JP2003028871A - Active solid-phase carrier and dna fragment detecting implement - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fixing method capable of stably combining a previously prepared DNA fragment, a nucleotide derivative such as an oligonucleotide, etc., to a surface of a solid-phase carrier of glass, silicon, or metal by a speedy reaction, a method for manufacturing a carrier for fixing a nucleotide derivative or its analogous body through the use of the fixing method, and an implement for detecting a nucleic acid fragment sample represented by a DNA chip. SOLUTION: In the active solid-phase carrier, a group of vinyl sulfonyl groups or their active precursor groups are fixed to the surface of the solid-phase carrier formed of glass, silicon, or metal each through a connecting group by covalent bonding through the use of a divinyl sulfonic compound, etc. In the detecting and fixing implement of the nucleic acid fragment sample, the nucleotide derivative such as an oligonucleotide, a polynucleotide, a peptide nucleic acid, etc., or its analogous body is combined with and fixed to the active solid- phase carrier by covalent bonding.

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. The invention is particularly applicable to DN
A high-density array-type detection tool (DNA detection chip) in which a large number of nucleotide derivatives or their analogs are aligned and immobilized on a solid-phase carrier surface at high density, which is useful for analyzing the base sequence of an A fragment sample, and the high-density array The present invention relates to a reactive solid phase carrier which can be advantageously used for producing a mold 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.

[0014]

DISCLOSURE OF THE INVENTION The present invention provides a nucleotide derivative such as an oligonucleotide, a polynucleotide, a peptide nucleic acid or the like, which has been prepared in advance, on the surface of a solid phase carrier made of glass, silicon or metal. Reactive solid phase carrier that can be particularly advantageously used for rapidly and stably binding and immobilizing a body, and a specific derivative comprising a nucleotide derivative or an analog thereof bound and immobilized to the reactive solid phase carrier. It is an object of the present invention to provide a tool for detecting DNA, RNA or a fragment thereof having a base sequence portion.

[0015]

According to the present invention, a group of vinylsulfonyl groups (separately referred to as vinylsulfone group or sulfonylvinyl group) may be formed on the surface of a solid support made of glass, silicon or metal. A) or a reactive precursor group thereof is covalently bonded and immobilized via a linking group, respectively.
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).

[0016]

[Of 4] _{-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 is obtained by adding the following formula (2) to a solid-phase carrier having a reactive group introduced on the surface and made of glass, silicon, or a metal.

[0021]

## STR00005 ## X ¹ --SO ₂ --L ² --SO ₂ --X ² --- (2)

[0022] [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 relates to a reactive solid group in which a group of vinylsulfonyl groups or their reactive precursor groups are covalently immobilized on the surface of a solid phase carrier made of glass, silicon or metal. To the surface of the phase carrier, a nucleotide derivative having a reactive group that reacts with the reactive group to form a covalent bond or an analogue thereof is brought into contact, through a linking group having a sulfonyl group. There is also a method for producing a solid phase carrier in which a nucleotide derivative or its analog is bound and immobilized on the surface of the carrier.

Typical examples of the nucleotide derivative or its analog immobilized on the solid phase carrier 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 to which the nucleotide derivative or its analog is immobilized is an oligonucleotide or a polynucleotide (DNA or its analog) 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 a fragment or RNA or a fragment thereof to generate 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.

[0028]

BEST MODE FOR CARRYING OUT THE INVENTION The reactive solid phase carrier of the present invention comprises a group of vinylsulfonyl groups or their reactive precursor groups linked to the surface of a solid phase carrier made of glass, silicon or metal, respectively. It has a structure in which it is bonded and fixed by a covalent bond via a group. As the reactive solid phase carrier of the present invention, a solid phase carrier in which a reactive group is introduced in advance is prepared, and this solid phase carrier reacts with the reactive group provided on the surface of the carrier to form a covalent bond. Produced by contacting with a compound having a reactive group at or near one end and a vinylsulfonyl group or a reactive precursor group of vinylsulfonyl group at or near the other end can do.

The solid phase carrier is a solid phase carrier made of glass, silicon or metal.

As the solid phase carrier used in the production of the reactive solid phase carrier of the present invention, a glass substrate, a silicon substrate, which has been conventionally used in the production of a DNA chip or has been proposed for the production of a DNA chip, An electrode substrate can be preferably used.

On the surface of the solid support, a polycationic compound (for example, poly-L) is used in order to bond and fix a bifunctional reactive compound such as a divinyl sulfone compound by a covalent bond.
-Lysine, polyethyleneimine, polyalkylamine and the like are preferable, and poly-L-lysine is more preferable), and coating treatment with a polymer having an amino group in the side chain (in this case, the surface of the solid phase carrier) The reactive group introduced is preferably an amino group). 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 a coating treatment with a polycationic compound, the amino group or mercapto group is introduced onto the surface of the solid support by electrostatic coupling between the polymer compound and the surface of the solid support. In the case of the surface treatment with the silane coupling agent, the amino group or the mercapto group is stably present on the surface of the solid phase carrier because it is bound and fixed to the surface of the solid phase carrier by a covalent bond. 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.

As the silane coupling agent having an amino group, γ-aminopropyltriethoxysilane, N-
It is preferable to use β (aminoethyl) -γ-aminopropyltrimethoxysilane or N-β (aminoethyl) -γ-aminopropylmethyldimethoxysilane, and it is particularly preferable to use γ-aminopropyltriethoxysilane.

Treatment with a polycationic compound may be combined with treatment with a 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 an ordinary solid phase carrier for a DNA chip, compartments or a large number of supposed regions are set in advance, and as described above, a difunctional sulfone compound or other bifunctional reaction is set for each region. 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 vinylsulfonyl 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.

[0038]

Embedded image -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 which 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.

[0044]

[Chemical 7]

[0045]

[Chemical 8]

"-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.

[0048]

[Chemical 9]

As L, in addition to the divalent linking group described above, 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 bifunctional reactive compound used to obtain the solid-phase support 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.

[0051]

Embedded image X ¹ --SO ₂ --L ² --SO ₂ --X ² --- (2)

[0052] [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 contacting the disulfone compound represented by the above formula (2) with the above-mentioned 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.

[0055]

[Chemical 11]

[0056]

[Chemical 12]

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

The method of synthesizing the disulfone compound used in the present invention is described in, for example, JP-B-47-2429 and JP-A-5-2429.
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 therefore can be easily and stably stabilized. 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.

Representative 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.

At one end of a nucleotide derivative such as an oligonucleotide or a DNA fragment or its analog, a reactive group which forms a covalent bond by reacting with the above vinylsulfonyl group or its reactive precursor group is introduced. . 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 or its analog having a reactive group 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 analogue from drying after spotting, it is dissolved in or dispersed in an aqueous liquid in which the nucleotide derivative or its analogue 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, it may be post-treated with ultraviolet rays, sodium borohydride or a Schiff reagent.
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 schematically shows a method for producing an oligonucleotide-immobilized solid-phase carrier, which is a typical embodiment of the present invention, and the structure of a typical oligonucleotide-immobilized solid-phase carrier. As the method for producing the solid phase carrier on which the oligonucleotide of the present invention is immobilized, when the disulfone compound represented by the above formula (2) is used, four types of production methods can be used depending on X ¹ and X ² .

In FIG. 1, an oligonucleotide in which both X ¹ and X ² of the disulfone compound of the 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 X ¹ is -Y. By substituting a reactive group (R) for the moiety,-(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 .

[0074] Step (1): to a solid support one surface active groups solid support (R) has been introduced (A), contacting the disulfone compound represented by the formula (I), the X ¹ - CR ¹ = CR
By adding a reactive group (R) to ² R ³ ,
The ^{R 3 R 2 C-R 1} HC-SO 2 -L-SO 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).

When the oligonucleotide 2 having a reactive group (Z) is spotted on the surface of the solid phase carrier (B) of FIG.
X ² or —Y of X ² and the reactive oligonucleotide fragment 2
However, unreacted X ² to which the oligonucleotide 2 is not bound is also present on the surface of the solid phase carrier (B). 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. It is preferable to mask X ² (that is, the halogen atom of X ² , for example). The mask treatment is preferably performed by bringing the surface of the solid support (C1) (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). Such an anionic compound reacts with a halogen atom of X ² ,
And negative charges (COO ⁻ , SO ₃ ⁻ , OSO ₃ ⁻ , PO ₃ ⁻ ,
Or PO ₂ ^-) can also be used any one as long as it has a, preferably an amino acid,
Particularly preferred is glycine or cysteine. Also, taurine can be preferably used.

The lifespan of the solid phase carrier on which the nucleotide derivative or its analog produced by the method of the present invention is fixed is usually several weeks for the cDNA fixed solid phase carrier on which 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.

RI and non-RI methods (fluorescence method, biotin method, chemiluminescence method, etc.) are known as labeling methods, but the fluorescence method is preferably used 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 or 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 characteristic of the hybridization using the solid phase carrier on which the nucleotide derivative or its analog is immobilized is that the amount of the 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.

[0085]

Example 1 Preparation of oligonucleotide-immobilized slide and measurement of immobilized amount of oligonucleotide The method of immobilizing the oligonucleotide of the present invention is represented by the reaction pathway of the oligonucleotide, and the reaction pathway is illustrated. 2 shows. In the figure, 1 represents a slide glass.

(1) Preparation of Solid Phase Carrier (B) Introducing Vinylsulfonyl Group 2% by weight of aminopropylethoxysilane (manufactured by Shin-Etsu Chemical Co., Ltd.) in ethanol solution, slide glass (25
(mm × 75 mm) was soaked for 10 minutes, taken out, washed with ethanol, and then dried at 110 ° C. for 10 minutes to prepare a silane compound-coated slide (A). Next, 5% by weight of this silane compound-coated slide (A) was used.
After soaking in a phosphate buffer (pH 8.5) solution of 1,2-bis (vinylsulfonylacetamide) ethane for 1 hour, it is taken out, washed with acetonitrile, and dried under reduced pressure for 1 hour to introduce a vinylsulfonyl group on the surface. A solid phase carrier (B) was obtained.

(2) Oligonucleotide spotting 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). Oligonucleotide (3'-CTAGTCTGTGA
AGTGTCTGATC-5 ') was dispersed in 0.1 M carbonate buffer (pH 9.3) to prepare an aqueous solution (1 x 10 ^-6 M, 1 µm).
L) was spotted on the slide (B) obtained in (1) above.
Immediately, apply the solid-phase carrier after spotting at 25 ° C and humidity of 90% to 1
After standing for a period of time, this solid phase carrier is added with 0.1 wt% SDS.
(Sodium dodecyl sulfate) and 2 x SSC (2 x SS
C: a solution obtained by diluting the stock solution of SSC by 2 times, and a mixed solution of SSC: standard salt-citrate buffer) twice, 0.2 ×
It was sequentially washed once with an 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 (C ) Got. When the fluorescence intensity on the surface of the solid phase carrier (C) was measured by a fluorescence scanning device, it was 2500, which was much larger than the background fluorescence intensity. Therefore, it can be seen that the oligonucleotide was efficiently immobilized on the slide glass by the immobilization method of the present invention.

Example 2 Detection of Complementary Target Oligonucleotide Sample (1) Preparation of Oligonucleotide-Immobilized Solid-Phase Carrier A 40-mer oligonucleotide (3′-TCCTCCATGTCCGGGG) having a 3′-terminal modified with an amino group was prepared.
AGGATCTGACACTTCAAGGTCTAG-
5 ') was dispersed in 0.1 M carbonate buffer (pH 9.3) to prepare an aqueous solution (1 x 10 ^-6 M, 1 µL), which was added to the solid phase carrier (B) obtained in Example (1). I spotted it. 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). 2 times diluted stock solution of SSC, mixed solution of SSC: standard salt-citrate buffer) twice, 0.2 × SSC aqueous solution 1 times
It was washed sequentially. 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 (C Got ').

(2) Detection of Complementary Target Oligonucleotide Sample 22mer target oligonucleotide sample (CTAGTCTGGTGA) having Cy5 (fluorescent label) bound to the 5'end.
AGTTCCAGATC-5 ′) dispersed in a hybridization solution (mixed solution of 4 × SSC and 10 wt% SDS, 20 μL) is spotted on the solid phase carrier (C ′) obtained in (1) above. Then, after protecting the surface with the microscope cover glass, 6 in the moisture chamber.
Incubated at 0 ° C. for 20 hours. Then, this product was mixed with 0.1 wt% SDS and 2 × SSC,
After sequentially washing with a mixed solution of 1 wt% SDS and 0.2 × SSC, and a 0.2 × SSC aqueous solution, 600 rpm
It was centrifuged for 20 seconds and dried at room temperature. When the fluorescence intensity on the surface of the slide glass was measured by a fluorescence scanning device, it was 1219, which was much larger than the background fluorescence intensity. Therefore, by using the oligonucleotide-immobilized solid-phase carrier prepared by the immobilization method of the present invention, the target oligonucleotide such as a target DNA fragment sample having complementarity with the oligonucleotide immobilized on the oligonucleotide-immobilized solid-phase carrier is used. It can be seen that the nucleotide sample can be efficiently detected.

Example 3 Preparation of oligonucleotide-immobilized solid phase carrier and measurement of immobilized amount of oligonucleotide (1) Preparation of amino group-introduced solid phase carrier (B1) 2% by weight aminopropylethoxysilane ( Add a glass slide (25) to the ethanol solution of Shin-Etsu Chemical Co., Ltd.
(mm × 75 mm) was soaked for 10 minutes, taken out, washed with ethanol, and then dried at 110 ° C. for 10 minutes to prepare a silane compound-coated slide (A). Then, the silane compound-coated solid phase carrier (A) was immersed in an acetonitrile solution in which chlorosulfonyl isocyanate (0.5 g) was dissolved in acetonitrile (1 mL) for 2 hours, then taken out, washed with acetonitrile, and depressurized for 1 hour. It was dried under to obtain a solid phase carrier (B1).

(2) Spotting of Oligonucleotide and Measurement of Fluorescence Intensity The 3'end and 5'end were respectively modified with an amino group and a fluorescent labeling reagent (FluoroLink Cy5-dCTP, manufactured by Amersham Pharmacia Biotech). Oligonucleotide (3'-CTAGTCTGTGA
AGTGTCTGATC-5 ') was dispersed in 0.1 M carbonate buffer (pH 8.0) to prepare an aqueous solution (1 x 10 ^-6 M, 1 µm).
L) was spotted on the solid phase carrier (B1) obtained in (1) above. Immediately, apply the solid-phase carrier after spotting at 25 ° C and humidity of 90%.
After standing for 1 hour at 0.1%
SDS (sodium dodecyl sulfate) and 2 x SSC (2 x
SSC: A solution obtained by diluting the stock solution of SSC by 2 times, SSC:
2 times with a mixed solution of standard salt-citrate buffer solution, 0.
It was sequentially washed once with a 2 × SSC aqueous solution. Then, the slide after the above washing is washed with a 0.1 M glycine aqueous solution (pH 1).
After immersing in 0) for 1 hour and 30 minutes, it was washed with distilled water and dried at room temperature to obtain a solid phase carrier (C1) on which the oligonucleotide was immobilized. When the fluorescence intensity of the surface of the solid phase carrier (C1) was measured by a fluorescence scanning device, it was 3210
And increased more than the background fluorescence intensity. Therefore, it is understood that the target oligonucleotide sample such as the target DNA fragment sample was efficiently bound and immobilized on the slide glass by the immobilization method of the present invention.

Example 4 Detection of Complementary Target Oligonucleotide Sample (1) Preparation of Oligonucleotide-Immobilized Solid-Phase Carrier As Example 1 except that an oligonucleotide whose 3 ′ end is not modified with a fluorescent labeling reagent is used. Similarly, a solid phase carrier (C1 ′) on which the oligonucleotide was immobilized was obtained. (2) Detection of Complementary Target Oligonucleotide Sample A 22-mer oligonucleotide sample (CTAGTCTGGAGATTTCCAG having Cy5 bound to the 5 ′ end).
ATC-5 ') for hybridization solution (4x
Mixed solution of SSC and 10 wt% SDS) (20μ
After being dispersed in L), the solid phase carrier (C1 ′) obtained in (1) above is spotted, the surface is protected by a microscope cover glass, and then 20 ° C. in a moisture chamber at 60 ° C.
Incubated for hours. Then, this was mixed with 0.1 wt% SDS and 2 × SSC, 0.1 wt% S.
Mixed solution of DS and 0.2xSSC, and 0.2xS
After sequentially washing with SC aqueous solution, it was centrifuged at 600 rpm for 20 seconds and dried at room temperature. When the fluorescence intensity on the surface of the slide glass was measured by a fluorescence scanning device, it was 1078.
And increased more than the background fluorescence intensity. Therefore, by using the oligonucleotide-immobilized solid phase carrier prepared by the immobilization method of the present invention,
It can be seen that a target oligonucleotide sample such as a target DNA fragment sample having complementarity with the oligonucleotide immobilized on the solid phase carrier can be efficiently detected.

[Example 5] Preparation of oligonucleotide-immobilized solid-phase carrier and measurement of immobilized amount of oligonucleotide Instead of chlorosulfonyl isocyanate, succinimidyl (4-vinylsulfonyl) benzoate (0.
A solid phase carrier (B2) was prepared in the same manner as in (1) of Example 3 except that 5 g) was used, and the same procedure as in (3) of Example 3 was performed except that this was used to prepare the oligonucleotide. A solid phase carrier (C2) on which was immobilized was obtained. Solid phase carrier (C
2) When the surface fluorescence intensity was measured by a fluorescence scanning device, it was 3250, which was much larger than the background fluorescence intensity. It can be seen that the oligonucleotide was efficiently immobilized on the slide glass by the immobilization method of the present invention.

Example 6 Detection of Complementary Target Oligonucleotide Sample (1) Preparation of Oligonucleotide-Immobilized Solid-Phase Carrier As Example 3 except that an oligonucleotide whose 3 ′ end is not modified with a fluorescent labeling reagent is used. Similarly, a solid phase carrier (C2 ′) on which the oligonucleotide was immobilized was obtained. (2) Detection of target oligonucleotide sample When the fluorescence intensity on the surface of the slide glass was measured by a fluorescence scanning device in the same manner as in Example 2 except that the solid phase carrier (C2 ′) obtained in (1) above was used, it was 2325. And increased more than the background fluorescence intensity.
Therefore, by using the oligonucleotide-immobilized solid-phase carrier prepared by the immobilization method of the present invention, a target oligonucleotide such as a target DNA sample having complementarity with the oligonucleotide immobilized on the oligonucleotide-immobilized solid-phase carrier. It can be seen that the sample can be detected efficiently.

Example 7 Preparation of Solid Phase Carrier Immobilizing Oligonucleotide and Measurement of Immobilization Level of Oligonucleotide (1) Synthesis of Vinyl Sulfone Compound-Silane Coupling Agent Conjugate 1.5 g Vinyl sulfonylacetoacetic acid in 75 mL tetrahydrofuran 2.2 g of 3-aminopropyltriethoxysilane and then 2.1 g of N, N-dicyclohexylcarbodiimide (DCC, a condensing agent).
Was added and stirred at room temperature for 2 hours. The obtained reaction mixture was concentrated and then purified by silica gel column chromatography (eluent: hexane / ethyl acetate = 10/1) to obtain the target conjugate.

(2) Production of Vinyl Sulfone Compound-Immobilized Substrate A 2% aqueous solution of the vinyl sulfone compound-silane coupling agent conjugate obtained by the above operation was prepared, and a slide glass (25 mm × 75 mm) was added to the aqueous solution. Soak for 1 minute, remove, wash with ethanol, and then at 110 ℃
And dried for 10 minutes to obtain a vinyl sulfone compound-immobilized substrate.

(3) Oligonucleotide spotting 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). Oligonucleotide (3'-CTAGTCTGTGA
AGTGTCTGATC-5 ') was dispersed in 0.1 M carbonate buffer (pH 8.0) to prepare an aqueous solution (1 x 10 ^-6 M, 1 µm).
L) was spotted on the solid phase carrier obtained in (2) 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 3 hours.
After soaking for 0 minute, the solid phase carrier was washed with distilled water and dried at room temperature to obtain a solid phase carrier on which the oligonucleotide was immobilized. When the fluorescence intensity on the surface of the solid phase carrier was measured by a fluorescence scanning device, it was 3500, which was much larger than the background fluorescence intensity. Therefore, it can be seen that the oligonucleotide was efficiently immobilized on the slide glass by the immobilization method of the present invention.

[Example 8] Preparation of oligonucleotide-immobilized solid-phase carrier and measurement of immobilized amount of oligonucleotide (1) Preparation of gold electrode having vinylsulfonyl group fixed on the surface Gold electrode whose surface was washed with acetone ( Surface area: 2.25m
² μL of an aqueous solution of 11-amino-1-undecathiol (1 mM) was dropped on the surface of m ² ), and the mixture was allowed to stand at room temperature for 10 hours while preventing the aqueous solution from drying. It was washed sequentially. Then, on the surface of the gold electrode, a phosphate buffer containing 3% of 1,2-bis (vinylsulfonylacetamido) ethane (pH 8.
2 μL of 5) was added dropwise and left at room temperature for 2 hours, and then the electrode surface was washed successively with distilled water and ethanol. Then 1
By drying under reduced pressure for an hour, a gold electrode having a vinylsulfonyl group bonded to the surface via a linking group was obtained.

(2) Immobilization of Oligonucleotide (Production of Electrochemical Analytical Element) Thymine 20 having an aminohexyl group introduced at the 5 ′ end to the gold electrode having a vinylsulfonyl group on the surface obtained in (1) above. Aqueous solution (1) of oligomeric oligonucleotide (T ₂₀ )
2 μL (00 picomoles / 1 μL) was added dropwise, the mixture was allowed to stand at room temperature for 1 hour, excess oligonucleotide (T ₂₀ ) was removed by washing, and then dried to prepare an electrochemical analysis element.

(3) Preparation of ferrocene-labeled oligonucleotide An aminohexyl group-linking oligonucleotide was obtained by binding an aminohexyl group linker to the 5'end of the adenine 20-mer (A ₂₀ ). Using the aminohexyl group-bound oligonucleotide obtained above, Analytic by Takenaka et al.
al Biochemistry, 218, according to the method described in 436-443 (1994), 5 'terminally prepare oligonucleotides adenine 20 mer labeled with ferrocene (F1-A _20).

(4) Detection of Complementary Target Oligonucleotide Sample On the surface of the electrochemical analysis element prepared in (2) above,
Adenine 20 whose 5'end is labeled with ferrocene
10 m containing the oligomer (F1-A ₂₀ ) of the monomer
2 μL of M Tris buffer solution (pH 7.5) was added dropwise,
Incubated at 25 ° C for 30 minutes. After the incubation is completed, the surface of the analysis element was washed with pure water to remove compounds F1-A ₂₀ unreacted. Differential pulse voltammetry (DVP) was performed at a voltage range of 100 to 700 mV using a 0.1 M potassium chloride-0.1 M acetic acid buffer solution (pH 5.60) solution as a measurement solution (38 ° C.) to obtain 460 mV. A response current derived from the compound F1-A ₂₀ was obtained at the applied voltage of. According to the above operation and results, the oligonucleotide is stably bound and immobilized on the surface of the gold electrode, and this electrode can be used to detect the complementary target nucleotide sample labeled with the electrochemical labeling substance. Was confirmed.

[0102]

INDUSTRIAL APPLICABILITY 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 analogue nucleotide is stably and rapidly immobilized on the surface of a solid phase carrier. be able to. Therefore, the solid phase carrier on which the nucleotide derivative or its analog prepared by the immobilization method of the present invention is immobilized is a very stable solid phase carrier on which the probe is hardly detached by hydrolysis. 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 to perform 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.

[Brief description of drawings]

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

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

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Yumiko Takeshita             3-11-46 Izumi, Asaka-shi, Saitama Fuji Photographic Group             Within Ilum Co., Ltd. (72) Inventor Junichi Yamanouchi             Fuji Photo, 210 Nakanuma, Minamiashigara City, Kanagawa Prefecture             Within Film Co., Ltd. (72) Inventor Yukio Sudo             3-11-46 Izumi, Asaka-shi, Saitama Fuji Photographic Group             Within Ilum Co., Ltd. (72) Inventor Osamu Soshimoto             3-11-46 Izumi, Asaka-shi, Saitama Fuji Photographic Group             Within Ilum Co., Ltd. F term (reference) 4B024 AA11 AA19 CA01 CA09 HA12                 4B029 AA07 BB20 CC03 FA15

Claims (16)

[Claims] 1. A reaction in which a group of vinylsulfonyl groups or reactive precursor groups thereof are covalently immobilized via a linking group on the surface of a solid-phase carrier made of glass, silicon, or a metal. Solid phase carrier. 2. The reactive solid phase carrier according to claim 1, wherein the linking body of the vinylsulfonyl group or its reactive precursor group and the linking group is represented by the following formula: in -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]. 3. The reactive solid phase carrier according to claim 2, wherein X is a vinyl group represented by —CH═CH ₂ . 4. The reactive solid phase carrier according to claim 2, wherein L is a linking group containing a divalent or higher valent atom other than carbon atoms. 5. L is —NH—, —S—, and —O—
The reactive solid phase carrier according to claim 2, which is a linking group having a linking site selected from the group consisting of: 6. 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 2. Carrier. 7. L is — (L ¹ ) _n —NHCH ₂ CH ₂ —
The reactive solid phase carrier according to claim 2, which is a linking group represented by [wherein L ¹ represents a linking group, and n is 0 or 1]. 8. The reactive solid phase carrier according to claim 6, wherein L ¹ is a linking group containing a group represented by —OSi—, and n is 1. Carrier. 9. A glass having a surface on which a reactive group is introduced,
A solid support made of either silicon or a metal has the following formula: ## STR2 ## X ¹ --SO ₂ --L ² --SO ₂ --X ² [wherein, X ¹ and X ² are independent of each other. ,-
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 hydrogen atom, alkali metal atom and ammonium group; and L ² represents a linking group]. The method for producing the reactive solid-phase carrier according to 1. 10. The method for producing a reactive solid phase carrier according to claim 9, wherein the reactive group introduced on the surface of the solid phase carrier is an amino group, a mercapto group or a hydroxyl group. 11. A reactive solid-phase carrier having a group of vinylsulfonyl groups or reactive precursor groups thereof fixed to each surface by covalent bonds on the surface of a solid-phase carrier made of glass, silicon, or metal. A nucleotide derivative or a derivative thereof having a reactive group that forms a covalent bond by reacting with the reactive group on the surface is brought into contact with the nucleotide derivative through a linking group having a sulfonyl group. A method for producing a solid-phase carrier in which an analogue is bound and immobilized on the surface of the carrier. 12. The method according to claim 11, wherein the nucleotide derivative or its analog is selected from the group consisting of oligonucleotides, polynucleotides and peptide nucleic acids. 13. A reactive solid-phase carrier having a vinylsulfonyl group represented by the following formula or a linking group of a reactive precursor group thereof and a linking group bonded and fixed, is used. Claim 11 or 12
The process according to: ## STR3 ## 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 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]. 14. 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 13. 15. A solid phase carrier having the nucleotide derivative or its analogue obtained by the method according to any one of claims 11 to 14 bound and immobilized on the surface of the carrier. 16. An oligonucleotide showing complementarity to the immobilized nucleotide derivative or its analogue in the presence of an aqueous medium, on the solid phase carrier to which the nucleotide derivative or its analogue of claim 15 is immobilized, or A method for binding and immobilizing complementary oligonucleotides or polynucleotides, which comprises contacting polynucleotides.