizing complementary to the mutant-type evaluation target nucleic acid; and 4) the mutant-type evaluation target nucleic acid and the wild type A bulge formed between a nucleic acid probe capable of hybridizing complementary to the nucleic acid to be evaluated,
The single nucleotide polymorphism in the nucleic acid to be evaluated is identified based on the fluctuation of the signal based on the compound capable of recognizing the bulge base of (B) bound to each of the detection method of [1],
[3] The detection method according to [1] or [2], wherein the compound has a naphthyridine ring,
[4] The detection method according to any one of [1] to [3], wherein the signal is fluorescence,
[5] The detection method according to any one of [1] to [4], wherein the compound is a 2,7-diaminonaphthyridine derivative compound,
[6] The 2,7-diaminonaphthyridine derivative compound is represented by the following formula (II):

The detection method according to the above [5], which is 2,7-diamino-1,8-naphthyridine represented by
[7] The nucleic acid probe of (A) is
i) Cytosine at a position adjacent to the 5 ′ or 3 ′ terminal side of the position capable of hybridizing complementarily to the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism A nucleic acid probe comprising a base sequence to which a residue or thymine residue is added and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue; or ii ) 5 ′ or 3 ′ at a position capable of hybridizing complementarily to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism on the antisense strand A cytosine residue or thymine residue is added at a position adjacent to the terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is a wild boar. A nucleic acid probe having a base sequence further substituted with
The detection method according to any one of [1] to [6],
[8] I ′) comprising at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position of the single nucleotide polymorphism, A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the pairing position, and / or II ′) containing at least one single nucleotide polymorphism 5 of the position which can hybridize complementarily to the antisense strand of the nucleic acid to be evaluated containing the wild-type nucleotide at the position where the single nucleotide polymorphism is present, and which matches the single nucleotide polymorphism on the antisense strand. A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 'or 3'end;
When,
I ″) comprising at least one single nucleotide polymorphism and capable of hybridizing complementarily to a nucleic acid to be evaluated containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and pairing with the single nucleotide polymorphism A nucleic acid probe having a nucleotide sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position to be selected, and / or II ″) at least one single nucleotide polymorphism, 5 ′ at the position where the single nucleotide polymorphism can be complementarily hybridized to the antisense strand of the nucleic acid to be evaluated containing the mutant nucleotide at the position where the single nucleotide polymorphism is present, and which matches the single nucleotide polymorphism on the antisense strand. Or a nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the 3 ′ end,
When,
A compound capable of recognizing a bulge base;
A kit for use in the method for detecting a single nucleotide polymorphism in a nucleic acid according to any one of [1] to [7], and [9] i ′) at least one single nucleotide 5 'or 3' terminal side of the position that can be complementarily hybridized to the nucleic acid to be evaluated containing the wild type nucleotide at the position where the single nucleotide polymorphism exists. A nucleic acid having a base sequence in which a cytosine residue or a thymine residue is added at a position adjacent to and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is substituted with an inosine residue probe,
ii ′) Complementary to the antisense strand of the nucleic acid to be evaluated comprising at least one single nucleotide polymorphism and containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and on the antisense strand Guanine to which a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where it is paired with a single nucleotide polymorphism and is adjacent to the cytosine residue or thymine residue A nucleic acid probe having a base sequence in which at least one of the residues is further substituted with an inosine residue;
When,
i ″) comprising at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a mutant nucleotide at the position of the single nucleotide polymorphism, and pairing with the single nucleotide polymorphism A cytosine residue or thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position to be detected, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is an inosine residue. A nucleic acid probe having a base sequence substituted by a group,
ii ″) Complementary hybridization to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and the antisense strand A cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism above, and is adjacent to the cytosine residue or thymine residue. A nucleic acid probe having a base sequence in which at least one of guanine residues is further substituted with an inosine residue;
When,
A kit for use in the method for detecting a single nucleotide polymorphism in a nucleic acid according to the above [8], comprising a compound capable of recognizing a bulge base;
About.

  According to the detection method of the present invention, a single nucleotide polymorphism in a nucleic acid can be easily detected with high sensitivity at a low cost without combining complicated steps such as labeling with a labeling substance. Excellent effect. Moreover, according to the kit of this invention, there exists the outstanding effect that the said detection method can be performed efficiently and simply at low cost.

FIG. 1 is a schematic diagram showing an example of a use example of a nucleic acid probe for a target nucleic acid. In the figure, the underlined bold letters indicate the positions of single nucleotide polymorphisms to be evaluated. A black circle indicates 2,7-diamino-1,8-naphthyridine.

FIG. 2 is a diagram showing the absorption spectrum and fluorescence spectrum of 10 μM 2,7-diamino-1,8-naphthyridine under various conditions. In the figure, the left panel shows an absorption spectrum at 300 nm to 450 nm. The right panel shows the fluorescence spectrum of 10 μM 2,7-diamino-1,8-naphthyridine. The excitation wavelength for fluorescence measurement has an absorption maximum in each absorption spectrum.

FIG. 3 is a diagram showing an absorption spectrum and a fluorescence spectrum of 10 μM 2,7-diamino-1,8-naphthyridine bound to adenine bulge, thymine bulge, cytosine bulge and guanine bulge. In the figure, the left panel shows an absorption spectrum at 300 nm to 450 nm. The right panel shows the fluorescence spectrum of 10 μM 2,7-diamino-1,8-naphthyridine. The excitation wavelength for fluorescence measurement has an absorption maximum in each absorption spectrum.

FIG. 4 shows a fluorescence spectrum based on the fluorescence of 2,7-diamino-1,8-naphthyridine bound to a bulge in the presence of a C or A allele. In FIG. 4, A allele is a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 1, T probe is a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 2, and G probe is shown in SEQ ID NO: 3. Nucleic acid containing the nucleotide sequence shown below, Callele is a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 4, and I probe is a nucleic acid containing the nucleotide sequence shown in SEQ ID NO: 5.

In one aspect, the present invention relates to a method for detecting a single nucleotide polymorphism in a nucleic acid. Specifically, the detection method of the present invention is (A) I) capable of hybridizing complementarily to a nucleic acid to be evaluated containing at least one single nucleotide polymorphism, and paired with the single nucleotide polymorphism. A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position, or II) antisense of a nucleic acid to be evaluated containing at least one single nucleotide polymorphism A cytosine residue or a thymine residue is added at a position adjacent to the 5 'or 3' end of the position that is capable of hybridizing to the strand in a complementary manner to the single nucleotide polymorphism on the antisense strand. A nucleic acid probe having a determined base sequence,
When,
(B) a compound capable of recognizing a bulge base;
(C) the nucleic acid to be evaluated;
Mix and
Detecting a signal based on the compound (B) that recognizes cytosine bulge or thymine bulge formed when the nucleic acid probe and the nucleic acid to be evaluated are hybridized;
It is a method characterized by evaluating the single nucleotide polymorphism.

  In the present invention, a compound containing a naphthyridine ring is shifted from the wavelength of the absorption maximum before binding by specifically binding to the bulge structure, and the fluorescence intensity depends on the type of base pair formed by the nucleotide adjacent to the bulge base. Is based on the inventors' surprising discovery that fluctuates. The present inventors can hybridize complementarily to a nucleic acid having a single nucleotide polymorphism, have a probe containing a bulge base adjacent to the position where the single nucleotide polymorphism is paired, and the single nucleotide polymorphism. Due to hybridization with nucleic acid, a duplex containing a bulge structure is formed, then a compound containing a naphthyridine ring is bound to a bulge base and fluorescence is measured, and the nucleotide at the position where the single nucleotide polymorphism exists is different. It was found that single nucleotide polymorphisms can be detected from fluctuations in fluorescence intensity.

  In the present specification, “base” refers to deoxyribonucleotide unless otherwise specified. Accordingly, in the present specification, unless otherwise specified, “cytosine” (C), “thymine” (T), “adenine” (A) and “guanine” (G) are each deoxyribonucleotide, , “2′-deoxycytidine”, “2′-deoxythymidine”, “2′-deoxyadenosine” and “2′-deoxyguanosine”.

  Further, in the present specification, “the nucleic acid to be evaluated containing at least one single nucleotide polymorphism” is confirmed to have at least one, more specifically, 1 to 5 single nucleotide polymorphisms. Unless otherwise specified, nucleotides present at single nucleotide polymorphism positions include both wild-type nucleotides and mutant nucleotides. Further, in the present specification, the “wild-type nucleotide at the position where the single nucleotide polymorphism is present” is a nucleotide at a site where the single nucleotide polymorphism has been confirmed, and the nucleotide at such a site in a so-called normal nucleotide sequence. The “mutant nucleotide at the position where the single nucleotide polymorphism is present” refers to a nucleotide at a site where a single nucleotide polymorphism has been confirmed, and a nucleotide at such a site in a so-called mutant nucleotide sequence.

  Further, in the present specification, “bulge base” means an unpaired base in double-stranded DNA, and for example, “buldycytosine” or “baldithymine” means one of the double-stranded DNAs. This refers to cytosine or thymine that is redundant with respect to single-stranded DNA and that does not form base pairs. Such a bulge structure by a bulge base is artificially formed by, for example, a single-stranded DNA comprising a single-stranded DNA comprising a specific base sequence and a base sequence further comprising one extra nucleotide at an arbitrary position in the base sequence. When hybridized with DNA, it can be generated as a duplex containing a bulge structure. In the present invention, a cytosine residue or a thymine residue added at a position adjacent to the 5 'or 3' end side of the position where the single nucleotide polymorphism is paired corresponds to the bulge base.

  Further, “cytosine bulge” or “thymine bulge” means a bulge generated by surplus of one cytosine or thymine with respect to the corresponding single-stranded DNA.

  In the present specification, “a compound capable of recognizing a bulge base” means a compound that stabilizes a duplex having a bulge base, for example, by forming a hydrogen bond with a bulge base present in the duplex. Examples include compounds having a naphthyridine ring, 2,6-diaminopyridine, and xanthine. Among them, a compound having a naphthyridine ring that emits fluorescence as a signal is preferable because of easy detection and evaluation.

  The detection method of the present invention is excellent in that a single nucleotide polymorphism can be detected without using a labeling substance such as a fluorescent substance by using a compound having a naphthyridine ring as a compound capable of recognizing a bulge base. Demonstrate. Therefore, in the detection method of the present invention, extension reaction, amplification reaction, enzyme reaction (for example, the extension reaction, amplification reaction, mismatch nucleic acid degradation reaction, etc.), examination of electrophoresis conditions, labeling with a labeling substance, etc. A single nucleotide polymorphism can be easily detected without performing a complicated process. Furthermore, since the compound containing the naphthyridine ring specifically binds to the bulge, the background signal level is reduced and single nucleotide polymorphism can be detected with high sensitivity.

  The compound containing the naphthyridine ring is a kind of nucleotide residue that is paired with a nucleotide residue adjacent to the bulge base, and the wavelength that shows the absorption maximum of the compound containing the naphthyridine ring is shifted by binding to the bulge. The fluorescence intensity varies depending on (that is, the allele). Therefore, the binding to the bulge can be evaluated by the shift of the wavelength showing such absorption maximum, and the single nucleotide polymorphism can be evaluated by the fluctuation of the fluorescence intensity.

  Examples of the compound containing the naphthyridine ring include the following (I):

(Wherein R ¹ and R ² each independently represent a primary amine residue, a secondary amine residue or a tertiary amine residue)
2,7-diaminonaphthyridine derivative compounds shown in the above. Examples of the primary amine residue include a —NH ₂ group. Examples of the secondary amine residue include a —NH (CH ₂ ) ₃ NH ₂ group, a —NH (CH ₂ ) ₂ NH ₂ group, a —NH (CH ₂ ) ₂ NH (CH ₃ ) group, and the like. Is mentioned. Furthermore, examples of the tertiary amine residue include a —N (CH ₃ ) (CH ₂ ) ₂ NH ₂ group. In the present invention, from the viewpoint of hydrogen bond formation with a bulge base, it is preferably a secondary amine residue, and more preferably, both R ¹ and R ² are secondary amine residues. It is desirable to be a group.

  Specific examples of the 2,7-diaminonaphthyridine derivative compound include the following formula (II):

2,7-diamino-1,8-naphthyridine shown in FIG. The 2,7-diamino-1,8-naphthyridine can be synthesized, for example, by the method described in JP-A-2004-262827. The 2,7-diamino-1,8-naphthyridine alone shows an absorption maximum at 376 nm under the condition of 10 mM sodium phosphate buffer (pH 7.0), and it becomes 396 nm by binding with cytosine bulge. The wavelength which shifts by 20 nm and shows the maximum value of the fluorescence intensity is shifted by about 32 nm from 398 nm to 430 nm.

  In the present invention, the same properties as 2,7-diamino-1,8-naphthyridine, that is, for example, generate fluorescence, have binding ability to bulge, and have an absorption maximum wavelength due to binding with bulge. May be used, and the derivative compound of 2,7-diamino-1,8-naphthyridine may be used as long as it exhibits the property that the fluorescence intensity changes depending on the allele. Examples of such derivative compounds include the following formula (III):

[Wherein R ³ and R ⁴ each independently represent a hydrogen atom or an amino group, and l, m and n each independently represents a natural number of 1 to 6,] (IV):

[Wherein, R ⁵ and R ⁶ each independently represent a hydrogen atom or an amino group, and o and p each independently represent a natural number of 1 to 6]
And the like.

In the compound represented by the formula (III), R ³ and R ⁴ are each independently a hydrogen atom or an amino group, generate fluorescence, have binding ability to bulge, and bind to bulge. From the viewpoint of sufficiently exhibiting the property that the absorption maximum wavelength shifts and the fluorescence intensity is changed by the allele, it is desirable that one of them is an amino group. Moreover, said l, m, and n are the natural numbers of 1-6 each independently. From the viewpoint of producing fluorescence, having the ability to bind to the bulge, and sufficiently exhibiting the property that the absorption maximum wavelength is shifted by the binding to the bulge and the fluorescence intensity is changed by the allele, the above l is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less, specifically, preferably 2 to 6 More preferably, it is 3-5, and more preferably 3-4. From the same viewpoint as described above, the m is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less. More specifically, it is preferably 2 to 6, more preferably 3 to 5, and still more preferably 3 to 4. Further, from the same viewpoint as described above, the n is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less. More specifically, it is preferably 2 to 6, more preferably 3 to 5, and still more preferably 3 to 4.

In the compound represented by the formula (IV), R ⁵ and R ⁶ are each independently a hydrogen atom or an amino group, generate fluorescence, have binding ability to bulge, From the viewpoint of sufficiently exhibiting the property that the absorption maximum wavelength is shifted by the bonding of and the fluorescence intensity is changed by the allele, it is preferable that at least one is an amino group. The o and p are each independently a natural number of 1 to 6. From the viewpoint of producing fluorescence, having the ability to bind to the bulge, and sufficiently exhibiting the property that the absorption maximum wavelength is shifted by the binding with the bulge and the fluorescence intensity is changed by the allele, the o is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less, specifically, preferably 2 to 6 More preferably, it is 3-5, and more preferably 3-4. From the same viewpoint, the p is preferably 2 or more, more preferably 3 or more, preferably 6 or less, more preferably 5 or less, and still more preferably 4 or less. Desirably, specifically, it is preferably 2 to 6, more preferably 3 to 5, and still more preferably 3 to 4.

  The hybridization when the nucleic acid probe of the present invention hybridizes complementarily to the nucleic acid to be evaluated containing at least one single nucleotide polymorphism or to the antisense strand of the nucleic acid to be evaluated is 1 mM to 1 M, preferably 10 to 10. It is desirable to carry out in a buffer solution containing 100 mM sodium chloride, pH 5-8, preferably pH 6-7, preferably phosphate buffer.

One embodiment of the nucleic acid probe according to I) of the present invention has a base sequence corresponding to the antisense strand of the nucleic acid to be evaluated containing a single nucleotide polymorphism, and is present at the site of the single nucleotide polymorphism in the nucleic acid to be evaluated. A nucleic acid to which a cytosine residue or a thymine residue is added at a position adjacent to a nucleotide that forms a base pair with a nucleotide to be paired, and one embodiment of the nucleic acid probe of the above II) is complementary to the nucleic acid probe of I) A nucleotide sequence corresponding to the sense strand of the nucleic acid to be evaluated containing a single nucleotide polymorphism, and a cytosine residue or thymine residue at a position adjacent to the nucleotide present at the site of the single nucleotide polymorphism. It is an added nucleic acid. Therefore, one embodiment of the probe of the above I) or II) is a base sequence corresponding to an antisense strand of a continuous base sequence including nucleotides in which I) single nucleotide polymorphism exists, and the single nucleotide polymorphism exists. A nucleic acid probe containing a nucleotide sequence to which a cytosine residue or a thymine residue is added adjacent to the 5 ′ or 3 ′ terminal side of the position on the antisense strand corresponding to the position, or II) a single nucleotide polymorphism It is a base sequence corresponding to a continuous base sequence including nucleotides present, and a cytosine residue or thymine residue is added adjacent to the 5 ′ or 3 ′ end side of the position corresponding to the position where the single nucleotide polymorphism exists. In other words, it can also be referred to as a nucleic acid probe containing a base sequence.

  Examples of the nucleic acid probe used in the present invention include a nucleic acid probe containing a wild-type nucleotide at the position where the single nucleotide polymorphism exists and a nucleic acid probe containing a mutant nucleotide at the position where the single nucleotide polymorphism exists.

  In the detection method of the present invention, a cytosine residue or a thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position where the nucleic acid probe used matches the single nucleotide polymorphism in the target nucleic acid. Another major feature is that it contains a base sequence. Therefore, in the detection method of the present invention, the nucleic acid probe is hybridized with the nucleic acid to be evaluated for single nucleotide polymorphism, so that a compound containing the naphthyridine ring is recognized and bound to form a bulge structure. Cytosine bulge or thymine bulge specific binding of a compound containing a ring can be induced, reducing the background signal level and enabling detection with high sensitivity. Here, the cytosine residue or thymine residue added adjacent to the 5 ′ or 3 ′ terminal corresponds to the “bulge base”. The bulge structure is generated when the nucleic acid probe has a bulge base when the nucleic acid probe is hybridized with the single nucleotide polymorphism nucleic acid to be evaluated. A compound containing a naphthyridine ring recognizes and binds to a bulge structure by forming a hydrogen bond with a bulge base.

In the nucleic acid probe, when the base adjacent to the bulge base is guanine, i) contains at least one single nucleotide polymorphism from the viewpoint of suppressing the quenching of the signal based on the compound capable of recognizing the bulge base. A cytosine residue or a thymine residue is added to a position adjacent to the 5 ′ or 3 ′ end side of the position that is complementary to the nucleic acid to be evaluated, and that matches the single nucleotide polymorphism; and An evaluation comprising a nucleic acid probe comprising a nucleotide sequence in which at least one guanine residue present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue, or ii) at least one single nucleotide polymorphism 5 ′ at a position capable of hybridizing complementary to the antisense strand of the target nucleic acid and paired with a single nucleotide polymorphism on the antisense strand, or 'A base in which a cytosine residue or thymine residue is added at a position adjacent to the terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue A nucleic acid probe having a sequence;
Is preferred.

One embodiment of the nucleic acid probe of i) or ii) is i) a nucleotide sequence corresponding to an antisense strand of a continuous nucleotide sequence containing nucleotides having a single nucleotide polymorphism, and the position of the single nucleotide polymorphism present A cytosine residue or a thymine residue is added adjacent to the 5 ′ or 3 ′ terminal side of the position corresponding to the antisense strand, and a guanine residue is present adjacent to the cytosine residue or thymine residue. A nucleic acid probe containing a nucleotide sequence in which at least one of the groups is further substituted with an inosine residue, or ii) a nucleotide sequence corresponding to a continuous nucleotide sequence including nucleotides having a single nucleotide polymorphism, A cytosine residue or thymine residue is added adjacent to the 5 ′ or 3 ′ terminal side of the position corresponding to the type existing position, and adjacent to the cytosine residue or thymine residue. A nucleic acid probe comprising a base sequence in which at least one of the existing guanine residues is further substituted with an inosine residue;
It is.

  That is, the nucleic acids i) and ii) are suitable when the base adjacent to the position where the single nucleotide polymorphism is present is cytosine.

  Therefore, in the detection method of the present invention, the type of the nucleic acid probe is appropriately selected according to the nucleotide adjacent to the position where the single nucleotide polymorphism to be evaluated is present and the base sequence around the position where the single nucleotide polymorphism is present. Can do.

The “nucleic acid probe containing a wild-type nucleotide at the position where the single nucleotide polymorphism exists” specifically includes I ′) at least one single nucleotide polymorphism, and the wild-type nucleotide exists at the position where the single nucleotide polymorphism exists. And a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end side of the position where it is paired with the single nucleotide polymorphism. A nucleic acid probe having a different base sequence,
II ′) Complementary to the antisense strand of the nucleic acid to be evaluated comprising at least one single nucleotide polymorphism and containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, on the antisense strand A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism of
i ′) containing at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position of the single nucleotide polymorphism, and pairing with the single nucleotide polymorphism A cytosine residue or thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is an inosine residue A nucleic acid probe having a base sequence substituted with
ii ′) Complementary to the antisense strand of the nucleic acid to be evaluated comprising at least one single nucleotide polymorphism and containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and on the antisense strand Guanine to which a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where it is paired with a single nucleotide polymorphism and is adjacent to the cytosine residue or thymine residue A nucleic acid probe having a base sequence in which at least one of the residues is further substituted with an inosine residue;
Etc.

Specifically, the “nucleic acid probe containing a mutant nucleotide at the position of the single nucleotide polymorphism” specifically includes I ″) at least one single nucleotide polymorphism, and the mutant at the position of the single nucleotide polymorphism. A cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism, and can be hybridized in a complementary manner to the nucleic acid to be evaluated including nucleotides. A nucleic acid probe having a determined base sequence,
II ″) the antisense strand comprising at least one single nucleotide polymorphism and capable of hybridizing complementary to the antisense strand of the nucleic acid to be evaluated containing the mutant nucleotide at the position of the single nucleotide polymorphism, A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ end side of the above single nucleotide polymorphism,
i ″) comprising at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a mutant nucleotide at the position of the single nucleotide polymorphism, and pairing with the single nucleotide polymorphism A cytosine residue or thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position to be detected, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is an inosine residue. A nucleic acid probe having a base sequence substituted by a group,
ii ″) Complementary hybridization to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and the antisense strand A cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism above, and is adjacent to the cytosine residue or thymine residue. A nucleic acid probe having a base sequence in which at least one of guanine residues is further substituted with an inosine residue;
Etc.

In the detection method of the present invention, more specifically,
1) A bulge formed between a wild-type evaluation target nucleic acid containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the wild-type evaluation target nucleic acid,
2) A bulge formed between a mutant nucleic acid to be evaluated containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the nucleic acid to be mutant-type evaluated;
3) a bulge formed between the wild-type evaluation target nucleic acid and a nucleic acid probe capable of hybridizing complementary to the mutant-type evaluation target nucleic acid; and 4) the mutant-type evaluation target nucleic acid and the wild type A bulge formed between a nucleic acid probe capable of hybridizing complementary to the nucleic acid to be evaluated,
The single nucleotide polymorphism in the nucleic acid to be evaluated is identified based on the variation of the signal based on the compound containing the naphthyridine ring of (B) bound to each of the nucleic acids. A schematic diagram showing one example of the use example of the nucleic acid probe for the target nucleic acid is shown in FIG.

  Specifically, for example, when a guanine-cytosine (GC) base pair is formed with a nucleic acid probe at the position where the single nucleotide polymorphism of the nucleic acid to be evaluated is present, the nucleic acid to be evaluated and the wild type probe or mutant type When the fluorescence based on the compound containing the naphthyridine ring (B) bound to the bulge formed between the probe and the probe is quenched to form an adenine-thymine (AT) base pair, Fluorescence based on the compound containing the naphthyridine ring of (B) bound to a bulge formed between a type probe and a mutant type probe can be detected. Here, if adenine is present at the position of the single nucleotide polymorphism, is it adenine? Strong fluorescence is observed at thymine (AT) base pair, adenine? With adenine (AA) mismatched base pairs, the fluorescence intensity is halved. On the other hand, if thymine is present at the position of single nucleotide polymorphism, thymine? Fluorescence is observed in adenine (TA) base pair, thymine? With thymine (TT) base pairing, the fluorescence is greatly reduced. In addition, when a nucleotide having a single nucleotide polymorphism and / or a nucleotide adjacent thereto is a cytosine residue, the guanine residue at the corresponding position of the nucleic acid probe is used instead of the inosine residue. Here, when a cytosine-inosine (CI) base pair is formed, the naphthyridine ring of (B) bound to a bulge formed between a nucleic acid to be evaluated and a wild type probe or a mutant type probe. Fluorescence based on compounds containing can be detected. When guanine is present at the position of single nucleotide polymorphism, cytosine is adjacent to guanine, and (YY ′) is (TA), cytosine bulge is replaced with guanine and YY ′ base at the position of single nucleotide polymorphism. Design the probe to be located between the pair to avoid fluorescence quenching by adjacent guanine bases. When guanine is present at the position where the single nucleotide polymorphism is present, cytosine is adjacent to guanine, and (YY ′) is (GC), cytosine bulge is 5 ′ of guanine at the position of the single nucleotide polymorphism. Or even if it is located on the 3 ′ side, fluorescence quenching cannot be avoided because guanine is adjacent. In this case, the typing of the strand containing cytosine existing at the single nucleotide polymorphism position of the opposite strand is considered. Therefore, according to the detection method of the present invention, single nucleotide polymorphisms can be evaluated based on the behavior of fluorescence or quenching depending on the type of base pair at the site where these single nucleotide polymorphisms exist.

  The length of the nucleic acid probe is 15 nucleotide residues or more, preferably 20 nucleotide residues or more, and 40 nucleotide residues from the viewpoint of sufficiently exhibiting sufficient stability and high sequence specificity for bulge DNA. The following, and preferably 30 nucleotide residues.

  In the detection method of the present invention, upon mixing (A) a nucleic acid probe, (B) a compound containing a naphthyridine ring, and (C) a nucleic acid to be evaluated for single nucleotide polymorphism, (A), (B) and ( C) is preferably (C) / (A) [molar ratio] of 1/5 to 1 from the viewpoint of sufficiently forming a double strand and obtaining sufficient fluorescence intensity. (B) [molar ratio] is 1/100 to 1/20, more preferably (C) / (A) [molar ratio] is 1/12 to 1, and (C) / (B) [ The molar ratio is preferably 1/50 to 1/30.

  The pH condition at the time of mixing efficiently detects the signal of a compound capable of recognizing a bulge base such as a compound containing a naphthyridine ring, the shift of the absorption maximum wavelength based on the binding of the compound to the bulge, and the fluorescence intensity. From the viewpoint of stability of nucleic acid, the pH is preferably 5 or more, more preferably 6 or more, and still more preferably 6.5 or more, and the compound containing the naphthyridine ring and the bulge base are sufficiently bonded. From the viewpoint of performing the process described above and exhibiting sufficient fluorescence intensity, the pH is preferably 9 or less, more preferably 8 or less, and even more preferably 7.5 or less.

  Moreover, in the case of the said mixing, a phosphate buffer, a tris hydrochloric acid buffer, etc. can be used, for example.

  In the detection method of the present invention, the mixture of the nucleic acid probe (A), the compound (B) containing a naphthyridine ring, and the nucleic acid to be evaluated for single nucleotide polymorphism (C) is (A). ) And (C) single nucleotide polymorphism target nucleic acid to be mixed to form a bulge structure, and then (B) a compound containing a naphthyridine ring may be further mixed, The nucleic acid probe of (A), the compound containing the naphthyridine ring of (B), and the nucleic acid to be evaluated for single nucleotide polymorphism of (C) may be mixed simultaneously.

  The fluorescence intensity based on a compound containing a naphthyridine ring bonded to a bulge base is cytosine bulge from the viewpoint of sufficiently distinguishing from the fluorescence derived from the compound containing the naphthyridine ring (B) not bonded to the bulge base. In this case, the fluorescence intensity is preferably 400 to 480 nm, more preferably around 430 nm.

  In another aspect, the present invention relates to a kit for use in the method for detecting a single nucleotide polymorphism in the nucleic acid of the present invention. The kit of the present invention contains the nucleic acid probe and the compound containing the naphthyridine ring. Since the kit of the present invention contains the nucleic acid probe and the compound containing the naphthyridine ring, the detection method of the present invention can be carried out efficiently and simply at low cost, and the single nucleotide sequence in the nucleic acid can be increased. The mold exhibits an excellent effect that it can be detected efficiently and with high sensitivity by a simple operation at a low cost.

  In the kit of the present invention, the type of the nucleic acid probe can be appropriately selected according to the nucleotide adjacent to the position of the single nucleotide polymorphism to be evaluated and the base sequence around the position of the single nucleotide polymorphism. Specifically, for example, when the nucleotide adjacent to the position of the single nucleotide polymorphism to be evaluated is a cytosine residue, the content of cytosine residues in the base sequence around the position of the single nucleotide polymorphism When i is high, the nucleic acid probe of i) and / or ii), specifically, the nucleic acid probe of i ′) and / or ii ′) and i ″) and / or ii ″) It may be a combination with a nucleic acid probe. When the nucleotide adjacent to the position of the single nucleotide polymorphism to be evaluated is a nucleotide residue other than cytosine, in the nucleotide sequence around the position of the single nucleotide polymorphism When the content of cytosine residues is low or does not contain cytosine residues, the nucleic acid probe of I) or II), specifically, the nucleic acid probe of I ′) and / or II ′) And I '' And / or II '') may be a combination of a nucleic acid probe.

  The kit of the present invention may appropriately contain a reagent for stably holding the nucleic acid probe, for example, a buffer or the like, a reagent for stably holding a compound containing the naphthyridine ring, and the like. Good.

  In addition, the kit of the present invention is suitable for performing the detection method of the present invention using all suitable reagents for performing detection methods such as an appropriate nucleic acid probe, a compound containing the naphthyridine ring, and other necessary reagents. It may be a form provided as a single container containing a different volume and / or form, in which a nucleic acid probe, a compound containing a naphthyridine ring, other necessary reagents, etc. are provided in separate containers. May be. In addition, such a kit may contain instructions describing the procedures for performing the detection method of the present invention using the components contained in the kit.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples.

  2,7-diamino-1,8-naphthyridine (10 μM) represented by the above formula (I) is replaced with 10 mM sodium phosphate buffer at pH 8.5, pH 7.0 or pH 5.0, respectively, or the following formula (V):

It is mixed with a 10 mM sodium phosphate buffer having a pH of 7.0 containing a duplex having cytosine bulge (30 μM), and absorption at wavelengths of 300 nm to 450 nm and fluorescence intensity at wavelengths of 350 nm to 500 nm are trade names: RF It measured with -5300PC (made by Shimadzu Corporation Corp.). The results are shown in FIG.

  As a result, as shown in FIG. 2, the wavelength of 2,7-diamino-1,8-naphthyridine having an absorption maximum at pH 7.0 is shifted from 364 nm to 394 nm by 30 nm due to binding with cytosine bulge. And the wavelength showing the maximum value of the fluorescence intensity is shifted from 398 nm to 430 nm by 32 nm. :

  Therefore, it was suggested that a nucleic acid forming a bulge can be detected by 2,7-diamino-1,8-naphthyridine. Further, from the result of FIG. 2, it was found that the detection can be performed efficiently under neutral conditions.

  2,7-diamino-1,8-naphthyridine (10 μM) represented by the above formula (I) in the presence of complete duplex (30 μM), adenine bulge, thymine bulge, cytosine bulge or guanine bulge (30 μM) In addition, the mixture was mixed with 10 mM sodium phosphate buffer at pH 7.0, and the absorption at a wavelength of 300 nm to 450 nm and the fluorescence intensity at a wavelength of 350 nm to 500 nm were measured with a trade name: RF-5300PC (manufactured by Shimadzu Corporation). did. The results are shown in FIG.

  As shown in FIG. 3, 2,7-diamino-1,8-naphthyridine (10 μM) is significantly longer when combined with cytosine bulge or thymine bulge than when combined with full duplex. You can see that it shifts. Furthermore, from the results shown in FIG. 3, it was suggested that the nucleic acid probe can efficiently detect single nucleotide polymorphisms by designing it to form cytosine bulge or thymine bulge.

  Formula (VI) below:

5 '-...- XY -...- 3' (evaluation symmetric nucleic acid) and 3 '-...- ZNW -...- 5' (nucleic acid) A duplex having a bulge structure (hereinafter referred to as “X_Y / ZNW”) formed from a probe) is T_A / ACT, T_A / ACG, T_C / ACI, T_C / ACT, or T_C / ACG was used. A allele (A allele) and C allele (SEQ ID NO: 4) and a complementary T probe (T probe) containing one extra cytosine residue at the 3 ′ end of the single nucleotide polymorphism site ( As shown in the left panel of FIG. 4, four duplexes containing cytosine bulges were generated by hybridization with SEQ ID NO: 2) and G probe (SEQ ID NO: 3). While cytosine bulges in two of these four duplexes are adjacent to Watson-Crick base pairs (T_A / ACT and T_C / ACG), cytosine bulges in the other two duplexes are mismatched AG base pairs and C Adjacent to T base pairs (T_A / ACG and T_C / ACT, respectively).

  The fluorescence intensity based on the binding of 2,7-diamino-1,8-naphthyridine and cytosine bulge was measured with a trade name: RF-5300PC (manufactured by Shimadzu Corporation). The results are shown in the right panel of FIG.

  As shown in the right panel of FIG. 4, regardless of the base pair adjacent to cytosine bulge, strong fluorescence was observed in the A allele duplex, and very weak luminescence was detected in the C allele duplex.

  On the other hand, the relative fluorescence intensity of the A allele duplex at 424 nm determined by the peak height was about 5.5 times the relative fluorescence intensity of the C allele duplex. In contrast to the CG base pair in T_C / ACG, the AG mismatch base pair in T_A / ACG did not quench the fluorescence of 2,7-diamino-1,8-naphthyridine bound to the adjacent cytosine bulge. . Absorbance measurement also revealed that 2,7-diamino-1,8-naphthyridine did not bind to cytosine bulge adjacent to the CT mismatch in T_C / ACT.

  2,7- to each duplex that combines two single nucleotide polymorphism alleles and two probes that are complementary to the allele and that contain bulge bases adjacent to the position corresponding to the single nucleotide polymorphism site. Diamino-1,8-naphthyridine was added, bound to the bulge structure, fluorescence measurement was performed by excitation at 394 nm, and it was suggested that the nucleotide base at the polymorphic site can be determined by variation in fluorescence intensity between each duplex. It was.

In these analyses, the fluorescence of 2,7-diamino-1,8-naphthyridine at 424 nm is 1) when 2,7-diamino-1,8-naphthyridine binds to cytosine bulge, and 2) to cytosine bulge. It was only observable when the adjacent base pair was not a CG base pair. Therefore, the C allele was detected as a negative signal of 2,7-diamino-1,8-naphthyridine fluorescence.

  Therefore, in the C allele, the guanine residue arranged corresponding to the position of the single nucleotide polymorphism in the G probe was replaced with the inosine residue in the I probe (SEQ ID NO: 5). As a result, the C allele was successfully detected as a positive signal. Since inosine does not quench the fluorescence of 2,7-diamino-1,8-naphthyridine, the fluorescence of 2,7-diamino-1,8-naphthyridine bound to cytosine bulge adjacent to the CI base pair (T_C / ACI) The intensity was about 10 times stronger than that bound to cytosine bulge adjacent to the CG base pair in T_C / ACG.

  As a result, by using 2,7-diamino-1,8-naphthyridine in combination with a nucleic acid probe that generates cytosine bulges adjacent to different base pairs, single nucleotide polymorphism typing can be performed in a homogeneous system. It was suggested that it can be done.

  Formula (VII) below:

5 '-...- AN ₁ -...- 3' (evaluated symmetric nucleic acid) and 3 '-...- TCN ₂ -...- 5' A cytosine bulge duplex was generated using a bulge structure formed from (nucleic acid probe) (hereinafter referred to as “A_N ₁ / TCN ₂ ”), and 2,7-diamino-1 was produced in the same manner as in Example 3. , The fluorescence intensity based on the combination of 8-naphthyridine and cytosine bulge was measured at an excitation wavelength at 410 nm. In formula (VII), N ₁ and N ₂ are independently any of T, A, C, or G, and all possible combinations of these four bases were used. In the formula (VII), oligonucleotides containing _{N 1} called _{N 1} allele, for example, if _{N 1} is A, as A allele. Similarly, it refers to an oligonucleotide containing _{N 2} and _{N 2} probe, for example, when _{N 2} is A, that A probe. Differential fluorescence intensity was obtained by subtracting the detector background signal reading from the fluorescence intensity peak reading obtained from each duplex. The results are shown in Table 1.

  As shown in Table 1, as in Example 3, the A allele duplex is totally intensely fluorescent. In contrast, only the fluorescence below the background signal was detected in the G allele duplex, and the difference fluorescence intensity was a negative value.

  Subsequently, based on the result of Table 1, the method of this invention was evaluated about the detection capability of the single nucleotide polymorphism for every base. Specifically, the fluorescence intensities obtained by four duplexes combining two different alleles in Table 1 and two probes having bases complementary to each polymorphic site were compared. The results are shown in Tables 2-7.

  From Tables 2 to 7, the variation in fluorescence intensity is significantly large for mutations from T to A, T to G, T to C, A to C, and A to G, so one is a wild type allele and the other is a mutation In the case of a type allele, four duplexes combined with the probe of the present invention having a base complementary to each polymorphic site are generated, and four duplexes are added by adding 2,7-diamino-1,8-naphthyridine It is suggested that the mutant allele can be identified based on the fluctuation of the fluorescence intensity.

Therefore, each allele and duplex were generated using an I probe in which N ₂ of formula (VII) was inosine instead of the G probe, and the fluorescence intensity was measured. The results are shown in Table 8.

  Table 8 shows that significantly higher fluorescence was obtained with the I probe than with the G probe in alleles other than G. Then, based on the results in Table 8, the fluorescence in the C to G, T to C, and A to C mutations were compared. The results are shown in Table 9, Table 10, and Table 11.

  From Table 9, it was found that the C to G mutation can be detected by using the I probe. Further, from Tables 10 and 11, the use of the I probe for the T-to-C and A-to-C mutations causes a greater variation in fluorescence intensity than when the G-probe is used, and the single nucleotide polymorphism of the present invention. It has been found that the detection method of detection gives better results.

  According to the present invention, a single nucleotide polymorphism in a nucleic acid can be detected with low sensitivity and high sensitivity by a simple operation. Therefore, inexpensive, simple and highly sensitive gene diagnosis, gene analysis, and the like are possible.

  SEQ ID NO: 1 is the sequence of synthetic DNA (A allele).

  SEQ ID NO: 2 is the sequence of synthetic DNA (T probe).

  SEQ ID NO: 3 is the sequence of a synthetic DNA (G probe).

  SEQ ID NO: 4 is the sequence of a synthetic DNA (C allele).

  SEQ ID NO: 5 is the sequence of synthetic DNA (I probe). N in the base number: 5 is inosine.

SEQ ID NO: 6 is the sequence of a synthetic DNA _{(N 1} allele). N in the base number: 9 is adenine, cytosine, guanine, or thymine.

SEQ ID NO: 7 is the sequence of the synthetic DNA _{(N 2} probe). N in the base number: 10 is adenine, cytosine, guanine, thymine, or inosine.

Claims (9)

(A) I) Complementary to a nucleic acid to be evaluated containing at least one single nucleotide polymorphism, and adjacent to the 5 ′ or 3 ′ terminal side of the position where the single nucleotide polymorphism is paired A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position, or II) capable of hybridizing complementary to the antisense strand of a nucleic acid to be evaluated containing at least one single nucleotide polymorphism A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism on the antisense strand,
When,
(B) a compound capable of recognizing a bulge base;
(C) the nucleic acid to be evaluated;
Mix and
Detecting a signal based on the compound (B) that recognizes cytosine bulge or thymine bulge formed when the nucleic acid probe and the nucleic acid to be evaluated are hybridized;
A method for detecting a single nucleotide polymorphism, comprising evaluating the single nucleotide polymorphism. 1) to 4) below:
1) A bulge formed between a wild-type evaluation target nucleic acid containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the wild-type evaluation target nucleic acid,
2) A bulge formed between a mutant nucleic acid to be evaluated containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and a nucleic acid probe capable of hybridizing complementarily to the nucleic acid to be mutant-type evaluated;
3) a bulge formed between the wild-type evaluation target nucleic acid and a nucleic acid probe capable of hybridizing complementary to the mutant-type evaluation target nucleic acid; and 4) the mutant-type evaluation target nucleic acid and the wild type A bulge formed between a nucleic acid probe capable of hybridizing complementary to the nucleic acid to be evaluated,
The detection method according to claim 1, wherein a single nucleotide polymorphism in the nucleic acid to be evaluated is identified on the basis of a change in signal based on a compound capable of recognizing the bulge base of (B) bound to each of the nucleic acids.   The detection method according to claim 1, wherein the compound has a naphthyridine ring.   The detection method according to claim 1, wherein the signal is fluorescence.   The detection method according to any one of claims 1 to 4, wherein the compound is a 2,7-diaminonaphthyridine derivative compound. The 2,7-diaminonaphthyridine derivative compound is represented by the following formula (II):

The detection method according to claim 5, which is 2,7-diamino-1,8-naphthyridine represented by formula (1). The nucleic acid probe of (A) is
i) Cytosine at a position adjacent to the 5 ′ or 3 ′ terminal side of the position capable of hybridizing complementarily to the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism A nucleic acid probe comprising a base sequence to which a residue or thymine residue is added and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is further substituted with an inosine residue; or ii ) 5 ′ or 3 ′ at a position capable of hybridizing complementarily to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and paired with the single nucleotide polymorphism on the antisense strand A cytosine residue or thymine residue is added at a position adjacent to the terminal side, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is a wild boar. A nucleic acid probe having a base sequence further substituted with
The detection method according to claim 1, wherein I ′) contains at least one single nucleotide polymorphism, and can hybridize complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and matches the single nucleotide polymorphism A nucleic acid probe having a base sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position, and / or II ′) at least one single nucleotide polymorphism, 5 ′ or 3 of the position that can be complementarily hybridized to the antisense strand of the nucleic acid to be evaluated containing a wild-type nucleotide at the position where the nucleotide polymorphism is present, and that matches the single nucleotide polymorphism on the antisense strand. A nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the terminal side,
When,
I ″) comprising at least one single nucleotide polymorphism and capable of hybridizing complementarily to a nucleic acid to be evaluated containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and pairing with the single nucleotide polymorphism A nucleic acid probe having a nucleotide sequence to which a cytosine residue or a thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position to be selected, and / or II ″) at least one single nucleotide polymorphism, 5 ′ at the position where the single nucleotide polymorphism can be complementarily hybridized to the antisense strand of the nucleic acid to be evaluated containing the mutant nucleotide at the position where the single nucleotide polymorphism is present, and which matches the single nucleotide polymorphism on the antisense strand. Or a nucleic acid probe having a base sequence to which a cytosine residue or thymine residue is added at a position adjacent to the 3 ′ end,
When,
A compound capable of recognizing a bulge base;
The kit for using for the detection method of the single nucleotide polymorphism in the nucleic acid of any one of Claims 1-7 formed by containing. i ′) containing at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a wild-type nucleotide at the position of the single nucleotide polymorphism, and pairing with the single nucleotide polymorphism A cytosine residue or thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is an inosine residue A nucleic acid probe having a base sequence substituted with
ii ′) Complementary to the antisense strand of the nucleic acid to be evaluated comprising at least one single nucleotide polymorphism and containing a wild-type nucleotide at the position where the single nucleotide polymorphism is present, and on the antisense strand Guanine to which a cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position where it is paired with a single nucleotide polymorphism and is adjacent to the cytosine residue or thymine residue A nucleic acid probe having a base sequence in which at least one of the residues is further substituted with an inosine residue;
When,
i ″) comprising at least one single nucleotide polymorphism, capable of hybridizing complementarily to a nucleic acid to be evaluated containing a mutant nucleotide at the position of the single nucleotide polymorphism, and pairing with the single nucleotide polymorphism A cytosine residue or thymine residue is added to a position adjacent to the 5 ′ or 3 ′ terminal side of the position to be detected, and at least one of the guanine residues present adjacent to the cytosine residue or thymine residue is an inosine residue. A nucleic acid probe having a base sequence substituted by a group,
ii ″) Complementary hybridization to the antisense strand of the nucleic acid to be evaluated containing at least one single nucleotide polymorphism and containing a mutant nucleotide at the position where the single nucleotide polymorphism is present, and the antisense strand A cytosine residue or thymine residue is added at a position adjacent to the 5 ′ or 3 ′ terminal side of the position that matches the single nucleotide polymorphism above, and is adjacent to the cytosine residue or thymine residue. A nucleic acid probe having a base sequence in which at least one of guanine residues is further substituted with an inosine residue;
When,
A kit for use in a method for detecting a single nucleotide polymorphism in a nucleic acid according to claim 8, comprising a compound capable of recognizing a bulge base.

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