JP5670396B2 - Nucleic acid probe set and method of using the same - Google Patents

Description

  The present invention relates to the field of genetic engineering, and more particularly to a nucleic acid probe set used for nucleic acid analysis and a method for using the same.

  In the method for detecting a target nucleic acid, a single-stranded nucleic acid probe that specifically hybridizes with the target nucleic acid is widely used. One method of detecting a target nucleic acid using a single-stranded nucleic acid probe is to label the nucleic acid probe with a fluorescent substance, and when this nucleic acid probe binds to the target nucleic acid, the fluorescence is quenched by the action of guanine in the target nucleic acid. In addition, there is a Q-probe method (Quenching Probe) that utilizes the phenomenon that fluorescence that has been quenched once emitted from the target nucleic acid. The Q-probe method has excellent advantages that the structure of the probe is simple, that trial and error are not required for the probe design, and that a highly accurate result is obtained (for example, Patent Document 1 and Patents). (Ref. 2).

Japanese Patent No. 3437816 JP 2002-119291 A

  However, in such a conventional single-stranded nucleic acid probe, a fluorescently labeled nucleic acid probe having a different base sequence must be prepared for each target nucleic acid. Such a fluorescently labeled nucleic acid probe is relatively expensive and has a problem that it takes a long time to synthesize. Therefore, an experiment using the nucleic acid probe is also costly and requires time for preparation. There is.

  Accordingly, an object of the present invention is to provide a nucleic acid probe that can solve the above-described problems of the prior art and can be prepared in a short time and at a low cost, and a method for using the same.

The above object is achieved by the present invention described below. That is, the present invention relates to one or two fluorescent probes (A) comprising an oligonucleotide containing a nucleotide (a) labeled with a fluorescence quencher (d) whose fluorescence intensity is attenuated when interacting with guanine ; Binding comprising an oligonucleotide having one or two fluorescent probe binding regions (b1) to which the fluorescent probe (A) hybridizes and a target nucleic acid binding region (b2) to hybridize with the target nucleic acid (C) When the fluorescent probe (A) hybridizes to the fluorescent probe binding region (b1) and the target nucleic acid (C) hybridizes to the target nucleic acid binding region (b2) to a guanine in nucleic acids comprising the target nucleic acid (C), the nucleotide (a) the labeled fluorescent quencher (d) Togaai Acts as the fluorescence intensity of the fluorescence quencher (d) is attenuated, a nucleic acid probe set, characterized in that the fluorescent probes (A) and the binding probe (B) is designed.

In the nucleic acid probe set of the present invention, the fluorescence quencher (d) is tetrabromosulfonefluorescein (TBSF), 2-oxo-6,8-difluoro-7-hydroxy-2H-1-benzopyran-3-carboxylic acid. acid (P acific Blue (TM)), rhodamine 6G, carboxy rhodamine 6G, carboxymethyl tetramethylrhodamine and 4,4-difluoro-5,7-dimethyl-4-bora -3a, 4a-diaza -s- indacene - Any one selected from the group consisting of 3-propionic acid ( BODIPY ™ -FL ) ; the one or two fluorescent probes (A) fluoresce the 5 ′ terminal nucleotide or the 3 ′ terminal nucleotide The oligonucleotide is labeled with a quencher (d); the one or two fluorescent probes (A) are 5 to 50 bases long; the target nucleic acid binding region (b2) is 5 to 60 bases In length Preferably there is.

The present invention also provides a detection how the target nucleic acid, characterized in that it comprises the following steps (1) and (2).
(1) a step of hybridizing the nucleic acid probe set of the present invention and a target nucleic acid (C),
(2) Next, measuring the fluorescence intensity of the complex of the hybridized nucleic acid probe set and the target nucleic acid (C) . This detection method may further include the following steps (3) and (4).
(3) changing the ratio between the nucleic acid probe set and the target nucleic acid and performing the steps (1) and (2);
(4) A step of comparing the fluorescence intensities obtained from the steps (2) and (3).

The present invention also provides a nucleic acid base sequence polymorphism analysis method characterized by including the following steps (1) to (4).
(1) a step of hybridizing the nucleic acid probe set of the present invention and a target nucleic acid (C),
(2) Next, measuring the temperature dependence of fluorescence intensity for the complex of the hybridized nucleic acid probe set and the target nucleic acid (C),
(3) A step of performing the steps (1) and (2) using another nucleic acid instead of the target nucleic acid,
(4) A step of comparing the temperature dependence of the fluorescence intensity obtained from the steps (2) and (3).

  The present invention also provides a method characterized by performing a melting curve analysis on the complex of the nucleic acid probe set of the present invention and the target nucleic acid (C).

  In the present invention, the fluorescent probe (A) comprises two fluorescent probes having different base sequences and labeled with different fluorescent substances, and the binding probe (B) has two different base sequences. The nucleic acid probe set of the present invention having a fluorescent probe binding region and an ABC-PCR method using the nucleic acid probe set are provided.

  Conventionally, nucleic acid probes used for various nucleic acid analysis methods have to be prepared for each target nucleic acid to be analyzed, which is labeled with an expensive fluorescent material. According to the present invention, an expensive fluorescent material is used. Therefore, it is not necessary to prepare the nucleic acid probe labeled with the target nucleic acid for each target nucleic acid, so that the nucleic acid probe can be provided at a lower cost and in a shorter time than the conventional nucleic acid probe.

It is a schematic diagram which shows the nucleic acid probe set complex of this invention before hybridizing with a target nucleic acid (C). It is a schematic diagram showing a nucleic acid probe set complex of the present invention hybridized with a target nucleic acid (C). It is a schematic diagram which expands and shows the peripheral part of the fluorescent substance (d) of FIG. It is the figure which measured the fluorescence intensity after adding a fixed quantity of the nucleic acid probe set of this invention to the solution from which a target nucleic acid concentration differs, and plotted the value. It is the figure which used the PCR reaction solution from which the amount of template DNA added initially differs for real-time PCR measurement, and plotted the Ct value about each solution. It is a figure which shows the melting curve of the nucleic acid probe set complex of this invention, and target nucleic acids. It is a figure which shows the negative first derivative curve of the melting curve of FIG. FIG. 6 is a schematic diagram showing the nucleic acid probe set of the present invention used in Example 4. It is a figure which shows the correlation with the correction | amendment fluorescence intensity value obtained by ABC-PCR experiment using the nucleic acid probe set of this invention, and the number of target nucleic acids before reaction.

  Next, the best mode for carrying out the present invention will be described with reference to the drawings. In the present invention, a complex obtained by hybridizing the fluorescent probe (A) and the binding probe (B) may be referred to as a “nucleic acid probe set complex”.

  In the present invention, the term “nucleotide” means a deoxyribonucleotide or ribonucleotide monomer, and the term “oligonucleotide” means an oligomer composed of nucleotides. It may be composed only of nucleotides or may contain both.

  The “target nucleic acid” in the present invention means a region of a base sequence that specifically hybridizes with the target nucleic acid binding region (b2) of the binding probe (B) constituting the nucleic acid probe set of the present invention. The case where it is part of a nucleic acid or gene is also included. The target nucleic acid may be of any type, and examples thereof include DNA, RNA, LNA, PNA, and artificially modified nucleic acid.

  An example of the nucleic acid probe set of the present invention is shown in FIG. 1, FIG. 2, and FIG. In these figures, (A) is a fluorescent probe, (B) is a binding probe, and the nucleic acid probe set of the present invention comprises the fluorescent probe (A) and the binding probe (B). (C) is an example of the target nucleic acid.

  FIG. 1 shows that the nucleic acid probe of the present invention has one fluorescent probe (A), one fluorescent probe binding region (b1), and one target nucleic acid binding region (b2), and before hybridization with the target nucleic acid (C). It is a schematic diagram which shows a set composite_body | complex. FIG. 2 is a schematic diagram showing a state in which the target nucleic acid (C) shown in FIG. 1 and the nucleic acid probe set complex of the present invention are hybridized. FIG. 8 is a schematic diagram showing the nucleic acid probe set complex of the present invention before hybridization with the target nucleic acid (C), which has two fluorescent probes (A) and two fluorescent probe binding regions (b1). .

  One or two fluorescent probes (A) constituting the nucleic acid probe set of the present invention (hereinafter sometimes simply referred to as “fluorescent probe (A)”) are nucleotides (a) labeled with a fluorescent substance (d) ). The base sequence of the fluorescent probe (A) is not particularly limited as long as it hybridizes with the fluorescent probe binding region (b1) of the binding probe (B), and is always irrespective of the type of target nucleic acid to be detected or analyzed. The same can be used. Thus, since the fluorescent probe (A) constituting the nucleic acid probe set of the present invention does not need to have a base sequence corresponding to a specific target nucleic acid, analysis of the target nucleic acid using the nucleic acid probe set of the present invention is performed. Is advantageous in that it is not necessary to prepare a fluorescent probe having an expensive fluorescent substance for each target nucleic acid to be detected or analyzed.

  On the other hand, the binding probe (B) constituting the nucleic acid probe set of the present invention has one or two fluorescent probe binding regions (b1) (hereinafter simply referred to as “fluorescent probe binding regions”) that hybridize with the fluorescent probe (A). b1) ”and a target nucleic acid binding region (b2) that hybridizes with the target nucleic acid (C).

  As shown in FIG. 2, the nucleic acid probe set of the present invention shown in FIG. 1 has a fluorescent substance in the fluorescent probe (A) when the nucleic acid probe set complex of the present invention and the target nucleic acid (C) are hybridized. It is designed so that the fluorescent character of the fluorescent substance (d) is changed by interaction between (d) and guanine in the nucleic acid containing the target nucleic acid (C).

  As long as the guanine is in the nucleic acid containing the target nucleic acid, it may be in the base sequence region of the target nucleic acid or outside the base sequence region of the target nucleic acid. When the guanine is present in the base sequence of the target nucleic acid and forms a base pair with the cytosine of the hybridized binding probe (B), the interaction between the fluorescent substance (d) and guanine is somewhat reduced. There is no particular problem, and if the guanine does not form a base pair because it is outside the base sequence region of the target nucleic acid, the interaction between the fluorescent substance (d) and guanine becomes easy. More preferred.

  Next, referring to FIG. 3, when the nucleic acid probe set complex of the present invention and the target nucleic acid (C) are hybridized, guanine in the nucleic acid containing the fluorescent substance (d) and the target nucleic acid (C) The conditions under which any nucleotide having a base (hereinafter abbreviated as “nucleotide ε”) can interact will be described. In the following description, a nucleotide having a guanine base at the 3 'end of the target nucleic acid (C) is referred to as nucleotide ε. FIG. 3 is an enlarged view of the peripheral portion of the fluorescent material (d) in FIG. The conditions under which the fluorescent substance (d) and nucleotide ε can interact depend on the length of the spacer connecting the fluorescent substance (d) and the nucleotide (a) labeled with the fluorescent substance (d), which will be described later, and are constant. Although not generalized, it can be expressed as follows.

  When the fluorescent probe (A) and the binding probe (B) are hybridized, the nucleotide in the fluorescent probe binding region (b1) that forms a base pair with the fluorescent probe (A), the target nucleic acid Let β be the nucleotide in the fluorescent probe (A) that forms a base pair with the nucleotide closest to the binding region (b2) (hereinafter abbreviated as “nucleotide α”). Let X denote the number (base) of the distance (β-a) between this nucleotide β and the nucleotide (a) labeled with a fluorescent substance. In FIG. 3, nucleotide α is a nucleotide having a thymine base at the 3 ′ end in the fluorescent probe binding region (b1), and nucleotide β is a nucleotide labeled with a fluorescent substance (d) in the fluorescent probe (A) ( It is a nucleotide having an adenine base adjacent to a), and X in this case is 1. Note that adjacent nucleotides are counted as “X = 1”, and one adjacent nucleotide is counted as “X = 2”.

  Next, nucleotides in the target nucleic acid binding region (b2) forming a base pair with the target nucleic acid (C) and closest to the nucleotide α (hereinafter abbreviated as “nucleotide γ”) and the nucleotide α. Y is the number of bases (γ−α) expressed by the number of bases. In FIG. 3, nucleotide γ is a nucleotide having a cytosine base at the 5 ′ end in the target nucleic acid binding region (b2), and Y in this case is 1. The count of Y is the same as that of X.

  In the case where the binding probe (B) and the target nucleic acid (C) are hybridized, the nucleotide in the target nucleic acid (C) that forms a base pair with the nucleotide γ is defined as δ. Let Z be the number that represents the distance (δ−ε) between the nucleotide δ and the nucleotide ε in terms of the number of bases. In FIG. 3, nucleotide δ is a nucleotide having a 3 ′ terminal guanine base in the target nucleic acid (C), and since this nucleotide is nucleotide ε, Z is 0. The count of Z is the same as that of X.

  When the nucleic acid probe set complex of the present invention and the target nucleic acid (C) are hybridized, the condition that the fluorescent substance (d) and the guanine of the nucleotide ε can interact is that the sum of X, Y and Z is The case where it becomes 6 or less is preferable. Although depending on the length of the spacer connecting the fluorescent substance (d) and the nucleotide (a) labeled with the fluorescent substance (d), the sum of X, Y and Z is more preferably 4 or less, most preferably 2 It is.

  In the present invention, the fluorescent substance (d) labeled on the fluorescent probe (A) is one that changes the fluorescent character of the fluorescent substance (d) when interacting with guanine. In the present invention, “fluorescent character” means fluorescence intensity, and “guanine and fluorescent substance interact to change the fluorescent character of fluorescent substance” means that guanine and fluorescent substance change. It means that the fluorescence intensity of the fluorescent substance in a state where it does not interact is different from the fluorescence intensity in a state where it interacts, and the degree thereof is not limited. Further, “quenching” of fluorescence means that the fluorescence intensity is attenuated when interacting with guanine as compared with the case where the fluorescent substance does not interact with guanine, and the degree thereof is not limited.

Examples of fluorescent substances that can be suitably used in the nucleic acid probe set of the present invention include fluorescein and its derivatives (eg, fluorescein-4-isothiocyanate (FITC), tetrachlorofluorescein, hexachlorofluorescein, tetrabromosulfone fluorescein (TBSF) and Derivatives thereof), EDANS (5- (2-aminoethyl) amino-1-naphthalenesulfonic acid), 6-JOE, Alexa Fluor (registered trademark) 488 (Molecular Probes), Alexa Fluor (registered trademark) 532 ( Molecular Probes) ) , Cy3 ( trademark; Amersham Biosciences), Cy5 ( trademark; Amersham Biosciences), 2-oxo-6,8-difluoro-7-hydroxy-2H-1-benzopyran-3 - carboxylic acid (Pacific Blue (TM; Molecular probes)), rhodamine 6G R6G) and derivatives thereof (eg, carboxyrhodamine 6G (CR6G), tetramethylrhodamine (TMR), tetramethylrhodamine isothiocyanate (TMRITC), x-rhodamine, carboxytetramethylrhodamine (TAMRA)), Texas Red (Molecular Probes) 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid ( BODIPY ™ -FL (Molecular Probes), BODIPY ( Trademarks) -R6G (Molecular Probes), BODIPY (trademark) 564 (Molecular Probes) and BODIPY (trademark) 581 (Molecular Probes).

Te tiger bromosulfonic fluorescein Among these, 2-oxo-6,8-difluoro-7-hydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue (TM)), rhodamine 6G, carboxy rhodamine 6G, carboxymethyl tetramethylrhodamine and 4,4-difluoro-5,7-dimethyl-4-bora -3a, it is used 4a- diaza -s- indacene-3-propionic acid (BODIPY (TM) -FL) More preferably, it is most preferred to use 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid ( BODIPY ™ -FL ) .

  The fluorescent probe (A) used in the nucleic acid probe set of the present invention can be used by consigning production to an oligonucleotide manufacturing contract company (for example, Tsukuba Oligo Service Co., Ltd., Ibaraki Prefecture). The method for labeling the oligonucleotide with the fluorescent substance is not particularly limited, and a conventionally known labeling method can be used. (Nature Biotechnology, 14, 303-308, 1996; Applied and Environmental Microbiology, 63, 1143-1147, 1997; Nucleic acids Research, 24, 4532-4535, 1996).

For example, when a fluorescent substance is bound to the 5 ′ terminal nucleotide, first, for example, — (CH ₂ ) _n —SH is introduced as a spacer into the 5 ′ terminal phosphate group according to a conventional method. A commercially available spacer can be used (for example, Midland Certified Reagent Company, USA). In this case, n is 3-8, preferably 6. A fluorescently labeled oligonucleotide can be obtained by binding a fluorescent substance having SH group reactivity or a derivative thereof to this spacer. The fluorescently labeled oligonucleotide can be purified by reverse phase chromatography or the like to obtain the fluorescent probe (A) of the present invention.

In addition, a fluorescent substance can be bound to the 3 ′ terminal nucleotide of the oligonucleotide. In this case, for example, — (CH ₂ ) _n —NH ₂ is introduced as a spacer into the OH group at the 3′-position C of ribose or deoxyribose. A commercially available spacer can also be used (for example, Midland Certified Reagent Company, USA). As another method, a phosphate group is introduced into the OH group at the 3′-position C of ribose or deoxyribose, and, for example, — (CH ₂ ) _n —SH is introduced as a spacer into the OH group of the phosphate group. In this case, n is 3 to 8, preferably 4 to 7.

An oligonucleotide labeled with a fluorescent substance can be synthesized by binding a fluorescent substance or a derivative thereof reactive to an amino group or an SH group to the spacer. The oligonucleotide can be purified by reverse phase chromatography or the like to obtain the fluorescent probe (A) of the present invention. As spacers - When introducing (CH _{2) n} -NH _2, kit reagents (e.g., Uni-link aminomodifier, Clontech) to use is convenient. Then, a fluorescent substance can be bound to the oligonucleotide according to a conventional method.

  The nucleotide (a) labeled with the fluorescent substance in the fluorescent probe (A) is not limited to the both terminal nucleotides of the oligonucleotide, and nucleotides other than the both terminal nucleotides can be labeled with the fluorescent substance (ANALYTICAL). BIOCHEMISTRY, 225, 32-38, 1998).

Although the fluorescent probe (A) constituting the nucleic acid probe set of the present invention can be prepared as described above, the preferred form of the fluorescent probe (A) is that the 5 ′ terminal or 3 ′ terminal nucleotide is labeled with the fluorescent substance (d). More preferably, the 5 ′ terminal nucleotide is labeled with BODIPY ™ -FL. The nucleotide base labeled with the fluorescent substance (d) is preferably guanine or cytosine.

  The fluorescent probe (A) constituting the nucleic acid probe set of the present invention may be any base sequence that hybridizes with the fluorescent probe binding region (b1) of the binding probe (B), and the base length is not particularly limited. If it is less than the base, it is not preferable in terms of low hybridization efficiency, and if it is more than 51 bases, it may not be preferable in that non-specific hybrids are likely to occur. Therefore, the fluorescent probe (A) preferably has 5 to 50 bases, more preferably 10 to 35 bases, and particularly preferably 12 to 30 bases.

  The base sequence of the fluorescent probe (A) may have nucleotides that are not complementary to the fluorescent probe binding region (b1) as long as it hybridizes with the fluorescent probe binding region (b1) of the binding probe (B). For example, like the fluorescent probe (A) of FIG. 1, it may have a nucleotide that is not complementary to the binding probe (B) at the 5 'end. On the other hand, the base sequence of the fluorescent probe binding region (b1) in the binding probe (B) only needs to be hybridized with the fluorescent probe (A), and the base length is the base length of the fluorescent probe (A). Depends on.

  The target nucleic acid binding region (b2) in the binding probe (B) may be a base sequence that hybridizes with the target nucleic acid (C), and its base length depends on the base length of the target nucleic acid, but it is 4 bases or less. If it is, it is not preferable in that the hybridization efficiency with the target nucleic acid (C) is low, and if it is 61 bases or more, it may not be preferable in that a non-specific hybrid is likely to be generated. Therefore, the target nucleic acid binding region (b2) preferably has 5 to 60 bases, more preferably 15 to 30 bases. The base sequence of the target nucleic acid binding region (b2) may have a base sequence that does not form a base pair with the target nucleic acid (C) as long as it hybridizes with the target nucleic acid (C).

  The nucleic acid probe set of the present invention can be used in various nucleic acid analysis methods. An example of a target nucleic acid detection method for determining whether or not a target nucleic acid is present in a solution using the nucleic acid probe set of the present invention is shown below.

  First, a solution for detecting a target nucleic acid (hereinafter abbreviated as “detection sample”) is serially diluted to prepare several types of solutions. A certain amount of each of the nucleic acid probe set of the present invention, that is, the fluorescent probe (A) and the binding probe (B) is added to these serially diluted detection samples, and then the added nucleic acid probe set complex of the present invention and the target nucleic acid are added. After adjusting the temperature of the solution to hybridize, the fluorescence intensity is measured. The temperature at which the probe set complex of the present invention is hybridized with the target nucleic acid varies depending on the melting temperature of the nucleic acid probe set complex of the present invention and the target nucleic acid (hereinafter referred to as “Tm1”) and other solution conditions. Is preferably in a temperature range in which sequence-specific hybridization between the nucleic acid probe set complex and the target nucleic acid occurs, and non-specific hybridization does not occur, preferably Tm1 to (Tm1-40) ° C. More preferably, it is Tm1- (Tm1-20) degreeC, More preferably, it is Tm1- (Tm1-10) degreeC. An example of such a preferred temperature is about 60 ° C.

  The melting temperature (hereinafter referred to as “Tm2”) of the fluorescent probe and the binding probe constituting the nucleic acid probe set of the present invention is preferably higher than Tm1 in order to reliably measure the fluorescence intensity. Is more preferably 5 ° C. or higher.

  When the target nucleic acid is not present in the detection sample, the same fluorescence intensity is observed in any of the detection samples diluted serially. On the other hand, when the target nucleic acid is present in the detection sample, fluorescence from the fluorescent substance of the nucleic acid probe set of the present invention is quenched by guanine in the nucleic acid containing the target nucleic acid. The degree of this quenching is changed by changing the ratio between the nucleic acid probe set and the target nucleic acid in the solution. Therefore, the presence of the target nucleic acid can be determined from the occurrence of fluorescence quenching by adding the nucleic acid probe set of the present invention to the detection sample serially diluted as described above and measuring their fluorescence intensity. Thus, the abundance of the target nucleic acid can be quantified from the magnitude of fluorescence quenching.

  The nucleic acid probe set of the present invention can also be used in a real-time PCR method. In the real-time PCR method, when the amplification product is quantified using the nucleic acid probe set of the present invention, the binding probe (B) is hybridized with the target nucleic acid using the base sequence amplified by PCR or a part thereof as the target nucleic acid. The base sequence of the target nucleic acid binding region (b2) is determined.

  The nucleic acid probe set of the present invention thus prepared is added to a PCR reaction solution, a PCR reaction is performed, and the fluorescence intensity is measured in each cycle of PCR. When the target nucleic acid in the reaction solution is amplified by the PCR reaction, the fluorescence due to the fluorescent substance of the nucleic acid probe set of the present invention is quenched by guanine in the nucleic acid containing the target nucleic acid. PCR amplification products can be quantified.

  Furthermore, the nucleic acid probe set of the present invention can also be used for analysis of nucleotide sequence polymorphisms of nucleic acids. Examples of base sequence polymorphisms that can be analyzed include single base polymorphisms from base nucleotide sequences, base substitutions, base deletions, and base insertions. An example of such an analysis method is shown below.

  In this analysis method, a solution containing the target nucleic acid (C) and a solution containing the nucleic acid to be analyzed are prepared using the base sequence serving as a reference as the target nucleic acid (C). The nucleic acid probe set of the present invention, that is, the binding probe (B) having the target nucleic acid binding region (b2) designed to hybridize with the target nucleic acid (C) and the fluorescent probe (A) were added to each solution. Thereafter, in each solution, the added nucleic acid probe set complex of the present invention and the target nucleic acid (C) or the nucleic acid to be analyzed are hybridized, and then the temperature dependence of the fluorescence intensity is measured. Specifically, the fluorescence intensity is measured for each temperature while changing the temperature of the solution from a low temperature to a high temperature.

  A plot of the measurement results against temperature is called a “melting curve”. By differentiating the melting curve of the solution containing the target nucleic acid with respect to the temperature, Tm1 between the nucleic acid probe set complex of the present invention and the target nucleic acid (C) can be easily determined as the temperature showing the extreme value. Such melting curve analysis can be performed by using a commercial program well known to those skilled in the art.

  The fluorescence intensity of the solution containing the target nucleic acid (C) is suppressed by a fluorescence quenching phenomenon due to guanine in the nucleic acid containing the target nucleic acid (C) at a low temperature. However, when the solution temperature is increased to around Tm1, the target nucleic acid is dissociated from the nucleic acid probe set complex of the present invention, and the degree of fluorescence quenching becomes weak, so that the fluorescence intensity increases rapidly. If there is a base sequence polymorphism in the base sequence of the nucleic acid to be analyzed, for example, a single base polymorphism from the base sequence of the target nucleic acid, base substitution, base deletion, base insertion, etc. Tm1 with the nucleic acid probe set complex of 1 shows a lower value than Tm1 between the target nucleic acid (C) and the nucleic acid probe set complex of the present invention. For this reason, by comparing the temperature dependence of the fluorescence intensity of the target nucleic acid and the nucleic acid probe set complex of the present invention and the temperature dependence of the fluorescence intensity of the nucleic acid to be analyzed and the nucleic acid probe set complex of the present invention, The base sequence polymorphism of the nucleic acid to be analyzed with respect to the base sequence of the target nucleic acid can be analyzed. As such an analysis procedure, melting curves can be compared with each other, but each melting curve is differentiated with respect to temperature, Tm1 is obtained as a temperature giving an extreme value, and comparison is made so that mutation can be easily performed. The presence or absence can be determined.

  In addition, in the nucleotide sequence of the nucleic acid to be analyzed, when a nucleotide having a guanine base that quenches the fluorescent substance of the fluorescent probe is mutated, the fluorescence intensity decreases due to the fluorescence quenching phenomenon at any temperature. Since it does not occur, the mutation can be identified from the melting curve.

  Conventionally, in melting curve analysis, it was necessary to prepare nucleic acid probes having different base sequences with expensive fluorescent labels for each target nucleic acid, and it took a long time to synthesize the nucleic acid probes. By using for melting curve analysis, it is not necessary to prepare expensive fluorescently labeled nucleic acid probes for each target nucleic acid, so preparation time for melting curve analysis can be shortened and melting curve analysis can be performed at a lower cost Can do.

  The nucleic acid probe set of the present invention described above is composed of a binding probe (B) having one fluorescent probe binding region (b1) and one fluorescent probe (A). As shown in FIG. 8, the probe set may have two fluorescent probe binding regions (b1). The fluorescent probe binding regions (b1) may have the same base sequence or different base sequences, but preferably have different base sequences. In this case, the number of fluorescent probes may be one, but it is preferable to have two fluorescent probes (A) having different base sequences. The structures of the fluorescent probe binding region (b1) and the fluorescent probe (A) in the nucleic acid probe set of the present invention shown in FIG. 8 are the same as described above.

  The above-described binding probe (B) has two fluorescent probe binding regions and the nucleic acid probe sets of the present invention having different base sequences are prepared by ABC-PCR (Alternately Binding probe Competitive PCR; Tani et al., Analytical Chemistry, printing preparations). It can be suitably used as an alternative to the AB probe conventionally used in the method.

  The ABC-PCR method disclosed by Tani et al. Is a kind of competitive PCR method. In addition to a nucleic acid containing a base sequence to be amplified (hereinafter abbreviated as “amplified sequence”) in a PCR reaction solution, an amplified sequence is used. Has a base sequence complementary to a part of the above amplified sequence, and both ends thereof are different in excitation wavelength and fluorescence wavelength. In this method, a nucleic acid probe (hereinafter abbreviated as “AB probe”) labeled with a fluorescent substance is added to perform a PCR reaction. When the AB probe is hybridized with a nucleic acid containing an amplification sequence, only one fluorescent substance is quenched by guanine in the nucleic acid, and when hybridized with an internal standard nucleic acid, both fluorescent substances are Since it is designed to be quenched by guanine in the standard nucleic acid, the amplification product by the PCR reaction can be quantified from the fluorescence intensities of the two fluorescent substances.

  The fluorescently labeled AB probe used in the above conventional ABC-PCR method must use a different probe for each base sequence to be amplified, but the nucleic acid probe set of the present invention is used in place of the AB probe. Thus, it is not necessary to prepare an expensive fluorescently labeled fluorescent probe for each base sequence to be amplified, so that the ABC-PCR method can be carried out at a lower cost.

An example of the nucleic acid probe set of the present invention suitable for use in such an ABC-PCR method is shown in FIG. In this case, the base sequence of the target nucleic acid binding region (b2) in the binding probe (B) is determined so as to hybridize with the target nucleic acid, with a part of the base sequence amplified by PCR as the target nucleic acid (C). When the nucleic acid probe set of the present invention is used in place of the AB probe in the ABC-PCR method, preferred combinations of fluorescent substances having different excitation wavelengths and different fluorescence wavelengths to be labeled on the two fluorescent probes constituting the nucleic acid probe set As an example, a combination of BODIPY (trademark) -FL and TAMRA can be mentioned.

  EXAMPLES Next, although an Example is given and this invention is demonstrated further more concretely, these Examples are only the illustrations of this invention, and do not intend limiting of this invention.

[Example 1] <Target nucleic acid detection test>
As shown in FIG. 1, an oligonucleotide having the same base sequence as a part of the human UCP1 gene is a target nucleic acid (SEQ ID NO: 1), and a fluorescent probe binding region having a base sequence complementary to the fluorescent probe on the 5 ′ end side (B1) has a binding probe (SEQ ID NO: 2) having a target nucleic acid binding region (b2) of a base sequence complementary to the target nucleic acid on the 3 ′ end side and a base sequence of SEQ ID NO: 3 and a 5 ′ terminal nucleotide A target nucleic acid detection test was performed using a nucleic acid probe set consisting of a fluorescent probe labeled with BODIPY (trademark) -FL (Molecular Probes).
Sequence number 1: tgaccacagtttgatcgagtg
SEQ ID NO: 2: tctcggagtccgggcgatcactcgatcaaactgtggtca-phosphate SEQ ID NO: 3: BODIPY (trademark) -FL-catcgcccggactccgaga-phosphate

  The binding probe and the fluorescent probe have a phosphate group at the 3 'end. The synthesis of the target nucleic acid, binding probe and fluorescent probe was outsourced to Tsukuba Oligo Service Co., Ltd. (Tsukuba City).

The final concentration of the target nucleic acid is 0, 0.1, 0.2, 0.00 in PCR buffer (containing 50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl ₂ , all at the final concentration, pH 8.7). Prepare 6 target nucleic acid solutions serially diluted to 3, 0.4 and 0.5 μM, and add fluorescent probe (final concentration 0.05 μM) and binding probe (final concentration 0.2 μM) to each, The fluorescence intensity of each solution was measured at 60 ° C. LightCycler (registered trademark) 480 (Roche) was used for measurement of fluorescence intensity. The measurement results are shown in FIG.

  As shown in FIG. 4, when the target nucleic acid is present in the solution, the fluorescence intensity decreases and the decrease in the fluorescence intensity is larger as the target nucleic acid concentration in the solution is higher than when the target nucleic acid is not present in the solution. It was confirmed. These results indicate that the presence of a target nucleic acid having a specific base sequence can be determined by adding the nucleic acid probe set of the present invention to the target nucleic acid solution and measuring the fluorescence intensity thereof. This suggests that it can be applied to quantification of target nucleic acids in solution.

[Example 2] <Quantitative real-time PCR experiment>
Five types of reaction solutions for real-time PCR containing 500 ng, 50 ng, 5 ng, 0.5 ng or 0.05 ng of human genomic DNA samples (Promega Corporation) as PCR template DNA were prepared. In each reaction solution, TITANIUM Taq DNA polymerase (Clontech), 4 types of dNTP (each final concentration 0.2 mM), forward primer (SEQ ID NO: 4, final concentration 0.5 μM), reverse A primer (SEQ ID NO: 5, final concentration 0.15 μM), a predetermined amount of TITANUM Taq PCR buffer (Clontech) and the nucleic acid probe set of the present invention are included.

In the nucleic acid probe set of the present invention, a part of UCP1 gene (SEQ ID NO: 1) in the human genome is a target nucleic acid, and a fluorescent probe binding region (b1) having a base sequence complementary to the fluorescent probe is provided on the 5 ′ end side. It has a binding probe (SEQ ID NO: 2, final concentration 0.2 μM) having a target nucleic acid binding region (b2) having a base sequence complementary to the target nucleic acid on the 3 ′ end side, and has a base sequence of SEQ ID NO: 3 'The terminal nucleotide is composed of a fluorescent probe (final concentration 0.05 μM) labeled with BODIPY ™ -FL (Molecular Probes). Each PCR reaction solution was prepared by making up to 20 μl with sterile water. By this PCR reaction, the nucleic acid containing the target nucleic acid is amplified.
Sequence number 4: agtggtggctaatgagagaa
Sequence number 5: aaggagtggcagcaagt

  The binding probe and the fluorescent probe have a phosphate group at the 3 'end. The target nucleic acid and nucleic acid probe were commissioned to Tsukuba Oligo Service Co., Ltd. (Tsukuba City), and the forward primer and reverse primer were commissioned to Japan Genetic Research Institute Co., Ltd. (Sendai City).

The reaction solution was subjected to the following PCR reaction using a real-time PCR apparatus (LightCycler (registered trademark) 480 (Roche)).
(1) Thermal denaturation step: 120 seconds at 95 ° C (2) Thermal denaturation step: 95 ° C, 10 seconds (3) Annealing step: 60 ° C, 20 seconds (4) Extension step: 72 ° C, 10 seconds Steps (2) to (4) were repeated after the thermal reaction step. The fluorescence intensity was measured in each annealing step of (3), and the first cycle number at which the fluorescence intensity became 95% or less of the fluorescence intensity at the fifth cycle (hereinafter abbreviated as Ct (cycle threshold) value). Asked. The Ct value is a value obtained by connecting the fluorescence intensity in each PCR cycle with a straight line.

  FIG. 5 shows a plot of the Ct value (average value of two measurements) versus the amount of each initially added template DNA. FIG. 5 reveals that there is a good correlation between the amount of template DNA added initially and the Ct value measured using the nucleic acid probe set of the present invention. That is, the results of this experiment show that in the real-time PCR method, the nucleic acid probe set of the present invention quantifies the template DNA containing the target nucleic acid contained in the reaction solution before the PCR reaction and the target nucleic acid contained in the reaction solution after the PCR reaction. It is very useful as a probe for quantification of amplification products containing

[Example 3] <Melting curve analysis of complex of nucleic acid probe set of the present invention and target nucleic acid>
A 21-base long nucleic acid (SEQ ID NO: 1), which is a part of the human UCP1 gene, is used as a target nucleic acid, and has a base sequence region complementary to the fluorescent probe on the 5 ′ end side and SEQ ID NO: 1 on the 3 ′ end side A binding probe (SEQ ID NO: 2) having a base sequence region complementary to the target nucleic acid and the nucleotide sequence of SEQ ID NO: 3 and the 5 ′ terminal nucleotide was labeled with BODIPY (trademark) -FL (Molecular Probes) A melting curve analysis of a complex of the nucleic acid probe set of the present invention and a target nucleic acid was performed using the nucleic acid probe set of the present invention comprising a fluorescent probe. Further, a melting curve analysis was similarly performed using a nucleic acid (SEQ ID NO: 6) in which the 17th base from the 5 ′ end side of the target nucleic acid was substituted with adenine instead of the target nucleic acid. In Example 3 below, the target nucleic acid and the nucleic acid of SEQ ID NO: 6 are collectively referred to as “target nucleic acids”.
Sequence number 6: tgaccacagtttgatcaagtg

  The binding probe and the fluorescent probe have a phosphate group at the 3 'end. The synthesis of the target nucleic acids and nucleic acid probes was outsourced to Tsukuba Oligo Service Co., Ltd. (Tsukuba City).

A PCR buffer solution (50 mM KCl, 10 mM Tris-HCl, 1.5 mM MgCl ₂ ) containing the nucleic acid probe set of the present invention (fluorescent probe (final concentration 0.05 μM) and binding probe (final concentration 0.2 μM)) is contained. All prepared 3 types of solutions, a solution in which the target nucleic acid or the nucleic acid of SEQ ID NO: 6 (both final concentration 0.1 μM) was added to the final concentration (pH 8.7) and a solution in which the target nucleic acids were not added. Was kept at 40 ° C. for 60 seconds, the nucleic acid probe set of the present invention and the target nucleic acid were hybridized, and then the fluorescence intensity was measured while raising the solution temperature from 40 ° C. to 80 ° C. The melting curve shown in FIG. 6 was obtained. In the figure, the melting curve of the solution containing the target nucleic acid of SEQ ID NO: 1 is shown as D, the melting curve of the solution containing the nucleic acid of SEQ ID NO: 6 is shown as E, and the melting curve of the solution containing no target nucleic acids is shown as F. Moreover, the negative first derivative curve of these melting curves is shown in FIG. For measurement of fluorescence intensity, LightCycler (registered trademark) 480 (Roche) was used.

  As a result of the experiment, by performing the melting curve analysis using the nucleic acid probe set of the present invention, the nucleic acid probe set of the present invention and the target nucleic acid are obtained from the negative first derivative curve of the melting curve shown by D and E in FIG. It was possible to clearly determine Tm1 of the complex with the class as a temperature showing the minimum. The Tm1 of the complex of the target nucleic acid of SEQ ID NO: 1 and the nucleic acid probe set, which is completely complementary to the target nucleic acid binding region, was about 64 ° C, but the SEQ ID NO: having a mismatched base pair with the target nucleic acid binding region The Tm1 of the complex of 6 nucleic acids and the nucleic acid probe set was lower, about 56 ° C. These results indicate that the nucleic acid probe set of the present invention can be suitably used as an alternative to the nucleic acid probes conventionally used in melting curve analysis.

[Example 4] <ABC-PCR experiment>
An ABC-PCR experiment was performed using the nucleic acid probe set of the present invention in which a part of the amoA gene (SEQ ID NO: 7) encoding ammonia oxidase of ammonia-oxidizing bacteria (Nitrosomonas europaea (NBRC 14298)) is the target nucleic acid. The effectiveness of the nucleic acid probe set of the present invention was evaluated.
Sequence number 7: gtcaccagccagttgcgtgtcagat

The nucleic acid probe set used in the ABC-PCR experiment is composed of one binding probe and two fluorescent probes as shown in FIG. The binding probe (SEQ ID NO: 8) is a 55-base long oligonucleotide having two fluorescent probe binding regions with different base sequences on the 5 ′ end side and 3 ′ end side and a target nucleic acid binding region in the center. is there. The fluorescent probe (A1-amoA) that hybridizes with the 5 ′ end side of the binding probe has the base sequence of SEQ ID NO: 9, and the 5 ′ terminal nucleotide is labeled with BODIPY (trademark) -FL (Molecular Probes). It is a 15-base long oligonucleotide having a phosphate group at the 3 ′ end.

On the other hand, the fluorescent probe (A2-amoA) that hybridizes with the 3 ′ terminal side of the binding probe has a base sequence of SEQ ID NO: 10 and has a 15 base length in which the 3 ′ terminal nucleotide is labeled with carboxytetramethylrhodamine (TAMRA). Oligonucleotide.
Sequence number 8: attcgggcgggcttaatctgacacgcaactggctggtgactttgggggggggttt
SEQ ID NO: 9: BODIPY (trademark) -FL-taagcccgcccgaat-phosphate SEQ ID NO: 10: aaaaccccccccccaaa-TAMRA

  In this example, as the internal standard nucleic acid, 2 bases on the 3 ′ end side (outside) adjacent to the 3 ′ end of the target nucleic acid in the amoA gene of the ammonia-oxidizing bacterium having the target nucleic acid are known methods (for example, (See Higuchi et al., Nucleic Acids Res., 1988, Vol. 16, pp. 7351-7367), and the amoA gene substituted with guanine was used. The fluorescent probe (A1-amoA) is not quenched when hybridized with a target nucleic acid as a nucleic acid probe set complex, but is quenched with the substituted guanine in the amoA gene when hybridized with an internal standard nucleic acid. The

The PCR reaction solution used for the ABC-PCR experiment was a predetermined amount of TITANIUM Taq DNA polymerase (Clontech) as a DNA synthase, a predetermined amount of TITANUM Taq PCR buffer (Clontech), a final concentration of 200 μM dATP, dCTP and dGTP. DUTP (Roche Diagnostics) at a final concentration of 600 μM, forward primer (SEQ ID NO: 11, final concentration 0.1 μM), reverse primer (SEQ ID NO: 12, final concentration 1 μM), binding probe (final concentration) 0.1 μM) and the above two types of fluorescent probes (both at a final concentration of 0.15 μM), uracil DNA glycosidase (0.25 U, Roche Diagnostics) to prevent contamination by amplification products, and internal standard nucleic acid Containing 10 ³ copies. Prepare a PCR reaction solution that does not contain the target nucleic acid and a PCR reaction solution that contains 10, 100, 300, 1,000, 3,000, 10,000, or 100,000 copies of the target nucleic acid. The female was up.
Sequence number 11: gghgactgggayttctgg
Sequence number 12: cctckgsaaagccttcttc

In the above sequences, H means A, C or T, Y means C or T, K means G or T, and S means G or C.
The above target nucleic acid is obtained from 0.5 g (wet weight) of ammonia-oxidizing bacteria (Nitrosomonas europaea (NBRC 14298), purchased from the National Institute of Technology and Evaluation, Biological Genetic Resources Division) cultured in NBRC liquid medium. DNA was extracted using a spin kit (Qbiogene), and this was used as genomic DNA purified using Microcon (registered trademark) YM-50 (Millipore).

In order to prevent contamination, during the preparation of the PCR reaction solution, the solution was kept at room temperature to cause a glycosidase reaction with uracil DNA glycosidase. After the solution was prepared, it was subjected to the following PCR reaction.
(1) Thermal denaturation step: 95 ° C for 120 seconds (2) Thermal denaturation step: 95 ° C, 45 seconds (3) Annealing step: 55 ° C, 60 seconds (4) Extension step: 72 ° C, 45 seconds (5) Extension Process: 72 degreeC, 45 second After the thermal reaction process of (1), after performing 60 cycles of process (2)-(4), the expansion | extension process of (5) was performed. ICycler (Bio-Rad Laboratories, Inc.) was used as the thermal cycler for the PCR reaction.

  After completion of the PCR reaction, the fluorescence intensity of the reaction solution was measured at 95 ° C. and 55 ° C. using SmartCycler (registered trademark, Sephaide). The fluorescence intensity of the fluorescent probe A1-amoA (hereinafter abbreviated as fluorescence intensity A1) was measured at an excitation wavelength of 450 to 495 nm and a detection wavelength of 505 to 537 nm, and the fluorescence intensity of the fluorescent probe A2-amoA (hereinafter referred to as fluorescence intensity A2). (Abbreviated) was measured at an excitation wavelength of 527 to 555 nm and a detection wavelength of 565 to 605 nm.

The obtained fluorescence intensity was substituted into the following formula 1, and corrected fluorescence intensity values were obtained for seven types of reaction solutions containing the target nucleic acid.
[Formula 1]
Fluorescence intensity correction value = [(G _{U, 55} / G _{U, 95} )-(G ₅₅ / G ₉₅ )] / [(R _{U, 55} / R _{U, 95} )-(R ₅₅ / R ₉₅ )]
here,
GU _{, 55} : fluorescence intensity at 55 ° C. of reaction solution not containing the target nucleic acid A1
GU _{, 95} : fluorescence intensity A1 of the reaction solution containing no target nucleic acid at 95 ° C.
G ₅₅ : fluorescence intensity A1 at 55 ° C. of the reaction solution containing the target nucleic acid
G ₉₅ : fluorescence intensity A1 of the reaction solution containing the target nucleic acid at 95 ° C.
R _{U, 55} : fluorescence intensity A2 of the reaction solution containing the target nucleic acid at 55 ° C.
R _{U, 95} : fluorescence intensity A2 of the reaction solution containing the target nucleic acid at 95 ° C.
R ₅₅ : fluorescence intensity at 55 ° C. of reaction solution not containing the target nucleic acid A2
R ₉₅ : fluorescence intensity A2 of the reaction solution not containing the target nucleic acid at 95 ° C.
It is.

  FIG. 9 is a graph in which the obtained corrected fluorescence intensity values are plotted on a semilogarithmic graph with the number of target nucleic acids before the PCR reaction taken on the horizontal axis. From this figure, even when an ABC-PCR experiment was performed using the nucleic acid probe set of the present invention, a graph (relational expression) that reverted to a right-angled hyperbola was obtained as in the case of using a conventionally used AB probe.

Next, using this relational expression as a calibration curve, an experiment was performed to calculate the number of target nucleic acids contained in the reaction solution before the PCR reaction from the fluorescence intensity of the reaction solution after the ABC-PCR reaction.
Three types of PCR reaction solutions similar to the ABC-PCR reaction solution were prepared except for the copy number of the target nucleic acid. As the reaction solution, 5 samples each of a solution containing 0, 2,000 or 6,000 copies of the target nucleic acid were prepared. The three kinds of PCR reaction solutions were subjected to 60 cycles of PCR reaction in the same manner as the ABC-PCR experiment, and the corrected fluorescence intensity value was determined by substituting the fluorescence intensity after the reaction into the above-mentioned formula 1, The number of target nucleic acids before the PCR reaction was calculated by fitting to a calibration curve. The results are shown in Table 1.

[Table 1]

  As is apparent from Table 1, the calculated value of the target nucleic acid number before the PCR reaction calculated from the fluorescence intensity of the reaction solution after the PCR reaction was in good agreement with the copy number of the target nucleic acid actually used. This result shows that the nucleic acid probe set of the present invention can be used for ABC-PCR experiments in the same manner as the conventionally used AB probe, and has high reliability.

  According to the present invention, a nucleic acid probe set capable of specifically analyzing a target nucleic acid is provided. In the nucleic acid probe set of the present invention, since it is not necessary to prepare an expensive fluorescently labeled nucleic acid probe for each target nucleic acid to be analyzed, a nucleic acid probe for a target nucleic acid is prepared at a lower cost and in a shorter time than conventional nucleic acid probes. Has the advantage of being able to. The nucleic acid probe set of the present invention can be applied to nucleic acid detection, quantification, polymorphism analysis, mutation detection and the like in the fields of medicine, molecular biology and agriculture.

A fluorescent probe B binding probe C target nucleic acid a nucleotide labeled with fluorescent substance b1 fluorescent probe binding region b2 target nucleic acid binding region d fluorescent substance α nucleotide α
β nucleotide β
γ nucleotide γ
δ nucleotide δ
ε nucleotide ε

Claims (11)

One or two fluorescent probes (A) comprising an oligonucleotide containing a nucleotide (a) labeled with a fluorescence quencher (d) whose fluorescence intensity attenuates when interacting with guanine, and the fluorescent probe (A A binding probe (B) composed of an oligonucleotide having one or two fluorescent probe binding regions (b1) to hybridize and a target nucleic acid binding region (b2) to hybridize with the target nucleic acid (C) Become
When the fluorescent probe (A) hybridizes to the fluorescent probe binding region (b1) and the target nucleic acid (C) hybridizes to the target nucleic acid binding region (b2), the target nucleic acid (C) The fluorescent probe (A) so that the fluorescence intensity of the fluorescence quenching substance (d) is attenuated by the interaction between the guanine in the nucleic acid containing the nucleic acid and the fluorescence quenching substance (d) labeled with the nucleotide (a). ) And the binding probe (B) are designed.   The fluorescence quencher (d) is tetrabromosulfonefluorescein (TBSF), 2-oxo-6,8-difluoro-7-hydroxy-2H-1-benzopyran-3-carboxylic acid (Pacific Blue ™), rhodamine From 6G, carboxyrhodamine 6G, carboxytetramethylrhodamine and 4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-s-indacene-3-propionic acid (BODIPY ™ -FL) The nucleic acid probe set according to claim 1, which is any one selected from the group consisting of:   The nucleic acid probe set according to claim 1, wherein the one or two fluorescent probes (A) are oligonucleotides in which 5'-terminal nucleotide or 3'-terminal nucleotide is labeled with a fluorescence quencher (d).   The nucleic acid probe set according to claim 3, wherein the one or two fluorescent probes (A) have a length of 5 to 50 bases.   The nucleic acid probe set according to claim 1, wherein the target nucleic acid binding region (b2) has a length of 5 to 60 bases. A method for detecting a target nucleic acid, comprising the following steps (1) and (2):
(1) a step of hybridizing the nucleic acid probe set according to claim 1 and a target nucleic acid (C);
(2) Next, measuring the fluorescence intensity of the complex of the hybridized nucleic acid probe set and the target nucleic acid (C). The method for detecting a target nucleic acid according to claim 6, further comprising the following steps (3) and (4).
(3) changing the ratio between the nucleic acid probe set and the target nucleic acid, and performing the steps (1) and (2);
(4) A step of comparing the fluorescence intensities obtained from the steps (2) and (3). A method for analyzing a nucleotide sequence polymorphism of a nucleic acid, comprising the following steps (1) to (4):
(1) a step of hybridizing the nucleic acid probe set according to claim 1 and a target nucleic acid (C);
(2) Next, measuring the temperature dependence of fluorescence intensity for the complex of the hybridized nucleic acid probe set and the target nucleic acid (C),
(3) In place of the target nucleic acid, the steps (1) and (2) are performed using another nucleic acid having a base sequence polymorphism having a different base sequence at the polymorphic site relative to the base sequence of the target nucleic acid. Process,
(4) A step of comparing the temperature dependence of the fluorescence intensity obtained from the steps (2) and (3). The method for analyzing a nucleic acid base sequence polymorphism according to claim 8, wherein the base sequence polymorphism is a single base polymorphism, base substitution, base deletion, or base insertion relative to the base sequence of the target nucleic acid.   A melting curve analysis is performed on the complex of the nucleic acid probe set according to claim 1 and a target nucleic acid (C).   The fluorescent probe (A) comprises two fluorescent probes with different base sequences and labeled with different fluorescent substances, and the binding probe (B) has two fluorescent probe binding regions with different base sequences. The nucleic acid probe set according to claim 1.

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