JP2003014745A - Reactive solid-phase carrier and dna fragment detecting tool - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fixing method by which a pre-prepared DNA fragment, a nucleotide derivative, such as the oligonucleotide, etc., or its analogue can be bonded densely and stably to the surface of a solid-phase carrier through a quick reaction, a method of manufacturing the solid-phase carrier for fixing the nucleotide derivative or its analogue utilizing the fixing method, and a detecting tool for detecting nuclei acid fragment samples, etc., represented by the DNA chip. SOLUTION: The reactive solid-phase carrier is constituted by fixing a group of vinylsulfonyl groups or their reactive precursor groups to the surface of a solid-phase carrier having a porous matrix containing pores having diameters between about 2 nm and about 1,000 nm in a porous area at porosity between about 10% and about 90% and having a thickness between about 0.01 μm and about 70 μm by performing covalent coupling through their coupling radicals.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a detection tool useful for analyzing the structure of biopolymers, and particularly for detecting a lot of biological origins useful for efficient analysis of gene expression, mutation, polymorphism and the like. The present invention relates to a detection tool in which a polymer substance or an analog thereof is aligned and fixed on the surface of a solid phase carrier having a porous substrate. In particular, the present invention is a high-density array-type detection tool in which a large number of nucleotide derivatives or their analogs, which are useful for analyzing the base sequence of a DNA fragment sample, are aligned and immobilized at high density on the surface of a solid-phase carrier having a porous substrate. (DNA detection chip), and a reactive solid phase carrier that can be advantageously used for producing a high-density array type detection tool.

[0002]

2. Description of the Related Art Technological development for efficiently analyzing gene functions of various organisms is rapidly progressing.
For the analysis of the nucleotide sequence of A or DNA fragment, DN
A detection tool called a chip, which has a large number of DNA fragments or nucleotide derivatives such as synthetic oligonucleotides immobilized on the surface of a solid substrate, is used. A detection molecule such as DNA or a fragment thereof or a synthetic oligonucleotide, such as a nucleotide derivative bound and immobilized on the surface of a solid substrate, is also called a probe molecule. A typical DNA chip is a microarray in which a large number of probe molecules are aligned and immobilized on a solid phase carrier such as a slide glass. The DNA chip-related technology relating to the production and use of this DNA chip is considered to be applicable to the detection of biomolecules other than DNA. Therefore, it is new to drug discovery research, the diagnosis of diseases and the development of prevention methods. Expected to provide the means.

The technology relating to the DNA chip has been embodied when a method for determining the base sequence of DNA by hybridization with an oligonucleotide was devised. This method was a method that could overcome the limitations of the nucleotide sequencing method using gel electrophoresis, but it did not reach practical use at the beginning.

After that, a DNA chip having the above-mentioned structure and a technique for producing the same were developed, and it became possible to efficiently investigate gene expression, mutation, polymorphism and the like in a short time. That is, a DNA fragment sample (also called a target DNA fragment) showing complementarity to a DNA fragment or an oligonucleotide on the prepared DNA chip is generally a DN.
It is detected by utilizing the hybridization between the DNA fragment or oligonucleotide on the A chip and the labeled DNA fragment sample.

In order to put the DNA chip production technique into practical use, a technique for aligning a large number of DNA fragments and oligonucleotides on the surface of a solid support at high density and stably is required.

As a method for producing a DNA chip, a method of directly synthesizing an oligonucleotide on the surface of a solid phase carrier (referred to as "on-chip method") and a DNA prepared in advance are prepared.
A method in which a fragment or an oligonucleotide is bound and immobilized on the surface of a solid phase carrier is known. As the on-chip method, the use of a protective group that is selectively removed by light irradiation is combined with the photolithography technology and solid-phase synthesis technology used in semiconductor manufacturing, and oligonucleotides in a predetermined minute matrix region are combined. A method of selectively synthesizing (referred to as "masking technique") is typical.

As a method for binding and immobilizing a DNA fragment or oligonucleotide prepared in advance to the surface of a solid support,
The following methods are known depending on the type of DNA fragment and the type of solid phase carrier. (1) The DNA fragment to be immobilized is cDNA (complementary DNA synthesized using mRNA as a template) or PCR product (cDNA
Is a DNA fragment amplified by the PCR method), the cDNA or PCR product is subjected to surface treatment with a polycationic compound (polylysine, polyethyleneimine, etc.) by a spotter device provided in the DNA chip manufacturing apparatus. It is general that the phase carrier is spotted and electrostatically bound to the solid carrier by utilizing the electric charge of the DNA fragment. As a method for treating the surface of the solid phase carrier, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group or the like is also used. In the surface treatment using this silane coupling agent, amino groups, aldehyde groups, etc. are immobilized on the surface of the solid phase carrier by covalent bonds, and therefore, compared to the case of the surface treatment with a polycationic compound, the solid treatment is more stable. It is fixed on the surface of the phase carrier.

As a modification of the above method utilizing the charge of the DNA fragment, the PCR product modified with an amino group is subjected to SSC.
A method has been reported in which it is suspended in (standard salt-citrate buffer), spotted on a silylated slide glass surface, incubated, and then treated with sodium borohydride and heat treatment in this order. However, there is a problem that it is difficult to obtain sufficient DNA fragment fixation stability by this fixation method. In DNA chip technology,
The limit of detection is important. Therefore, it is possible to improve the detection limit of hybridization between the DNA fragment probe and the labeled sample nucleic acid fragment by allowing the DNA fragment to be stably bound and immobilized on the surface of the solid phase carrier in a sufficient amount (that is, at high density). Have a direct impact.

(2) When the oligonucleotide (probe molecule) to be immobilized is a synthetic oligonucleotide,
First, an oligonucleotide into which a reactive group is introduced is synthesized, and the oligonucleotide is spotted on the surface of a solid-phase carrier that has been surface-treated in advance to form a reactive group, and the oligonucleotide is covalently bonded to the surface of the solid-phase carrier. A method of binding and fixing by using is also known. For example, a method of reacting an amino group-introduced oligonucleotide on a slide glass having an amino group introduced on the surface thereof in the presence of PDC (p-phenylene diisothiocyanate), and reacting the slide glass with an aldehyde group-introduced oligonucleotide. The method is known. These two methods have the advantage that the oligonucleotide is stably bound and immobilized on the surface of the solid phase carrier, as compared with the method (1) in which the charge of the DNA fragment is used to immobilize by electrostatic binding. However, in the method in which PDC is present, the reaction between PDC and the amino group-introduced oligonucleotide is slow, and in the method in which the aldehyde group-introduced oligonucleotide is used, the stability of the Schiff base as a reaction product is low (hence, hydrolysis). Is likely to occur).

In recent years, as a probe molecule for a DNA chip, instead of an oligonucleotide or a polynucleotide (including a synthesized oligonucleotide or polynucleotide and a DNA molecule or a DNA fragment, and an RNA molecule or an RNA fragment), A technique using an oligonucleotide analog called PNA (peptide nucleic acid) has also been proposed. A method using a combination of avidin and biotin is also known as a method for covalently immobilizing the PNA on the solid-phase substrate (Japanese Patent Laid-Open No. Hei 11).
-332595 publication). This publication also describes a technique of utilizing a surface plasmon resonance (SPR) biosensor as a solid phase substrate. DNA with probe molecule immobilized on surface plasmon resonance biosensor
The DNA fragment bound and immobilized on the surface of the chip through hybridization can be detected by utilizing the surface plasmon resonance phenomenon.

It is also known to use a charge coupled device (CCD) as a substrate for a DNA chip (Nuclei
c Acids Research, 1994, Vol.22, No.11, 2124-212
Five).

Japanese Unexamined Patent Publication (Kokai) No. 4-228076 (corresponding to US Pat. No. 5,387,505) discloses that a target DNA having a biotin molecule attached thereto is bound to a substrate having an avidin molecule immobilized on the surface to separate the target DNA. The technique to do is described.

Japanese Examined Patent Publication No. 7-43380 (corresponding to US Pat. No. 5,049,962) discloses a detection tool for use in a ligand-receptor assay, which describes microporous polymer particles having reactive groups on the surface. Analytical devices having receptor molecules bound to the surface are described.

International publication WO00 / 61282 describes a porous solid phase carrier. By mainly applying particles of an inorganic substance to the surface, the thickness from the support is 0.0
It is 1 to 70 μm, and the porosity is 10 to 90%. The use of this porous solid phase carrier has an advantage that the amount of biological polymer immobilized can be increased because the surface area becomes large.

[0015]

DISCLOSURE OF THE INVENTION The present invention is intended to stably and densely bind a previously prepared nucleotide derivative such as an oligonucleotide, a polynucleotide, a peptide nucleic acid or the like or an analogue thereof to a solid-phase carrier surface at a high density. A reactive solid phase carrier which can be used particularly advantageously, and
It is an object of the present invention to provide a tool for detecting DNA, RNA or a fragment thereof having a specific base sequence part, which is obtained by binding and immobilizing a nucleotide derivative or its analog to the reactive solid phase carrier.

[0016]

According to the present invention, a group of vinylsulfonyl groups (also referred to as vinylsulfone group or sulfonylvinyl group) or its reactivity are formed on the surface of a solid-phase carrier having a porous substrate. There is a reactive solid phase carrier in which precursor groups are bonded and fixed by covalent bonds via respective linking groups. The porous substrate in the present invention is
The porous region has a pore diameter of about 2 nm to about 1000 nm, a porosity of about 10% to about 90% and about 0.01 μm to about 7 μm.
It has a thickness of 0 μm.

The porous substrate may be composed of an organic polymer or an inorganic substance. When the porous substrate is composed of an inorganic substance, the porous substrate is silicon,
It preferably contains alumina or titanium. The above-mentioned vinyl sulfonyl group or the linking body of its reactive precursor group and the linking group is preferably represented by the following formula (1).

-L-SO ₂ -X (1)

[In the above formula, X is -CR ¹ = C
R ² R ³ or —CHR ¹ —CR ² R ³ Y; R ¹ , R
² and R ³ are, independently of each other, a hydrogen atom and a carbon atom number of 1
An atom or a group selected from the group consisting of an alkyl group having 6 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms and having an alkyl chain having 1 to 6 carbon atoms. the expressed; Y is a halogen _{^{atom, -OSO 2 R 11, -OCOR 12}} , -OSO 3 represents M, and the atom or group selected from the group consisting of quaternary pyridinium group; R ¹¹ is the number of carbon atoms 1 Represents a group selected from the group consisting of an alkyl group having 6 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms and having an alkyl chain having 1 to 6 carbon atoms. R ¹² represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is a hydrogen atom,
Represents an atom or group selected from the group consisting of alkali metal atoms and ammonium groups; and L represents a linking group].

In the above formula (1), X is -CH =
Desirably a vinyl group represented by CH ₂ ,

L is preferably a linking group containing a divalent or higher valent atom other than carbon atoms. As L, -NH
A linking group having a linking site such as-, -S-, or -O- is desirable. As an example of L, −
_{^{(L 1) n -NH- (CR}} 1 R 2) 2 - or - (L ¹⁾ _n -
A linking group represented by S- (CR ¹ R ² ) _2- [wherein R ¹ and R ² have the same meanings as described above, L ¹ represents a linking group, and n is 0 or 1] be able to. In particular, L _{^{is, - (L 1) n -NHCH}} 2 CH 2 - [ where, L ¹ represents a linking group, and n is 0 or 1] desirably is a linking group represented by. The above L ¹ is preferably a linking group containing a group represented by —OSi—.

The reactive solid phase carrier of the present invention comprises a solid phase carrier having a porous substrate having a reactive group introduced on the surface thereof, represented by the following formula (2):

X ¹ --SO ₂ --L ² --SO ₂ --X ²

[0024] [In the above formula, X ¹ and X ² are independently from each other, -CR ¹ = CR ² R ³ or -CHR ¹ -CR,
² R ³ Y (reactive precursor group); R ¹ , R ² and R ³
Are, independently of each other, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total carbon atom number of 7 having an alkyl chain having 1 to 6 carbon atoms. To 26 represent an atom or group selected from the group consisting of aralkyl groups; Y is a halogen atom,
OSO ₂ R ¹¹ , —OCOR ¹² , —OSO ₃ M, and an atom or a group selected from the group consisting of quaternary pyridinium groups; R ¹¹ represents an alkyl group having 1 to 6 carbon atoms,
R ¹² represents a group selected from the group consisting of an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms and having an alkyl chain having 1 to 6 carbon atoms; Represents a group selected from the group consisting of an alkyl group having 1 to 6 atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is selected from the group consisting of a hydrogen atom, an alkali metal atom and an ammonium group. Which represents an atom or a group; and L ² represents a linking group].

A typical example of the disulfone compound represented by the above formula (2) is 1,2-bis (vinylsulfonylacetamido) ethane.

The reactive group introduced on the surface of the solid phase carrier is preferably an amino group, a mercapto group or a hydroxyl group.

The present invention also forms a covalent bond by reacting with a reactive solid group on the surface of a reactive solid phase carrier having a group of vinylsulfonyl groups or reactive precursor groups thereof fixed by a covalent bond. To produce a solid phase carrier having a nucleotide derivative or an analog thereof bound and immobilized on the surface of a carrier via a sulfonyl group-containing linking group. There is also a method.

Representative examples of the nucleotide derivative or its analog immobilized on a solid-phase carrier having a porous substrate include oligonucleotides, polynucleotides, and peptide nucleic acids. These nucleotide derivatives or analogs thereof include vinylsulfonyl groups such as an amino group, an imino group, a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, or a carboximide group at or near one end of the molecule. Those having a reactive group that forms a covalent bond that reacts with the group or its reactive precursor group are utilized.

The above-mentioned solid phase carrier having a porous substrate on which the nucleotide derivative or its analog is immobilized is an oligonucleotide or a polynucleotide which is complementary to the immobilized nucleotide derivative or its analog in the presence of an aqueous medium. The complementary oligonucleotide or polynucleotide can be immobilized by contacting nucleotides (DNA or a fragment thereof, or RNA or a fragment thereof) and causing hybridization. The complementary oligonucleotide or polynucleotide to be immobilized preferably has a detectable label (eg, fluorescent label) attached thereto so that the immobilization can be detected from the outside. The present invention also utilizes a solid phase carrier having a porous substrate having the above nucleotide derivative or its analog bound and fixed on the surface of the carrier, or a solid phase carrier to which the above-mentioned complementary oligonucleotide or polynucleotide is bound and fixed. The present invention also relates to a method for identifying or screening genes.

[0030]

BEST MODE FOR CARRYING OUT THE INVENTION The reactive solid-phase carrier of the present invention comprises a group of vinylsulfonyl groups or reactive precursor groups thereof covalently bonded to the surface of a solid-phase carrier having a porous substrate through linking groups. It has a configuration in which it is coupled and fixed by. The reactive solid phase carrier of the present invention is prepared by preparing a solid phase carrier having a porous substrate in which reactive groups have been introduced in advance, and reacting this solid phase carrier with the reactive groups provided on the surface of the carrier. And a compound having a reactive group forming a covalent bond at one end or near the end and a vinylsulfonyl group or a reactive precursor group of the vinylsulfonyl group at the other end or near the end. It can be manufactured by contacting.

The solid phase carrier is preferably a substrate having a smooth surface. Examples of the material of the solid phase carrier include non-porous substances such as glass and plastic.

Typical methods of forming the porous regions are by addition of material (eg deposition) and removal of material (eg selective etching). In the addition method, a porous region is formed on the surface of the underlying substrate to increase the effective surface area. The porous region can be formed by depositing the following substances, optionally using a solvent and a catalyst. The porous region may be, for example, colloidal silica, tetramethoxysilane (TMO) typically used in sol-gel processes.
It can be formed from organosilicon compounds such as S), metal alkoxides, silsesquioxanes or other silanes or combinations of these materials. For these precursors,
The morphology (pore size, porosity, thickness) of the formed porous region can be adjusted by appropriately selecting parameters such as the composition, concentration, pH, aging time and temperature of the solution. Inorganic materials other than those mentioned above (for example, materials based on aluminum or titanium) can also be used in the same manner as above.

Alternatively, the porous region matrix can be templated. In the templating process, a material such as a polymer is deposited with a matrix and then burned out, leaving behind a porous structure with selected properties.
The templating material may be any of polymers such as polystyrene latex, polymers in solution, or combinations of these materials. The heat burn-off process is typically carried out by heating in air and can be carried out at temperatures from 150 ° C. or higher to the melting temperature (or glass transition temperature) of the material forming the matrix of the porous layer. The matrix material can be fired after the templated material is burned out. By varying the time and temperature of the firing process, varying degrees of densification and pore characteristics can be achieved.

The porous region is formed by spin coating, dip coating, extraction (aerosol), individual spots deposited on the surface,
It can be formed on the surface of the underlying substrate by a variety of processes, including the use of barriers (physical or chemical) to specifically deposit the coating on channels, pads, spots or patterned surfaces. The thickness of the porous region can be adjusted by varying either or both the precursor and the layer forming conditions. For example, the thickness can be adjusted depending on the concentration of the reagent used. Similarly, the layer thickness can be increased by providing multiple depositions. During application of successive reagents, further processing may be performed such as baking the substrate to remove solvent from the film prior to application of the next agent. The thickness of the film can also be changed by adjusting the speed of rotation or deposition,
For example, slower speeds produce thicker films and faster speeds produce thinner films. In addition, varying the draw speed from the dip coating bath affects the layer thickness, with slower speeds producing thinner films and higher speeds producing thicker films. It is also possible to influence the film thickness by controlling the solution conditions. When using, for example, the TMOS technique, the solution can initiate gelation, which increases the viscosity and thus the thickness of the deposited layer.

The coating, particles or other components can be spun on the surface of the substrate, the substrate can be dipped in a solution containing the above-mentioned agent, the agent can be sprayed or otherwise applied to the surface of the substrate. The substrate can be treated to create high porosity areas on the entire surface of the substrate or only at selected locations, such as by spotting reagents in grids, circular spots, areas, cells or any desired shape.
It is also possible to utilize degradation techniques to increase the porosity of the substrate. For example, the porous regions can be etched into the surface of the substrate (ie, the material of the surface of the underlying substrate material). This surface can be prepared as an etched glass, such as a phase-separated sodium borosilicate glass. In certain embodiments of the degradation procedure to form a porous matrix, the pore size can also be controlled by the annealing time and temperature of the annealing step performed before etching (longer annealing, higher Temperature increases pore size). The depth of the porous region can also be controlled by etching parameters (solution concentration, composition, time, etc.) according to the substrate material.

Examples of the porous substance include porous ceramics, porous activated carbon, woven fabrics, non-woven fabrics, filter papers, membrane films, etc. When the substance itself is porous, it is sprayed onto a solid phase carrier or an adhesive. Can be pasted with. It is also possible to coat the precursor of the porous substance on the solid phase carrier to make it porous on the solid phase surface. The Solgel reaction is a typical example. Colloidal silica is generally used, but not limited to this.

As the organic polymer which constitutes the porous substrate, monosaccharides such as glucose and fructose or their derivatives, polysaccharides such as dextran, amyloses and starch or their derivatives such as carboxymethylsta -Chi. Polymeric polymers such as PVA, polyacrylic acid, cellulose, carboxymethyl cellulose, and polyacetal are mentioned.

On the surface of the solid support having a porous substrate,
In order to bond and fix a bifunctional reactive compound such as a divinyl sulfone compound by a covalent bond, a polycationic compound (eg, poly-L-lysine, polyethyleneimine, polyalkylamine or the like is preferable, and poly-L-
It is more preferable that it is lysine), etc., and a coating treatment with a polymer having an amino group in its side chain (in this case, the reactive group introduced to the surface of the solid-phase carrier is an amino group)
It is desirable to do. Alternatively, the surface of the solid phase carrier can be contact-treated with a surface-treating agent having a reactive group that reacts with the surface of the solid phase carrier, such as a silane coupling agent, and separately, a reactive group having a reactive group such as an amino group.

In the case of the coating treatment with a polycationic compound, the surface of the solid support having a porous substrate is introduced with an amino group or a mercapto group on the surface of the solid support by electrostatic coupling between the polymer compound and the surface of the solid support. On the other hand, in the case of surface treatment with a silane coupling agent,
Since it is bound and fixed by covalent bond to the surface of the solid support,
The amino group or mercapto group is stably present on the surface of the solid support. In addition to the amino group and the mercapto group, an aldehyde group, an epoxy group, a carboxyl group, or a hydroxyl group can be preferably introduced. Examples of the silane coupling agent having an amino group include γ-aminopropyltriethoxysilane, N-β (aminoethyl) -γ-aminopropyltrimethoxysilane and N-β (aminoethyl) -γ-aminopropylmethyldimethoxysilane. Is preferably used, and it is particularly preferable to use γ-aminopropyltriethoxysilane.

The treatment with the polycationic compound may be combined with the treatment with the silane coupling agent. By this method, electrostatic interaction between the solid phase carrier having low hydrophobicity or hydrophilicity and the DNA fragment can be promoted. A layer made of a hydrophilic polymer having a charge or a layer made of a crosslinking agent may be further provided on the surface of the solid support treated with the polycationic compound. By providing such a layer, it is possible to reduce the unevenness of the solid-phase support treated with the polycationic compound.
Depending on the type of solid phase carrier, it is possible to incorporate a hydrophilic polymer or the like into the carrier, and a solid phase carrier treated in this way can also be preferably used.

On the surface of a solid-phase carrier for a normal DNA chip, a plurality of pre-divided areas or presumed areas are set, and as described above for each area, a bifunctional reaction such as a divinyl sulfone compound is carried out. A reactive group capable of reacting with the reactive compound is introduced in advance. The surface of each region of the solid phase carrier to be used is equipped with a reactive group such as the above-mentioned amino group, mercapto group, or hydroxyl group. As described above, the surface treatment with a silane coupling agent or the method of coating and coating the surface of the solid support with a polymer having a reactive group such as an amino group in the side chain is used to Introduced.

When the solid phase carrier having a reactive group is brought into contact with a bifunctional reactive compound such as a divinyl sulfone compound, the reactive group reacts with the bifunctional reactive compound to form a covalent bond. Is formed, the reactive group portion of the solid phase carrier is extended to form a reactive chain having a vinylsulfonyl group or a reactive precursor group at or near the tip thereof, whereby the reactive solid phase carrier of the present invention is formed. Is generated.

In the reactive solid phase carrier of the present invention, the linking group of the vinyl sulfonyl group or its reactive precursor group and the linking group introduced on the surface of the solid phase carrier is represented by the following formula (1). Is desirable.

-L-SO ₂ -X (1)

In the above formula (1), X is -CR ¹
= Represents a CR ² R ³ or -CHR ¹ -CR ² R ³ Y (reactive precursor group). R ¹ , R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an alkyl group having 1 to 6 carbon atoms. It represents an aralkyl group having 7 to 26 total carbon atoms having a chain. Examples of the alkyl group having 1 to 6 carbon atoms include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, and an n-hexyl group, and a methyl group is particularly preferable. . Examples of the aryl group include a phenyl group and a naphthyl group. It is preferred that R ¹ , R ² and R ³ are all hydrogen atoms.

Y is --OH, --OR ⁰ , --SH, NH ₃ ,
NH ₂ R ⁰ (wherein R ⁰ is a group such as an alkyl group excluding a hydrogen atom) is a group substituted by a nucleophile, or a group capable of leaving as “HY” by a base, Examples include halogen atom, -OSO ₂ R
¹¹ , —OCOR ¹² , —OSO ₃ M, or a quaternary pyridinium group (R ¹¹ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, or a carbon atom having 6 to 20 carbon atoms). Represents an aralkyl group having 1 to 6 alkyl chains and having 7 to 26 total carbon atoms; R
¹² represents an alkyl group having 1 to 6 carbon atoms or a halogenated alkyl group having 1 to 6 carbon atoms; M
Represents a hydrogen atom, an alkali metal atom or an ammonium group).

L represents a divalent or more divalent linking group that links the solid phase carrier or the linking group bonded to the solid phase carrier and the above-mentioned —SO ₂ —X group. However, L may be a single bond. As the divalent linking group, an alkylene group having 1 to 6 carbon atoms, and 3 to 1 carbon atoms
An aliphatic ring group having 6 to 6 carbon atoms, an arylene group having 6 to 20 carbon atoms, and a heterocyclic group having 2 to 20 carbon atoms, which contains 1 to 3 heteroatoms selected from the group consisting of N, S and P,
-O -, - S -, - SO -, - SO 2 -, - SO 3 -, -
It is preferably a group formed by combining one or more groups selected from the group consisting of NR ¹¹ —, —CO— and combinations thereof. R ¹¹ is a hydrogen atom, an alkyl group having 1 to 15 carbon atoms, an aryl group having 6 to 20 carbon atoms, or an alkyl group having 1 to 6 carbon atoms and having 7 to 21 carbon atoms. It is preferably an aralkyl group, more preferably a hydrogen atom or an alkyl group having 1 to 6 carbon atoms, and particularly preferably a hydrogen atom, a methyl group or an ethyl group. L is ^{-NR 11 -, - SONR 11 -} , - CONR
¹¹ -, - NR ¹¹ COO-, and -NR ¹¹ CONR ¹¹ -
In the case of a group formed by combining two or more groups selected from the group consisting of, R ^{11 s} may combine with each other to form a ring.

The alkyl group of R ^11, aralkyl group having an aryl group, and R ¹¹ of R ¹¹ may have a substituent. Examples of such a substituent include a hydroxyl group, an alkoxy group having 1 to 6 carbon atoms, an alkenyl group having 1 to 6 carbon atoms, a carbamoyl group having 2 to 7 carbon atoms, and 1 to 6 carbon atoms. Alkyl group having 7 to 16 carbon atoms, aryl group having 6 to 20 carbon atoms, sulfamoyl group (or its Na salt, K salt, etc.), sulfo group (or its Na salt, K salt, etc.) ), A carboxylic acid group (or its Na salt, K salt, etc.), a halogen atom, an alkenylene group having 1 to 6 carbon atoms, an arylene group having 6 to 20 carbon atoms, a sulfonyl group, and a combination thereof. An atom or group selected from the group can be mentioned.

Preferred specific examples of the above-mentioned "-X" group are shown below. Moreover, the example of the group which can be used as “—L—SO ₂ —X” is also shown below.

[0050]

[Chemical 1]

[0051]

[Chemical 2]

"-X" means (X1),
(X2), (X3), (X4), (X7), (X8),
(X13) or (X14) is preferable,
More preferably, it is (X1) or (X2). Particularly preferred is a vinyl group represented by (X1).

Preferred specific examples of L are shown below. However,
a is an integer of 1 to 6, preferably 1 or 2, and particularly preferably 1. b is an integer of 0 to 6, and is preferably 2 or 3.

[0054]

[Chemical 3]

As L, in addition to the above-mentioned divalent linking group, a hydrogen atom of the alkylene group of the above formula is -SO ₂ CH =
A group substituted with a CH ₂ group is also preferable.

The difunctional reactive compound used for obtaining the solid phase carrier having the vinylsulfonyl group or the reactive precursor group represented by the above formula (1) fixed by a covalent bond is represented by the following formula ( The disulfone compound represented by 2) can be advantageously used.

X ¹ --SO ₂ --L ² --SO ₂ --X ² (2)

[0058] [In the above formula, X ¹ and X ² are independently from each other, -CR ¹ = CR ² R ³ or -CHR ¹ -CR,
² R ³ Y (reactive precursor group); R ¹ , R ² and R ³
Are, independently of each other, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total carbon atom number of 7 having an alkyl chain having 1 to 6 carbon atoms. To 26 represent an atom or group selected from the group consisting of aralkyl groups; Y is a halogen atom,
OSO ₂ R ¹¹ , —OCOR ¹² , —OSO ₃ M, and an atom or a group selected from the group consisting of quaternary pyridinium groups; R ¹¹ represents an alkyl group having 1 to 6 carbon atoms,
R ¹² represents a group selected from the group consisting of an aryl group having 6 to 20 carbon atoms, and an aralkyl group having 7 to 26 carbon atoms and having an alkyl chain having 1 to 6 carbon atoms; Represents a group selected from the group consisting of an alkyl group having 1 to 6 atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is selected from the group consisting of a hydrogen atom, an alkali metal atom and an ammonium group. Represents an atom or group; and L ² represents a linking group].

That is, the reactive solid phase carrier of the present invention can be easily produced by bringing the disulfone compound represented by the above formula (2) into contact with the above solid phase carrier, for example, in an aqueous atmosphere. You can

Representative examples of the disulfone compound preferably used in the present invention are shown below. The disulfone compound may be used as a mixture of two or more kinds.

[0061]

[Chemical 4]

[0062]

[Chemical 5]

A typical example of the disulfone compound represented by the above formula (2) is 1,2-bis (vinylsulfonylacetamido) ethane [corresponding to S1 above].
Can be mentioned.

The synthesis method of the disulfone compound used in the present invention is described, for example, in JP-B-47-2429 and JP-A-47-2429.
0-35807, JP-A-49-24435, 53
The details are described in various publications such as No. 41551 and No. 59-18944.

Utilizing the reactive solid phase carrier obtained as described above, DNA, RNA, DNA fragments, or R
A detection tool for detecting and immobilizing naturally occurring polynucleotides or oligonucleotides such as NA fragments (generally D
In order to make (what is called NA chip)
A nucleotide derivative or an analogue thereof having a reactive group such as an amino group which forms a covalent bond by reacting the above-mentioned reactive solid-phase carrier with a vinylsulfonyl group or a reactive precursor group thereof on the surface of the carrier. A contact method is used. That is, in this way, a detection tool (so-called DNA chip) provided with a probe molecule composed of a desired nucleotide derivative or its analog can be produced.

The vinylsulfonyl group or its reactive precursor group bonded to the surface of the solid-phase substrate of the present invention through a covalent bond has high resistance to hydrolysis, and thus can be easily and stably prepared. Stable covalent bond that can be stored and has amino group in advance, or has reactive group such as amino group introduced, reacts rapidly with the reactive group of nucleotide derivative or its analog. Can be formed.

Typical examples of the nucleotide derivative or its analog used as a probe molecule include oligonucleotides, polynucleotides and peptide nucleic acids. These nucleotide derivatives or their analogs are of natural origin (DNA, DN
A fragment, RNA, or RNA fragment) or a synthetic compound. Further, as the nucleotide derivative or its analog, a compound called LNA (J. A.
m. Chem. Soc. 1998, 120, 13252-13253).

When a DNA fragment is used as the probe molecule, it can be divided into two types depending on the purpose. In order to investigate the expression of a gene, it is preferable to use cDNA, a part of cDNA, or a polynucleotide such as EST. The function of these polynucleotides may be unknown, but in general, based on the sequences registered in the database, PCR is carried out using a cDNA library, a genomic library or the entire genome as a template.
It is amplified and prepared by the method (hereinafter referred to as "PCR product"). Those which are not amplified by the PCR method can also be preferably used. Further, in order to examine a mutation or polymorphism of a gene, it is preferable to synthesize various oligonucleotides corresponding to the mutation or polymorphism based on a known standard sequence and use this. Furthermore, for the purpose of analyzing the base sequence, it is preferable to synthesize 4 ⁿ (n is the base length) kinds of oligonucleotides and use them. The base sequence of the DNA fragment is preferably determined in advance by a general base sequence determination method. The DNA fragment is preferably a 2 to 50 mer, and particularly preferably a 10 to 25 mer.

A reactive group which forms a covalent bond by reacting with the above-mentioned vinylsulfonyl group or its reactive precursor group is introduced into one end of a nucleotide derivative such as an oligonucleotide or a DNA fragment or its analog. . Such reactive groups include amino groups, imino groups, hydrazino groups, carbamoyl groups, hydrazinocarbonyl groups,
Alternatively, it is preferably a carboximide group, and particularly preferably an amino group. These reactive groups are usually bonded to the oligonucleotide or DNA fragment via a crosslinker. As a crosslinker,
For example, an alkylene group or N-alkylamino-
An alkylene group is used, but a hexylene group or N
-Methylamino-hexylene group is preferred,
A hexylene group is particularly preferable. Since the peptide nucleic acid (PNA) has an amino group, it is usually unnecessary to newly introduce a reactive group.

The contact between the nucleotide derivative having a reactive group or its analog and the reactive solid phase carrier is usually carried out by spotting an aqueous solution of the nucleotide derivative or its analog on the surface of the reactive solid phase carrier. It is carried out by. Specifically, a nucleotide derivative having a reactive group or an analog thereof is dissolved or dispersed in an aqueous medium to prepare an aqueous solution, and the aqueous solution is dispensed into a 96-well or 384-well plastic plate, It is preferable that the poured aqueous liquid is dropped onto the surface of the solid phase carrier using a spotter device or the like.

In order to prevent the nucleotide derivative or its analog from drying after spotting, it is dissolved in or dispersed in an aqueous liquid in which the nucleotide derivative or its analog is dissolved.
High boiling materials may be added. The substance having a high boiling point is a substance that can be dissolved in an aqueous liquid in which the nucleotide derivative or its analogue after spotting is dissolved or dispersed, and is used as a sample such as a nucleic acid fragment sample to be detected (target nucleic acid fragment). Without interfering with the hybridization of
Further, it is preferable that the substance is not so viscous. Such substances include glycerin, ethylene glycol, dimethylsulfoxide and low molecular weight hydrophilic polymers. Examples of hydrophilic polymers include polyacrylamide, polyethylene glycol, and sodium polyacrylate. The molecular weight of this polymer is preferably in the range of 10 ^{3 to} 10 ⁶ . As the substance having a high boiling point, it is more preferable to use glycerin or ethylene glycol, and it is particularly preferable to use glycerin. The concentration of the substance having a high boiling point is preferably in the range of 0.1 to 2% by volume, and particularly preferably in the range of 0.5 to 1% by volume, in the aqueous solution of the nucleotide derivative or its analog.

For the same purpose, the solid phase carrier after spotting the nucleotide derivative or its analog is
It is also preferable to place it in an environment having a humidity of 0% or more and a temperature range of 25 to 50 ° C.

After the spotting of the nucleotide derivative having a reactive group or its analog, post-treatment with ultraviolet rays, sodium borohydride or Schiff reagent may be carried out.
These post-treatments may be performed by combining a plurality of types, and it is particularly preferable to perform a combination of heat treatment and ultraviolet treatment. These post-treatments are particularly effective when the surface of the solid support is treated only with the polycationic compound. It is also preferable to carry out incubation after the spotting. After the incubation, it is preferable to wash and remove the unreacted nucleotide derivative or its analog.

The fixed amount (number) of the nucleotide derivative or its analog is 10 ^{2 to} 1 with respect to the surface of the solid phase carrier.
It is preferably in the range of 0 ⁵ types / cm ² . The amount of the nucleotide derivative or its analog is 1 to 10 ^-15.
It is preferably in the range of mol and the weight is preferably several ng or less. By spotting, the aqueous solution of the nucleotide derivative or its analog is fixed in the form of dots on the surface of the solid support. The dot shape is almost circular.
The fact that there is no change in shape is important for quantitative analysis of gene expression and analysis of single nucleotide mutations. The distance between the dots is preferably in the range of 0 to 1.5 mm, and particularly preferably in the range of 100 to 300 μm. The size of one dot is 50 to 3 in diameter.
It is preferably in the range of 00 μm. The amount of the nucleotide derivative or its analog adhering to the surface of the solid phase carrier is preferably in the range of 100 pL to 1 μL, and particularly preferably in the range of 1 to 100 nL.

FIG. 1 shows the orientation of a typical embodiment of the present invention.
Method for producing gonucleotide-fixed solid-phase carrier and typical method
Schematic representation of the structure of an oligonucleotide-immobilized solid-phase carrier
You Porous group to which the oligonucleotide of the present invention is immobilized
As a method for producing a solid phase carrier having quality, the above formula (2) is used.
When the disulfone compound represented by ¹Oh
And X²There are four types of manufacturing methods available.

In FIG. 1, an oligonucleotide in which both X ¹ and X ² of the disulfone compound of formula (2) are —CHR ¹ —CR ² R ³ Y (reactive precursor group) is immobilized. (A) of the solid phase carrier (C1), and X ¹ is -C
HR ¹ -CR ² R ³ Y, wherein X ² is -CR ¹ = CR ² R ³
Oligonucleotide-immobilized solid phase carrier (C2)
The manufacturing method (b) of is shown. However, X ¹ is solid phase carrier 1
Assuming that the reactive group (R) introduced on the surface of the first reacts with the group, X ¹ may be —CR ¹ = CR ² R ³ . Hereinafter, “X ¹ ” will be described as a group that first reacts with the reactive group (R) introduced on the surface of the solid phase carrier 1.

The manufacturing methods (a) and (b) will be described. Step (1): The disulfone compound represented by the formula (1) is brought into contact with the solid phase carrier 1 [solid phase carrier (A)] in which the reaction active group (R) is introduced on the surface of the carrier, and the -Y portion of X1 is contacted. By substituting the reactive group (R) for-(CR ¹ R ² )
The _{n -SO 2 -L-SO 2 -X} 2 group introduced into the surface of the solid support.

Step (2): Introduced in Step (1)-
The reactive group (Z) is obtained by bringing the oligonucleotide 2 having the reactive group (Z) at one end into contact with X ² of the (CR ¹ R ² ) _n —SO ₂ —L—SO ₂ —X ² group. Is added, or the -Y of X ² is contacted with the oligonucleotide 2 to substitute the reactive group (Z).

The carrier for immobilizing probe molecules of the present invention comprises X ¹
Is a group that first reacts with the reactive group (R) introduced on the surface of the solid phase carrier 1, the X ¹ thereof is -CR ¹ = CR ²
It may be produced by the following method using R ³ .

Step (1): Solid phase carrier (A) having active groups (R) introduced on the surface of the solid phase carrier 1 having a porous substrate.
Is contacted with a disulfone compound represented by the formula (I),
To -CR ¹ = CR ² R ³ of X ^1, by addition of reactive group ^{(R), -R 3 R 2} CR 1 HC-SO 2 -L-S
The O ₂ -X ² group introduced into the surface of the solid support.

Step (2): X of the —R ³ R ² C—R ¹ HC—SO ₂ —L—SO ₂ —X ² group introduced in step (1).
^The reactive group (Z) is added to ² by contacting an oligonucleotide having the reactive group (Z) at one end, or the oligonucleotide is contacted with the -Y portion of X ² . Thereby substituting this reactive group (Z).

The oligonucleotide having a reactive group (Z) at one end is a compound represented by 2 in FIG. The cross-linker (Q) is not essential, but it is generally present between the reactive group (Z) and the phosphate group for the convenience of preparing the reactive oligonucleotide 2. -Phosphate ester group-NNNN ... N
N represents an oligonucleotide. R ⁴ is a group determined by the reaction between the reactive group (R) and X ^1, and Z ¹ is a group determined by the reaction between X ² and the reactive group (Z).

Solid support having porous substrate of FIG. 1 (B)
2 having a reactive group (Z) on its surface
On the solid phase carrier (B), the reaction between X ² or —Y of X ² and the reactive oligonucleotide fragment 2 occurs.
There is also unreacted X ^{2 on} the surface of which the oligonucleotide 2 is not bound. Such X ² may cause a non-specific reaction in the subsequent hybridization with a labeled nucleic acid fragment sample, and may possibly measure non-specific binding. X
It is preferable to mask ² (that is, the halogen atom of X ² , for example). The mask treatment is performed on the solid phase carrier (C
It is preferable to carry out by bringing the surface of 1) (or (C2)) into contact with an anionic compound having an amino group or a mercapto group. Since the oligonucleotide 2 has a negative charge, it is possible to prevent the oligonucleotide 2 from reacting with unreacted X ² by generating a negative charge also on the surface of the solid phase carrier (C).
Examples of such anionic compounds include those which react with the halogen atom of X ² and have a negative charge (COO ⁻ , SO ₃ ⁻ , OS).
Any one having O ₃ ⁻ , PO ₃ ⁻ , or PO ₂ ⁻ ) can be used, but an amino acid is preferable, and glycine or cysteine is particularly preferable. Also, taurine can be preferably used.

The life of the solid phase carrier on which the nucleotide derivative or its analog prepared by the method of the present invention is fixed is usually several weeks for the cDNA fixed solid phase carrier on which the cDNA is fixed. The solid-phase carrier in which is immobilized has a longer period of time. The solid phase carrier to which the nucleotide derivative of the present invention or an analog thereof is immobilized is used for monitoring gene expression, determining a nucleotide sequence, mutation analysis, polymorphism analysis and the like. The principle of detection is hybridization with a labeled sample nucleic acid fragment described below.

As the labeling method, RI method and non-RI method (fluorescence method, biotin method, chemiluminescence method, etc.) are known, but it is preferable to use the fluorescence method in the present invention. As the fluorescent substance used for the fluorescent label, any substance can be used as long as it can bind to the base portion of the nucleic acid, and a cyanine dye (for example, Cy3, Cy5, etc. of commercially available Cy Dye ^™ series), rhodamine 6G A reagent, N-acetoxy-N ² -acetylaminofluorene (AAF) or AAIF (iodine derivative of AAF) can be used.

As the sample nucleic acid fragment, a nucleic acid fragment sample such as a DNA fragment sample or an RNA fragment sample whose sequence or function is unknown is usually used.

The nucleic acid fragment sample is preferably isolated from a eukaryotic cell or tissue sample for the purpose of examining gene expression. If the sample is genomic, it is preferably isolated from any tissue sample except red blood cells. Any tissue except red blood cells is preferably peripheral blood lymphocytes, skin, hair, semen and the like. If the sample is mRNA, mR
It is preferable to extract from a tissue sample in which NA is expressed. mRNA is labeled with dNTP by a reverse transcription reaction.
(“DNTP” means a deoxyribonucleotide whose base is adenine (A), cytosine (C), guanine (G) or thymine (T)), and is preferably used as a labeled cDNA. It is preferable to use dCTP as the dNTP because of its chemical stability. MRN required for one-time hybridization
The amount of A is several μg or less, though it varies depending on the amount of the spotted liquid and the labeling method. When the DNA fragment on the solid phase carrier on which the nucleotide derivative or its analog is immobilized is oligo DNA, it is desirable that the nucleic acid fragment sample has a low molecular weight. Since it is difficult to selectively extract mRNA in prokaryotic cells, it is preferable to label total RNA.

The nucleic acid fragment sample is preferably obtained by performing PCR of the target region in a reaction system containing a labeled primer or labeled dNTP for the purpose of investigating gene mutation and polymorphism.

For hybridization, an aqueous solution prepared by dissolving or dispersing a labeled nucleic acid fragment sample, which had been dispensed in a 96-well or 384-well plastic plate, was immobilized with the nucleotide derivative of the present invention or its analog. Preference is given to carrying out by spotting on a solid support. The amount of spotting is preferably in the range of 1 to 100 nL. Hybridization is preferably carried out in the temperature range of room temperature to 70 ° C. and in the range of 6 to 20 hours. After the completion of hybridization, it is preferable to wash with a mixed solution of a surfactant and a buffer solution to remove the unreacted nucleic acid fragment sample. As the surfactant, sodium dodecyl sulfate (S
It is preferred to use DS). As the buffer solution, a citrate buffer solution, a phosphate buffer solution, a borate buffer solution, a Tris buffer solution, a Good's buffer solution, or the like can be used, but a citrate buffer solution is particularly preferred.

A feature of hybridization using a solid phase carrier on which a nucleotide derivative or its analog is immobilized is that the amount of labeled nucleic acid fragment sample used can be extremely reduced. Therefore, it is necessary to set the optimum conditions for hybridization depending on the chain length of the nucleotide derivative or its analog immobilized on the solid phase carrier and the type of the labeled nucleic acid fragment sample. For analysis of gene expression, it is preferable to carry out hybridization for a long time so that a gene with low expression can be sufficiently detected. For the detection of single nucleotide mutation, it is preferable to carry out hybridization for a short time. In addition, by preparing two kinds of nucleic acid fragment samples labeled with fluorescent substances different from each other and using them simultaneously for hybridization, the same DN can be obtained.
There is also a feature that the expression amount can be compared and quantified on the A fragment fixed solid phase carrier. The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.

[0091]

Examples Example 1: Preparation of slides on which DNA fragments are fixed,
Measurement of immobilized amount of DNA fragment The preparation and performance of a reactive solid phase carrier having a porous substrate, and a comparison with a reactive solid phase carrier having no porous substrate as a control are shown below.

(1) Preparation of porous solid phase carrier (A) 15% suspension 15 of colloidal silica (SNO-TEX PS-S [NISSAN CHEMICAL INDUSTRIES] / average particle size about 10 nm)
g, methanol 13g, water 5g, TMOS 1g,
Stir for 10 minutes and rest for 1 hour. The solution was filtered at 0.22 micron. A slide glass was immersed in this solution and dried at room temperature for 2 hours. Then at 70 ℃ for 1 hour,
Heated at 100 ° C. for 2 hours. This glass slide is reacted with 200 ml of a 2 mass% solution of Shin-Etsu Silicone KBE903 (Shin-Etsu Chemical Co., Ltd.) for 3 minutes using a commercially available slide washer. After completion of the reaction, the plate is washed with 200 ml of ultrapure water for 1 minute (using a slide washer). While exchanging the ultrapure water, the above-mentioned washing condition is repeated twice. After washing with water and drying for 10 minutes in a dryer at 45 ° C,
Put in an oven set at 0 ° C. and heat-treat for 10 minutes.
After cooling, a 3 mass% 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution is reacted for 120 minutes using a slide washer. After the reaction is completed, the product is washed with ultrapure water for 20 seconds × 3 times. 25 ° C for drying
The drying was performed for 30 minutes to obtain a porous solid phase carrier (A).

A film refractive index of about 1.3 was obtained by measuring the deflection of the surface of the porous solid phase carrier (A). Since the refractive index of the porous film is the volume average of the solid phase and the pore space, an estimated value of the porosity can be obtained. From the above, the porosity of the above film was estimated to be about 30 to 40%. The film thickness is about 65.
It was requested to be 00 angstrom. Pore size is calculated by assuming three equal size particles in a triangular layer:
Guessed. In this arrangement, the pore diameter is about 0.15 times the diameter of the particles, so in the case of the above 10 nm particles, the smallest pore is estimated to be on the order of 2 nm. The above measurement was performed in the same manner as the method described in International Publication WO00 / 61282.

(2) Spotting of DNA fragment and measurement of fluorescence intensity The 3'end and 5'end were modified with an amino group and a fluorescent labeling reagent (FluoroLink Cy5-dCTP, manufactured by Amersham Pharmacia Biotech). DNA fragment (3'-TCCTCCATGTCCGGGG
AGGATCTGACACTTCAAGGTCTAG-
5 ') was dispersed in 0.1 M carbonate buffer (pH 9.3), and an aqueous solution (1 × 10 ^-6 M, 1 μL) was spotted on the slide (A) obtained in (1) above. Immediately after the spotted solid phase carrier was left at 25 ° C. and 90% humidity for 1 hour, the solid phase carrier was mixed with 0.1 wt% SDS (sodium dodecyl sulfate) and 2 × SSC (2 × SSC: SSC). The undiluted solution was washed twice with a mixed solution of a solution obtained by diluting the stock solution with SSC: standard salt-citrate buffer solution, and once with a 0.2 × SSC aqueous solution. Then, the washed slide is immersed in a 0.1 M glycine aqueous solution (pH 10) for 1 hour and 30 minutes, washed with distilled water, and dried at room temperature to obtain a solid phase carrier (B ) Got. The fluorescence intensity on the surface of the solid phase carrier (B) was 25690 as measured by a fluorescence scanning device.

(3) Preparation and performance of comparative control slides Glass slides made by Shin-Etsu Silicone KBE903 (Shin-Etsu Chemical Co., Ltd.)
200 ml of a 2% by weight solution of the above is reacted for 3 minutes using a commercially available slide washer. After the reaction is completed, use 200 ml of ultrapure water for 1
Rinse with water for a minute (using slide washer). While exchanging the ultrapure water, the above-mentioned washing condition is repeated twice. After washing with water, and after drying for 10 minutes in a dryer at 45 ℃, 110
Place in an oven set at ℃ and heat for 10 minutes. After cooling, the mixture is reacted with 3% by weight of 1,2-bis (vinylsulfonylacetamide) ethane / pH 8.0 borate buffer solution using a slide washer for 120 minutes. After the reaction,
Wash with ultrapure water for 20 seconds x 3 times. Drying is performed for 30 minutes with a dryer set at 25 ° C. The same evaluation as in (2) above was performed and a fluorescence intensity of 1900 was obtained.

From the above results, it can be seen that the immobilization method of the present invention efficiently and efficiently immobilized the DNA fragments on the slide glass.

Example 2 Detection of Sample DNA Fragment (1) Preparation of Solid Phase Carrier Immobilizing Porous DNA Fragment 40 mer DNA Fragment (3'-TCCTCCCATGTCCGGGGGAGGA with 3'End Modified by Amino Group)
TCTGACACTTCAAGGTCTAG-5 ′) was dispersed in 0.1 M carbonate buffer (pH 9.3) to prepare an aqueous solution (1 × 10 ⁻⁶ M, 1 μL), and the porous solid phase obtained in (1) of Example 1 was used. It was spotted on the carrier (A). Immediately after leaving the solid-phase carrier after spotting at 25 ° C. and humidity of 90% for 1 hour,
This solid phase carrier was mixed with 0.1 wt% SDS (sodium dodecyl sulfate) and 2 x SSC (2 x SSC: SSC stock solution).
The solution was diluted twice, a mixed solution of SSC: standard salt-citrate buffer) and washed twice with a 0.2 × SSC aqueous solution. Then, slide the slides after washing as described above.
After immersing in a 1 M glycine aqueous solution (pH 10) for 1 hour and 30 minutes, it was washed with distilled water and dried at room temperature to obtain a solid phase carrier (C) on which a DNA fragment was immobilized.

(2) Detection of sample DNA fragment 22 mer sample oligonucleotide (CTAGTCTGTGAAGTTCCAG) having Cy5 bound to the 5'end.
ATC-5 ') for hybridization solution (4x
Mixed solution of SSC and 10 wt% SDS) (20μ
What was dispersed in L) was spotted on the solid phase carrier (C) obtained in (1) above, the surface was protected with a microscope cover glass, and then the mixture was incubated in a moist chamber at 60 ° C. for 20 hours. . Then, add 0.1 wt% SDS
And 2 × SSC mixed solution, 0.1 wt% SDS and 0.1.
After sequentially washing with a mixed solution of 2 × SSC and a 0.2 × SSC aqueous solution, the mixture was centrifuged at 600 rpm for 20 seconds and dried at room temperature. The fluorescence intensity on the surface of the slide glass was 19,600 when measured with a fluorescence scanning device.

Samples DN were also tested using comparative control slides.
When the A fragment was detected, the fluorescence intensity was 1500. Porous D produced by the immobilization method of the present invention
It can be seen that by using the NA fragment-immobilized solid phase carrier, a sample DNA fragment having a complementarity with the immobilized DNA fragment can be detected with high sensitivity.

[0100]

By utilizing the immobilization method of the present invention, a probe of a nucleotide derivative such as an oligonucleotide, a polynucleotide, or a peptide nucleic acid or its analog nucleotide is densely formed on the surface of a solid support having porosity. And it can be fixed stably. Therefore, the solid phase carrier to which the nucleotide derivative or its analog prepared by the immobilization method of the present invention is
It becomes a very stable solid-phase carrier in which separation of the probe due to hydrolysis hardly occurs. In particular, when an amino group or the like is introduced into the surface of the solid phase carrier by using a silane coupling agent, both the binding of the amino group and the like to the surface of the solid phase carrier and the binding of the probe are covalent bonds. The probe can be firmly fixed on the solid phase carrier. The stable immobilization of the probe makes it possible to obtain a detection tool having a high detection limit that can be effectively used for gene analysis and the like.

As one example thereof, by using the oligonucleotide-immobilized solid-phase carrier prepared according to the present invention and carrying out hybridization with a sample nucleic acid fragment, a probe immobilized on the oligonucleotide-immobilized solid-phase carrier can be obtained. A nucleic acid fragment sample having complementarity can be detected with high sensitivity. In addition, non-specific adsorption of sample nucleic acid fragments can be prevented by spotting a probe sample such as an oligonucleotide on the surface of the reactive solid phase carrier and then treating the surface of the solid phase carrier with an anionic compound such as glycine. ,
This has a great effect on highly sensitive detection of nucleic acid fragment samples having complementarity.

[0102]

[Sequence list] SEQUENCE LISTING <110> Fuji Photo Film Co. Ltd., <120> A reactive solid support and an apparatus for detection of DNA fra gment <130> A11244MA <160> 2

[0103] <210> 1 <211> 22 <212> DNA <213> Artificial DNA <400> 1 ctagtctgtg aagtgtctga tc 22

[0104] <210> 2 <211> 40 <212> DNA <213> Artificial DNA <400> 2 tcctccatgt ccggggagga tctgacactt caaggtctag 40

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a typical oligonucleotide-immobilized solid phase carrier of the present invention and a method of immobilizing a typical oligonucleotide of the present invention.

FIG. 2 is a schematic diagram showing a fixing method of Example 1.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12Q 1/68 G01N 31/22 121P G01N 31/22 121 33/58 A 33/58 37/00 102 37 / 00 102 C12N 15/00 ZNAF F term (reference) 2G042 AA01 BD19 CA10 CB03 DA08 DA09 DA10 EA01 FA11 FB07 FC01 FC02 FC04 FC06 HA02 HA08 2G045 BA01 BA13 BB21 CA17 CB01 CB11 AFB12 A16 AFB12 A16 AFB12 A16 AFB12 A16 A02 FB02 A16A02 FB02 A16A02 CA01 HA12 4B029 AA07 AA21 AA23 BB15 BB20 CC03 FA01 FA09 4B063 QA01 QA18 QQ42 QQ53 QR32 QR55 QR82 QS34 QX01

Claims (25)

[Claims] 1. A porous region of about 2 nm to about 1000 n.
m pore size, about 10% to about 90% porosity and about 0.0
A group of vinylsulfonyl groups or their reactive precursor groups are covalently immobilized via a linking group on the surface of a solid-phase carrier having a porous substrate with a thickness of 1 μm to about 70 μm. Phase carrier. 2. The reactive solid phase carrier according to claim 1, wherein the porous substrate is composed of an organic polymer. 3. The reactive solid phase carrier according to claim 1, wherein the porous substrate is composed of an inorganic substance. 4. The reactive solid phase carrier according to claim 1, wherein the porous substrate contains silicon, alumina or titanium. 5. The reactive solid phase carrier according to any one of claims 1 to 4, wherein the linking body of the vinylsulfonyl group or its reactive precursor group and the linking group is represented by the following formula: in L-SO ₂ -X [above formula, X represents a -CR ¹ = CR ² R ³ or ^{-CHR 1 -CR 2 R 3 Y;} R 1, R 2 and R ^3,
Independently of each other, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total carbon atom number of 7 to 26 having an alkyl chain having 1 to 6 carbon atoms. Represents an atom or group selected from the group consisting of aralkyl groups;
^{_{O 2 R 11, -OCOR 12,}} -OSO 3 M, and represents an atom or group selected from the group consisting of quaternary pyridinium group; R ¹¹ is an alkyl group having carbon atoms 1 to 6, carbon atoms 6 to 20 aryl groups and 1 carbon atom
7 to 2 total carbon atoms having an alkyl chain of 6 to 6
6 represents a group selected from the group consisting of 6 aralkyl groups;
R ¹² represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is a hydrogen atom, an alkali metal atom or an ammonium group. Represents an atom or group selected from the group consisting of; and L represents a linking group]. 6. The reactive solid phase carrier according to claim 5, wherein X is a vinyl group represented by —CH═CH ₂ . 7. The reactive solid phase carrier according to claim 5, wherein L is a linking group containing a divalent or higher atom other than carbon atoms. 8. L is —NH—, —S—, and —O—
The reactive solid phase carrier according to claim 5, which is a linking group having a linking site selected from the group consisting of: 9. L is — (L ¹ ) _n —NH— (CR ¹ R ² )
₂ - or _{^{- (L 1) n -S- (}} CR 1 R 2) 2 - [ where, R ¹
And R ² have the same meanings as described above, L ¹ represents a linking group, and n is 0 or 1], the reactive solid phase according to claim 5. Carrier. 10. L is — (L ¹ ) _n —NHCH ₂ CH ₂ —
The reactive solid phase carrier according to claim 5, which is a linking group represented by [wherein L ¹ represents a linking group, and n is 0 or 1]. 11. The reactive solid phase carrier according to claim 9, wherein L ¹ is a linking group containing a group represented by —OSi—, and n is 1. 12. The solid phase carrier comprises a glass substrate, a resin substrate,
The reactive solid phase carrier according to claim 1, which is a sheet-like substrate selected from the group consisting of a glass substrate or a resin substrate surface-treated with a silane coupling agent, and a glass substrate or a resin substrate having a coating layer on the surface. . 13. A solid phase carrier, a silicate glass substrate, a silicate glass substrate surface-treated with a silane coupling agent,
The reactive solid phase carrier according to claim 12, which is a sheet-shaped substrate selected from the group consisting of a silicate glass substrate coated with an organic coating layer. 14. A solid phase carrier having a surface on which a reactive group has been introduced, has the following formula: X ¹ —SO ₂ —L ² —SO ₂ —X ² [wherein, X ¹ and X ² are independent of each other. And-
CR ¹ ═CR ² R ³ or —CHR ¹ —CR ² R ³ Y; R ¹ , R ² and R ³ are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a carbon atom. Represents an atom or a group selected from the group consisting of an aryl group having 6 to 20 atoms and an aralkyl group having 7 to 26 total carbon atoms and having an alkyl chain having 1 to 6 carbon atoms; Y
It is a halogen ^{_{atom, -OSO 2 R 11, -OCOR 12}} , -
OSO ₃ M, and an atom or group selected from the group consisting of quaternary pyridinium groups; R ¹¹ is an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a carbon atom. Represents a group selected from the group consisting of aralkyl groups having a total number of carbon atoms of 7 to 26 and having an alkyl chain of 1 to 6; R ¹² represents an alkyl group having 1 to 6 carbon atoms and a number of carbon atoms of 1 to 6; Represents a group selected from the group consisting of 1 to 6 halogenated alkyl groups; M
Represents an atom or a group selected from the group consisting of a hydrogen atom, an alkali metal atom and an ammonium group; and L ² represents a linking group]. The method for producing the reactive solid-phase carrier according to 1. 15. The method for producing a reactive solid phase carrier according to claim 14, wherein the reactive group introduced on the surface of the solid phase carrier is an amino group, a mercapto group or a hydroxyl group. 16. A reactive solid phase carrier having a porous substrate on which a group of vinylsulfonyl groups or reactive precursor groups thereof are immobilized by covalent bonds, respectively, and reacting with the reactive groups to share the same. A solid phase in which the nucleotide derivative or its analog is bound and immobilized on the surface of a carrier through a linking group having a sulfonyl group, which is characterized by contacting a nucleotide derivative or its analog with a reactive group that forms a bond. Method for producing carrier. 17. The method according to claim 16, wherein the nucleotide derivative or its analog is selected from the group consisting of an oligonucleotide, a polynucleotide, and a peptide nucleic acid. 18. A reactive solid phase carrier having a porous substrate, in which a vinyl sulfonyl group represented by the following formula or a linking group of its reactive precursor group and a linking group is bonded and fixed. the method according to claim 16 or 17, characterized by using a: in -L-SO ₂ -X [above formula, X is, -CR ¹ = CR ² R ³ or -CHR ¹ -CR ² R ³ represents Y; R ¹ , R ² and R ³ are
Independently of each other, a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an aryl group having 6 to 20 carbon atoms, and a total carbon atom number of 7 to 26 having an alkyl chain having 1 to 6 carbon atoms. Represents an atom or group selected from the group consisting of aralkyl groups;
^{_{O 2 R 11, -OCOR 12,}} -OSO 3 M, and represents an atom or group selected from the group consisting of quaternary pyridinium group; R ¹¹ is an alkyl group having carbon atoms 1 to 6, carbon atoms 6 to 20 aryl groups and 1 carbon atom
7 to 2 total carbon atoms having an alkyl chain of 6 to 6
6 represents a group selected from the group consisting of 6 aralkyl groups;
R ¹² represents a group selected from the group consisting of an alkyl group having 1 to 6 carbon atoms and a halogenated alkyl group having 1 to 6 carbon atoms; M is a hydrogen atom, an alkali metal atom or an ammonium group. Represents an atom or group selected from the group consisting of; and L represents a linking group or a single bond]. 19. X is --CR ¹ = CR ² R ³ [R ¹ , R ²
And R ³ have the same meanings as described above], and the production method according to claim 18. 20. A solid-phase carrier having a porous substrate having the nucleotide derivative or its analog obtained by the method according to any one of claims 15 to 18 bound and immobilized on the surface of the carrier. 21. Complementary to the immobilized nucleotide derivative or its analog in the presence of an aqueous medium, on a solid phase carrier having a porous substrate to which the nucleotide derivative or its analog of claim 20 is immobilized. And a method for binding and immobilizing complementary oligonucleotides or polynucleotides, which comprises contacting the oligonucleotides or polynucleotides shown in FIG. 22. The method of claim 21, wherein a complementary label is attached to the complementary oligonucleotide or polynucleotide. 23. An oligonucleotide or a polynucleotide which shows complementarity to the immobilized nucleotide derivative or its analogue on the solid phase carrier having a porous substrate on which the nucleotide derivative or its analogue is immobilized. A solid-phase carrier to which complementary oligonucleotides or polynucleotides are bound and immobilized, characterized in that nucleotides are complementarily bound. 24. The solid phase carrier according to claim 23, wherein a detectable label is bound to the complementary oligonucleotide or polynucleotide. 25. A solid phase carrier having a porous substrate having the nucleotide derivative or analogue thereof according to claim 20 bound and immobilized on the surface of a carrier, or the complementary oligonucleotide or polynucleotide according to claim 23. Characterized by utilizing a fixed solid phase carrier,
Gene identification or screening method.