JP2007130019A - Support for immobilizing dna - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a support used as a DNA library by immobilizing a DNA, and particularly a support suitable for reproducing a DNA by DNA amplification reaction. <P>SOLUTION: The DNA is immobilized on the surface of the support made of one kind or more of materials selected from constituent elements including amorphous carbon and graphite. A hydroxyl group may be bonded to the surface of the support or a group obtained by bonding a hydrocarbon group to a carboxyl group and having the carboxy group at its terminal end may be bonded to the surface of the support through an ester bond or a peptide bond. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a substrate for immobilizing DNA, more particularly to a chemically modified substrate, and more particularly to a chemically modified substrate having a hydroxyl group, a carboxyl group or the like at its terminal.

Conventionally, in a DNA amplification reaction or the like, in order to obtain a specific amount of target DNA,
1) Increase the temperature of the sample to 95 ° C. in order to undo the hydrogen bond of the double-stranded DNA.
2) The sample temperature is then lowered to 45 ° C. to recombine with primers for replicating DNA.
3) The temperature of the sample is raised to 74 ° C. in order to further replicate the DNA by extending the primer with a thermostable polymerase.
It was necessary to repeat the heat cycle of 1) to 3).
In such a DNA amplification reaction, a sample is placed in a synthetic resin container or the like, and the container is accommodated in an aluminum block to perform the heat cycle.

However, the heat cycle takes a lot of time, and it takes several hours to obtain the target amount of DNA. In addition, since the temperature control accuracy is low, DNA other than the target DNA is also replicated.
In view of such problems, it is a technical object of the present invention to provide an optimum substrate that can easily immobilize DNA and replicate DNA by a DNA amplification reaction.

The substrate of the present invention is a substrate for immobilizing DNA, and is characterized by comprising one or a plurality of constituent elements composed of amorphous carbon, amorphous carbon, and graphite.
The substrate is desirably chemically modified having a polar group, a hydroxyl group or a carboxyl group at the terminal. It is also desirable that the carboxyl group is bonded to the substrate surface via an ester bond, and it is also desirable that the carboxyl group is bonded to the substrate surface via a peptide bond.

The substrate for immobilizing the DNA of the present invention is made of a carbon material such as amorphous carbon, amorphous carbon, graphite, etc., and since carbon is present on the surface layer, the surface is easily chemically modified with a hydroxyl group, a carboxyl group, or the like. The DNA can be easily immobilized and is optimal for a chip for replicating DNA by a DNA amplification reaction. Further, when the surface of the substrate of the present invention is contaminated, it can be hydrolyzed to regenerate the chemical modification.

The substrate used in the present invention has a carbon material such as amorphous carbon, amorphous carbon, graphite, or the like as a constituent element, and the formation method thereof can be selected as appropriate. Preferably, the microwave plasma CVD method, ECRCVD method, high frequency Plasma CVD, IPC, DC sputtering, ECR sputtering, ion plating, arc ion plating, EB vapor deposition, resistance heating vapor deposition, and the like. For example, amorphous carbon containing hydrogen obtained by steaming a resist film made of a polyimide-based material may be used. Further, graphite powder mixed with a resin to be a slurry and baked and hardened may be used. In the present invention, one or a combination of the above components may be used.

It is also desirable that the surface of the substrate of the present invention is intentionally roughened. This is because such a rough surface can increase the surface area and immobilize a large amount of DNA. The shape of the substrate is not particularly limited, such as a flat plate shape, a spherical shape, or a polygonal shape. Further, it may be a composite of these substrates and other substances. The substrate of the present invention may be present on the surface layer.

Next, a specific group is added (chemical modification) to the surface of the substrate. This chemical modification facilitates the immobilization of DNA on the surface of the substrate. Specific groups added (chemically modified) to the substrate surface and having a polar group at the terminal include groups such as a hydroxyl group, a carboxyl group, a sulfate group, a cyano group, a nitro group, a thiol group, and an amino group. In addition, organic carboxylic acids are also included.
Moreover, a carboxyl group is good also as a group which has a carboxyl group at the terminal through another hydrocarbon group between base | substrates. Here, the hydrocarbon group having 0 to 10 carbon atoms is preferable for immobilizing DNA. Acids that can become hydrocarbon groups include monocarboxylic acids such as formic acid, acetic acid, and propionic acid, dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, maleic acid, fumaric acid, and phthalic acid, and trimellitic acid. And polyvalent carboxylic acids.

When the substrate of the present invention is applied to a DNA amplification reaction, there are two application cases: hydrolysis resistance is required and hydrolysis needs to be regenerated by chemical modification.
When hydrolysis resistance is required, it is preferable to bond a group having a carboxyl group bonded to the end of the hydrocarbon group to the substrate surface via a peptide bond in order to impart alkali resistance.

On the other hand, when it is necessary to hydrolyze the generated chemical modification and regenerate it, the group having a carboxyl group bonded to the end of the above-mentioned hydrocarbon group is converted to an ester bond so that it can be hydrolyzed with an alkaline solution. It is preferable to bond to the substrate surface via

As a method of bonding the hydroxyl group to the substrate surface at the end of the hydrocarbon group, the substrate surface is oxidized with oxygen plasma and then treated with water vapor, or the substrate surface is chlorinated by irradiating with ultraviolet light in chlorine gas and then an alkaline solution. Examples of the method include hydrolyzing and hydroxylating in the substrate, and a method of oxidizing the substrate surface with oxygen plasma and then chlorinating and then hydrolyzing and hydroxylating in an alkaline solution.

In addition, as a method of bonding a carboxyl group-bonded group to the end of a hydrocarbon group to the substrate surface via a peptide bond, the substrate surface is chlorinated by irradiating with ultraviolet light in chlorine gas, and then irradiated with ultraviolet light in ammonia gas. Then, after amination, a method of reacting with carboxylic acid chloride in a non-aqueous solvent and then neutralizing in a weak alkaline solution can be mentioned.

In addition, as a method of bonding a group having a carboxyl group bonded to the terminal of a hydrocarbon group to the substrate surface via an ester bond, the substrate surface is chlorinated by irradiating with ultraviolet light in chlorine gas and then carboxylic in a non-aqueous solvent. A method of reacting with acid soda and then neutralizing in a weak acid solution, or oxidizing the substrate surface with oxygen plasma, then chlorinating, hydrolyzing in an alkaline solution and hydroxylating, and then in a non-aqueous solvent A method of reacting with carboxylic acid chloride and then neutralizing in a weak alkaline solution can be mentioned.

Hereinafter, the present invention will be described in detail with reference to examples.
Example 1
Using a microwave plasma CVD method, a diamond disk containing non-diamond carbon having a diameter of 64 mm, a thickness of 0.1 mm, and a surface roughness Ra of 0.5 mm was vapor-phase synthesized. As a result of analyzing this disk in an area of 10 mm × 10 mm by Raman spectroscopy, the peak ratio of diamond carbon (complete diamond) and non-diamond carbon (incomplete diamond) was investigated. Was confirmed. A 10 mm square sample was cut out from the disk containing non-diamond carbon by laser.
In Example 1, after oxidizing the sample surface with oxygen plasma excited by microwaves, placing the sample in a separable flask, substituting the atmosphere in the flask with water vapor, and heating to 400 ° C. while flowing water vapor. After holding for 30 minutes, it was allowed to cool. Next, the sample was taken out and dried to obtain a substrate having a hydroxyl group at the terminal.
Moreover, when the peak intensity of hydrogen and hydroxyl groups after sample cutting, oxygen plasma treatment, and steam treatment was measured by SIMS, the peak intensity ratio of hydroxyl groups when the hydrogen peak intensity was 1 was as shown in the following table. The value shown in FIG.

Table 1
-----------------------
Peak intensity ratio of hydroxyl group -----------------------
0.11 after cutting out the sample
After oxygen plasma treatment 0.61
After steaming 1.09

As shown above, the peak intensity ratio of the hydroxyl group was increased by the oxygen plasma treatment and the subsequent steam treatment, and it was confirmed that the substrate surface was chemically modified with the hydroxyl group.

(Example 2)
Using a microwave plasma CVD method, a diamond disk containing non-diamond carbon having a diameter of 64 mm, a thickness of 0.1 mm and a surface roughness Ra of 0.3 mm was vapor-phase synthesized. As a result of analyzing this disc by an Raman spectroscopic analysis method in an area of 10 mm × 10 mm, all were non-diamond carbon (incomplete diamond). A 10 mm square sample was cut out from this disk by laser.
This sample was placed in a separable flask, and the atmosphere in the flask was replaced with argon gas. Then, an Hg-Xe lamp was used while flowing chlorine gas at a flow rate of 1 SCCM, and ultraviolet light having a main wavelength of 3600 angstroms was applied for 60 minutes. Irradiated to chlorinate the sample surface. After replacing the atmosphere with argon gas, a sample was taken out, boiled in a 10% by weight aqueous sodium hydroxide solution for 15 minutes, further washed with water and dried to obtain a substrate having a hydroxyl group at the end.
Moreover, when the peak intensity of hydrogen, hydroxyl group and chlorine group was measured by SIMS before and after chlorination and after sodium hydroxide treatment, the peak intensity ratio of hydroxyl group and chlorine group when the hydrogen peak intensity was set to 1. The values shown in Table 2 below were obtained.

Table 2
-----------------------
Peak intensity ratio -----------------------
Hydroxyl group Chlorine group -----------------------
Before chlorination 0.11 −
After chlorination 0.17 0.47
After sodium hydroxide treatment 0.45 0.31

As shown above, the peak intensity ratio of the hydroxyl group was increased by the chlorination treatment and the subsequent sodium hydroxide treatment, and it was confirmed that the substrate surface was chemically modified with the hydroxyl group. Moreover, since the chlorine group was reducing, it was confirmed that it was substituted with a hydroxyl group.

(Example 3)
Using a microwave plasma CVD method, a diamond disk having a diameter of 64 mm, a thickness of 0.1 mm and a surface roughness Ra of 0.5 mm and a mixture of complete and incomplete diamond was vapor-phase synthesized. As a result of analyzing this disc by Raman spectroscopy in an area of 10 mm × 10 mm, the peak ratio of diamond carbon to non-diamond carbon (incomplete diamond) was investigated, and the presence of non-diamond carbon could be confirmed. . A 10 mm square sample was cut out from the diamond disk containing non-diamond carbon by laser, the surface was oxidized with oxygen plasma excited by microwaves, and then the sample surface was chlorinated. After replacing the atmosphere with argon gas, a sample was taken out, boiled in a 10% by weight aqueous sodium hydroxide solution for 15 minutes, further washed with water and dried to obtain a substrate having a hydroxyl group at the end.
Moreover, when the peak intensity of hydrogen, hydroxyl group, and chlorine group after surface polishing, after oxygen plasma treatment, after chlorination, and after sodium hydroxide treatment was measured by SIMS, the peak intensity of hydrogen was set to 1. The peak intensity ratio between the hydroxyl group and the chlorine group was as shown in Table 3 below.

Table 3
-----------------------
Peak intensity ratio -----------------------
Hydroxyl group Chlorine group -----------------------
Before oxygen plasma treatment 0.11 −
After oxygen plasma treatment 0.67 −
After chlorination 0.19 0.44
After sodium hydroxide treatment 0.50 0.30

As shown above, the peak intensity ratio of hydroxyl groups was increased by oxygen plasma treatment, subsequent chlorination, and subsequent sodium hydroxide treatment, confirming that the substrate surface was chemically modified with hydroxyl groups. . Moreover, since the chlorine group was reducing, it was confirmed that it was substituted with a hydroxyl group.

Example 4
A graphite disk is formed by sputtering, and a 10 mm square sample is cut out with a laser and placed in a separable flask. The atmosphere in the flask is replaced with argon gas, and then chlorine gas is supplied at a flow rate of 1 SCCM. The sample surface was chlorinated by irradiating it with ultraviolet light having a dominant wavelength of 3600 angstroms for 60 minutes while using an Hg-Xe lamp. After the atmosphere in the flask was again replaced with argon gas, 100 ml of a 1% by weight sodium sebacate N, N-dimethylformamide solution was added, a condenser was placed in the separable flask, and the mixture was refluxed for 2 hours. Next, the sample was taken out, washed with a 1% by weight acetic acid aqueous solution, further washed with acetone, and then dried to obtain a substrate having a carboxyl group at the terminal to which sebacic acid was bonded via an ester bond.
In addition, when the peak intensity of hydrogen, hydroxyl group and chlorine group before and after chlorination and after treatment with sodium sebacate was measured by SIMS, the peak intensity ratio of hydroxyl group and chlorine group when the hydrogen peak intensity was 1. The values shown in Table 4 below were obtained.

Table 4
-----------------------
Peak intensity ratio -----------------------
Hydroxyl group Chlorine group -----------------------
Before chlorination 0.11 −
After chlorination 0.17 0.47
After treatment with sodium sebacate 0.35 0.31

As shown above, the peak intensity ratio of the hydroxyl group is increased by chlorination followed by treatment with sodium sebacate, and absorption derived from carbon-hydrogen stretching vibration on the sample surface using the FTIR method. When the strength and the absorption strength derived from the carbon-oxygen stretching vibration were measured, all the absorption strengths were increased (the increase rate of the absorption strength of the sample with respect to the surface blank was about 30%). From this, it was confirmed that the substrate surface was chemically modified with a group in which a carboxyl group was bonded to the end of the hydrocarbon group of sebacic acid. Moreover, since the chlorine group was reducing, it was confirmed that it was substituted with a hydroxyl group.

(Example 5)
A resist film made of a polyimide material was steamed and baked to form a thin plate made of amorphous carbon containing hydrogen. A 10 mm square sample was cut from the thin plate with a laser, and the surface was oxidized with oxygen plasma excited by microwaves, and then the sample surface was chlorinated. After substituting the atmosphere with argon gas, the sample was taken out and boiled in a 10% by weight aqueous potassium hydroxide solution for 15 minutes to replace the sample surface with hydroxyl groups. After drying, the sample was placed in a separable flask equipped with a condenser equipped with a calcium chloride drying tube at the top, 50 ml of chloroform and 1 g of triethylamine were added, and the atmosphere in the flask was replaced with argon gas. Subsequently, a solution prepared by dissolving 10 g of succinyl chloride in 50 ml of chloroform was gradually added while cooling the separable flask with ice. After refluxing for 4 hours, the sample was taken out, washed with a 10% by weight aqueous potassium carbonate solution, further washed with acetone, and dried to obtain a substrate having a carboxyl group at the end, to which malonic acid was bonded via an ester bond. It was.
In addition, the peak intensity of hydrogen and hydroxyl groups before and after oxygen plasma treatment, after chlorination, after hydroxylation, and after succinyl chloride treatment was measured by SIMS. The ratio was a value shown in Table 5 below.

Table 5
-----------------------
Peak intensity ratio of hydroxyl group -----------------------
Before oxygen plasma treatment 0.11
After oxygen plasma treatment 0.66
0.19 after chlorination
0.65 after hydroxylation
0.46 after succinyl chloride treatment

As shown above, the peak intensity ratio of the hydroxyl group is increased by the oxygen plasma treatment, the subsequent chlorination, the subsequent hydroxylation, and the subsequent succinyl chloride treatment, and the FTIR method is used to When the absorption intensity derived from the carbon-hydrogen stretching vibration and the absorption intensity derived from the carbon-oxygen stretching vibration were measured, both absorption strengths were increased (the rate of increase of the absorption intensity with respect to the surface blank of the sample was About 25%). From this, it was confirmed that the surface of the substrate was chemically modified with a group in which a carboxyl group was bonded to the end of the hydrocarbon group of malonic acid.

(Example 6)
Mixing resin powder with graphite powder and adding a solvent to form a slurry, vaporizing the solvent, gas phase synthesis of a graphite disk with a diameter of 64 mm, a thickness of 0.1 mm and a surface roughness Ra of 0.3 mm did. As a result of analyzing this disc by an Raman spectroscopic analysis method in an area of 10 mm × 10 mm, all were non-diamond carbon (amorphous diamond). A 10 mm square sample was cut out from this disk by laser. The surface of the sample was hydrogenated with hydrogen plasma excited by microwaves, and then the sample surface was chlorinated. After replacing the atmosphere in the separable flask in which the sample is placed with argon gas, the sample was irradiated with ultraviolet light having a dominant wavelength of 3600 angstroms for 60 minutes using an Hg-Xe lamp while flowing ammonia gas at a flow rate of 1 SCCM. The surface was aminated. After the atmosphere was replaced with argon gas, a condenser having a calcium chloride drying tube was placed on the top of the separable flask, 50 ml of chloroform was added, and the atmosphere was again replaced with argon gas.

Subsequently, a solution prepared by dissolving 10 g of succinyl chloride in 50 ml of chloroform was gradually added while cooling the separable flask with ice. After refluxing for 4 hours, the sample was taken out, washed with a 10% by weight aqueous potassium carbonate solution, further washed with acetone, and then dried to obtain a substrate having malonic acid bonded via a peptide bond and having a carboxyl group at the terminal. It was.
In addition, the peak intensity of hydrogen, hydroxyl group, and chlorine group before and after chlorination, after amination, and after succinyl chloride treatment was measured by SIMS. The intensity ratio was a value shown in Table 6 below.

Table 6
-----------------------
Peak intensity ratio -----------------------
Hydroxyl group Chlorine group -----------------------
Before chlorination 0.11 −
After chlorination 0.19 0.45
After amination 0.16 0.10
After succinyl chloride treatment 0.55 0.10

As shown above, the peak intensity ratio of the hydroxyl group is increased by the hydrogen plasma treatment, the subsequent chlorination, the subsequent hydroxylation, and the subsequent succinyl chloride treatment, and the FTIR method is used to When the absorption intensity derived from the carbon-hydrogen stretching vibration and the absorption intensity derived from the carbon-oxygen stretching vibration were measured, both absorption strengths were increased (the rate of increase of the absorption strength relative to the surface blank of the sample was About 25%).
From this, it was confirmed that the substrate surface was chemically modified with a group in which a carboxyl group was bonded to the end of the hydrocarbon group of malonic acid.
When a DNA amplification reaction was carried out using a chemically modified substrate having a carboxyl group at the end obtained as in Examples 1 to 6, a target amount of DNA could be obtained in about 1 hour. It was.

Furthermore, using the chemically modified substrate of the present invention, the terminal base of the oligonucleic acid was fixed to the terminal hydroxyl group or terminal carboxyl group by hydrogen bonding, and further, DNA having a complementary base sequence to this oligonucleic acid was fixed, It can also be used as a DNA library chip. Further, instead of DNA, nucleotides, oligonucleotides, DNA fragments and the like can be immobilized on the diamond surface to form a library.

Since the substrate of the present invention uses a carbon material, it is easy to immobilize DNA on the substrate. For example, the DNA amplification reaction operation can be extremely facilitated.
Further, since the surface of the substrate of the present invention is chemically modified with a hydroxyl group, a carboxyl group or the like, it is possible to stabilize the immobilization of DNA and is optimal for a chip for replicating DNA by a DNA amplification reaction.
In addition, when the surface of the substrate of the present invention is contaminated, it can be hydrolyzed to regenerate the chemical modification, thereby saving an expensive DNA chip.

Claims (4)

A substrate for immobilizing DNA consisting of one or a plurality of constituent elements composed of amorphous carbon, amorphous carbon, and graphite. 2. The substrate according to claim 1, which is chemically modified having a polar group, a hydroxyl group or a carboxyl group at its terminal. The substrate according to claim 2, wherein the carboxyl group is bonded to the surface of the substrate through an ester bond. The substrate according to claim 2, wherein the carboxyl group is bonded to the surface of the substrate through a peptide bond.