JP2001128697A - Dna fragment immobilized solid-phase carrier, method for immobilizing dna fragment and method for detecting nucleic acid fragment - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an immobilization method capable of bonding a previously prepared DNA fragment to the surface of a solid carrier by a quick reaction and making a reaction product stably maintain a bond, to obtain a DNA fragment immobilized solid-phase carrier and to provide a method for detecting a nucleic acid fragment. SOLUTION: This DNA fragment immobilized solid-phase carrier is characterized in that the DNA fragment is immobilized through a triazine group to the surface of a solid-phase carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a large number of DNs which are very useful for simultaneous analysis of gene expression, mutation, polymorphism, etc.
The present invention relates to a method for immobilizing DNA fragments on a solid-phase carrier surface, which is necessary for producing a high-density array (DNA chip) in which A fragments and oligonucleotides are arranged on a solid-phase surface.

[0002]

2. Description of the Related Art Technology for efficiently analyzing the functions of all genes of various organisms has been developed. DNA chips are
This is a microarray in which many DNA molecules are arranged on a solid support such as a slide glass, and is very useful for simultaneous analysis of gene expression, mutation, polymorphism, and the like. The DNA chip technology using the DNA chip is applicable to biomolecules other than DNA, and is expected to provide new means for drug discovery research, disease diagnosis and development of preventive methods, and the like.

[0003] DNA chip technology has been embodied in
Method for Determining DNA Base Sequence by Hybridization with Oligonucleotide (SBH: sequ
Encoding by hybridization was invented (Drmanac, R. et.
al. , Genomics, 4, page 114 (1
989)). Although SBH was a method capable of overcoming the limitations of base sequencing using gel electrophoresis, it did not reach practical use.

[0004] Subsequently, a DNA chip fabrication technique was developed, and so-called HTS (high-throughput), which efficiently and efficiently examines gene expression, mutation, polymorphism and the like in a short time.
t screening) has become possible (Fodo
r, S. P. A. , Science, 251, page
767 (1991) and Schena, M .; , Sc
issue, 270, page 467 (199
5)).

[0005] However, in order to put the DNA chip production technology into practical use, a DNA chip production technology for aligning a large number of DNA fragments and oligonucleotides on the surface of a solid support is required. DNA on prepared DNA chip
Fragments are generally detected by hybridization with a labeled sample nucleic acid fragment.

As a method for producing a DNA chip, a method of directly synthesizing a DNA fragment on the surface of a solid support (referred to as an “on-chip method”), a method of immobilizing a DNA fragment prepared in advance on the surface of a solid support, It has been known. The on-chip method combines the use of protecting groups that are selectively removed by light irradiation with the photolithography and solid-phase synthesis techniques used in semiconductor manufacturing to define a specific area of a tiny matrix. A method of performing selective synthesis (referred to as “masking technique”) is typical.

A method for immobilizing a previously prepared DNA fragment on the surface of a solid support is as follows, depending on the type of the DNA fragment and the type of the solid support. (1) The DNA fragment to be fixed is cDNA (complementary DN synthesized using mRNA as a template).
In the case of A) or PCR products (DNA fragments obtained by amplifying cDNA by PCR), these are surface-coated with polycations (polylysine, polyethyleneimine, etc.) using a spotter device provided in a DNA chip producing device. It is common to spot onto the surface of the treated solid support and electrostatically bond to the solid support using the charge of DNA. As a method for treating the surface of the solid support, a method using a silane coupling agent having an amino group, an aldehyde group, an epoxy group, or the like is also used (Geo, Z. et al., Null).
eic Acids Research, 22,545
6-5465 (1994)). In this case, since amino groups, aldehyde groups, and the like are introduced into the surface of the solid support by covalent bonds, they are more stably present on the surface of the solid support than in the case of polycations.

As a modification of the method utilizing the charge of DNA, a PCR product modified with an amino group is suspended in SSC (standard saline-citrate buffer), and this is spotted on the surface of a silylated slide glass. After incubation, a method of sequentially performing a treatment with sodium borohydride and a heat treatment has been reported (Schena, M. eta).
l. Proc. Natl. Acad. Sci. USA
93, 10614-10619 (1996)). However, this fixing method has a problem that sufficient stability is not always obtained. In DNA chip technology, the detection limit is important. Therefore, stably immobilizing DNA in a sufficient amount on the surface of the solid support directly leads to improvement in the detection limit of hybridization between DNA and a labeled sample nucleic acid fragment.

(2) When the DNA fragment to be fixed is a synthetic oligonucleotide, an oligonucleotide having a reactive group introduced therein is synthesized, and the oligonucleotide is spotted on the surface-treated solid support and covalently bonded. ("Protein / Nucleic acid / Enzyme", Vol. 43, (1998), 2004-2)
011, Lamture, J.M. B et al. , Nu
cl. Acids Res. , 22,2121-212
5, 1994, and Guo, Z .; , Et al. , N
ucl. Acids Res. , 22,5456-54
65, 1994). For example, a method of reacting an amino group-introduced oligonucleotide with a slide glass having an amino group introduced thereto in the presence of PDC (p-phenylenediisothiocyanate) and a method of reacting an aldehyde group-introduced oligonucleotide with the slide glass are known. Have been. In the above two methods, the oligonucleotide is stably immobilized on the surface of the solid support as compared with the method (1) utilizing the charge of DNA. 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 using an aldehyde group-introduced oligonucleotide, the stability of the reaction product, the Schiff base, is low (in general, hydrolysis is difficult). Easily occur).

[0010]

DISCLOSURE OF THE INVENTION The present invention relates to an immobilization method capable of stably binding a previously prepared DNA fragment to the surface of a solid support by a rapid reaction, a DNA fragment-immobilized solid support, and a nucleic acid fragment. The challenge is to provide a method for detecting

[0011]

The present invention resides in a DNA fragment-immobilized solid phase carrier characterized in that a DNA fragment is immobilized on the surface of a solid phase carrier via a triazine group.

In the DNA fragment-immobilized solid phase carrier of the present invention, the triazine group is preferably immobilized on the surface of the solid phase carrier by an imino bond.

The present invention also provides (1) contacting a 2,4-dihalogenated triazine compound optionally having a substituent at the 6-position with a solid support having a reactive group introduced on its surface. Introducing a 4-halogenated triazinyl group which may have a substituent at the 6-position on the surface of the solid-phase carrier; and (2) introducing a 4-halogenated triazinyl group which may have a substituent at the 6-position. A step of sequentially condensing a halogen atom of a halogenated substituted triazinyl group with the reactive group of a DNA fragment having a reactive group at one end, wherein D
There are also methods for fixing NA fragments.

A preferred embodiment of the method for immobilizing a DNA fragment on the surface of a solid support according to the present invention is as follows. (A) after condensing a halogen atom of a 4-halogenated triazinyl group optionally having a substituent at the 6-position with the reactive group of a DNA fragment having a reactive group at one end, An anionic compound having an amino group is brought into contact with the surface of the solid support on which the fragments are condensed. (Ii) The solid support having the reactive group introduced therein is the solid support having the amino group introduced therein. (C) An amino group-introduced solid support is prepared by bringing a silane coupling agent having an amino group into contact with the surface of the solid support. (D) A DNA fragment whose base sequence is known is used.

The present invention further provides an aqueous solution obtained by dissolving or dispersing a nucleic acid fragment sample labeled with a fluorescent substance on the DNA fragment-immobilized solid phase carrier of the present invention, followed by incubating the DNA fragment. Detecting a hybrid DNA formed by a DNA fragment and a nucleic acid fragment sample immobilized on the fragment-immobilized solid phase carrier, wherein the hybrid DNA is complementary to the DNA fragment immobilized on the DNA fragment-immobilized solid phase carrier. There is also a method for detecting a nucleic acid fragment having a property.

[0016]

FIG. 1 schematically shows a solid support for immobilizing DNA fragments and a method for immobilizing DNA fragments according to the present invention.
"DNA fragment" means a DNA fragment having no active group, unless otherwise specified.

The DNA fragment-immobilized solid support of the present invention (C)
Is a solid support on which a DNA fragment is immobilized via a triazine group. The DNA fragment-immobilized solid support (C) is obtained by bringing the triazine compound (1) into contact with the active group-introduced solid support (A) in which the active group (R) is introduced on the surface of the solid support 1. Triazine fixed solid support (B)
Then, a DNA fragment (2) having an active group (Z) at one end is allowed to act. In practice, one of the halogen atoms (X) bonded to the triazine group reacts with the active group (R) to form a covalent bond, whereby the triazine compound (1) is bonded to the solid support 1. Here, L ¹ is determined by the reaction between the active group (R) and the halogen atom (X), and Z ¹ is determined by the reaction between the halogen atom (X) and the active group (Z). -Phosphate group -NNNN ... NN is D
Represents the NA fragment. Y represents a crosslinker. DNA
The fragment (2) may not have the crosslinker (Y), but for the sake of preparation of the DNA fragment (2) having the active group (Z) at one end, the active group (Z) and the linker are not used. It is generally present between an acid ester group.

Hereinafter, each step of FIG. 1 will be described.

As the solid phase carrier, a carrier having low hydrophobicity or low hydrophilicity is preferable. In addition, even if the surface has unevenness and low flatness, it can be preferably used. Materials for the solid support include ceramics or new ceramics such as glass, cement, and porcelain, polymers such as polyethylene terephthalate, cellulose acetate, bisphenol A polycarbonate, polystyrene, and polymethyl methacrylate, silicon, activated carbon, porous glass, and porous materials. Examples include ceramics, porous silicon, porous activated carbon, woven or knitted fabric, nonwoven fabric, filter paper, short fibers, and porous materials such as membrane filters. The pore size of the porous material is preferably in the range of 2 to 1000 nm, and 2 to 50 nm.
It is particularly preferred that it is in the range of 0 nm. The material of the solid support is particularly preferably glass or silicon. This is due to the ease of surface treatment and the ease of analysis by electrochemical methods. The thickness of the solid support is 1
It is preferably in the range of 00 to 2000 μm.

The surface of the solid support is preferably a polycation (for example, poly-L-lysine, polyethyleneimine, polyalkylamine, etc.) for immobilizing DNA fragments, and is poly-L-lysine. (In this case, the active group (R) introduced into the surface of the solid support is an amino group) or a silane having an active group (R) introduced into the surface of the solid support. The contact treatment is preferably performed with a coupling agent, and particularly preferably the contact treatment is performed with a silane coupling agent having an active group (R). The active group (R) is preferably an amino group. In the case of a polycation, the amino group is introduced to the surface of the solid support by electrostatic bonding, whereas in the case of a silane coupling agent, the amino group is introduced by a covalent bond. Stable on the carrier surface. In addition to the amino group, an aldehyde group, an epoxy group, a carboxyl group, a hydroxyl group or a thiol group can be preferably introduced.

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

The treatment using a polycation may be combined with the treatment using a silane coupling agent. It is possible to promote electrostatic interaction between the DNA fragment and the solid support having low hydrophobicity or low hydrophilicity. A layer made of a charged hydrophilic polymer or the like or a layer made of a cross-linking agent may be further provided on the surface of the solid support that has been subjected to the polycation treatment. By providing such a layer, it is possible to reduce unevenness of the solid support that has been subjected to the polycation treatment. Depending on the type of the solid phase carrier, a hydrophilic polymer or the like can be contained in the carrier, and a solid phase carrier that has been subjected to such treatment can be preferably used.

The triazine compound used in the present invention is represented by the following formula (I).

[0024]

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In the above compound, R ¹ is a hydrogen atom,
Halogen atom (F, Cl, Br, etc.), hydroxyl group, -OM,
Alkoxy group of 1 to 20 carbon atoms, an alkyl group having 1 to 20 carbon atoms, -NR ² R ³ and -NHCOR ⁴
Represents an atom or group selected from the group consisting of X is
It represents a halogen atom and is preferably F, Cl or Br, and particularly preferably Cl.

M is an alkali metal atom (Na, K, etc.),
Alternatively, an ammonium group which may have a substituent
Preferably. Ammonium which may have a substituent
The NH (C_TwoH_Five)_Three, NH (CH_TwoCH
OHCH_Three)_Three, NH_ThreeC_TwoH_Five, NH_Three(N-C_ThreeH₇), N
H_Three(N-C_FourH₉), NH_Two(C_TwoH_TwoOH)_Two, NH
_Two(C_TwoH_Five)_Two, NH_Two(I-C_ThreeH₇)_Two, NH_Two(N-
C_ThreeH₇)_Two, NH_TwoCH_Three(N-C_FourH₉) , NH_Two(CH
_Three)_Two, NH_Two(N-C_FourH₉)_Two, NHC_TwoH_Five(I-C _Three
H₇) Or NHCH_Three(N-C_FourH₉)_TwoTo mention
Can be. M is a sodium atom or potassium
Particularly preferred is an atom. 1 to 20 carbon atoms
An alkoxy group and an alkyl having 1 to 20 carbon atoms
Any of the groups may have a substituent. Above alkoki
As the methoxy group, methoxy, ethoxy, n-propoxy,
n-butoxy group, benzyloxy group, or n-dec
And a luoxy group. The above alkyl group
Methyl, ethyl, n-propyl, n-butyl
Tyl, n-hexyl, n-dodecyl, methoxy
A butyl group or a butoxyethyl group;
You. Examples of the alkoxy group include a methoxy group and an ethoxy group.
Group or 2-methoxyethoxy group
Preferably, the alkyl group is a methyl group or
Particularly preferred is an n-butyl group.

R ² and R ³ independently represent a hydrogen atom, an alkyl group having 1 to 20 carbon atoms, or an aryl group having 6 to 20 carbon atoms. Both the alkyl group and the aryl group may have a substituent. Preferred alkyl groups are the same as the above-described alkyl groups. Preferred examples of the aryl group include a phenyl group, a p-tolyl group, and a p-methoxyphenyl group. -NR ² Preferred specific examples of R ³ include, -NH _2, -NHCH ₃ or -NHC ₂
H ₅ can be mentioned.

R ⁴ is a hydrogen atom, having 1 to 20 carbon atoms.
An aryl group having 6 to 20 carbon atoms, an alkylthio group having 1 to 20 carbon atoms, or an arylthio group having 6 to 20 carbon atoms. The alkyl group, the aryl group, the alkylthio group and the arylthio group each may have a substituent, and the preferred alkyl group and aryl group are the same as the above-described alkyl group and aryl group, respectively. . -NH
Preferred specific examples of COR ⁴ include —NHCOCH ₃ ,
Or it can be mentioned -NHCOC ₆ H _5.

Examples of the substituent which R ¹ , R ² , R ³ and R ⁴ may have include an alkoxy group having 1 to 12 carbon atoms, an alkylamide group having 1 to 12 carbon atoms and a carbon atom having 1 to 12 carbon atoms. An alkylsulfonamide group having 1 to 12 carbon atoms, an alkoxycarbonyl group having 2 to 13 carbon atoms, and 6 carbon atoms
To 20 arylamide groups, an arylsulfonamide group having 6 to 20 carbon atoms, an aryloxycarbonyl group having 7 to 21 carbon atoms, a hydroxyl group, a nitro group, a halogen atom, and an atom selected from the group consisting of a carboxylic acid group and Group.

Representative examples of the triazine compound preferably used in the present invention are shown below.

[0031]

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[0032]

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[0033]

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[0034]

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As the triazine compound, (T1)
Most preferably, cyanuric chloride represented by the following formula is used.

The synthesis method of the triazine compound (I) used in the present invention is described in JP-B-47-6151 and JP-B-47-151.
33380, 54-25411, JP-A-56-1
Details are described in each gazette such as No. 30740. Also, JP-B-53-2726, JP-A-50-61219, JP-A-56-27135, JP-A-56-60430, and JP-A-57-260, which have a structure similar to that of the triazine compound (I).
The compounds described in Japanese Patent No. 40244 can also be used effectively in the present invention.

In the present invention, the triazine compound (I)
May be used as a mixture of two or more.

In the fixing method of the present invention, a 2,4-dihalogenated triazine optionally having a substituent at the 6-position is added to the solid support (A) having a reactive group (R) introduced on its surface. Contacting the solid support (A) to introduce a 4-halogenated triazinyl group which may have a substituent at the 6-position, and a halogen atom of the 4-halogenated triazinyl group and DNA The step of condensing the fragment (2) with the reactive group (Z) is performed sequentially. Here, the triazine compound 6
The substituent at the position means R ¹ in the general formula (I). As shown in FIG. 1, a halogenated triazine is covalently bonded to the solid support 1 and has a D group having an active group (Z) at one end.
Since the NA fragment (2) also covalently binds, the immobilized DNA
The fragments have high stability. As described above, since the active group (R) is particularly preferably an amino group,
It is preferable that the triazine-immobilized solid phase carrier is one in which a halogenated triazine and a solid phase carrier are bound via an imino group (-NH-).

The DNA fragment can be divided into two types depending on the purpose. To examine gene expression, the cD
It is preferable to use a polynucleotide such as NA, a part of cDNA, or EST. These polynucleotides may be of unknown function, but are generally amplified by PCR using a cDNA library, genomic library or whole genome as a template based on sequences registered in a database. (Hereinafter, referred to as “PCR product”). Those not amplified by the PCR method can also be preferably used. In addition, in order to examine gene mutations and polymorphisms, it is preferable to synthesize various oligonucleotides corresponding to the mutations and polymorphisms based on a known sequence as a standard, and use them. Furthermore, in the case of nucleotide sequence analysis, it is preferable to use 4 ⁿ (n is the length of a base) synthesized oligonucleotides. 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.

An active group (Z) is preferably introduced at one end of the DNA fragment in order to condense with the halogen atom (X) of the triazinyl group. In the description of FIG. 1 described above, a DNA fragment having an active group (Z) is shown. Such an active group (Z) includes an amino group, an imino group,
It is preferably a hydrazino group, a carbamoyl group, a hydrazinocarbonyl group, or a carboximide group, and particularly preferably an amino group.

The crosslinker (Y) is preferably an alkylene group or an N-alkylamino-alkylene group, more preferably a hexylene group or an N-methylamino-hexylene group, and particularly preferably a hexylene group. preferable.

The DNA fragment can be spotted by dissolving or dispersing the DNA fragment in an aqueous medium in 96 wells or 3 wells.
The dispensing is preferably performed by dispensing into an 84-well plastic plate and dropping the dispensed aqueous liquid onto the surface of the solid support using a spotter or the like.

When the DNA fragment was spotted on the surface of the solid support (B) in FIG. 1, the halogen atom (X) of the triazinyl group was
Condensation reaction with the DNA fragment (2) occurs, but unreacted triazinyl groups to which the DNA fragment (2) does not condense also exist on the surface of the solid support (B). Since such a triazinyl group may cause a non-specific reaction in hybridization with a nucleic acid fragment sample, the triazinyl group (that is, the halogen atom (X) of the triazinyl group) is masked beforehand. Preferably. The mask treatment is preferably performed by bringing an anionic compound having an amino group into contact with the surface of the solid support (C). Since the DNA fragment (2) has a negative charge,
This is to prevent the DNA fragment (2) from reacting with the unreacted triazinyl group by generating a negative charge also on the surface of the solid support (C). As such an anionic compound, it reacts with an unreacted halogen atom (X) of a triazinyl group and has a negative charge (COO ⁻ , SO ₃ ⁻ ,
Any compound having OSO ₃ ⁻ , PO ₃ ⁻ , or PO ₂ ⁻ ) can be used, but is preferably an amino acid, and particularly preferably glycine. Taurine can also be preferably used.

In order to prevent drying of the DNA fragment after spotting,
A substance having a high boiling point may be added to the aqueous liquid in which the DNA fragment is dissolved or dispersed. As a substance with a high boiling point,
It is preferably a substance which can be dissolved in an aqueous liquid in which a DNA fragment is dissolved or dispersed, and which does not hinder hybridization with a sample nucleic acid fragment and has low viscosity. Such materials can include glycerin, ethylene glycol, dimethyl sulfoxide and low molecular weight hydrophilic polymers. Examples of the hydrophilic polymer include polyacrylamide, polyethylene glycol, and sodium polyacrylate. The molecular weight of the polymer is preferably in the range of 10 ^{3 to} 10 ⁶ . As the substance having a high boiling point, glycerin or ethylene glycol is more preferably used, and glycerin is particularly preferably used. The concentration of the substance having a high boiling point is determined in the aqueous liquid of the DNA fragment.
It is preferably in the range of 0.1 to 2% by volume,
It is particularly preferred that it is in the range of 5 to 1% by volume. For the same purpose, it is also preferable to place the solid phase carrier after spotting the DNA fragment in an environment with a humidity of 90% or more and a temperature range of 25 to 50 ° C.

After spotting the DNA fragment, post-treatment with ultraviolet light, sodium borohydride or Schiff's reagent may be performed. These post-treatments may be performed in combination of a plurality of types, and particularly preferably performed in combination of the heat treatment and the ultraviolet treatment. These post-treatments are particularly effective when the surface of the solid support is treated only with the polycation. After spotting, it is also preferable to carry out incubation. After the incubation, it is preferable to wash and remove unspotted DNA fragments.

The DNA fragment is applied to the surface of the solid support at 1
It is preferably in the range of 0 ^{2 to} 10 ⁵ types / cm ² . The amount of the DNA fragment is in the range of 1 to 10 ^-15 mol, and the weight is preferably several ng or less. By spotting, the aqueous liquid of the DNA fragment is fixed in the form of dots on the surface of the solid support. The shape of the dot is almost circular. The absence of variation in shape is important for quantitative analysis of gene expression and analysis of single nucleotide mutation. The distance between the dots is preferably in the range of 0 to 1.5 mm, 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 spotting is
It is preferably in the range of 100 pL to 1 μL,
It is particularly preferred that it is in the range of 100 to 100 nL.

The life span of the DNA fragment-immobilized solid phase carrier produced by the above-described process is determined by the cD in which the cDNA is immobilized.
It takes several weeks with the NA-immobilized solid phase carrier and even longer with the oligo DNA-immobilized solid phase carrier on which the oligo DNA is immobilized. These DNA fragment-immobilized solid supports are used for monitoring gene expression, determining the base sequence, analyzing mutations, analyzing polymorphisms, and the like. The principle of detection is hybridization with a labeled sample nucleic acid fragment described below.

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

As a sample nucleic acid fragment, it is preferable to use a DNA fragment sample or an RNA fragment sample whose sequence or function is unknown. The sample nucleic acid fragment is preferably isolated from eukaryotic cell or tissue samples for the purpose of examining gene expression. If the sample is a genome, it is preferably isolated from any tissue sample except red blood cells. Preferably, any tissue other than red blood cells is peripheral blood lymphocytes, skin, hair, semen and the like. If the sample is mRNA, it is preferably extracted from a tissue sample in which mRNA is expressed. mRNA is labeled dN by reverse transcription reaction.
It is preferable that TP ("dNTP" means a deoxyribonucleotide whose base is adenine (A), cytosine (C), guanine (G) or thymine (T)) is incorporated into a labeled cDNA. As dNTP, it is preferable to use dCTP for chemical stability. MRN required for one hybridization
The amount of A varies depending on the amount of the liquid and the labeling method, but is preferably several μg or less. When the DNA fragment on the DNA fragment-immobilized solid phase carrier is an oligo DNA, it is desirable that the sample nucleic acid fragment be reduced in molecular weight. In prokaryotic cells, it is difficult to selectively extract mRNA, so that
A is preferably labeled. For the purpose of examining gene mutation or polymorphism, the sample nucleic acid fragment is preferably obtained by PCR of the target region in a reaction system containing a labeled primer or a labeled dNTP.

In the hybridization, an aqueous liquid, in which a labeled sample nucleic acid fragment is dissolved or dispersed, dispensed into a 96-well or 384-well plastic plate is placed on the DNA fragment-immobilized solid support prepared above. It is preferable to carry out by spotting. The amount of spotting is
It is preferably in the range of 1 to 100 nL. Hybridization is preferably carried out in a temperature range from room temperature to 70 ° C. and for a time in the range from 6 to 20 hours.
After the completion of the hybridization, it is preferable to wash with a mixed solution of a surfactant and a buffer to remove unreacted sample nucleic acid fragments. It is preferable to use sodium dodecyl sulfate (SDS) as the surfactant. Buffers include citrate buffer, phosphate buffer,
A borate buffer, a Tris buffer, a Good buffer, and the like can be used, but a citrate buffer is particularly preferable.

The feature of the hybridization using the DNA fragment-immobilized solid phase carrier is that the amount of the labeled sample nucleic acid fragment used is extremely small. Therefore, it is necessary to set optimal conditions for hybridization depending on the chain length of the DNA fragment immobilized on the solid support and the type of the labeled sample nucleic acid fragment. For gene expression analysis, it is preferable to perform hybridization for a long time so that low-expressed genes can be sufficiently detected. For detection of a single nucleotide mutation, it is preferable to perform hybridization for a short time.
Another feature is that by preparing two types of sample nucleic acid fragments labeled with different fluorescent substances and simultaneously using them for hybridization, the expression levels can be compared and quantified on the same DNA fragment-immobilized solid phase carrier.

[0052]

[Example 1] Production of DNA fragment-immobilized solid phase carrier
And method for measuring the amount of immobilized DNA fragment
Is represented by the reaction pathway of the DNA fragment,
The reaction route is shown in FIG. In the figure, 1 is a slide glass
Represent. (1) Preparation of solid support (A) coated with poly-L-lysine
Add sodium hydroxide (50 g) to distilled water (150 mL)
Slurry in ethanol and ethanol (200 mL).
Id glass (25mm x 75mm) for 1 hour
And washed with ethanol. Then 10% by volume
Soaked in poly-L-lysine (Sigma) aqueous solution for 1 hour
Remove afterwards, centrifuge in plate centrifuge and dry at room temperature
did. (2) Preparation of triazine-immobilized solid support (B) Solid support coated with poly-L-lysine obtained in (1) above
(A) was treated with 10% by volume of cyanuric chloride (100 μg / m
L) Acetone-0.1 M phosphate buffer (pH 7.0)
Dipped in the mixed solution (100 mL) for 20 minutes and removed
Thereafter, the plate was washed with 0.1 M phosphate buffer (pH 7.0),
Dry for 3 hours under reduced pressure to prepare the triazine-immobilized solid support (B)
did. (3) Spotting of DNA Fragments and Measurement of Fluorescence Intensity The 3 'end and 5' end are amino groups and fluorescent labels, respectively.
Reagent (FluoroLink Cy5-dCTP, Ama
Siam Pharmacia Biotech)
DNA fragment (3'-CTAGTCTGTGGAAGTG
TCTGATC-5 ') in 0.1 M carbonate buffer (pH
9.3) aqueous liquid (1 × 10 ^-6M, 1μ
L) is the triazine-immobilized solid support obtained in (2) above.
(B) was spotted. Immediately, 25 drops of solid support after spotting
After standing at 90 ° C. and 90% humidity for 1 hour,
0.1% by weight SDS (sodium dodecyl sulfate) and 2 ×
SSC (2 × SSC: A solution obtained by diluting the stock solution of SSC two-fold
Solution, SSC: mixed solution with standard salt-citrate buffer)
And twice with a 0.2 × SSC aqueous solution. Next
Then, the solid support after the above-mentioned washing is dissolved in 0.1 M glycine aqueous solution.
After immersion in liquid (pH 10) for 1 hour 30 minutes, distilled water
And dried at room temperature.
(C) was obtained. The fluorescence intensity on the surface of the solid support (C)
It was 1048 when measured with a fluorescent scanning device.
Was. By the immobilization method of the present invention, DNA fragments can be efficiently used.
It can be seen that it was fixed to the slide glass.

Example 2 Detection of Sample DNA Fragment (1) Preparation of DNA Fragment-Immobilized Solid Phase Carrier The procedure of Example 1 was repeated except that a DNA fragment whose 3 ′ end was not modified with a fluorescent labeling reagent was used. A solid support (C ′) on which the DNA fragment was immobilized was obtained. (2) Detection of Sample DNA Fragment A 22-mer sample oligonucleotide (GATCAGACACTTCACAGAC) with Cy5 bound to the 5 'end
TAG-5 ′) with a hybridization solution (4 ×
A mixed solution of SSC and 10% by weight of SDS) (20 μl)
What was dispersed in L) was spotted on the solid support (C ′) obtained in the above (1), the surface was protected with a microscope cover glass, and then incubated at 60 ° C. for 20 hours in a Moisture chamber. did. Then, this is 0.1% by weight SD
After sequentially washing with a mixed solution of S and 2 × SSC, a mixed solution of 0.1% by weight SDS and 0.2 × SSC, and a 0.2 × SSC aqueous solution, centrifuged at 600 rpm for 20 seconds and dried at room temperature. did. The fluorescence intensity on the surface of the solid support was measured by a fluorescence scanning apparatus and was found to be 455. By using the DNA fragment-immobilized solid phase carrier prepared by the immobilization method of the present invention, a sample DN having complementarity with the DNA fragment immobilized on the DNA fragment-immobilized solid phase carrier is obtained.
It can be seen that the A fragment can be detected.

[0054]

According to the present invention, DNA can be deposited on the surface of a solid support.
The fragments can be fixed stably and quickly. In particular, when an amino group is introduced to the surface of a solid support using a silane coupling agent, both the amino group binding to the solid support surface and the DNA fragment binding are covalent bonds. The fragments can be fixed. The stable immobilization of DNA fragments leads to the preparation of a DNA fragment-immobilized solid phase carrier having a high detection limit that can be effectively used for gene analysis and the like. As one example, by using the DNA fragment-immobilized solid phase carrier prepared according to the present invention and performing hybridization with a sample nucleic acid fragment, the DNA fragment immobilized on the DNA fragment-immobilized solid phase carrier is complemented. Can be detected with high sensitivity. In addition, after the DNA fragment is spotted, the non-specific adsorption of the sample nucleic acid fragment can be prevented by treating the surface of the solid support with an anionic compound such as glycine. Effective for high-sensitivity detection.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a typical solid support for a DNA fragment of the present invention and a method for immobilizing a typical DNA fragment of the present invention.

FIG. 2 shows the method of bringing a triazine-immobilized solid-phase carrier into contact with a fluorescently labeled DNA fragment having an amino group at one end thereof.
It is a schematic diagram which shows the method of fixing an NA fragment.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 C12N 15/00 ZNAA (72) Inventor Osamu Seshmoto 3-11- Izumi, Asaka-shi, Saitama 46 Fuji Photo Film Co., Ltd. F-term (reference) 4B024 AA11 AA20 CA01 CA09 HA09 HA14 4B029 AA07 AA21 AA23 BB20 CC03 CC13 FA15 4B063 QA01 QA11 QQ42 QR32 QR56 QR83 QS03 QS34 QS36 QS39 QX02

Claims (8)

[Claims] 1. A DNA fragment-immobilized solid phase carrier comprising a DNA fragment immobilized on a surface of a solid phase carrier via a triazine group. 2. The method according to claim 1, wherein the triazine group is immobilized on the surface of the solid support by an imino bond.
2. The solid support for immobilizing a DNA fragment according to item 1. (1) A 2,4-dihalogenated triazine compound which may have a substituent at the 6-position is brought into contact with a solid support having a reactive group introduced on its surface, and Introducing a 4-halogenated triazinyl group optionally having a substituent at the 6-position onto the surface of the carrier; and (2) a 4-halogenated triazinyl group optionally having a substituent at the 6-position And a DN having a reactive group at one end
A method for immobilizing a DNA fragment on the surface of a solid support, comprising sequentially performing a step of condensing the reactive group of the A fragment with the reactive group. 4. The method according to claim 3, wherein after the step (2), an anionic compound having an amino group is brought into contact with the surface of the solid support on which the DNA fragment is condensed. Fixing method. 5. The method for immobilizing a DNA fragment according to claim 3, wherein the solid support having the reactive group introduced therein is a solid support having an amino group introduced therein. 6. The solid support to which an amino group has been introduced, which is produced by bringing a silane coupling agent having an amino group into contact with the surface of the solid support. The method for immobilizing a DNA fragment according to the above. 7. The method for immobilizing a DNA fragment according to claim 3, wherein a DNA fragment whose base sequence is known is used. 8. An aqueous solution obtained by dissolving or dispersing a nucleic acid fragment sample labeled with a fluorescent substance on the DNA fragment-immobilized solid phase carrier according to claim 1 or 2, and then incubating the DNA. Detecting a hybrid DNA formed by a DNA fragment and a nucleic acid fragment sample immobilized on the fragment-immobilized solid phase carrier, wherein the hybrid DNA is complementary to the DNA fragment immobilized on the DNA fragment-immobilized solid phase carrier. A method for detecting a nucleic acid fragment having a property.