WO2005080958A1

WO2005080958A1 - Method of detecting double-stranded dna having specific sequence

Info

Publication number: WO2005080958A1
Application number: PCT/JP2005/002687
Authority: WO
Inventors: Norihiko Minoura; Masayo Ogiso
Original assignee: National Institute Of Advanced Industrial Science And Technology
Priority date: 2004-02-23
Filing date: 2005-02-21
Publication date: 2005-09-01
Also published as: JPWO2005080958A1; JP4423397B2

Abstract

A double-stranded DNA-containing sample is electrophoresed by using a polymer gel, which has a pore having a template in the shape of a double-stranded DNA molecule imprinted thereon and further having a substituent recognizing a base pair of the double-stranded DNA in the pore, as an electrophoresis medium. Thus, the double-stranded DNA can be conveniently and quickly detected and/or separated in the double-stranded state as such.

Description

Specification

Method for detecting double-stranded DNA with specific sequence

Technical field

The present invention relates to a novel electrophoresis medium for base-specifically detecting double-stranded DNA, a method for producing the same, and a method for detecting double-stranded DNA using the electrophoresis medium. Background art

[0002] The technology of discriminating and detecting DNA having a specific base sequence from other DNA is expected to be applied to disease prevention, diagnosis, drug discovery research, and the like, and is attracting attention in the medical field. I have.

In order to detect a DNA base sequence, it is common to use a DNA sequencing method called a dideoxy method (see Non-Patent Document 1). This method utilizes the principle that when repairing single-stranded DNA to double-stranded DNA, dideoxynucleotides are incorporated in the same manner as deoxynucleotides, blocking 3'-OH and stopping repair there. are doing. According to the type of dideoxynucleotide triphosphate to be added, a DNA chain terminated at the position where it appears is obtained and analyzed by sequencing gel. Since the autosequencer used for the analysis is expensive and requires skill in sample preparation, the dideoxy method is also used at the thorny experimental research level. A DNA microarray (see Non-Patent Document 2) has been developed as a more convenient method.

[0003] This method is based on the hybridization of DNA as a basic principle. Specifically, a single-stranded DNA that is complementary to a base sequence to be detected is immobilized on a substrate. This method involves complementary binding to single-stranded DNA previously labeled with a fluorescent substance or isotope and detection of the sample DNA bound to the single-stranded DNA immobilized on the substrate by fluorescence or isotope. . This DNA microarray can be used for clinical diagnosis because it can compare and analyze the base sequences of different sample DNAs.

Non-Patent Document 1: F. Sanger, S. Nicklen, A.R.Coulson, Proc. Natl. Acad. Sci. U.S.A., 74,

5463 (1977)

Non-Patent Document 2: Kenichi Yamashita, Shiori Takenaka, Makoto Takagi, Bunseki, 5, 227-232 (2002)] Problems the invention is trying to solve

[0004] As described above, the dideoxy method requires expensive equipment and requires techniques for sample preparation, so it is expensive and is not a routine technique. In addition, to detect the presence or absence of DNA of a specific base sequence using a DNA microarray, the double-stranded DNA that is the sample must first be denatured to single-stranded DNA. In addition, in order to prepare a DNA chip, it is necessary to previously immobilize a single-stranded DNA complementary to a base sequence to be detected on a substrate such as a slide glass or silicon. In addition, during the hybridization process, a mismatch between the DNA immobilized on the substrate and the sample DNA may occur.Therefore, it is necessary to optimize the hybridization conditions and reduce the mismatch. Takes time.

[0005] The above problem is a problem due to the use of single-stranded DNA. If double-stranded DNA can be used as it is for the sample and there is a means for detection, it is not necessary to denature the sample DNA or immobilize the single-stranded DNA on the substrate. Also, since no hybridization is performed, no mismatch between DNAs occurs. On the other hand, currently used double-stranded DNA analysis methods rely solely on electrophoresis utilizing differences in base length.If double-stranded DNAs with different base sequences have the same base length, However, the two cannot be distinguished, detected or separated. Therefore, there has been a demand for a method for base-specific detection and separation without denaturing double-stranded DNA into single-stranded DNA.

Means for solving the problem

[0006] The inventors of the present invention have conducted intensive studies to obtain a method for base-specific detection of double-stranded DNA as double-stranded DNA, and as a result, recognized and captured double-stranded DNA having a specific base sequence. By creating a polymer material by molecular imprinting and using it as an electrophoresis medium, double-stranded DNA can be base-specifically identified, detected, and further separated. And completed the present invention. That is, the present invention is as shown below.

[0007] (1) A double-stranded DNA molecule is formed of a polymer gel having pores imprinted as a triangle and having a substituent that recognizes the base pair of the double-stranded DNA in the pores. A medium for electrophoresis, characterized by (2) The electrophoretic medium according to claim 1, wherein the substituent that recognizes a base pair of the double-stranded DNA has the following chemical structure.

[Chemical 1]

(However, in the formula, Y represents CH or N.)

(3) The electrophoretic medium according to claim 1 or 2, wherein the polymer gel is a crosslinked polyacrylamide gel.

(4) A method for producing an electrophoresis medium for identifying double-stranded DNA, comprising the following steps (a) to (c).

(a) mixing a double-stranded DNA with a functional monomer having a substituent that recognizes the base pair of the double-stranded DNA, and allowing the functional monomer to self-assemble to the base pair of the double-stranded DNA The process

(b) a step of polymerizing the obtained self-assembled product and a polymer gel-forming monomer,

(c) removing double-stranded DNA from the formed polymer gel to form a polymer gel on which the double-stranded DNA molecules used in step (a) are imprinted;

(5) The double-stranded DNA-containing sample is dropped on the electrophoresis medium according to any one of claims 13 to 13, and electrophoresed, and imprinted in a polymer gel of the electrophoresis medium. The same double-stranded DNA as the imprinted one in the sample was used as an index, with the index of shortening the migration distance caused by the double-stranded DNA in the sample being captured in the pores corresponding to the double-stranded DNA molecule. A method for detecting and / or separating double-stranded DNA, which comprises detecting.

The invention's effect [0008] The present invention uses a polymer gel on which double-stranded DNA molecules are imprinted as a medium for electrophoresis to detect and / or separate double-stranded DNA in a sample in a base-specific manner. This method does not require expensive equipment unlike the conventional dideoxy method, does not require special techniques for sample preparation, and has been a problem in DNA detection using microarrays. The purpose is not to perform laborious operations such as denaturation of double-stranded DNA into single-stranded DNA, immobilization of single-stranded DNA to a substrate, no mismatching, and setting of hybridization conditions. This makes it possible to easily and quickly detect and / or separate DNA having a base sequence of: Brief Description of Drawings

FIG. 1 is a schematic view showing an outline of a process for producing an imprinted polymer gel of the present invention.

FIG. 2 is a schematic diagram showing an outline of a method for detecting double-stranded DNA in a test sample of the present invention.

[Figure 3] Figure 3 shows the correlation between the migration distance of the standard sample for electrophoresis in imprinted gel media and polyacrylamide gel media, which was used to detect DNA with the same base sequence as type I molecule in the DNA mixed sample. It is a graph.

BEST MODE FOR CARRYING OUT THE INVENTION

[0010] The method for detecting and / or separating double-stranded DNA having a specific sequence according to the present invention comprises the steps of preparing a polymer material having a molecule-capturing ability, and using the prepared polymer material as an electrophoresis medium. And performing DNA electrophoresis to detect DNA.

In the step of preparing a polymer material having a molecule capturing ability, a technique of molecular imprinting is used. The molecular imprinting method is a method for obtaining a polymer (host polymer) having vacancies formed by using a guest molecule as a 铸 type, and is roughly classified into a guest molecule and a functional monomer non-covalently bonded to the guest molecule. And then subjecting the monomer forming the host polymer to a polymerization reaction, followed by removing the guest molecule, and performing a polymerization reaction between the functional monomer covalently bonded to the guest molecule and the monomer forming the host polymer. After that, there is a method in which the covalent bond between the guest molecule and the functional monomer is cleaved to remove the guest molecule. In the present invention, the force S that can use the above two methods, the former method is substantially preferred. In the present invention, a double-stranded DNA molecule for detection or separation is used as it is without denaturing as a guest molecule,

In addition, a functional group that recognizes a base pair of the double-stranded DNA as a functional monomer, has a substituent that is non-covalently bonded to the base pair, and is capable of polymerizing with a monomer that forms a host polymer. Is used.

Examples of the substituent that recognizes the base pair of the double-stranded DNA and binds non-covalently to the base pair include the following substituents.

[Chemical 1]

H H

H / N 丫 N 丫

(However, in the formula, Y represents CH or N.)

Further, the monomer for forming the host polymer may be any monomer that can form a hydrophilic gel that can be used as a medium for electrophoresis.

Therefore, for example, when forming a polyacrylamide gel as a host polymer, use acrylamide as a monomer, N, N'-methylenebisacrylamide, N, N, diaryltartaric diamide, bisatarylylcysteamine, etc. A cross-linking agent may be used. In this case, the functional group that forms the host polymer in the functional monomer is a functional group that can be copolymerized with atalinoleamide, for example, a butyl group, an aryl group, an atalyloyl group, a (meth) atalyloyloxy group, Butadienyl group, acrylamide group, cyanoacryloyloxy group and the like. Examples of such a functional monomer compound are represented by the following general formula.

[Chemical 2]

(Wherein, X is a group copolymerizable with acrylamide, and Y represents CH or N.

)

[0012] The substituent of the above [Chemical formula 1] recognizes a base pair consisting of adenine and thymine (Α · Τ) in the double-stranded DNA molecule, and non-covalently bonds to the base pair by hydrogen bonding. Therefore, the functional monomer having the substituent self-assembles with the double-stranded DNA.

In the present invention, in order to obtain an electrophoresis medium for detecting a double-stranded DNA molecule, a double-stranded DNA to be detected is mixed with the above functional monomer to obtain a self-assembly, and the self-assembly is obtained. The functional monomer portion is subjected to a polymerization reaction with the monomer forming the polymer gel, and then the double-stranded DNA is removed from the polymerization reaction product.

Thereby, a double-stranded DNA molecular shape force S-imprinted polymer gel is obtained. The polymer gel has 錡 -shaped holes corresponding to the double-stranded DNA in the structure thereof, and the surface of the holes has a hole of the double-stranded DNA derived from the functional monomer.置換 There is a substituent that recognizes a base pair and binds non-covalently to the base pair.

[0013] The electrophoresis medium composed of this polymer gel is used for detecting and separating or separating double-stranded DNA having a target base sequence from a DNA sample. To detect double-stranded DNA in a sample, a sample containing the double-stranded DNA is dropped on the cathode side of the electrophoresis medium, and electrophoresis is performed. The double-stranded DNA in the sample moves to the anode side, but when the double-stranded DNA having the target base sequence is present in the sample, the double-stranded DNA is a polymer constituting the electrophoresis medium. As a result of being trapped in the 铸 -shaped holes corresponding to the shape of the double-stranded DNA molecule imprinted in the gel and hindering migration, electrophoresis The distance becomes shorter. Therefore, by detecting this electrophoresis distance, it is possible to detect the presence or absence of double-stranded DNA having the target base sequence in the sample without denaturing it to single-stranded DNA, and to shorten this electrophoresis distance. By collecting the obtained double-stranded DNA from the electrophoresis medium, a double-stranded DNA having a target base sequence can be isolated from the sample.

In the present invention, the target double-stranded DNA can be distinguished from other double-stranded DNA in a base sequence-specific manner. The reason is as follows.

There are two types of base pairs in double-stranded DNA: Α · Τ and C'G. Their arrangement or the content ratio of each base pair varies. For example, when detecting a DNA having a base sequence of A (T) _G (C) _G (C) _G (C) -G (C) -A (T) as double-stranded DNA (note that , A (T) represents the base pair Α · Τ in the double-stranded DNA, and G (C) represents the same base pair G ′ C.), based on the double-stranded DNA, a functional monomer The above-mentioned polymer gel is prepared using a monomer that recognizes the base pair {Α}. In this case, on the surface of the pores of the polymer gel imprinted with the molecular shape of the double-stranded DNA in the polymer gel as 铸, the position corresponding to the position of 者· A substituent that recognizes Τ will be placed.

Therefore, if a double-stranded DNA having the above sequence is present in the test sample, the position of the base pair {Α} of the double-stranded DNA is in the 内 -shaped hole in the polymer gel. It matches the position of the substituent that recognizes Α · Τ, and is firmly captured by self-assembly. On the other hand, in the sample, for example, a double-stranded DNA having a base sequence of A (T) -G (C) -G (C) -A (T) -G (C) -G (C) is formed. Even if it is present, the position of the substituent in the pore and the position of Α · Τ in the double-stranded DNA do not completely match, even if they partially match. This difference appears as a difference in the capturing ability of the polymer gel, and is reflected in the migration distance by electrophoresis. The capturing ability of such a polymer gel also varies depending on the content ratio of base pairs of double-stranded DNA. Accordingly, the present invention provides a double-stranded DNA specifically having a base sequence by providing a substituent for identifying a specific base pair on the inner surface of a pore in a polymer gel, which is not limited only to the molecular shape of double-stranded DNA. It distinguishes strand DNA, and is based on a completely new finding that discrimination can be carried out simply and easily, especially using the migration distance by electrophoresis as an index. It is.

Further, with respect to the method for detecting and separating the double-stranded DNA of the present invention, referring to FIGS. 1 and 2, 2-vinyl-4,6-diamino-1,3,3 as a functional monomer. This will be described more specifically by taking, as an example, the case where acrylamide is used as a monomer for forming a polymer gel using 5-triazine (VDAT).

[0017] As described above, the present invention provides (1) a step of preparing a polymer gel having a molecule-capturing ability, and (2) electrophoresis using the prepared polymer gel to perform double-stranded formation in a test sample. It consists of detecting and Z or separating the DNA.

(1) In the present invention, first, a polymer gel having a double-stranded DNA recognition site having a sequence to be detected is synthesized. In this step, a double-stranded DNA (type II double-stranded DNA) having the same sequence as the target molecule and a functional monomer that specifically interacts with the A and base pairs of type II double-stranded DNA, Step of preparing a self-assembly with bullet-4,6-diamino-1,3,5-triazine (VDAT) (first step), electrophoresis gel for electrophoresis between the self-assembly and monomer of polymer gel raw material A step of forming a polymer gel containing type II double-stranded DNA and VDAT by polymerization in a polymerization vessel (second step); removing type II double-stranded DNA from the polymer gel; It is subdivided into a step (third step) for creating a recognition site for the target double-stranded DNA (see Fig. 1).

[0019] In the first step, an operation of mixing type I double-stranded DNA and VDAT and self-assembling VDAT to the base pair of type II double-stranded DNA is performed (Fig. 1, Self-assembly). The type II double-stranded DNA used in this step has the type II of the recognition site created in the polymer gel and has the same sequence as the double-stranded DNA for the purpose of detection and separation. As a solution for dissolving type II double-stranded DNA and VDAT, water such as distilled water, purified water, ultrapure water, etc., as well as a pH buffer comprising various salt solutions, Tris-HCl, etc. are used. These lysates must be at a concentration and pH that does not cause denaturation of type II double-stranded DNA. The mixing ratio of type II double-stranded DNA and VDAT differs depending on the base sequence of the DNA used. Preferably, the molar ratio of Α · Τ base pairs: VDAT in the DNA is in the range of 20: 1 1: 1. More preferably, the molar ratio of A · Τ base pair: VDAT is in the range of 10: 1 to 1: 1. If the molar ratio of Α · Τ base pairs to VDAT is greater than 20: 1, the interaction between the VDAT and the recognition site created in the electrophoresis gel in the subsequent operation will increase. Since the number of action points is reduced, the recognition effect is reduced.

[0020] When the molar ratio of Α · Τ base pairs to VDAT is larger than 1: 1, an excessive amount of VDAT that cannot self-assemble with 铸 -type double-stranded DNA is generated. An excessive amount of VDAT is irregularly immobilized at a position other than the recognition site in the electrophoresis gel during the polymerization of the polymer raw material monomer in the second step, and causes nonspecific adsorption. After mixing the type II double-stranded DNA and VDAT, the mixture is allowed to stand at room temperature or in a refrigerator for 2 hours or more, for example, to obtain a self-assembly in which VDAT is bound to the A · A base pair of the type II double-stranded DNA.

[0021] In the second step, the self-assembled solution of the type II double-stranded DNA prepared in the first step and VDAT, acrylamide as a raw material monomer for a polymer gel, and N, Ν'-methylenebisatarylamide as a cross-linking agent are used. Prepare a mixed solution for polymerization, fill the electrophoresis gel polymerization vessel for electrophoresis, and perform polymerization (indicated as polymerization reaction in Fig. 1). The concentration of the self-assembly solution in the solution for polymerization is preferably 15 v / v% or more and 30 v / v% or less. More preferably, it is 20v / v% or more and 30v / v% or less. If it is less than 15ν / ν%, the number of recognition sites in the generated polymer gel will decrease, and a sufficient recognition effect cannot be obtained. On the other hand, if it exceeds 30v / v%, the polymerization product becomes brittle.

[0022] A mixture of atalinoleamide and Ν, Ν'-methylenebisacrylamide (weight ratio of acrylamide: Ν, Ν'-methylenebisacrylamide 19: 1) is more than 3.5ν / ν% in the solution for polymerization. Mix as follows. The mixing amount is appropriately selected depending on the molecular length of the target double-stranded DNA. The self-assembled solution was mixed with acrylamide, which had previously expelled oxygen in the solution under reduced pressure, and a mixed solution of Ν, Νmethylenebisacrylamide to prepare a polymerization solution. % Aqueous ammonium persulfate solution and polymerization accelerators Ν, Ν, Ν ', Ν tetramethylenediamine are added, and polymerized in a electrophoresis gel polymerization vessel for electrophoresis to obtain a polymer gel.

In the third step, 錡 -type double-stranded DNA is removed from the polymer gel obtained in the second step, and a desired DNA molecule shape 錡 -shaped hole (double-stranded DNA (Recognition site). Specifically, the polymer gel obtained in the second step is placed in an electrophoresis apparatus, and an appropriate buffer such as a tris-borate buffer, a tris-acetate buffer, or a tris-phosphate buffer is used. Pre-energize at a current of 210 mA for about 3 hours to remove type II double-stranded DNA (electrophoretic extraction in Fig. 1). In this way imprinted polymer Get a gel. In addition, all the steps so far are carried out in the same manner without adding double-stranded type II DNA to obtain a non-imprinted polymer gel.

[0024] (2) In the step of detecting DNA using the polymer gel that has passed through the third step, electrophoresis is performed using the imprinted polymer gel as a medium with a test sample solution, and the separated two Visualize the single-stranded DNA by staining. In this step, a conventional method of DNA electrophoresis is applied, but the above-described imprinted polymer gel is used instead of the conventional electrophoretic gel. After electrophoresis, the DNA separated in the imprinted polymer gel is stained with ethidium bromide to visualize the migration band of DNA, and then the DNA that has been captured by the recognition site of the polymer gel and has migrated shorter is detected. (See Figure 2).

[0025] In this step, first, a non-imprinted polymer gel on which the double-stranded DNA to be detected is not imprinted (the polyacrylamide conventionally used for separating double-stranded DNA due to a difference in molecular length) is used. Prepare two gels (A in Fig. 2) and two imprinted polymer gels (B in Fig. 2) on which the double-stranded DNA to be detected is imprinted. For each polymer gel, apply a double-stranded DNA of known molecular length (A and B in Fig. 2 on the left) and a test sample (A and B in Fig. 2 on the right) from the top of each polymer gel. Perform electrophoresis. After staining the migrating band of DNA with etidumimbu Myd, the migration distance of the detected migration band was measured (corresponding to a-f in the figure), and the migration distance of double-stranded DNA of known molecular length was set to 1.00. Calculate the relative movement distance of the test sample (b / a, c / a, e / d, f / d in Fig. 2).

[0026] In the case of an electrophoretic band derived from DNA not to be detected, the relative movement distances in the A and B polymer gels are the same (c / a = f / d), whereas In the case of the electrophoresis band, the relative movement distance in the B polymer gel is smaller than the relative movement distance in the A polymer gel (b / a> e / d). Therefore, it is determined that the migrating band of e having a smaller relative movement distance is the DNA to be detected. As a simpler method, it is possible to stain the migration band of DNA with ethidium bromide and then visually determine the difference in the relative movement distance between the polymer gels.

As a method for more accurately detecting a double-stranded DNA to be detected, a sample in which two or more double-stranded DNAs of known molecular lengths are mixed is prepared by using the above-described double-stranded DNA sample of known molecular length. After measuring the migration distance of the migration band using the same method as above, apply it to each polymer gel. There is a method to create a calibration curve of migration distance and molecular length. Using this calibration curve, the molecular length of the test sample for each polymer gel is determined. The molecular length of the double-stranded DNA in the test sample determined from the calibration curve of the imprinted polymer gel is larger than the molecular length of the double-stranded DNA in the test sample determined from the calibration curve of the non-imprinted polymer gel These correspond to the double-stranded DNA to be detected. Furthermore, the target double-stranded DNA can be isolated and analyzed by collecting the stained electrophoretic band.

The electrophoresis method of the present invention is based on the difference in the ability of a polymer gel on which double-stranded DNA is imprinted to capture double-stranded DNA in addition to the separation using the difference in migration distance depending on the molecular length. The difference of the migration distance is used. Therefore, the affinity of the imprinted double-stranded DNA for 铸 -shaped holes differs depending on the similarity of the base sequences of various double-stranded DNAs. Therefore, according to the electrophoresis method of the present invention, the base sequence can be specifically identified even for double-stranded DNA of the same size, not only for detection and separation of the target double-stranded DNA, and these can be used. Can be separated, and analysis dependent on the base sequence can be performed, which cannot be performed by conventional electrophoresis.

[0029] Furthermore, in the above-described electrophoresis method of the present invention, a non-imprinted polymer gel on which double-stranded DNA is not imprinted and a polymer gel on which double-stranded DNA to be detected is imprinted are separated. The double-stranded DNA to be detected and the polymer gel imprinted with the double-stranded DNA can be installed in parallel on the same electrophoresis apparatus and analyzed simultaneously using the same buffer solution. This is a method for detecting and separating single-stranded DNA.

Example

Next, the present invention will be described in more detail with reference to Examples, but it is not intended to limit the present invention.

(Example 1)

1. Preparation of self-assembled body fluid

Poly dA-poly dT (molecular length 1033 bp) was prepared as a type II double-stranded DNA. poly dA-poly dT (0.1 A units), VDAT (0.8 μM) with 0.2 M NaCl in 50 mM HEPES buffer (ρΗ7.3)

260

And left in the refrigerator overnight. 2. Preparation of gel for electrophoresis (imprint gel)

Dissolve 38 g of acrylamide and 2 g of Ν, Ν'-methylenebisacrylamide in MilliQ water.

100 ml (acrylamide-BIS mixture). 0.44 ml of this acrylamide-BIS mixed solution was dissolved in Tris-borate buffer (0.045 M Tris, 0.045 M boric acid, 0.00125 MEDTA, pH 8.3) to make a total volume of 4 ml. After degassing the solution under reduced pressure, 1 ml of the self-assembled liquid was mixed, 7.5 μl of a 10 w / v% ammonium persulfate aqueous solution, and ^ -tetramethylethylenediamine (TEMED) 2.4 a 1 were further mixed. did. 2 ml of the above solution was placed in two glass gel tubes for electrophoresis gel polymerization (0.4 cm inside diameter, 14.5 cm long) with the bottom end stopped by parafilm, layered with distilled water, allowed to stand at room temperature, and polymerized. .

3. Removal of type II double-stranded DNA

According to the above procedure, the polymerized imprinted polymer gel was set on the electrophoresis apparatus, and Tris-borate buffer (0.089 M Tris, 0.089 M boric acid, 0.0025 M EDTA, pH 8.3) was applied to the cathode side and the cathode side. The solution was filled in a buffer solution tank, and a current of 5 mA was supplied for about 3 hours at a constant current to remove double-stranded 铸 -molecule DNA.

[0033] 4. Electrophoresis experiment

As the electrophoresis sample DNA, a size standard DNA (molecular length of 100 bp) and poly dA-poly dT (the same as double-stranded DNA of type III, 1033 bp) were prepared. Mix 4 μl of each electrophoresis sample with sample buffer (50 w / v% glycerin, 10 mM EDTA, 0.05 w / v% bromophenol blue) 2 / i 1 and 14 μl of MilliQ water, and mix 6 μl of the mixture. Each was applied to the cathode side of the imprinted polymer gel. The current was passed until the blue dye of bromophenol blue migrated to the anodic end of the gel at a constant 5 mA. After energization, the imprinted polymer gel was removed from the electrophoresis gel polymerization glass tube and immersed in a 0.5 μg / ml bromide solution of aqueous broth for 30 minutes at room temperature to stain the separated DNA band. Thereafter, the gel was washed twice with distilled water for 30 minutes, and the gel was irradiated with a UV light to confirm a DNA band stained with a bromide tube.

[Comparative Example 1] Electrophoresis using polyacrylamide gel (analysis method based on molecular length of double-stranded DNA, conventional method)

The same operation as in Example 1 was performed, except that in Example 1-2, a 50 mM HEPES buffer solution (pH 7.3) containing 0.2 M NaCl was used instead of 1 ml of the self-assembled aggregate solution. (Example 2)

The migration distance between the size standard DNA (molecular length 100 bp) and poly dA-poly dT (molecular length 1033 bp) obtained in Example 1 and Comparative Example 1 was measured. The results are shown as relative migration distances when the migration distance (cm) of the size standard DNA was set to 1.00 (Table 1).

[Table 1] Migration distance (cm)

Size standard DNA Poly dA-poly dT Relative migration distance Example 1 1.60 1.30 0.81

Comparative Example 1 1.40 1.28 0.91 The relative migration distance between the size standard DNA of Comparative Example 1 and poly dA-poly dT was 0.91, whereas the relative migration distance was smaller when the imprinted polymer gel used in Example 1 was used. It was found that the distance was 0.81, which was smaller than Comparative Example 1. Therefore, it was proved that the imprinted polymer gel recognized the same DNA as the type I molecule and shortened its migration distance. By selecting electrophoretic bands with shorter electrophoresis distances, it is possible to detect double-stranded DNA having the same base sequence as the type I molecule.

(Example 3)

Detection of DNA to be detected in DNA mixed sample 1. Electrophoresis using imprinted gel

a) Preparation of electrophoresis medium imprinted with 560 bp phage DNA fragment

A 560 bp fragment of phage DNA was detected in the same manner as in Example 13 except that a 560 bp fragment of phage DNA was used instead of poly dA-poly dT as type II double-stranded DNA. Electrophoresis gel medium (imprint gel) was prepared. b) Electrophoresis experiment As standard sample for electrophoresis, size standard DNA (100bp-1000bp, 100bp Molecular Ruler, total DNA concentration 100μg / ml) 8 / i1, sample buffer (50w / v ° / W lyserin, 10mM EDTA, 0.05w / v% Bromophenol blue) 4 / i 1 and 28 μl of MilliQ water were used as a DNA mixed sample to obtain 8 μl of 560 bp fragment of λ phage DNA (1.0 A Units) and 700 bp fragment of λ phage DNA (1.7 bp). A Units) 8 μ \ poly dA-poly dT (1.5 A

Units) A mixture of 8 μl, sample buffer 4 μl, MilliQ water 12 μl was prepared, and these samples were applied in 19 μl aliquots on the cathode side of the imprint gel medium. The operation was the same as in 4.

2. Electrophoresis using polyacrylamide gel (analysis method based on the molecular length of double-stranded DNA, conventional method)

In the process of la) above, a 50 mM HEPES buffer (ρΗ7.3) containing 0.2 M NaCl was used in place of the 560 bp fragment of λ phage DNA. And the same operation as in the above lb) was performed using the polyacrylamide electrophoresis medium.

3.Result

The migration distance of the electrophoresis standard sample obtained in each of the above electrophoresis experiments 1 and 2 was measured, and a correlation graph of the migration distance between the imprint gel medium and the polyacrylamide gel medium was created (FIG. 3). The migration distance obtained from each DNA in the DNA mixed sample was plotted on this correlation graph, and the deviation from the correlation line was confirmed.

If the test sample contains the same base sequence DNA as the type II molecular DNA used when preparing the imprint gel, the migration distance becomes shorter due to the capturing effect of the recognition site in the imprint gel medium. The resulting plot will be shifted upward from the correlation line. On the other hand, if the test sample contains DNA that is different from the type II molecular DNA used when preparing the imprint gel, the plot obtained from the DNA is the standard sample for electrophoresis used when preparing the correlation graph. On the correlation line Will ride.

As a result of the examination, among the three plots obtained from the DNA mixed sample, the 700 bp fragment of phage DNA (indicated by b in Fig. 3) and poly dA-poly dT (indicated by a in Fig. 3) were correlated. While the plot was on a straight line, the plot obtained from the 560 bp fragment of λ phage DNA (indicated by c in FIG. 3) was shifted upward from the correlation line. Therefore, by confirming the difference in the correlation graph force, it was found that it was possible to examine whether or not there was a DNA having the same base sequence as the type II molecular DNA used in preparing the imprint gel in the DNA mixed sample.

Claims

The scope of the claims

[1] A double-stranded DNA molecule is formed of a polymer gel having pores imprinted as a triangle and having a substituent that recognizes the base pair of the double-stranded DNA in the pores. Electrophoresis medium

[2] The electrophoretic medium according to [1], wherein the substituent that recognizes a base pair of the double-stranded DNA has the following chemical structure.

[Chemical 1]

(Where Y represents CH or N)

[3] The electrophoretic medium according to claim 1 or 2, wherein the polymer gel is a crosslinked polyacrylamide gel.

[4] A method for producing a medium for electrophoresis for detecting double-stranded DNA, comprising the following steps (a) to (c):

(c) a step of removing double-stranded DNA from the formed polymer gel

[5] A double-stranded DNA-containing sample is dropped on the electrophoresis medium according to any one of claims 13 to 13 to perform electrophoresis, and imprinted in a polymer gel of the electrophoresis medium. The double-stranded DNA in the sample is trapped in the holes corresponding to the double-stranded DNA molecule shape. A method for detecting double-stranded DNA, comprising detecting and / or separating the same double-stranded DNA as that imprinted in a sample, using the shortened migration distance as an index.