JP2007078399A - Solid support and DNA chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a solid support body for manufacturing a DNA chip (gene detecting microarray) having a high immobilization amount and high coupling strength of a DNA or an oligonucleotide. <P>SOLUTION: This solid support body of the present invention is covalent-bonded with a group expressed by a general formula (1): -NH-CO-X-OY on a carrier surface. X represents a 1-50 C of alkylene group allowing substitution with an alkyl group, or a group expressed by a general formula (2): -(R<SP>1</SP>-R<SP>2</SP>)<SB>n</SB>-, and Y represents hydrogen or dimethoxy trityl group, in the general formula (1). R<SP>1</SP>represents a methylene group allowing substitution with an alkyl group, O or NR<SP>3</SP>; R<SP>2</SP>represents a methylene group allowing substitution with an alkyl group; R<SP>3</SP>represents hydrogen or an alkyl group; and n represents 1-25 of integer, in the general formula (2). <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a solid support, and specifically to a solid support for immobilizing nucleic acid molecules. In particular, the present invention relates to a solid support for producing a DNA chip having a high immobilized amount of nucleic acid molecules and high binding strength.
The present invention also relates to a DNA chip and a gene detection microarray using the solid support.

  In recent years, technological development for analyzing all gene functions of living organisms has progressed. As an analysis means for such development, a DNA chip has been used. A DNA chip is usually in the form of a microarray in which a large number of DNA fragments are immobilized on a solid support such as a slide glass, and a DNA fragment sample that is complementary to the DNA fragments immobilized on the DNA chip is hybridized. To be immobilized on a DNA chip and used as a detection method. As means for detecting the formed hybrid, a method using a fluorescent label or a radioactive label previously bound to a DNA fragment sample, a method using an intercalator having a fluorogenic group or a conductive group to be incorporated into the hybrid, etc. are known. ing.

  In order to put the technique using a DNA chip as described above into practical use, a technique for immobilizing many DNA fragments and oligonucleotides on the surface of a solid support is necessary. As a method for immobilizing DNA fragments and oligonucleotides on the surface of a solid support, for example, the method described in Non-Patent Document 1 is known.

  The method described in Non-Patent Document 1 uses a silane coupling agent containing an ethylene glycol linker to treat the surface of glass, which is a solid support, and synthesizes a DNA fragment or oligonucleotide using this linker as a foothold. Is the method. In the linker included in the DNA chip obtained by the method described in Non-Patent Document 1, DNA (oligonucleotide) is used during ammonia treatment when immobilizing DNA (oligonucleotide) or during subsequent chemical treatment. ) Would cause a significant desorption.

Nucleic Acid Resarch Vol.20, No.7, 1679-1687 (1992)

  As described above, the conventional DNA chip is not necessarily sufficient in the amount of DNA or oligonucleotide immobilized on the solid support and the binding strength, but the amount of DNA or oligonucleotide immobilized is higher, It has been desired to develop a DNA chip having a higher binding strength of DNA or oligonucleotide.

Accordingly, an object of the present invention is to provide a solid support for producing a DNA chip having a high immobilized amount of DNA or oligonucleotide and a high binding strength.
Another object of the present invention is to provide a DNA chip and a gene detection microarray having a high immobilized amount of DNA or oligonucleotide and a high binding strength.

In order to achieve the above object, as a result of intensive studies, the present inventors have found that a solid support obtained by covalently bonding a group (linker) having a specific structure can achieve the above object.
The present invention has been made based on the above findings, and provides a solid support in which a group represented by the following general formula (1) is covalently bonded to the surface of a carrier.
-NH-CO-X-OY (1)
(In the general formula (1), X represents an alkylene group having 1 to 50 carbon atoms which may be substituted with an alkyl group, or a group represented by the following general formula (2), and Y represents Represents hydrogen or dimethoxytrityl group.)
-(R ¹ -R ² ) _n- (2)
(In the general formula (2), R ¹ represents a methylene group optionally substituted with an alkyl group, O or NR ³ , R ² represents a methylene group optionally substituted with an alkyl group, and R ³ represents Represents hydrogen or an alkyl group, and n represents an integer of 1 to 25.)

  The present invention also provides a DNA chip and a gene detection microarray in which a nucleic acid molecule is bonded to a hydroxyl group on the solid support.

  ADVANTAGE OF THE INVENTION According to this invention, the solid support body which can manufacture the DNA chip and the microarray for gene detection with the fixed amount and binding strength of DNA or oligonucleotide are obtained.

Hereinafter, the solid support of the present invention will be described first.
The solid support of the present invention is a solid support for immobilizing nucleic acid molecules, and can be used for producing a DNA chip (microarray for gene detection) as described later.
The solid support of the present invention is formed by covalently bonding a group represented by the following general formula (1) to the surface of a carrier.
-NH-CO-X-OY (1)
In the general formula (1), (i) X represents an alkylene group having 1 to 50 carbon atoms which may be substituted with an alkyl group, or (ii) represented by the following general formula (2). And Y represents hydrogen or a dimethoxytrityl group.

First, the case (i) will be described.
In the above general formula (1), X is an alkylene group which may be substituted with an alkyl group. The alkylene group is an alkylene group having 1 to 50 carbon atoms, preferably 10 to 25 carbon atoms. When the number of carbon atoms of the alkylene group is less than 1, the amount of DNA or oligonucleotide (hereinafter also simply referred to as “nucleic acid”) immobilized when a DNA chip (microarray for gene detection) is prepared. On the other hand, if the alkylene group has more than 50 carbon atoms, it becomes difficult to obtain the reaction material.

  Moreover, as said alkyl group, a C1-C5 alkyl group is mentioned, for example, For example, a methyl group, an ethyl group, n-propyl group, isopropyl group, n-butyl group, isobutyl group, tert- A butyl group, n-pentyl group, isopentyl group, etc. are mentioned.

Next, the case (ii) will be described. In the case of (ii), X is a group represented by the following general formula (2).
-(R ¹ -R ² ) _n- (2)
In the general formula (2), R ¹ represents a methylene group optionally substituted with an alkyl group, O or NR ³ , R ² represents a methylene group optionally substituted with an alkyl group, and R ³ represents hydrogen Or an alkyl group is represented, n represents the integer of 1-25.
Examples of the alkyl group in the general formula (2) include those described above, and examples thereof include an alkyl group having 1 to 5 carbon atoms, such as a methyl group, an ethyl group, an n-propyl group, and an isopropyl group. , N-butyl group, isobutyl group, tert-butyl group, n-pentyl group, isopentyl group and the like. Moreover, n represents the integer of 1-25, Preferably it is an integer of 2-10. When n is smaller than 1, as in the case of (i), when a DNA chip (gene detection microarray) is prepared, DNA or oligonucleotide (hereinafter, also simply referred to as “nucleic acid” in this specification) On the other hand, when the number of carbon atoms of the alkylene group is larger than 25, the reaction material is difficult to obtain.

Examples of the group represented by the general formula (1) in the solid support of the present invention include, but are not limited to, those represented by the following formula (3).
—NH—CO— (CH ₂ ) ₁₅ —OH (3)

  The solid support of the present invention is formed by covalently bonding the group represented by the general formula (1) described above to the support surface. As the carrier to be used, those conventionally used for producing a DNA chip and a gene detection microarray can be used without particular limitation. Examples of the carrier to be used include silicon; glass such as microporous glass and porous glass; metal; magnetic beads whose surface is covered with glycine methacrylate with ferrite as a core; plastic (for example, polyester resin, polyethylene resin, polypropylene resin, Acrylonitrile butadiene styrene resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, vinyl chloride resin). The shape of the carrier may be any shape such as a plate shape (substrate shape), a bead shape, a thread shape, a spherical shape, a polygonal shape, and a powder shape.

  As the carrier, a microporous glass (CPG) is preferably used because a DNA chip (microarray for gene detection) produced using the solid support of the present invention can be used for the probe-on-carrier method. . The probe-on-carrier method means a technique used for detecting SNPs, for example, by using a DNA probe bound to a carrier without separating the probe molecule from the carrier. Glass is optimal. The size of CPG used is preferably a particle size of 500 to 5,000 mm. When using a plate-shaped carrier as the carrier, the width is usually about 0.1 to 100 mm, the length is about 0.1 to 100 mm, and the thickness is about 0.01 to 10 mm.

  The carrier may have a surface treatment layer formed of diamond-like carbon. Diamond-like carbon (DLC) is a general term for imperfect diamond structures that are a mixture of diamond and carbon, and the mixing ratio is not particularly limited. When forming the surface treatment layer by diamond-like carbon, the thickness of the layer is preferably 1 nm to 10 μm.

In order to covalently bond the group represented by the general formula (1) to the carrier, an amino group is preferably bound to the surface of the carrier. Therefore, it is preferable to use a carrier having an amino group bonded to the surface or capable of bonding an amino group.
There is no restriction | limiting in particular as a method to couple | bond an amino group with a support | carrier, A conventionally well-known method is mentioned, For example, the method of processing a support | carrier with a silane coupling agent is mentioned. A silane coupling agent refers to a compound having both an organic functional group capable of reacting with an organic compound such as a resin and a portion capable of binding an inorganic compound such as glass via a siloxane bond.

  Examples of the silane coupling agent used include γ-aminopropyltoximethoxysilane, N-β (aminoethyl) γ-aminopropyltrialkoxysilane, N-β (aminoethyl) γ-aminopropylmethyldialkoxysilane, γ-aminopropyltrialkoxysilane, γ-aminopropylmethyldialkoxysilane, N, N-dimethylaminopropyltrialkoxysilane, N-methylaminopropyltrialkoxysilane, N-phenyl-γ-aminopropyltrialkoxysilane, etc. Can be mentioned.

  When the carrier surface is treated with a silane coupling agent, the carrier is preferably washed in advance. As a method for cleaning the carrier, for example, many methods such as cleaning with water, cleaning with a chemical solution, cleaning with plasma, cleaning with UV ozone, and the like are known. Cleaning with is preferred. Although an appropriate cleaning method varies depending on the type of the carrier, for example, when glass is used as the carrier, a cleaning method using a sodium hydroxide aqueous solution having a predetermined concentration is preferable.

  The method for treating the carrier with a silane coupling agent is not particularly limited, and examples thereof include a dipping method (dipping method), a spin coating method, a spray coating method, a water surface casting method, and the like. An immersion method capable of uniform treatment is preferred. In this case, the treatment is preferably performed by immersing the carrier in a solution of the silane coupling agent having a concentration of about 0.05 to 2% by mass, and washing away the solution containing the excess silane coupling agent after the reaction is completed. The concentration and the coating method are not limited to these.

Next, the manufacturing method of the solid support body of this invention is demonstrated. Although there is no restriction | limiting in particular in the manufacturing method of the solid support body of this invention, Manufacturing with the following method is preferable.
The portion obtained by removing the amino group of the group represented by the general formula (1), which is a linker, is bonded to the carrier obtained by the above-described process and having the amino group bonded to the surface. Specifically, a compound represented by the following general formula (4) is reacted with triethylamine and 4,4′-dimethoxytrityl chloride to obtain a compound represented by the following general formula (5).
HO-CO-X-OY (4)
In the general formula (4), X and Y are the same as those in the general formula (1).
_{Et 3 N + HO-CO-} X-O-DMTr (5)
In the general formula (5), X is the same as in the general formula (1), Et represents an ethyl group, and DMTr represents a dimethoxytrimethyl group.

  The above reaction can be carried out in a solvent such as pyridine with stirring at room temperature (about 25 ° C.) for about 4 to 10 hours. The compound represented by the general formula (5) obtained by the above reaction can be purified by a purification method such as column chromatography, recrystallization, sublimation or the like.

Next, the compound represented by the general formula (5) obtained as described above is covalently bonded to the surface of the carrier. In order to bind the compound represented by the general formula (5) to the surface of the carrier, the compound represented by the general formula (5) is dissolved in a solvent such as dichloromethane, tetrahydrofuran, N, N-dimethylformamide, and the carrier is dissolved in this solvent. It can be carried out by dipping and stirring at room temperature (about 25 ° C.) for about 8 to 24 hours.
After the compound represented by the general formula (5) is bonded to the surface of the carrier, the solid support of the present invention in which Y is hydrogen can be obtained by removing the dimethyltrimethyl group. There is no restriction | limiting in particular in the method of removing a dimethyltrimethyl group, It can implement by a conventionally well-known method.

  Next, the DNA chip (or microarray for gene detection) of the present invention will be described. The DNA chip (or microarray for gene detection) of the present invention is formed by binding nucleic acid molecules to the hydroxyl groups on the solid support of the present invention described above. As the nucleic acid molecule, any of DNA, RNA and the like may be a nucleic acid molecule, and the type can be selected according to the purpose. In order to examine gene expression, it is preferable to select a polynucleotide such as cDNA, cDNA fragment, or EST. As the polynucleotide to be used, those whose function is unknown may be used. However, the polynucleotide is amplified by the PCR method using a cDNA library, genomic library or whole genome as a template based on the sequence registered in the database. May be prepared. In order to examine gene mutations and polymorphisms, various oligonucleotides corresponding to mutations and polymorphisms may be synthesized based on known standard sequences and used.

  Examples of a method for binding a nucleic acid molecule to a hydroxyl group or dimethoxytrityl group on a solid support include a method for binding a DNA prepared in advance and a method for direct synthesis on a solid support. Examples of the method for directly synthesizing DNA on a solid support include the phosphoramidite method. Although there is no particular limitation on the spot diameter of DNA on the surface of the carrier when a DNA chip (gene detection microarray) is produced, it is usually about 50 to 200 μm.

The DNA chip (microarray for gene detection) of the present invention can be used for a method for identifying a nucleic acid in a sample.
As a method, first, a sample is hybridized with the DNA chip of the present invention (gene detection microarray). In hybridization, a sample can be added to the nucleic acid fixed to the DNA chip (microarray for gene detection), for example, about 0.1 μM to 100 μM. Moreover, although the hybridization conditions differ depending on the type of nucleic acid, for example, at a temperature of 0 to 60 ° C., for example, about 1 to 30 minutes.

After hybridization is completed, washing is performed 2 to 5 times with a washing solution suitable for the type of DNA chip (microarray for gene detection). As described above, the DNA chip (microarray for gene detection) of the present invention can be used for nucleic acid identification and gene detection. Examples of the gene detection technique include real-time PCR and the like in addition to the above-described DNA chip and gene detection microarray, and the DNA chip (gene detection microarray) of the present invention can be used in the above-described technique.
The sample used in the above-described method is not particularly limited, and examples thereof include cell extracts, body fluids such as blood, PCR products, oligonucleotides, and the like.

Hereinafter, the present invention will be described in more detail with reference to examples. Needless to say, the scope of the present invention is not limited to such examples.
Example 1
Synthesis of triethylammonium 16-O- (4,4′-dimethoxytrityl) hexadecanoate Triethylamine (11 mmol, 1.5 mL) was dissolved in pyridine (100 ml) in which 16-hydroxyhexadecanoic acid (10 mmol, 2.7 g) was dissolved. ) And then 4,4′-dimethoxytrityl chloride (11 mmol, 3.7 g) was added and stirred at room temperature for 6 hours. Next, methanol (10 ml) was added and the mixture was stirred for 5 minutes. The reaction solution was diluted with ethyl acetate (500 ml), and then extracted with 500 ml of saturated brine three times. Next, the organic phase was recovered, and the recovered organic phase was dried over anhydrous sodium sulfate and filtered, and then the solvent was distilled off under reduced pressure to obtain a crude product. The obtained crude product was purified by silica gel column chromatography (hexane, ethyl acetate, 1% triethylamine) to obtain a 95% yield of triethylammonium 16-O- (4,4′-dimethoxytrityl) hexadecanoate. Got in.
¹ H NMR (CDCl ₃ ): 1.14 (t, 9H, J = 7.3 Hz), 1.21-1.45 (m, 28H), 2.21 (t, 2H, J = 7.6 Hz), 2.87 (dd, 6H, J = 7.3 Hz, 14.6 Hz), 3.01 (t, 2H, J = 8.9 Hz), 7.90 (d, 2H, J = 8.9 Hz),

Example 2
Microporous glass (CPG) having a pore size of 2000 mm was heated at 250 ° C. for 1 hour under reduced pressure. After replacing the system with argon gas, a mixed solvent of anhydrous toluene (99 mL), γ-aminopropyltrimethoxysilane (0.1 mL), and n-butanol (0.9 mL) was added. Subsequently, heating under reflux was performed for 15 minutes, the mixed solvent was removed by decantation, and CPG was washed twice with anhydrous toluene. Again, anhydrous toluene (100 mL) was added and heated to reflux for 12 hours, and then CPG was suction filtered and dried under reduced pressure to obtain an aminopropyl CPG solid phase carrier.

  The aminopropyl CPG solid phase carrier (500 mg 52 μmol) obtained as described above was sufficiently dried. Fully dried aminopropyl microporous glass (CPG) solid phase support, triethylammonium 16-O- (4,4′-dimethoxytrityl) hexadecanoate obtained in Example 1 (175 mg, 260 μmol) and Dicyclohexylcarbodiimide (DCC, 268 mg, 1.3 mmol) was dissolved in dichloromethane (5 mL) and stirred at room temperature for 12 hours for reaction. After completion of the reaction, the solid support was filtered, washed with acetonitrile, dried and added to a solution of acetic anhydride (0.5 ml) and DMAP (5 mg) dissolved in pyridine (4.5 mL). Next, the solution was stirred at room temperature (25 ° C.) for 3 hours, and then the solid support was filtered again and washed with acetonitrile to obtain a solid support. When the amount of linker introduced into the solid support was determined by colorimetric determination of the trityl group, the amount of linker introduced was 21 μmol / g. The density of dimethoxytrimethyl group of the obtained solid support was measured by DMTr cation assay and found to be 14.4 μmol / g.

Comparative Example 1
According to the method described in Non-Patent Document 1, it was introduced via a Southern type linker, and then a DMtr group was introduced to obtain a solid support. The linker introduced into the solid phase carrier is a group represented by the following formula (6).
_{-O-Si (R) 2 CH} 2 CH 2 CH 2 OCH 2 CH (OH) CH 2 - (OCH 2 CH 2) n -ODMTr (6)
In the above formula (6), R is a methoxy group and n is 6.
When the density of dimethoxytrimethyl groups in the obtained solid support was measured by DMTr cation assay, the density of DMTr groups was 10 μmol / g.

Example 3
The solid supports obtained in Example 2 and Comparative Example 1 were each immersed in 28% ammonia water and allowed to stand at room temperature (25 ° C.) for 8 hours. After 8 hours, the solid support was taken out from the ammonia water, and the density of dimethoxytrimethyl groups was measured for each. In the solid support obtained in Example 2, the density of dimethoxytrimethyl groups was reduced, that is, the elimination was 4%, whereas in the solid support obtained in Comparative Example 1, dimethoxytrimethyl groups were reduced. The decrease in density (desorption) was 96%. From this result, it can be seen that the solid support of the present invention is difficult to desorb the linker even by treatment with aqueous ammonia, that is, the solid support of the present invention has high immobilization amount and binding strength of nucleic acid molecules. Recognize. For this reason, the elimination of oligonucleotides is suppressed to a minimum even by chemical treatment. Therefore, when applied to a gene analysis device such as a DNA chip (microarray for gene detection), accuracy is greatly improved over the conventional one. And higher quality control is possible.

Example 4
A 14-mer DNA oligomer (DNA probe) was bound to the solid support obtained in Example 2 by a phosphoramidite method via a hexaethylene glycol linker, and a CPG (DNA chip) bound to the DNA probe. Got. As a protecting group for the amino group in the base part, a phenoxyacetyl group for adenine, an acetyl group for cytosine, a 4-isopropylphenoxyacetyl group for guanine, and a standard synthesis cycle of ABI392 was used in the chain extension process. .

The synthesized DNA probe is as follows.
Probe Wild: 5'-d (GCCTCCGGTTCAT) (SEQ ID NO: 1)
Probe HSC-4: 5'-d (GCCTCTGGTTCAT) (SEQ ID NO: 2)
Probe Ca-9: 5'-d (GCCTCCAGTTCAT) (SEQ ID NO: 3)
A diagram of the obtained probe is shown in the following formula (7).

Example 5
Three types of fluorescent oligonucleotides represented by the following SEQ ID NOs: 4 to 6 were prepared according to a conventional method. All of the fluorescent oligonucleotides represented by SEQ ID NOs: 4 to 6 have fluorescein bound to the 3 ′ end.
Fluorescent oligonucleotide Wild: GGCATGAACCGGAGGCCCAT (SEQ ID NO: 4)
Fluorescent oligonucleotide HSC-4: GGCATGAACCAGAGGCCCAT (SEQ ID NO: 5)
Fluorescent oligonucleotide Ca-9: GGCATGAACTGGAGGCCCAT (SEQ ID NO: 6)

  A solution (250 nM oligonucleotide, 100 mM phosphate buffer, 1 M sodium chloride, pH 7.0) of a fluorescent oligonucleotide having the base sequence represented by SEQ ID NOs: 4 to 6 was prepared. Next, the CPG to which the DNA probe obtained in Example 4 was bound was sufficiently dried, and then the CPG was immersed in 0.25 mL of the fluorescent oligonucleotide solution. After stirring the fluorescent oligonucleotide solution at a temperature of 50 ° C. for 10 hours, the fluorescent oligonucleotide solution was removed, and CPG was added to 0.25 mL of a phosphate buffer (100 mM phosphate buffer, 1M sodium chloride, pH 7.0), Washing was performed by stirring at a temperature of 50 ° C. for 1 hour. After completion of the washing, the phosphate buffer was removed and CPG fluorescence was measured.

  A combination of a DNA probe having the base sequence represented by SEQ ID NO: 1 and an oligonucleotide having SEQ ID NO: 4, a DNA probe having the base sequence represented by SEQ ID NO: 2 and an oligonucleotide having SEQ ID NO: 5 A combination of a DNA probe having a base sequence represented by SEQ ID NO: 3 and an oligonucleotide having a base sequence represented by SEQ ID NO: 6 is a perfect match of both and a DNA having a base sequence represented by SEQ ID NO: 1 The combination of the probe and the oligonucleotide having SEQ ID NO: 5 is a CA mismatch, the combination of the DNA probe having the base sequence represented by SEQ ID NO: 1 and the oligonucleotide having the SEQ ID NO: 6 is a GT mismatch, DNA pro having the base sequence represented by SEQ ID NO: 2 The combination of the DNA probe having the nucleotide sequence represented by SEQ ID NO: 2 and the oligonucleotide having the SEQ ID NO: 6 is TG, G A combination of a DNA probe having a base sequence represented by -T mismatch, SEQ ID NO: 3 and an oligonucleotide having SEQ ID NO: 4 is a DNA probe having a base sequence represented by A-C mismatch, SEQ ID NO: 3 and a sequence The combination with the oligonucleotide having the number: 5 shows an A-C, C-A mismatch.

  The measurement results are shown in FIG. FIG. 1 is a graph showing the results of fluorescence measurement, and the horizontal axis represents relative fluorescence brightness. On the left side, the types of DNA probes used are shown, and the results of fluorescence measurement with fluorescent oligonucleotides having the base sequences represented by SEQ ID NOs: 4 to 6 are shown. As is clear from FIG. 1, the fluorescence brightness was high in the perfectly matched combination, and the fluorescence brightness was lower in the mismatch than in the perfect match.

It is a graph which shows the result of a fluorescence measurement.

Claims (4)

A solid support in which a group represented by the following general formula (1) is covalently bonded to the surface of a carrier.
-NH-CO-X-OY (1)
(In the general formula (1), X represents an alkylene group having 1 to 50 carbon atoms which may be substituted with an alkyl group, or a group represented by the following general formula (2), and Y represents Represents hydrogen or dimethoxytrityl group.)
-(R ¹ -R ² ) _n- (2)
(In the general formula (2), R ¹ represents a methylene group optionally substituted with an alkyl group, O or NR ³ , R ² represents a methylene group optionally substituted with an alkyl group, and R ³ represents Represents hydrogen or an alkyl group, and n represents an integer of 1 to 25.) The solid support according to claim 1, wherein the carrier is microporous glass. A DNA chip comprising a nucleic acid molecule bound to a hydroxyl group on the solid support according to claim 1. A microarray for gene detection, wherein a nucleic acid molecule is bonded to a hydroxyl group on the solid support according to claim 1 or 2.

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