JP2009011183A - Method for detecting dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To eliminate the formation of a recombined DNA produced, when a sample DNA is prepared or electrophoresis of the sample DNA is carried out in a capillary tube, and to provide a method for detecting the DNA with which exact quantitative analysis can be made. <P>SOLUTION: The method for detecting the DNA includes carrying out the electrophoresis of the sample DNA constituted of a double-stranded DNA, composed of a specific base sequence and/or a double-stranded DNA, in which a part of the base sequence of the double-stranded DNA is different and separating the sample DNA. Further, the method for detecting the DNA includes a step of filling a capillary with a conjugate, constituted of pyrrole-imidazole polyamide and polyethylene glycol, a step of injecting the sample DNA into the capillary filled with the conjugate, and a step of applying a voltage across both ends of the capillary and carrying out electrophoresis of the sample DNA. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a DNA analysis method for detecting a difference in a part of a base sequence in DNA.

  In recent years, rapid progress in molecular biology has made it possible to accurately understand the relationship between various diseases and genes, and attention has been focused on gene-targeted medicine.

  In particular, SNP of DNA (abbreviation of single nucleotide polymorphism, generally referred to as “single nucleotide polymorphism”) has attracted attention. It is in the specificity that SNP has. SNPs are commonly found in humans and animals, but SNPs vary from individual to individual even in the same species. That is, by examining the differences in SNPs, it is possible to predict the morbidity rate of each individual's disease, the effect and sensitivity to the drug, and perform medical care tailored to the individual. Furthermore, it is considered that the parent-child relationship between humans and animals can be specified.

  As a method for examining DNA having different SNPs and some base sequences, there are a gene diagnosis apparatus and a gene diagnosis method using affinity capillary electrophoresis. (For example, refer to Patent Document 1.) In this method, first, a conjugate DNA in which a polymer compound is bound to a probe DNA complementary to a target sequence is prepared. Since this conjugate DNA is a polymer compound, it has a property that it is difficult to move in the capillary. The conjugated DNA is filled in a capillary tube that is coated so that electroosmotic flow does not occur, and single-stranded sample DNA is injected from the cathode side and a voltage is applied. At this time, migration of sample DNA having a sequence completely complementary to the conjugated DNA is hindered by interaction with the filled conjugated DNA. At this time, by comparing the difference in affinity in hybridization with the conjugate DNA between the sample DNA having SNP and the sample DNA having a completely complementary sequence, the DNA having SNP is compared. Can be clearly identified.

When discriminating the SNP of the sample DNA using the conjugate DNA to which the polymer compound is bound, the sample DNA is put into a single-stranded state in order to bind the sample DNA and the conjugate DNA (hereinafter referred to as hybrid). To do. As sample DNA, genomic DNA is extracted from a biological sample, amplified by PCR using a primer DNA labeled with a fluorescent dye, and then denatured into a single strand (referred to as sample DNA preparation). The adjusted sample DNA is injected into a capillary tube filled with the conjugate DNA, and a PCR product amplified by electrophoresis is optically detected by a detection unit.
JP 2002-340858 A

  However, in the conventional technique, when the sample DNA is prepared or when the sample DNA is electrophoresed in the capillary tube, a part of the complementary strand in which the PCR product is denatured and the sample DNA are recombined to return to the double strand. A phenomenon occurs. When these sample DNAs are separated by electrophoresis, the unbound DNA that does not bind to the conjugate DNA, the recombined DNA that becomes a double strand rebound to the double strand, and the bound DNA that binds to the conjugate DNA are in this order. A peak is detected. The peak value due to the recombined DNA is a value close to the peak value of the unbound DNA to be measured, and shows a value several times larger than the bound DNA peak to be measured next. In addition, the position where the peak of recombined DNA occurs largely moves depending on the conditions, so that there is a problem that accurate measurement cannot be performed.

  The present invention has been made in view of the above problems, and provides a DNA detection method capable of performing accurate quantitative analysis by eliminating the generation of recombined DNA that occurs when sample DNA is electrophoresed in sample tubes or in capillary tubes. The purpose is to provide.

  In order to solve the conventional problems, the DNA detection method of the present invention comprises double-stranded DNA containing a specific base sequence and / or double-stranded DNA in which a part of the base sequence of the double-stranded DNA is different. In a DNA detection method for separating sample DNA by electrophoresis, a step of filling a capillary with a conjugate of pyrrole imidazole polyamide and polyethylene glycol, and a step of injecting the sample DNA into the capillary filled with the conjugate And a step of applying a voltage to both ends of the capillary to cause electrophoresis of the sample DNA.

  According to the DNA detection method of the present invention, since the sample DNA can be detected as double-stranded DNA, no recombined DNA is generated, and the peak of the recombined DNA that has become unnecessary noise during measurement is detected. Generation can be eliminated. Therefore, the peak of non-binding DNA and the peak of binding DNA, which have been difficult to quantitatively analyze, can be measured independently, and more precise DNA analysis is possible. Furthermore, by directly detecting double-stranded DNA, a complicated sample DNA preparation step is not required, and DNA can be detected quickly.

  Hereinafter, embodiments of the DNA detection method of the present invention will be described in detail with reference to the drawings.

(Embodiment 1)
First, a double-stranded DNA containing a specific base sequence and / or a sample DNA consisting of a double-stranded DNA containing a base sequence with a part of the specific base sequence different by electrophoresis is separated by electrophoresis. A method for detecting DNA will be described.

  FIG. 2 schematically shows the configuration of a DNA apparatus for detecting DNA according to the present invention. In FIG. 2, a capillary 1 is filled with a conjugate 10 containing a buffer solution, and a sample DNA 9 is filled at the end on the negative electrode 2 side. A buffer 6 is contained in the container 4 and the container 5, the positive electrode 3 is inserted into the container 5, and the negative electrode 2 is inserted into the container 4. Further, the end of the capillary 1 is inserted into the container 4 and the container 5. There is a detection unit 7 on the side of the container 5 of the capillary 1, and a double-stranded DNA containing a specific base sequence that has moved through the capillary 1 and a double-stranded DNA containing a base sequence in which a part of the specific base sequence is different. It can be detected. The positive electrode 3 and the negative electrode 2 are connected to a power supply control unit 8, and a predetermined voltage can be applied by the power supply control unit 8. The operation and action of the DNA detection apparatus configured as described above will be described below.

  First, the sample DNA of the embodiment of the present invention will be described. The sample DNA to be analyzed in the embodiment of the present invention amplifies a target DNA portion using a method such as PCR using DNA obtained from a plant, animal, or human cell or blood as a template. These PCR products are suitable in length so that the conjugate described later does not bind to multiple sites of the PCR product, and is preferably about 40 bp to 100 bp. At this time, a fluorescent label is added to the 5 ′ end of the primer DNA on the forward side for performing PCR, and when the DNA is amplified by PCR, a PCR product in which a labeling agent is bound to the 5 ′ end of one strand is obtained. Produced. In the present embodiment, it is assumed that the fluorescent label is Cy5.

  The pyrrole-imidazole polyamide used in the conjugate of the present invention is a substance that can specifically bind to a specific base sequence region contained in double-stranded DNA. By combining the pyrrole (Py) site and the imidazole (Im) site constituting the pyrrole-imidazole polyamide, the pyrrole-imidazole polyamide can bind to double-stranded DNA having an arbitrary base sequence. It is known that Im / Py pairs can recognize and bind GC base pairs, Py / Im pairs recognize CG base pairs, and Py / Py pairs recognize AT base pairs or TA base pairs. For example, the pyrrole-imidazole polyamide shown in FIG. 1 is composed of an Im / Py pair, an Im / Py pair, an Im / Py pair, and a Py / Im pair as shown in FIG. Therefore, this pyrrole imidazole can bind to a double-stranded DNA region having a base sequence of 5′-GGGC-3 ′ · 3′-CCCG-5 ′.

  The pyrrole-imidazole polyamide can be specifically bound to a DNA base sequence having an arbitrary length by changing the number of pairs of Im and Py. However, when the number of base pairs to which pyrrole-imidazole polyamide binds is 9 or more, the binding power to the target specific base sequence increases, so that it binds to a base sequence that is one base different from the target base sequence. This is not preferable because of the possibility. On the other hand, if the number of base pairs to which pyrrole-imidazole polyamide binds is 3 or less, it is not preferable because it may also bind to a target base sequence region and a non-target base sequence existing in a position other than that region. . Accordingly, the number of base pairs to which pyrrole-imidazole polyamide is bonded is preferably in the range of 4 to 8.

  The conjugate is prepared by combining the pyrrole-imidazole polyamide described above and polyethylene glycol. Polyethylene glycol is a substance that undergoes electrophoresis at a slower rate than the sample DNA during electrophoresis in order to allow sufficient interaction between the sample DNA and the conjugate during electrophoresis, and the conjugate flows out of the capillary. It has a function as a weight that does not. For this reason, the weight average molecular weight of polyethylene glycol is preferably 2,000 or more. On the other hand, if the molecular weight of polyethylene glycol is too large, the transfer rate of polyethylene glycol in the reaction solution decreases during the reaction of pyrrole-imidazole polyamide and polyethylene glycol. Therefore, the contact opportunity to pyrrole imidazole polyamide decreases, and the reaction efficiency of polyethylene glycol to pyrrole imidazole polyamide decreases. In order to prevent a decrease in reaction efficiency, the weight average molecular weight of polyethylene glycol is preferably 150,000 or less. Therefore, the weight average molecular weight of the polyethylene glycol used in the present invention is preferably in the range of 2,000 to 150,000.

  The conjugate may be synthesized using, for example, a peptide solid phase synthesis method, which is a known method for chemically synthesizing peptides and proteins. In peptide solid phase synthesis, a solid phase carrier such as polystyrene polymer gel beads modified with amino groups or the like, and a Py or Im monomer unit modified with a protecting group are used. Details of the monomer unit will be described later. First, a solid phase carrier, a Py monomer unit or Im monomer unit to be introduced, and HATU (0- (7-azobenzotriazol-1-yl) -1,1,3,3-tetramethyluronium which is a condensing agent Hexafluorophosphate) and DIEA (N, N-diisopropylethylamine) as a reaction solvent are mixed to react the monomer unit with the solid support. Next, the solid phase carrier is treated with a 20% piperidine / DMF solution to remove the protecting group of the monomer unit. By repeating the above-described reaction of the monomer unit and removal of the protecting group, the desired pyrrole-imidazole polyamide is synthesized on a solid support. Furthermore, NHS-PEG is reacted to obtain the desired conjugate. Finally, the conjugate is obtained by cutting out the conjugate synthesized by treatment with a strong acid solution from the solid surface. The details of the synthesis method are described in patent documents and the like (for example, International Publication No. WO2003-000683 etc.), and will be omitted.

  The concentration of the conjugate is determined with respect to the concentration of the sample DNA in order to satisfactorily separate double-stranded DNA containing a specific base sequence and / or double-stranded DNA containing a part of the specific base sequence. It is appropriate to make it 50 to 600 times.

  The conjugate prepared as described above is put into a buffer (for example, Tris-Borate buffer 50 mM pH 7.4) and injected into the capillary. In order to prevent the electroosmotic flow generated during electrophoresis from occurring in this capillary, it is preferable to coat the inner wall of the capillary with acrylamide or the like. Prevention of electroosmotic flow is not limited to coating with acrylamide or the like, and other methods may be used as long as electroosmotic flow can be suppressed. For example, a commercially available coating capillary may be used. The capillary may be a plastic fine channel made of a material that does not generate electroosmotic flow and having the same size as the capillary.

  The amount of sample DNA injected into the capillary must not be too large or too small. If the injection amount is too large, the sample DNA may be broadened during electrophoresis, and the sample DNA may not be electrophoretically separated. If the injection amount is too small, it is difficult to perform the sample DNA injection operation with good reproducibility. Therefore, the amount of sample DNA injected into the capillary is preferably 1/5 to 1/10 of the effective length of the capillary. The voltage and current applied to the capillary for allowing the sample DNA to move to the positive electrode side by electrophoresis are preferably 6 kV and 3 to 16 μA, respectively, when a 25 cm long capillary is used.

  The sample DNA is detected via a fluorescent dye Cy5 labeled at the 5 ′ end on the forward side of the primer. The detection unit is configured to be able to irradiate excitation light (630 nm) and detect fluorescence (660 nm), and detect the presence of sample DNA by detecting Cy5 which is a fluorescent dye corresponding to the excitation and fluorescence wavelength. can do.

  As described above, the DNA detection method according to the first embodiment can avoid the generation of recombined DNA and can eliminate the generation of a peak that causes noise. Moreover, since preparation of sample DNA becomes unnecessary, DNA can be detected rapidly.

Hereinafter, a specific embodiment of the present invention will be described in detail as a first embodiment. Note that the present invention is not limited to this.
(Production of Example 1)
[Preparation of PCR products]
PCR amplification was performed using TaKaRa Ex Taq (TaKaRa). As a template, Mannenbosi, a kind of wheat, and Ichibanboshi, a single-base mutant of Mannenbosi, were used. The forward primer was 5 ′-(Cy5) -CAAGCATGTCGCATCGAAAC-3 ′ (forward primer, Sequence Listing 1) labeled with a fluorescent dye, in this case Cy5, and the reverse primer was 5′-TGCTGCCTTGGCGAGGTAC-3 ′ (reverse Primers and Sequence Listing 2) were added to a final concentration of 500 nM, respectively.

  The reaction cycle is as follows. 95 ° C-10 minutes, (95 ° C-30 seconds, 55 ° C-30 seconds, 72 ° C-30 seconds) 30 cycles, 72 ° C-5 minutes. As a result, 50 bp PCR products of Mannenbosi and Ichibanboshi having the sequences shown below were prepared.

Mannenbosi PCR product 5'-CAAGCATGTCGCATCCGAAACGCCCGGGCCGCCGTACCTCGCCAAGCAGCA-3 '(Mannenbosi PCR product, Sequence Listing 3)
Ichibamboshi PCR product 5′-CAAGCATGTCGCATCGAACGCCCCGAGCCGCCGTACCCTCGCCAAGCAGCA-3 ′ (Ichibamboshi PCR product, Sequence Listing 4)
(Synthesis of monomer unit)
The Py monomer unit 30 and Im monomer unit 31 used for the solid phase synthesis were synthesized according to the procedure shown in FIG. Note that commercially available reagents and solvents used for the reaction and purification were used. Abbreviations of the reagents used are as follows. Dimethylformamide (DMF), diisopropylethylamine (DIEA), triisopropylsilane (TIS), trifluoroacetic acid (TFA), carbonyldiimidazole (CDI), 4-dimethylaminopyridine (DMAP).

  (1) Synthesis of 1-methyl-2-trichloroacetylpyrrole (22): 20 (90.3 g, 1.10 mol) of trichloroacetyl chloride (200.0 g, 1.10 mol) in a solution of methylene chloride (600 ml) A methylene chloride (200 ml) solution was added dropwise over 3 hours. At this time, nitrogen gas was sprayed into the solution to remove hydrogen chloride generated during the reaction. After stirring overnight, the solvent was distilled off under reduced pressure. The residue was subjected to silica gel column chromatography, and trichloroacetyl 22 (189.7 g, 76%) was obtained from the eluate of ethyl acetate-hexane (1:10, v / v).

(2) Synthesis of 1-methyl-4-nitro-2-trichloroacetylpyrrole (24): A solution of 22 (45.2 g, 0.200 mol) in acetic anhydride (200 ml) was cooled to −40 ° C. and kept at the same temperature. Fuming nitric acid (18.5 ml, 0.360 mol) was added dropwise. After stirring at room temperature for 2 hours, isopropanol was added, and the precipitated solid was separated by filtration to obtain a nitro compound 24 (27.2 g). Further, the filtrate was distilled off under reduced pressure, and the resulting residue was subjected to silica gel column chromatography to obtain 24 (10.7 g) from the ethyl acetate-hexane (1:10, v / v) eluate. . (Total yield 70%)
(3) Synthesis of 1-methyl-4-nitro-2-trichloroacetylimidazole (25): 21 (16.4 g, 0) in a solution of trichloroacetyl chloride (36.3 g, 0.20 mol) in methylene chloride (120 ml) .20 mol) in methylene chloride (80 ml) was added dropwise over 2 hours. After stirring for 4 hours, triethylamine (20.2 g, 0.20 mol) was added dropwise under ice cooling. After distilling off the solvent under reduced pressure, the residue was subjected to silica gel column chromatography to obtain trichloroacetyl derivative 23 (23.2 g, 51%) from the eluate of ethyl acetate-hexane (1: 1, v / v). It was.

(4) Synthesis of methyl 4-nitro-1-methylpyrrole-2-carboxylate (26): DMAP (0.50 g, 4.3) in a solution of 24 (32.4 g, 0.12 mol) in methanol (140 ml). 55 mmol) was added and stirred for 40 minutes. Thereafter, the precipitate was filtered off and washed with methanol to obtain 26 (18.9 g). In the filtrate, the solvent was distilled off under reduced pressure, and then the same operation was repeated to obtain 26 (2.3 g). (Total yield 97%)
(5) Synthesis of methyl 4-nitro-1-methylimidazole-2-carboxylate (27): DMAP (0.500 g, 4.55 mmol) in a solution of 25 (70.0 g, 256 mmol) in methanol (500 ml) Was added and stirred for 2 hours. Thereafter, the precipitate was filtered off and washed with diethyl ether to obtain 27. The filtrate was evaporated under reduced pressure, and the same operation was repeated twice to obtain 27 (46.0 g, 97%).

(6) Synthesis of 1-methyl-4-aminopyrrole-2-carboxylic acid methyl ester hydrochloride (28): 26 (15.3 g, 83.0 mmol) in methanol and dichloromethane (150 ml, 1: 2, v / v ) Dissolved in a mixed solvent, suspended by adding 10% palladium carbon (3 g), and stirred for 2 days under a hydrogen atmosphere. Thereafter, the mixture was filtered through Celite to remove palladium on carbon, and 10% hydrochloric acid was added to the filtrate. The resulting precipitate was filtered off to obtain 28 (4.71 g). The filtrate was further distilled off under reduced pressure, and recrystallized with ethyl acetate-methanol to obtain 28 (6.86 g). (Total yield 11.6 g, 73%)
(7) Synthesis of 1-methyl-4-aminoimidazole-2-carboxylic acid methyl ester hydrochloride (29): To a solution of 27 (20.0 G, 108 mmol) in dichloromethane (300 ml) was added 10% palladium carbon (5 g). Suspended and stirred for 1 day under hydrogen atmosphere. Thereafter, the mixture was filtered through Celite to remove palladium on carbon, and the filtrate was acidified with 10% hydrochloric acid. The precipitate was collected by filtration to obtain 29 (19.8 g, 96%).

  (8) Synthesis of 4-[(9-fluorenylmethoxycarbonyl) amino] -1-methyl-2-pyrrolecarboxylic acid (30), which is a Py monomer unit: distilled 28 (10.9 g, 57.2 mmol) Dissolved in water (80 ml) and sodium hydroxide (9.2 g) was added. After stirring overnight, the mixture was neutralized with 1N hydrochloric acid and evaporated under reduced pressure. The residue was dissolved in a mixed solvent of water and ethylene glycol dimethyl ether (100 ml, 1: 1, v / v) and used for the next reaction. After sodium carbonate (5.3 g) was dissolved in this solution, 9-fluorenylmethyl chloroformate (17.8 g, 68.6 mmol) was added. After stirring overnight, the precipitate was filtered off to give 30 (12.3 g, 34.1 mmol). Further, the filtrate was added to a mixed solution (1: 1, v / v) of 1M aqueous sodium carbonate and diethyl ether. The precipitate was filtered off to obtain 30 (3.4 g). The aqueous layer was acidified with 10% hydrochloric acid and extracted with ethyl acetate. The organic layer was distilled off under reduced pressure and recrystallized with hexane-dioxane to obtain 30 (1.6 g) as a Py monomer unit (total yield: 17.3 g, 84%).

  (9) Synthesis of 4-[(9-fluorenylmethoxycarbonyl) amino] -1-methyl-2-imidazolecarboxylic acid (31), which is an Im monomer unit: 29 (8.24 g, 41.9 mmol) was distilled. Dissolved in water (60 ml) and sodium hydroxide (4.2 g) was added. After stirring overnight, the mixture was neutralized with 1N hydrochloric acid and evaporated under reduced pressure. The residue was dissolved in a mixed solvent of water and ethylene glycol dimethyl ether (200 ml, 1: 1, v / v) and used for the next reaction. After sodium hydrogen carbonate (14.1 g) was dissolved in this solution, 9-fluorenylmethylsuccinimidyl carbonate (16.9 g, 50.1 mmol) was added. After stirring overnight, the precipitate was filtered off to give 31 (10.8 g, 29.7 mmol). Further, the filtrate was acidified with 10% hydrochloric acid, and the resulting precipitate was filtered off and washed with ethyl acetate to obtain 31 (1.2 g, 3.30 mmol) as an Im monomer unit (total). Yield 12.0 g, 79%).

[Synthesis of conjugate]
Various pyrrole imidazole polyamides were synthesized using the obtained monomer units 30 and 31. The synthesis protocol is shown in Table 1.

  Solid phase synthesis was performed using a peptide synthesizer (Applied Biosystems, Pioneer). A commercially available Wang Resin preloaded with Fmoc-β-alanine was used as the solid phase carrier. Further, 4 equivalents of HATU, DIEA, and Py · Im monomer units were used with respect to the active terminal of the solid phase carrier. Prior to synthesis, the solid support was swelled in DMF for 30 minutes and then packed in a Pioneer synthesis column. In the synthesis, the Fmoc group of β-alanine preloaded on the carrier was first deprotected by treating with 20% piperidine in DMF for 5 minutes. Thereafter, the carrier was washed with methanol for 50 seconds, and Py or Im monomer units to be introduced, HATU and DIEA were passed through the column, cycled for 60 minutes, and then washed again with methanol for 40 seconds. These cycles of deprotection and extension were taken as one cycle, and the monomer units were condensed in order according to the target polyamide sequence. Then, after deprotecting in the same manner, PEG-NHS having a molecular weight of 20,000, which is 4 equivalents of polyamide, was reacted at room temperature for 6 hours in a DMF solution to modify the PEG on the polyamide. After completion of the reaction, the solid phase carrier is taken out from the column, dried under reduced pressure, transferred into a 50 ml eggplant flask, added with 5 ml of 95% TFA, 2.5% TIS, 2.5% water and stirred for 30 minutes. It was cut out from the carrier. Purification was performed by HPLC using a 0.1% TFA aqueous solution and acetonitrile to obtain conjugate 11 (0.11 g) shown in FIG. The resulting conjugate 11 was dissolved in Tris-Borate buffer 50 mM (pH 7.4) to prepare a conjugate of 10 uM.

[SNP measurement by capillary electrophoresis]
FIG. 2 shows the configuration of the fluorescence capillary electrophoresis apparatus for performing DNA detection described in the first embodiment. The container 4 and the container 5 contain a buffer solution 6 (Tris-Borate buffer 50 mM pH 7.4), the positive electrode 3 is inserted into the container 5, and the negative electrode 2 is inserted into the container 4. Has been. Further, the end of the capillary 1 is inserted into the container 4 and the container 5. As the capillary 1, a capillary tube having an inner diameter of 100 μm (manufactured by Otsuka Electronics Co., Ltd.) coated with the inner wall of the capillary was used. Then, as shown in FIG. 2, sample DNA 9 (Mannenbosi 2.5 uM, Ichibanboshi 5.0 uM) was injected into the capillary 1 filled with the conjugate 11 containing the buffer 6 (Tris-Borate buffer pH 7.4). Next, a voltage of 6 kV is applied between the negative electrode 2 and the positive electrode 3 by the power supply control unit 8, and the sample DNA 9 in the capillary 1 is electrophoresed at a measurement temperature of 25 degrees. The sample DNA 9 was separated by the difference in affinity between the DNA and unbound DNA for the conjugate 11. In the detector 7, the fluorescent dye Cy5 labeled on the primer DNA was irradiated with laser excitation light 630nm, and the fluorescence emitted from Cy5 was detected with a photomultiplier at a wavelength of 660nm. In FIG. 4, the graph which measured the sample DNA in the present Example 1 is shown.

(Comparison example creation)
As a comparative example, a DNA detection method using the conjugate shown in FIG. As the sample DNA to be measured, single-stranded DNA obtained by denaturing the same fluorescently labeled PCR product as in Example 1 was used, and measurement was performed with the same apparatus as in FIG. The measurement results are shown in FIG.

[Contrast between Example 1 and Comparative Example]
In the comparative example, as shown in FIG. 5, the peak of the primer, the peak of unbound DNA, the peak of recombined DNA, and the peak of bound DNA are detected from the earlier time. The peak value of recombined DNA shows a fluorescence intensity that is 74% more than the peak value of unbound DNA, indicating that accurate measurement is difficult. On the other hand, in Example 1 of the present invention, as shown in FIG. 4, the peak of the primer, the peak of non-binding DNA, and the peak of binding DNA are detected from the earliest time. The peak disappears. This shows that the conjugate according to the present invention has an effect of significantly suppressing unnecessary peaks as compared with a conventional single-stranded conjugate.

  As described above, the DNA detection method of Example 1 was able to avoid the generation of recombined DNA and eliminate the occurrence of noise peaks. In addition, since it was not necessary to prepare sample DNA, DNA could be detected quickly.

  The DNA detection method according to the present invention prevents noise peaks from occurring in SNP measurement, and is useful for improving peak quantification and sensitivity. In addition, the present invention is expected to be diverted to a small SNP detection device using a microchip, and it is considered that rapid diagnosis of genes by Point (Point of Care Testing) using the present invention is also possible.

The figure which showed the structural formula of the conjugate 11 in this invention The figure which showed simply the structure of the apparatus which performs DNA detection in this invention The figure which showed notionally the coupling | bonding to the DNA of the conjugate 11 in this invention The graph which showed the waveform detected when measuring sample DNA when Example 1 of this invention is applied A graph showing waveforms detected when measuring sample DNA prepared in a comparative example, which is a conventional method The figure which showed the synthesis scheme of Py monomer unit 30 in this Example 1, and Im monomer unit 31 Diagram showing structural formula of conjugate in conventional method

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Capillary 2 Negative electrode 3 Positive electrode 4 1st container 5 2nd container 6 Buffer 7 Detection part 8 Power supply control part 9 Sample DNA
10, 11 Conjugate 20 1-methylpyrrole 21 1-methylimidazole 22 1-methyl-2-trichloroacetylpyrrole 23 1-methyl-2-trichloroacetylimidazole 24 1-methyl-4-nitro-2-trichloroacetylpyrrole 25 1-methyl-4-nitro-2-trichloroacetylimidazole 26 methyl 4-nitro-1-methylpyrrole-2-carboxylic acid ester 27 methyl 4-nitro-1-methylimidazole-2-carboxylic acid ester 28 1-methyl- 4-Aminopyrrole-2-carboxylic acid methyl ester hydrochloride 29 1-methyl-4-amino-imidazole-2-carboxylic acid methyl ester hydrochloride 30 4-[(9-fluorenylmethoxycarbonyl) amino] -1- Methyl-2-pyrrolecarbo Acid 31 4-[(9-Fluorenylmethoxycarbonyl) amino] -1-methyl-2-imidazolecarboxylic acid

Claims (3)

In a DNA detection method in which a sample DNA comprising a double-stranded DNA containing a specific base sequence and / or a double-stranded DNA having a part of the base sequence of the double-stranded DNA is separated by electrophoresis,
Filling a capillary with a conjugate of pyrrole-imidazole polyamide and polyethylene glycol;
Injecting the sample DNA into the capillary filled with the conjugate;
Applying a voltage to both ends of the capillary to cause electrophoresis of the sample DNA;
A DNA detection method comprising: The DNA detection method according to claim 1, wherein the conjugate is represented by a general formula of [Chemical Formula 1].

However, X is N-methylpyrrole or 1-methylimidazole, R shows arbitrary structures, Z shows polyethyleneglycol, L shows arbitrary structures, L 'shows CO-NH-, L ″ Represents —CH ₂ —CH ₂ —CH ₂ —CO—NH—, and n is in the range of 4 to 8. The DNA detection method according to claim 1 or 2, wherein the polyethylene glycol of the conjugate has a weight average molecular weight of 2,000 to 150,000.

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