JP2012231727A - Fto gene polymorphism detection probe - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for detecting rs9939609 of FTO gene polymorphism by specifying an effective probe for detecting rs9939609 of FTO gene polymorphism, and to provide a kit therefor.SOLUTION: There is provided a probe for detecting polymorphism of FTO gene, which comprises an oligonucleotide comprising a base sequence of 10-65 base length containing base numbers 401-410 of a specific base sequence, and having homology to a specific base sequence except that the base corresponding to the 410th base is cytosine, wherein the base corresponding to the base number 410 is labeled with a fluorochrome.

Description

  The present invention relates to a method for detecting rs9939609 of an FTO gene polymorphism, and a nucleic acid probe and kit therefor.

Obesity is known to cause diabetes and metabolic syndrome. In Japan, metabolic syndrome is a diagnostic item in health examinations.
SNP (single nucleotide polymorphism) of FTO gene is known as SNP related to obesity. It has been reported that SNP (rs9939609) of FTO gene (fat mass and obesity-associated gene) is associated with body mass index (BMI) and obesity (Non-patent Document 1).
Thus, investigating SNP polymorphisms in the FTO gene is very important for diagnosis of obesity, diabetes, metabolic syndrome, and the like.

The following methods are known as methods for detecting a base polymorphism.
Non-Patent Document 1 describes a method (KASPar method) in which an amplification product is obtained using a primer having an additional sequence, a fluorescence detection signal of a FRET probe is obtained, and SNP typing is performed. However, this method requires two FRET probes necessary for the reaction and is costly, and requires two or more types of primers, which requires design effort and cost. There was a problem of need.
Non-Patent Document 2 describes a method of performing SNP typing by obtaining a fluorescence detection signal using a TaqMan probe. However, this method requires two TaqMan probes necessary for the reaction and is costly. The TaqMan probe requires modification and is expensive, and results analysis requires expertise and expertise. .
Non-Patent Document 3 describes a method of performing SNP typing by obtaining a detection signal by the Invader method. However, this method has a problem that it requires labor and specialized skills for the experimental operation, and it takes time to obtain the result.
Non-Patent Document 4 describes a method of performing SNP typing from the difference in restriction enzyme cleavage pattern of an amplification product by RFLP method. However, this method has a problem in that it has a risk of affecting the result of the experiment due to contamination because it deals with the amplification product, and it takes time to perform the experiment and takes time to obtain the result.

  On the other hand, a method is known in which a region containing a mutation is amplified by PCR, a melting curve analysis is performed using a nucleic acid probe labeled with a fluorescent dye, and the mutation is analyzed based on the result of the melting curve analysis (patent) References 1, 2). However, in these documents, regarding probe design, when a quenching probe whose terminal portion is labeled with a fluorescent dye is hybridized with a target nucleic acid, a plurality of probe-nucleic acid hybrid base pairs are at least one G in the terminal portion. There is only teaching to design to form C pairs. In addition, these methods have a problem that they cannot be implemented with all arbitrary sequences.

JP 2001-286300 A JP 2002-119291 A

Science 2007; 316: 889-94 N Engl J Med 2008; 359: 2558-66 J Hum Genet 2008; 53: 546-553 J Clin Endocrinol Metab 2008; 93: 1501-1505

  An object of the present invention is to identify a probe effective for detecting rs9939609 of FTO gene polymorphism, to provide a method for detecting rs9939609 of FTO gene polymorphism, and to provide a kit therefor.

  The present inventor has found that the mutation can be detected by designing a probe based on a specific region containing rs9939609 of the FTO gene polymorphism and performing a melting curve analysis using the probe, thereby completing the present invention. It was.

That is, the present invention is as follows.
(1) A probe for detecting a polymorphism of the FTO gene, which is a base sequence having a length of 10 to 65 bases including base numbers 401 to 410 in the base sequence shown in SEQ ID NO: 1 or 2, and SEQ ID NO: 1 or 2 except that the base corresponding to the 410th base in 2 is homologous to SEQ ID NO: 1 or 2, except that the base corresponding to the base number 410 is an oligonucleotide labeled with a fluorescent dye. A polymorphism detection probe.
(2) The polymorphism detection probe according to (1), wherein the oligonucleotide has a base corresponding to the base number 410 labeled with a fluorescent dye at the first to third positions counted from the 3 ′ end.
(3) The polymorphism detection probe according to (1), wherein the oligonucleotide has a base corresponding to base number 410 labeled with a fluorescent dye at the 3 ′ end.
(4) Any one of (1) to (3), wherein the oligonucleotide emits fluorescence when it does not hybridize to the target sequence and decreases or increases in fluorescence intensity when hybridized to the target sequence. The polymorphism detection probe according to Item.
(5) The polymorphism detection probe according to (4), wherein the oligonucleotide emits fluorescence when it does not hybridize to the target sequence and decreases in fluorescence intensity when hybridized to the target sequence.
(6) The probe for detecting a polymorphism according to any one of (1) to (5), wherein the oligonucleotide has a base length of 10 to 47.
(7) The probe for detecting a polymorphism according to any one of (1) to (6), wherein the oligonucleotide has a base length of 10 to 34.
(8) The polymorphism detection probe according to any one of (1) to (7), wherein the oligonucleotide has a base length of 15 to 27.
(9) The probe for detecting a polymorphism according to any one of (1) to (8), wherein the probe is a probe for melting curve analysis.
(10) The polymorphism detection probe according to any one of (1) to (9), wherein the oligonucleotide is represented by SEQ ID NO: 4.
(11) A method for detecting a polymorphism of an FTO gene using the probe for detecting a polymorphism according to any one of (1) to (10).
(12) The probe for detecting a polymorphism according to any one of (I) (1) to (10) and a single-stranded nucleic acid in a sample are contacted, and the fluorescently labeled oligonucleotide and the single-stranded nucleic acid To obtain a hybrid formed body,
(II) dissociating the hybrid by changing the temperature of the sample containing the hybrid, and measuring a change in fluorescence signal based on the dissociation of the hybrid;
(III) determining the Tm value, which is the dissociation temperature of the hybrid, based on the change in the signal; and (IV) the presence or absence of a polymorphism in the FTO gene based on the Tm value. Determining the abundance ratio,
The polymorphism detection method according to (11), comprising:
(13) The polymorphism detection method according to (12), further comprising amplifying the nucleic acid before or simultaneously with the step (I).
(14) By detecting the polymorphism in the FTO gene by the polymorphism detection method according to any one of (11) to (13), and based on the presence or absence of the detected polymorphism, obesity and diabetes And / or determining the likelihood of metabolic syndrome.
(15) A polymorphism detection kit comprising the polymorphism detection probe according to any one of (1) to (10).
(16) The polymorphism detection kit according to (15), further comprising a primer that can be amplified using a region containing a sequence to which the oligonucleotide hybridizes in the base sequence shown in SEQ ID NO: 1 as a template.
(17) The polymorphism detection kit according to (16), wherein the primer is the primer set forth in SEQ ID NOs: 7 and 8.
(18) The polymorphism detection kit according to (16) or (17), further comprising any primer or probe described in SEQ ID NOs: 11 to 16.

By adding the probe of the present invention and performing melting curve analysis (Tm analysis), SNP typing of rs9939609 of the FTO gene polymorphism becomes possible.
The probe of the present invention has high specificity and high detection sensitivity.
By using the method of the present invention, there is almost no risk of contamination since it is not necessary to take out the amplification product even when PCR is performed. Also, the method of the present invention is easy to automate because it is simple to operate.
By the method of the present invention, the FTO gene polymorphism rs9939609, the B3AR gene polymorphism rs4994 and the UCP1 gene polymorphism rs1800592 can also be detected simultaneously.
By using the probe of the present invention, the working time and reaction time can be shortened, and the FTO gene polymorphism rs9939609 can be easily detected.

TAMRA (3T-FTO-F1, 5T-FTO-F2, 5T-FTO-F3) per unit time in Tm analysis for FTO-R1-WT and FTO-R1-mt (complementary strand oligo) of Example 1 The amount of change in fluorescence intensity (d fluorescence intensity increase / t) is on the vertical axis, and the temperature is on the horizontal axis. In the following charts, the relationship between the vertical axis and the horizontal axis is the same. In Tm analysis for specimen A (purified genome) of Example 2, Pacific Blue (3PB-FTO-F1) (left figure) and BODIPY FL (5FL-B3AR-wt-R6-15) (middle figure) were used as probes. TAMRA (5T-UCP1-G-F4-17) (right figure) was used. In the Tm analysis of the specimen B (purified genome) of Example 2, Pacific Blue (3PB-FTO-F1) (left figure) and BODIPY FL (5FL-B3AR-wt-R6-15) (center figure) were used as probes. TAMRA (5T-UCP1-G-F4-17) (right figure) was used.

<1> Probe of the Present Invention and Detection Method of the Present Invention The probe of the present invention is a probe for detecting a polymorphism of the FTO gene, which comprises base numbers 401 to 410 in the base sequence shown in SEQ ID NO: 1 or 2. A base sequence of 65 bases in length, which has homology to SEQ ID NO: 1 or 2 except that the base corresponding to the 410th base in SEQ ID NO: 1 or 2 is cytosine, and the base corresponding to base number 410 is fluorescent A polymorphism detection probe comprising an oligonucleotide labeled with a dye.
Here, FTO gene polymorphism rs9939609 is the 401st base of SEQ ID NOs: 1 and 2. This rs number indicates the registration number of the dbSNP database (//www.ncbi.nlm.nih.gov/projects/SNP/) of National Center for Biotechnology Information.

The probe of the present invention is the above in the nucleotide sequence shown in SEQ ID NO: 1 (sequence having a wild type base of the FTO gene) or the nucleotide sequence shown in SEQ ID NO: 2 (sequence having a variant (polymorphic) base of the FTO gene). Other than having the specified sequence, it can be prepared in the same manner as the probes described in Patent Documents 1 and 2.
“Homology” in the present application refers to a base having a sequence having a homology of 80% or more, more preferably 90% or more, and most preferably 95% or more to a complementary strand of a base sequence in a specific base sequence. Point to an array.
The length of the probe of the present invention is 10 to 65 bases, preferably 10 to 47 bases, more preferably 10 to 34, and most preferably 15 to 27.
An example of a base sequence of a probe for detecting rs9939609 of the FTO gene polymorphism used in the present invention is 5′-tgtgaattt r gtgatgcac-3 ′ (SEQ ID NO: 3). In the sequence, “r” represents a or t. More preferably, it is 5′-tgtgaatttagtgatgcac-3 ′ (SEQ ID NO: 4).
As the fluorescent dye, those described in Patent Documents 1 and 2 can be used, and specific examples include Pacific Blue (trademark), FAM (trademark), TAMRA (trademark), BODIPY (trademark) FL, and the like. . The method of binding the fluorescent dye to the oligonucleotide can be performed according to a conventional method, for example, the methods described in Patent Documents 1 and 2.

The probe of the present invention preferably emits fluorescence of the fluorescent dye when not hybridized to the target sequence, and decreases or increases fluorescence of the fluorescent dye when hybridized to the target sequence. More preferably, it is a quenching probe that emits fluorescence of a fluorescent dye when not hybridized and quenches fluorescence of the fluorescent dye when hybridized.
In the probe of the present invention, the first to third bases counted from the 5 ′ or 3 ′ end are preferably labeled with a fluorescent dye, and more preferably the 3 ′ end is labeled with a fluorescent dye. . In the present specification, “1st to 3rd counting from the 5 ′ end” refers to 5 ′ end as the first, and “1st to 3rd counting from the 3 ′ end” to 3 ′ The end is counted as the first.
The base labeled with the fluorescent dye in the probe of the present invention is the 410th base in SEQ ID NO: 1 or 2.

The detection method of the present invention uses the probe of the present invention and includes the following steps. (I) Contacting the polymorphism detection probe of the present invention and a single-stranded nucleic acid in a sample, and hybridizing the fluorescently labeled oligonucleotide and the single-stranded nucleic acid to obtain a hybrid,
(II) dissociating the hybrid by changing the temperature of the sample containing the hybrid, and measuring a change in fluorescence signal based on the dissociation of the hybrid;
(III) determining the Tm value, which is the dissociation temperature of the hybrid, based on the change in the signal; and (IV) the presence or absence of a polymorphism in the FTO gene based on the Tm value. Determine the abundance ratio.

  The detection method of the present invention can be carried out according to the usual methods of nucleic acid amplification and melting curve analysis (Tm analysis) other than using the probe of the present invention. The detection method of the present invention may include amplifying the nucleic acid before or simultaneously with the step (I).

  As a method for nucleic acid amplification, a method using a polymerase is preferred, and examples thereof include PCR, ICAN, LAMP and the like. When amplification is performed by a method using a polymerase, amplification is preferably performed in the presence of the probe of the present invention. It is easy for those skilled in the art to adjust the amplification reaction conditions and the like according to the probe used. As a result, only the Tm value of the probe is analyzed after amplification of the nucleic acid, so that it is not necessary to purify the amplification product after the reaction is completed. Therefore, there is no worry of contamination by amplification products. Moreover, since it can detect with the same apparatus as an apparatus required for amplification, it is not necessary to move a container. Therefore, automation is also easy.

  In the present invention, the DNA in the sample may be single-stranded DNA or double-stranded DNA. When the DNA is double-stranded DNA, for example, prior to the hybridization step, it preferably includes a step of dissociating the double-stranded DNA in the sample by heating. By dissociating the double-stranded DNA into single-stranded DNA, hybridization with the detection probe becomes possible in the next hybridization step.

  In the present invention, the addition ratio (molar ratio) of the probe of the present invention with respect to the DNA in the sample is not limited, but since a sufficient detection signal can be secured, it is preferably 1 or less with respect to the DNA in the sample, 0.1 times or less is more preferable. In this case, the DNA in the sample may be, for example, the sum of the detection target DNA in which the detection-target polymorphism has occurred and the non-detection target DNA in which the polymorphism has not occurred, or the detection-target polymorphism. The total of the amplification product containing the detection target sequence in which the occurrence of the polymorphism and the amplification product containing the non-detection target sequence in which the polymorphism does not occur may be used. In addition, although the ratio of the detection target DNA in the DNA in the sample is usually unknown, as a result, the addition ratio (molar ratio) of the probe depends on the detection target DNA (amplification product including the detection target sequence). On the other hand, it is preferably 10 times or less, more preferably 5 times or less, and further preferably 3 times or less. Moreover, the lower limit in particular is not restrict | limited, For example, it is 0.001 times or more, Preferably it is 0.01 times or more, More preferably, it is 0.1 times or more.

  The ratio of the probe of the present invention to the DNA may be, for example, a molar ratio with respect to double-stranded DNA or a molar ratio with respect to single-stranded DNA.

  The Tm value will be described. When a solution containing double-stranded DNA is heated, the absorbance at 260 nm increases. This is because hydrogen bonds between both strands in double-stranded DNA are unwound by heating and dissociated into single-stranded DNA (DNA melting). Based on this phenomenon, the melting temperature Tm is generally defined as the temperature at which the absorbance reaches 50% of the total increase in absorbance.

  In the present invention, the measurement of the signal fluctuation accompanying the temperature change for determining the Tm value can be performed by measuring the absorbance at 260 nm based on the principle described above, but is based on the signal of the label added to the probe of the present invention. It is preferable to measure a signal that varies depending on the state of hybridization between the DNA and the probe. For this reason, it is preferable to use the aforementioned labeled probe as the probe of the present invention. Examples of the labeled probe include a fluorescently labeled oligonucleotide probe that emits fluorescence when it does not hybridize to the target sequence and decreases (quenches) when it hybridizes to the target sequence, or hybridizes to the target sequence Fluorescently-labeled oligonucleotide probes that emit fluorescence when not and increase in fluorescence intensity when hybridized to a target sequence. If the probe is like the former, no signal is shown when a hybrid (double-stranded DNA) is formed with the detection target sequence, or the signal is weak, but the signal is shown when the probe is released by heating. Or the signal increases. In the case of the latter probe, a signal is shown by forming a hybrid (double-stranded DNA) with the detection target sequence, and the signal decreases (disappears) when the probe is released by heating. Therefore, by detecting a change in the signal based on this label under signal-specific conditions (absorbance, etc.), it is possible to determine the progress of melting and the Tm value as in the case of the absorbance measurement at 260 nm. The labeling substance in the labeling probe is, for example, as described above, but a fluorescent dye-labeled probe is preferable.

  The nucleic acid used as a template for nucleic acid amplification is not particularly limited as long as it contains nucleic acid, and examples thereof include blood, oral mucosa suspension, somatic cells such as nails and hair, germ cells, milk, ascites Derived from or can be derived from any biological source such as fluid, paraffin embedded tissue, gastric fluid, gastric lavage fluid, peritoneal fluid, amniotic fluid, cell culture. The template nucleic acid can be used as obtained from the source either directly or after pretreatment to modify the properties of the sample, for example using a genome purified from blood. it can.

  Hereinafter, the case where PCR is used will be further described as an example. The primer pair used for PCR can be set in the same manner as the primer pair setting method in ordinary PCR, except that the region where the probe of the present invention can hybridize is amplified. The length and Tm value of the primer are usually 12-mer to 40-mer, 40-70 ° C, preferably 16-mer to 30-mer, 55-60 ° C. The length of each primer in the primer pair may not be the same, but the Tm of both primers is preferably substantially the same (usually, the difference is within 2 ° C.). The Tm value is a value calculated by the nearest base pair (Nearest Neighbor) method. Examples of primer pairs include those consisting of primers having the base sequences shown in SEQ ID NOs: 7 and 8.

  PCR is preferably performed in the presence of the probe of the present invention used in the present invention. Thereby, Tm analysis can be performed without purifying the amplification product after completion of the amplification reaction. It is easy for those skilled in the art to adjust the Tm value of the primer and the reaction conditions for PCR according to the probe used.

  The Tm analysis can be performed according to a usual method except that the fluorescence of the fluorescent dye of the probe of the present invention is measured. The fluorescence can be measured by measuring light having an emission wavelength using excitation light having a wavelength corresponding to the fluorescent dye. The rate of temperature increase in Tm analysis is usually 0.1-1 ° C./second. The composition of the reaction solution for performing Tm analysis is not particularly limited as long as hybridization between the probe and a nucleic acid having a sequence complementary to the base sequence is possible, but usually the monovalent cation concentration is 1.5-5 mM, pH is 7-9. Since the reaction solution of the amplification method using DNA polymerase such as PCR normally satisfies this condition, the amplified reaction solution can be used as it is for Tm analysis.

  Detection of rs9939609 of FTO gene polymorphism based on the result of Tm analysis can be performed according to a usual method. Detection in the present invention includes detection of the presence or absence of mutation and determination of the abundance ratio of nucleic acids having polymorphisms.

By using the probe and polymorphism detection method of the present invention, a polymorphism in the FTO gene can be detected, and the ease of applying obesity, diabetes and / or metabolic syndrome is evaluated based on the presence or absence of the detected polymorphism. Can do. Specifically, if the base of rs9939609 of the FTO gene is T / T, it is wild type, and obesity, diabetes and / or metabolic syndrome is difficult, but if it is T / A, A / A, it is obese. Suggested to be prone to diabetes and / or metabolic syndrome.
In the method of the present invention, the FTO gene polymorphism rs9939609 (T> A), the β3 adrenergic receptor (B3AR) gene polymorphism rs4994 (T> C), and the uncoupling protein 1 (UCP1) gene polymorphism rs1800592 (A> G) can be detected simultaneously, and if there is a polymorphism (risk allele) of the B3AR gene and / or UCP1 gene, it is likely to be obese, diabetic and / or metabolic syndrome It is suggested.

  The base sequence of a probe that can detect the gene sequence of rs4994 (T> C) of the B3AR gene polymorphism and the polymorphism of the mutation site is described in, for example, JP-A-2004-313120. Specifically, a nucleic acid probe whose 3 ′ end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when hybridized, and comprising a nucleic acid comprising the base sequence of SEQ ID NOs: 8-12 of JP-A-2004-313120 It is a probe.

  It is possible to detect the polymorphism of the B3AR gene polymorphism rs4994 (T> C) and its mutation site and the UCP1 polymorphism rs1800592 (A> G) gene sequence and its polymorphism The base sequence of the probe is described in, for example, WO2008 / 066164. Specifically, a nucleic acid probe whose 3 ′ end is labeled with a fluorescent dye and the fluorescence of the fluorescent dye decreases when it is hybridized, the bases of SEQ ID NOs: 83, 95, 120, 127, and 139 of WO2008 / 066164 A nucleic acid probe consisting of a sequence. SEQ ID NOs: 83, 120, and 127 are B3AR gene polymorphism detection probes, and SEQ ID NOs: 95 and 139 are UCP1 gene polymorphism detection probes.

<2> Kit of the Present Invention The kit of the present invention is a kit for use in the detection method of the present invention. This kit is characterized by including the polymorphism detection probe of the present invention. The kit of the present invention can also be used to determine diseases associated with diabetes or obesity.
The probe is as described above for the probe of the present invention.

In addition to the probe, the detection kit of the present invention may further contain reagents necessary for performing nucleic acid amplification in the detection method of the present invention, particularly primers for amplification using DNA polymerase.
In the detection kit of the present invention, the probe, primer and other reagents may be separately accommodated, or a part of them may be a mixture.
The detection kit of the present invention may further contain a probe and a primer for detecting rs4994 (T> C) of the B3AR gene polymorphism and rs1800592 (A> G) of the UCP1 gene polymorphism.
Hereinafter, the present invention will be specifically described by way of examples. However, these examples are illustrative and are not limited thereto.

Example 1 (when FTO probe performance is confirmed with a complementary strand oligonucleotide as a detection target)
Based on the base sequence containing the rs9939609 site of the polymorphism of the FTO gene (SEQ ID NO: 1 (wild type)), probes having C at the terminal end (corresponding to the mutant type) shown in Table 1 were designed. In Table 1, the position of the probe indicates the base number in the base sequence shown in SEQ ID NO: 2. P at the 3 ′ end indicates phosphorylation. Labeling with TAMRA was performed according to a conventional method.
Table 1 shows the sequences of complementary strand oligonucleotides (corresponding to wild type (SEQ ID NO: 9) and mutant type (SEQ ID NO: 10)) used as detection targets. In Table 1, the position of the oligo indicates the base number in the base sequence shown in SEQ ID NO: 2.

  The Tm value is Meltcalc 99 free from http://www.meltcalc.de/download.htm. The software setting conditions are Oligo conc [μM] 0.2, Na eq. [MM] 50, DMSO [%]. Calculated.

Tm analysis was performed using a fully automatic SNPs inspection device (trade name i-densy (trademark), manufactured by Arkray). The following solution was added to a reaction tube dedicated to i-densy and overlaid with 30 μL of mineral oil. The above-mentioned SEQ ID NOs: 9 and 10 were used as complementary strand oligonucleotides for the probe. The conditions for Tm analysis are as follows.
The excitation wavelength and detection wavelength in Tm analysis were 520 to 555 nm and 585 to 700 nm (TAMRA), respectively.

As a result of Tm analysis using the probe of Table 1, as for 3T-FTO-F1 (SEQ ID NO: 4), the TAMRA peaks are clearly seen around 54 ° C. (wild type) and 60 ° C. (mutant type). However, for 5T-FTO-F2 (SEQ ID NO: 5) and 5T-FTO-F3 (SEQ ID NO: 6), the TAMRA peak was not observed with sufficient intensity (FIG. 1).
Accordingly, any probe sequence may be used as long as C at the 5 ′ or 3 ′ end of the probe is fluorescently labeled, and the 410th C is fluorescently labeled as in the probe of SEQ ID NO: 4. It is understood that it is important.

Example 2 (When purified genome is used as a detection target and Tm analysis is performed consistently after PCR amplification. In addition to the FTO gene, when B3AR and UCP1 primers and probes are used for simultaneous detection by multiple reactions)
Based on the nucleotide sequence (SEQ ID NO: 1 (wild type)) containing the polymorphic rs9939609 site of the FTO gene, the primers shown in Table 6 were designed so that the polymorphic site could be amplified. In Table 6, the position of the primer indicates the base number in the base sequence shown in SEQ ID NO: 1.
PCR and Tm analysis were performed using a fully automatic SNPs inspection device (trade name i-densy (trademark), manufactured by ARKRAY). As a sample, 4 μL of human genome (100 copies / μL) (sample A or sample B) purified from whole blood was used (400 copies / test). The conditions for PCR and Tm analysis are as follows.
Excitation and detection wavelengths in Tm analysis are 365-415 nm and 445-480 nm (Pacific Blue), 420-485 nm and 520-555 nm (BODIPY FL), respectively, or 520-555 nm and 585- 700 nm (TAMRA).

  In addition to FTO gene polymorphism, the following were used as primers and probes for detecting B3AR gene polymorphism and UCP1 gene polymorphism.

As a result of evaluation of the FTO gene by the fluorescence of Pacific Blue, as for Sample A, the Pacific Blue peak was observed at around 52 ° C. (FIG. 2A), and for Sample B, the Pacific Blue peaks were 52 ° C. and 58 ° C. It was seen in the vicinity (FIG. 3A).
Moreover, as a result of evaluating the β3AR gene by fluorescence of BODIPY FL, BODIPY FL peaks were observed at around 52 ° C. and 62 ° C. for specimen A (FIG. 2B), and BODIPY FL peaks were also obtained for specimen B. It was seen around 52 ° C. and 62 ° C. (FIG. 3B).
Furthermore, as a result of evaluating the UCP gene by fluorescence of TAMRA, TAMRA peaks were observed at around 51 ° C. and 58 ° C. for sample A (FIG. 2C), and TAMRA peaks were around 51 ° C. for sample B Only seen (FIG. 3C).

From the results of FIGS. 2A and 3A, when the probe shown in SEQ ID NO: 4 in Table 6 was used, a change in fluorescence intensity that could be analyzed by Tm analysis was observed for the FTO gene polymorphism rs9939609 (T> A). . That is, T / A type specimen B has two peaks around 52 ° C. and 58 ° C., and T / T type specimen A has only one peak around 52 ° C. There is a change in the pattern of change in fluorescence intensity. In addition, it can be seen from these results that there is only one peak around 52 ° C. when the reaction is performed with the human genome of T / T type.
Furthermore, the B3AR gene polymorphism rs4994 (T> C) also showed a change in fluorescence intensity that can be analyzed by Tm analysis. That is, T / C type specimens A and B have two peaks near 52 ° C. and 62 ° C., and there is a change in the pattern of the change amount of the inherent fluorescence intensity.
Furthermore, a change in fluorescence intensity that can be analyzed by Tm analysis was also observed for rs1800592 (A> G) of the UCP1 gene polymorphism. That is, the G / A type specimen A has two peaks at around 51 ° C. and 58 ° C., and there is a change in the inherent fluorescence intensity change pattern. Also, from these results, it can be seen that when the reaction is performed with the sample B of A / A type, there is only one peak near 51 ° C.
Therefore, the FTO gene polymorphism rs9939609 (T> A), the B3AR gene polymorphism rs4994 (T> C) and the UCP1 gene polymorphism rs1800592 (A) can be obtained by using the probes of SEQ ID NOs: 4, 15, and 16 simultaneously. > G) can be detected simultaneously.

  By using the probe of the present invention, obesity and / or diabetes and metabolic syndrome can be diagnosed.

Claims (18)

A probe for detecting a polymorphism of the FTO gene, which is a base sequence having a length of 10 to 65 bases including base numbers 401 to 410 in the base sequence shown in SEQ ID NO: 1 or 2, and 410 in SEQ ID NO: 1 or 2 Except for the base corresponding to the 2nd base being cytosine, it is homologous to SEQ ID NO: 1 or 2, and the base corresponding to the base number 410 comprises an oligonucleotide labeled with a fluorescent dye. Type detection probe. 2. The polymorphism detection probe according to claim 1, wherein the oligonucleotide has a base corresponding to base number 410 labeled with a fluorescent dye at positions 1 to 3 counted from the 3 ′ end. The probe for detecting a polymorphism according to claim 1, wherein the oligonucleotide has a base corresponding to base number 410 labeled with a fluorescent dye at the 3 'end. The polymorphism according to any one of claims 1 to 3, wherein the oligonucleotide emits fluorescence when it does not hybridize to the target sequence, and decreases or increases in fluorescence intensity when hybridized to the target sequence. Probe for detection. The probe for detecting a polymorphism according to claim 4, wherein the oligonucleotide emits fluorescence when it does not hybridize to the target sequence, and the fluorescence intensity decreases when it hybridizes to the target sequence. The probe for detecting a polymorphism according to any one of claims 1 to 5, wherein the oligonucleotide has a base length of 10 to 47. The probe for detecting a polymorphism according to any one of claims 1 to 6, wherein the oligonucleotide has a base length of 10 to 34. The probe for detecting a polymorphism according to any one of claims 1 to 7, wherein the oligonucleotide has a base length of 15 to 27. The probe for polymorphism detection according to any one of claims 1 to 8, wherein the probe is a probe for melting curve analysis. The polymorphism detection probe according to any one of claims 1 to 9, wherein the oligonucleotide is represented by SEQ ID NO: 4. The polymorphism detection method of the FTO gene using the probe for a polymorphism detection of any one of Claims 1-10. (I) The probe for detecting a polymorphism according to any one of claims 1 to 10 and a single-stranded nucleic acid in a sample are brought into contact, and the fluorescently labeled oligonucleotide and the single-stranded nucleic acid are hybridized. Obtaining a hybrid,
(II) dissociating the hybrid by changing the temperature of the sample containing the hybrid, and measuring a change in fluorescence signal based on the dissociation of the hybrid;
(III) determining the Tm value, which is the dissociation temperature of the hybrid, based on the change in the signal; and (IV) the presence or absence of a polymorphism in the FTO gene based on the Tm value. Determining the abundance ratio,
The polymorphism detection method according to claim 11, comprising: 13. The polymorphism detection method according to claim 12, further comprising amplifying the nucleic acid before or simultaneously with the step (I). A polymorphism in the FTO gene is detected by the polymorphism detection method according to any one of claims 11 to 13, and obesity, diabetes and / or metabolic syndrome is based on the presence or absence of the detected polymorphism. Determining the ease of taking. A polymorphism detection kit comprising the polymorphism detection probe according to claim 1. The polymorphism detection kit according to claim 15, further comprising a primer that can be amplified using a region containing a sequence to which the oligonucleotide hybridizes in the base sequence shown in SEQ ID NO: 1 as a template. The polymorphism detection kit according to claim 16, wherein the primer is the primer set forth in SEQ ID NOs: 7 and 8. Furthermore, the polymorphism detection kit of Claim 16 or 17 containing the primer or probe in any one of sequence number 11-16.