WO2006104260A1

WO2006104260A1 - Nucleic acid microarray, process for production of the same, and substrate for nucleic acid microarray

Info

Publication number: WO2006104260A1
Application number: PCT/JP2006/307284
Authority: WO
Inventors: Noritsugu Umehara; Yoichi Akagami; Masaru Kato; Jun Imai; Hiroshi Yoshida; Nobutaka Kusaba
Original assignee: National University Corporation Nagoya University; Akita Prefecture; Incorporated National University Iwate University; Nipro Corporation
Priority date: 2005-03-31
Filing date: 2006-03-30
Publication date: 2006-10-05
Also published as: JPWO2006104260A1; JP5238250B2

Abstract

Disclosed are a nucleic acid microarray which can immobilize a nucleic acid thereon satisfactorily without the need of the immobilization through a covalent bond provided by introducing a specific reactive group to a substrate or the nucleic acid; a substrate for the nucleic acid microarray; and others. A nucleic acid microarray comprising a plastic substrate and a nucleic acid immobilized on the substrate, at least the surface of the substrate containing an inorganic filler selected from the group consisting of an oxide filler, a hydroxide filler, a carbonate filler and a sulfate filler; a nucleic acid microarray comprising a plastic substrate which has been treated with plasma or an ion beam and a nucleic acid immobilized on the substrate, the at least the surface of the substrate containing an inorganic filler selected from the group consisting of an oxide filler, a hydroxide filler, a carbonate filler and a sulfate filler; a process for producing the nucleic acid microarray; a substrate for the nucleic acid microarray; and a method for detecting a nucleic acid using the nucleic acid microarray.

Description

Specification

Nucleic acid microarray, method for producing the same, and substrate for nucleic acid microarray

The present invention relates to a nucleic acid microarray, a method for producing the same, and a substrate for nucleic acid microarrays. More specifically, the present invention relates to a plastic nucleic acid microarray, a method for producing the same, and a base material used therefor.

Background art

With the progress of genome analysis projects, all base sequences of genomes of various organisms have been deciphered, and based on these base sequence information, new expression for examining gene expression patterns and gene product functions at the genome level. Projects are underway. The DNA chip was developed as a means to dramatically advance these functional analysis projects, and DNA chip technology is used for dienotyping of all genes such as humans, mice, and yeast, and gene expression analysis. Yes.

Currently used methods for producing a DNA chip (or DNA microarray) are roughly classified into the Affymetrix method and the Stanford method. A DNA chip based on the former method is produced by combining photolithography and light irradiation chemical synthesis to synthesize oligonucleotides of about 20 to 25 mer on a glass substrate at a high density (US Patent No. 1). 5 4 4 5 9 3 4 Specification, U.S. Pat.No. 5 7 4

No. 4,300,5 and U.S. Pat. No. 5,700,037). A DNA chip by the latter method is typically produced by spotting a pre-prepared DNA fragment on a slide glass at a high density (US Pat. No. 5,880,071).

5 2 2 and International Publication No. 9 8 1 8 9 6 1 (see pamphlet). On the other hand, biochips using various plastic resins instead of glass substrates are also being studied (see Japanese Patent Application Laid-Open No. 2 0 0 5-1 0 0 0 4) Untreated resin has DNA binding properties The method of modifying the surface of the resin substrate is low. For example, Japanese Patent Application Laid-Open No. 2 005 5-10 0 0 0 4 discloses a method for introducing an aldehyde group as a method for modifying the surface of a substrate, in order to increase the reactivity with an aldehyde group. Describes that it is necessary to introduce an amino group into DNA.

Disclosure of the invention

However, the above-mentioned DNA chip is expensive to manufacture with a glass substrate chip and is expensive. Although resin-made chips are advantageous in terms of cost, the fixation of DNA to the base material is inferior to that of glass substrates, and the strength of the fixation and the improvement of analysis power have not been resolved.

An object of the present invention is to provide a nucleic acid microarray, a base material for a nucleic acid microarray, and the like that can sufficiently fix a nucleic acid without introducing a specific reactive group into a substrate or a nucleic acid and performing a covalent bond. is there.

The inventors of the present invention have found that fixing the base material and the DNA is strengthened by applying a specific treatment to the plastic base material, and have completed the present invention.

That is, the present invention is as follows.

(1) A nucleic acid microarray in which a nucleic acid is immobilized on a plastic substrate, and at least a surface portion of the substrate is provided with an oxide-based filter, a hydroxide-based filter, a carbonate-based filter, and a sulfate. A nucleic acid microarray comprising an inorganic filler selected from the group consisting of a series filler.

(2) The nucleic acid microarray according to (1), wherein the inorganic filler is titanium dioxide.

(3) The nucleic acid microarray according to (1) or (2), wherein the plastic is polypropylene, polymethylmetatalylate, polycarbonate, or polyethylene terephthalate.

(4) A nucleic acid microarray in which a nucleic acid is immobilized on a plasma-treated or ion beam-treated plastic substrate, and an oxide-based filter, a hydroxide-based filter, and a carbonate on at least the surface of the substrate. A nucleic acid microarray comprising an inorganic filler selected from the group consisting of a series filler and a sulfate series filler.

(5) The nucleic acid microarray according to (4), wherein the inorganic filler is titanium dioxide. (6) The nucleic acid microarray according to (4) or (5), wherein the plastic is polypropylene, polymethyl methacrylate, polycarbonate, or polyethylene terephthalate.

(7) The nucleic acid microarray according to any one of (4) to (6), wherein the substrate has a surface roughness Ra (JISB 060 1- 1 994) of 0.06 to 0.5 μπι.

(8) Plasma treatment or ion beam treatment of a plastic substrate containing an inorganic filler at least on the surface, and

A step of immobilizing nucleic acid on the surface of the plastic substrate after the treatment

A method for producing a nucleic acid microarray comprising:

(9) The manufacturing method according to (8), wherein the plasma treatment step is performed in an inert gas atmosphere, and the ion beam treatment step is performed using an inert gas as an ionized gas.

(1 0) The production method according to (9), wherein the inert gas is nitrogen gas.

(11) The production method according to any one of (8) to (10), wherein the nucleic acid has a length of 50 bases or more.

(1 2) The method according to any one of (8) to (11), wherein the step of fixing the nucleic acid is performed by UV irradiation.

(1 3) A base material for nucleic acid microarrays made of plastic, comprising an oxide-based filler, a hydroxide-based filter, a carbonate-based filter, and a sulfate-based filter on at least a surface portion of the substrate. A substrate comprising an inorganic filler selected from the group consisting of:

(14) The substrate according to (1 3), wherein the inorganic filler is titanium dioxide.

(15) The substrate according to (13) or (14), wherein the surface of the substrate is further subjected to plasma treatment or ion beam treatment.

(16) The substrate according to (15), wherein the substrate has a surface roughness R a (J I S B 060 1 — 1 9 94) of from 0.06 to 0.5 / zm.

(1 7) The nucleic acid microarray according to any one of (1) to (7) or the above (8) to (1 2) a step of contacting a nucleic acid microarray obtained by the production method according to any one of the above and a sample containing the nucleic acid to be analyzed; and

Detecting the nucleic acid hybrid formed by the contacting step

A method for detecting a nucleic acid having a specific base sequence.

(18) The detection method according to the above (17), wherein the nucleic acid hybrid is labeled.

(19) The detection method according to the above (18), wherein the label is alkaline phosphatase.

Brief Description of Drawings

Figure 1 compares the fixability of DNA for polypropylene substrates with and without titanium dioxide.

Figure 2 shows a comparison of DNA fixability between a substrate made of polyethylene terephthalate containing titanium dioxide and a substrate not containing it.

Figure 3 shows the color development of a DNA microarray using a polycarbonate substrate containing titanium dioxide.

BEST MODE FOR CARRYING OUT THE INVENTION

The nucleic acid microarray of the present invention is a nucleic acid microarray in which a nucleic acid is immobilized on a plastic substrate, and an oxide-based filler, a hydroxide-based filter, and a carbonate-based filter on at least a surface portion of the substrate. And an inorganic filler selected from the group consisting of sulfate-based fillers.

In the present invention, the nucleic acid microarray refers to a nucleic acid (also referred to as a probe) mounted on the surface of a plastic substrate.

Specific examples of the resin used for the plastic substrate of the nucleic acid microarray of the present invention include polyolefin resins such as polyethylene, polypropylene, polypentene, polymethylpentene, and saturated cyclic polyolefin; acryl resins such as polymethyl methacrylate; polycarbonate; polyacetal ; Ethylene ■ Vinyl alcohol copolymer resin; Polystyrene, AS (acrylonitrile 'styrene) tree Polystyrene resins such as oil, ABS (acrylonitrile butadiene styrene) resin; vinyl chloride resins such as polyvinyl chloride and polyvinylidene chloride; cellulose acetate; polyphenylene ether resin; polyphenylene nord resin; Resin; Polyetheretherketone resin; Thermosetting resin such as phenol resin, urea resin, melamine resin, unsaturated polyester resin, polyimide, epoxy resin, moon; Polyester terephthalate such as polyethylene terephthalate and polybutylene terephthalate; polylactic acid And biodegradable resins such as starch-derived resins such as corn.

Of the above, polypropylene, polymethylmethacrylate, polycarbonate and polyethylene terephthalate are preferred because they are inexpensive and easily available. In terms of the environment, it is preferable to use biodegradable resins or resin materials that have established recycling methods (eg, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, etc.), but this is not a limitation. Absent.

In the present invention, the inorganic filler is a plastic auxiliary material that is generally added to a resin as an extender, and depending on the type, the viscosity increases, the molding compression ratio decreases, the opacity increases, and the light resistance. Effects such as improvement of mechanical properties, improvement of mechanical characteristics, and improvement of appearance may be added. From the viewpoint of easy visual detection of light emission detection in the nucleic acid microarray, those that improve opacity and those that improve the appearance are preferred, but are not limited thereto.

Examples of the inorganic filler include spherical particles, fibers, and flakes, and spherical particles are preferable from the viewpoint of dispersibility of the inorganic filler in the resin. The average particle diameter of the spherical particles is preferably from 0.2 to 5.0 O / zm, more preferably from 0.5 to 2.0 m, from the viewpoint of dispersibility of the inorganic filler in the resin. The average particle size of the inorganic filler is a value measured by a light scattering particle size distribution measuring device. The inorganic filler in the present invention is selected from the group consisting of oxide fillers, hydroxide fillers, carbonate fillers, and sulfate fillers. Examples of oxide fillers include titanium dioxide, silica, diatomaceous earth, alumina, iron oxide, zinc oxide, magnesium oxide, etc .; hydroxide fillers such as aluminum hydroxide, magnesium hydroxide, etc .; carbonates Examples of the filler include calcium carbonate, magnesium carbonate, and dolomite; examples of the sulfate-based filler include barium sulfate, calcium sulfate, and ammonium sulfate. The inorganic filler may be used singly or in combination of two or more.

The inorganic filler described above can impart a positive charge, which is considered to be advantageous for immobilization of nucleic acids, to the substrate surface. Furthermore, an oxide-based filler is preferable, and titanium dioxide, which is excellent as a pigment, is more preferable.

The blending amount of the inorganic filler is appropriately set according to the type of the resin and the inorganic filler in which the inorganic filler is blended, but from the viewpoint of maintaining the shape of the base material satisfactorily with respect to 100 parts by weight of the resin. The inorganic filler is 20 parts by weight or less, preferably 10 parts by weight or less.

By blending the inorganic filler, the affinity for the nucleic acid on the surface of the hydrophobic plastic is increased. The hydrophobicity in the present invention refers to a property having a contact angle 0 with water described later of about 65 ° or more. Furthermore, if the hydrophobic plastic is subjected to a surface treatment described later, the wettability with respect to an aqueous solution in which nucleic acid fragments are dissolved or dispersed in an aqueous medium is improved, so that the nucleic acid can be sufficiently fixed. Become.

The inorganic filler should just be contained in the surface part of the said base material at least. Therefore, the inorganic filler may be contained in the entire base material, or may be unevenly distributed on the surface portion, the unevenly distributed portion may be the entire surface portion, and the surface portion on which the nucleic acid is immobilized. It may be a partial region.

As a specific embodiment, a base material containing an inorganic filler is formed on the whole base material formed by molding a plastic containing an inorganic filler; a plastic molded plate not containing an inorganic filler contains an inorganic filler. Examples include, but are not limited to, a multi-layer substrate in which plastic molded plates are laminated. When laminating plastic molded plates containing inorganic filler, it is recommended to fix nucleic acids. You may laminate | stack only on the fixed part.

The substrate can be produced according to a known method. For example, resin and inorganic fillers and other additives as necessary are mixed and kneaded with a mixer, Henschel, extruder, kneader or roll, etc., and molded by extrusion molding, injection molding, compression molding or calendar molding, etc. can do. Among these, from the viewpoint of production easiness and cost, it is preferable to produce the resin and the inorganic filler by coextrusion, but there is no limitation thereto. Furthermore, the base material can be produced by cutting into an appropriate size if necessary.

Specifically, when the plastic substrate is a polypropylene substrate, for example, about 10 parts by weight of titanium dioxide fine particles are mixed with 100 parts by weight of a polypropylene resin, and are formed into a substrate by coextrusion. A method, but not limited to this.

The size of the substrate can be appropriately determined according to the purpose of use of the microarray, but from the viewpoint of ease of handling at the bedside, it is about 1 to 100 mm ² , preferably about 1 0: L 0 0 O mm ²

The plastic substrate is preferably subjected to a surface treatment if necessary. By this surface treatment, an appropriate surface roughness of the substrate surface can be obtained, wettability to an aqueous solution in which nucleic acid fragments are dissolved or dispersed in an aqueous medium is improved, and nucleic acid can be sufficiently fixed. Become.

Examples of the surface treatment method include ion beam treatment or plasma treatment, preferably ion beam treatment using an inert gas as an ionization gas or plasma treatment in an inert gas atmosphere.

The ion beam treatment refers to ion implantation and film formation by the energy generated by ionizing inert gas, irradiating the surface of the plastic substrate with high-speed acceleration by applying voltage, and colliding with it. A surface treatment method that imparts the effect of improving etching and wettability.

As the inert gas in the ion beam processing, the nucleus after the ion beam processing is used. From the viewpoint of increasing the efficiency of acid fixation, nitrogen gas and argon gas are preferred. When using DNA or RNA having a phosphate skeleton (which is a polyanion), nitrogen can be more firmly fixed by improving the wettability of the surface of the plastic substrate and ion charging of the surface. Gas is more preferred.

The method of ion beam processing is not particularly limited. For example, a device equipped with an ECR ion gun that uses cyclotron resonance (ECR) of electrons in a magnetic field under reduced pressure. The ion beam obtained from the above can be irradiated with an acceleration energy of about 300 to 700 V at a dose of about 1.0 X 10 ° ² ozZcm ² . Examples of the apparatus used include, but are not limited to, a small ECR ion shower apparatus (EIS-200 ER) manufactured by Erio Nitas. The number of such treatments is not particularly limited as long as desired wettability and surface roughness are obtained, but from the viewpoint of suppressing wettability deterioration, 0.25 to 2.0 X 10 ²⁰ io nZc per time. The dose of m ² is preferably irradiated at least 5 times or more, preferably 10 times or more, but is not limited thereto.

The plasma treatment is performed by irradiating a solid surface with plasma generated by the ionizing action of the inert gas by discharging in an inert gas atmosphere, etching the surface, improving wettability, and introducing functional groups. The process which provides effects, such as. Examples of the discharge include corona discharge (high-pressure and low-temperature plasma), arc discharge (high-pressure and high-temperature plasma), and glow discharge (low-pressure and low-temperature plasma). Of these, corona discharge is preferred from the viewpoint of surface treatment reactivity. However, it is not limited to this. Examples of the inert gas in the plasma treatment include nitrogen gas, argon gas, oxygen gas, helium gas, neon gas, and xenon gas. From the viewpoint of increasing the efficiency of nucleic acid fixation after plasma treatment, nitrogen gas and argon Gas is preferred. When DNA or RNA having a phosphate skeleton (which is a polyanion) is used, an amine group and / or an amino group are generated on the surface of the plastic substrate, thereby improving the wettability of the surface and ions on the surface. Depending on the charge, yo Nitrogen gas is more preferable because it can be fixed more firmly.

The plasma treatment method is not particularly limited. For example, when corona discharge is used, the base material is placed in a sealed container, and a nitrogen gas atmosphere (about 10 to about 50 to about 50 to 50 ° C.) is used. Under 20 Pa), discharge at an output of about 100-50 OW and process for about 100-1 000 seconds. Examples of the apparatus used include, but are not limited to, a small high-performance plasma surface treatment apparatus (PDC 200 series) manufactured by Yamato Scientific. The number of such treatments is not particularly limited as long as desired wettability and surface roughness are obtained, but is usually about 1 to 10 times.

By the ion beam treatment or plasma treatment, the wettability of the surface of the plastic substrate is increased. The wettability can be determined by measuring the contact angle Θ between the plastic substrate and water. The contact angle 0 of water after the treatment is 60 ° or less, preferably 40 ° or less, particularly preferably 20 ° or less, from the viewpoint of nucleic acid fixation strength. The water contact angle Θ on the surface of the base material before treatment is, for example, about 80 ° for polymethyl metatalylate and about 76 ° for polycarbonate. The contact angle 0 is a value measured with an optical microscope after 0.25 μL of distilled water was dropped. By performing the ion beam treatment or plasma treatment, the surface roughness of the plastic substrate can be adjusted. The surface roughness R a (JISB 060 1-1 9 94) of the plastic substrate is preferably from 0.06 to 0.5 zm from the viewpoint of making nucleic acid fixation stronger, and from 0.08 to 0. 45 μm is preferred. The surface roughness Ra is a value measured with a laser microscope.

A nucleic acid microarray can be produced by immobilizing nucleic acids on the substrate obtained as described above.

The nucleic acid (also referred to as a probe) in the present invention refers to DNA, RNA, PNA or a derivative thereof. The nucleic acid (probe) may be naturally derived, artificially synthesized, single stranded or double stranded. The DNA may be natural or synthetic DNA, cDNA reverse-transcribed from mRNA, etc. It is not particularly limited. The RNA is not particularly limited, such as mRNA, cRNA or rRNA. PNA refers to a molecule having a nucleobase in the peptide backbone. The derivative means a product obtained by appropriately modifying the DNA, RNA, or PNA within the scope of the object of the present invention.

Among the DNA, RNA, and PNA, single-stranded DNA is preferable in terms of production cost, the point that various enzymes can be used, and a wide range of applications, but is not limited thereto. Examples of the scope of application include prediction of drug sensitivity of individual patients, determination of pathogenic bacteria or resistance, and determination of individual constitution, and generally refers to applications in a field called tailor-made medicine. However, it is not limited to this.

Here, the nucleic acid microarray of the present invention is generally called a DNA chip when the nucleic acid (probe) is a single-stranded DNA.

The type of nucleic acid (probe) mounted on the nucleic acid microarray of the present invention can be appropriately set according to the purpose of use. For example, when it is used to predict individual drug susceptibility, a single nucleotide polymorphism (SNP) is generally detected, so the number of polymorphisms at each polymorphic site to be detected is detected. Is the sum of More specifically, when predicting the susceptibility of the osteoporosis therapeutic agent disclosed in Japanese Patent Application Laid-Open No. 2000-3 50 600, the B sm I restriction fragment length polymorphism on intron 8 of the VDR gene, Ap o E gene Hh a I restriction fragment length polymorphism Detects the Xb a I restriction fragment length polymorphism on intron 1 of the ER gene. 2 types of B sm I restriction fragment length polymorphism on intron 8 of the VDR gene, 3 types of Hha I restriction fragment length polymorphism of the Ap o E gene, Xba on intron 1 of the ER gene Since there are two I restriction fragment length polymorphisms, the total is 7. Therefore, the types of nucleic acids (probes) mounted on the nucleic acid microarray can be set to seven types. Furthermore, a nucleic acid having an arbitrary base sequence may be mounted as a control. Although a specific example has been described above, the present invention is not limited to this. The length of the nucleic acid (probe) in the present invention is at least 50 bases as long as it can be easily immobilized on a plastic substrate by UV irradiation and can hybridize with the nucleic acid to be measured. That's fine. Among these, from the viewpoint of nonspecific adsorption frequency, it is 50 to 500 bases, preferably about 200 to 40 bases.

As the nucleic acid to be immobilized, in the case of a nucleic acid derived from nature, a nucleic acid prepared by isolation and purification by a known method and cleaving to a predetermined length with an enzyme or the like can be used if necessary. In the case of a synthetic nucleic acid, those prepared by a known synthesis method can be used depending on the type of nucleic acid. In the case of DNA, those prepared by reverse transcription reaction or polymerase chain reaction can be suitably used.

However, the length of the base sequence necessary for detection is generally about 10 to 30 bases. In such a case, the above-mentioned length of 50 bases or more can be achieved by adding a sequence that does not affect the detection to the end of the nucleic acid sequence prepared as described above. For example, an oligomer selected from the group consisting of poly dT, poly d A, poly d C and poly d G can be added. From the viewpoint of adsorption strength and nonspecific adsorption frequency, poly d T oligomer can be added. It is preferable to add it. The poly dT oligomer can be added by terminal transferase or the like, but is not limited thereto.

The nucleic acid is immobilized by preparing a solution of the nucleic acid, spotting the solution on the surface of the plastic substrate, preferably the surface of the plastic substrate after the ion beam treatment or plasma treatment, and irradiating with UV. . As the solution, an aqueous solvent such as a phosphate buffer is generally used. In addition, the spotting method is not particularly limited, and examples thereof include a method using a dispenser (such as SMP-3 manufactured by Musashi Engineering Co., Ltd.). In the present invention, by using a plastic substrate containing an inorganic filler at least on the surface portion, preferably after the substrate is subjected to ion beam treatment or plasma treatment.

1) Oxide filler, hydroxide filler, carbonate filler and sulfate The inorganic filler selected from the fillers imparts a positive charge to the substrate surface, which is considered to favor the immobilization of nucleic acids, more preferably

2) By increasing the hydrophilicity of the plastic substrate surface, the wettability of the plastic substrate to the solution is improved,

3) By making the surface of the plastic substrate rough and increasing the surface area, the surface shape suitable for nucleic acid fixation can be produced.

Under these conditions, the nucleic acid can be easily fixed.

For the UV irradiation, UV having a wavelength of about 250 to 350 nm is usually used, and irradiation is performed for about 1 to 10 minutes with about 5000 to 8 000 μwZcm ² , but is not limited thereto.

After the fixation of the nucleic acid (probe), washing or prokking treatment may be performed from the viewpoint of reducing nonspecific adsorption.

The nucleic acid microarray of the present invention thus obtained can be suitably used for the following detection method of the present invention.

The nucleic acid microarray of the present invention can be used in a method for detecting a nucleic acid having a specific base sequence. The detection method includes a step of bringing the microarray into contact with a sample containing a nucleic acid to be analyzed, and a step of detecting a nucleic acid hybrid formed by the contact step. Hereinafter, each process will be described.

(1) Contact process

(1-1) Pretreatment of nucleic acid (target) to be analyzed

As the nucleic acid (target) to be analyzed, a DNA fragment sample or RNA fragment sample whose sequence or function is unknown can be used. The target nucleic acid can be isolated from a living cell or tissue sample for the purpose of examining gene expression. When the target nucleic acid is mRNA, it is preferably converted to cDNA by reverse transcription. For the purpose of investigating gene mutation and polymorphism, the target nucleic acid is preferably amplified by a reaction system containing a labeled primer or labeled dNTP, but is not limited thereto. The method for amplifying the target nucleic acid is not particularly limited, such as PCR method, LAMP method, and Invader method, and can be amplified using reagents and equipment suitable for each method.

In order to facilitate detection of the nucleic acid during amplification of the target nucleic acid, labeling is preferred. As labeling methods, there are RI method and non-RI method, but it is preferable to use non-RI method because of easy handling. Non-RI methods include fluorescent labeling and enzyme labeling. Any fluorescent label can be used as long as it can bind to the base part of the nucleic acid, such as cyan dyes (eg, Cy 3 and Cy 5 in the Cy Dye ™ series), rhodamine 6 G reagent. , N-acetoxy_N ₂ —acetylyl nofnoleolene (AAF), AA IF (AAF's iodine derivative), and the like are not limited thereto. Examples of enzyme labels include, but are not limited to, alkaline phosphatase, peroxy, and nandase.

(1-2) Hybridization

The hybridization can be carried out by spotting a target nucleic acid, preferably an aqueous solution in which a labeled target nucleic acid is dissolved or dispersed, onto the nucleic acid microarray of the present invention. The amount of spotting is preferably in the range of 1 to 100 μL from the viewpoint of facilitating detection visually or with an apparatus, but is not limited thereto. Hybridization may be performed under conditions that allow the target nucleic acid and the nucleic acid immobilized on the array to form a hybrid. Usually, it is preferably carried out in a temperature range of about 25 to 70 ° C. and in a range of about 10 minutes to 24 hours, but is not limited thereto. After completion of hybridization, wash with washing solution to remove unreacted target nucleic acid. Examples of the washing solution include those containing a surfactant in a buffer solution.

(2) A step of detecting the nucleic acid hybrid formed by the contacting step The detection method can be appropriately set according to the label. In the case of RI labeling, it can be detected by autoradiography. In the case of fluorescent labels, light with an excitation wavelength suitable for each fluorescent label is irradiated and the fluorescence intensity is measured. Can be detected. In the case of enzyme labeling, measurement can be performed by adding a substrate for each enzyme. When alkaline phosphatase is used, for example, p-nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolinolephosphate p-toluidine salt (BC IP) are added as substrates. The force that can be measured visually or with a CCD camera optodensitometer is not limited to this.

The detection method based on the combination of the Al force phosphatase label and the substrate NB TZB C IP can be suitably used for clinical diagnosis because the measurement result can be visually observed. The nucleic acid microarray of the present invention can be used for various applications such as gene expression monitoring, dienotyping, and genetic polymorphism screening. In clinical practice, it is expected to be applied to clinical diagnosis and tailor-made medicine. Example

Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to these examples.

Example 1

Substrate preparation

A PP substrate containing an inorganic filler was prepared by a co-extrusion machine using 500 g of polypropylene (PP) as a resin and 50 g of titanium dioxide particles having an average particle diameter of about 500 μπι as an inorganic filler.

Ion beam irradiation

A small ECR ion shower device (EI S-200 ER, manufactured by Erio Nitas) was used as the ion beam source. Mounting the PP substrate to the substrate holder, after evacuating the chamber foremost about ^{5. 0 X 10- 3 ~ 3. 0} X 1 0- 4 P a, was introduced into the chamber one nitrogen 2. 38 sccm . Nitrogen ions were extracted by irradiating a 50 W microwave in the plasma source and accelerated at a voltage of 600 eV to irradiate the PP substrate surface.

DNA probe As the DNA probe, the DNA probe shown in SEQ ID NOS: 1 and 2 (provided by Sigmajienosis Japan) was used. The DNA probe of SEQ ID NO: 1 is an estrogen receptor allele (X ba I polymorphism) that is cleaved by the restriction enzyme X ba I in the intron region between the first and second etasons. (X-type) can be detected. On the other hand, the DNA probe of SEQ ID NO: 2 can detect a sequence (X type) that is not cleaved by the restriction enzyme X ba I among the estrogen receptor alleles (X ba I polymorphism). Polythymine was added to these DNA probes using terminal transferase (manufactured by New England Bio 1ab) at the 3 and terminal ends. Specifically, 41 1 for terminal transferase (SO unit Z zl), 10 xl for deoxythymidine triphosphate (10 pmol Z / l), DNA probes of SEQ ID NOS: 1 and 2 (5 O mM) ) 4 μ 1, 5 μ 1 of NEB uffer 4 attached to the product, and 50 μ 1 of purified water containing 5 μ 1 of the force codylate buffer supplied with the product. By adding 20 XSSC buffer 50 / i 1 attached to the product, a polythymine-added nucleic acid probe (average length of about 400 bp) 2 pmol / μ 1 was obtained.

X-type detection probe (SEQ ID NO: 1): tctggagttg ggatga

x-type detection probe (SEQ ID NO: 2): gtggtctaga gttggg

Immobilization of DNA probe

Next, the polythymine-added nucleic acid probe of SEQ ID NO: 1 or 2, respectively,

1; 0 p m o 1 / i 1, diluted with 10 X S S C buffer attached to the product, and applied to each of the ion beam treated PP substrates by 0. 3 1

Nucleic acid microarrays were produced by immobilizing 2 nm UV light for 2 minutes.

Comparative Example 1

In Example 1, a DNA microarray was produced in the same manner as in Example 1 except that no inorganic filler was contained. Experimental example 1: Inspection using DNA microarray 1

Increase in genomic DNA of specimens

A forward primer having the base sequence shown in SEQ ID NO: 3 and a reverse primer having the base sequence shown in SEQ ID NO: 4 and conjugated with piotin at the 5 'end. The nucleotide sequence containing the XbaI polymorphism was amplified by the PCR method using Taq DNA polymerase (Roche Diagnostats). In the amplification reaction, the denaturation process was 94 ° C for 30 seconds, the annealing process was 55 ° C for 20 seconds, the chain extension process was 72 ° C for 20 seconds, and 30 cycles for one cycle.

Forward primer (SEQ ID NO: 3): gttccaaatg tcccagccgt

Reno primer (Care [| number 4): cctgcaccag aatatgttac c

Nucleic acid detection

To 20 μL of the amplified DNA solution, add a solution (20 μL) of sodium hydroxide (5 Μ) and ethylenediamine tetraacetic acid (0.05 Μ), stir well, and let stand for 5 minutes. It was denatured into a main chain. Solution containing denatured DNA, solution containing sodium dodecyl sulfate (0.01 w_v%), sodium chloride (1.8 w / v%) and sodium citrate (1. OwZv%) (l mL) In addition, one microarray of Example 1 or Comparative Example 1 was infiltrated and shaken at a reaction temperature of 45 ° C. for 30 minutes for hybridization. Then add alkaline phosphatase-labeled streptavidin, and then add p-nitroblue tetrazolium (NBT) and 5-promo-4-chloro-3-indolyl phosphate ρ-toluidine salt (BCIP). A color reaction with alkaline phosphatase bound to the sample bound to the probe was performed. That is, when nucleic acid detection is accurate, the DNA probe containing the sequence of SEQ ID NO: 1 does not develop color, and the DNA probe containing the sequence of SEQ ID NO: 2 develops color.

Figure 1 shows the result of color development. The DNA microarray prepared from the PP substrate containing titanium dioxide of Example 1 can be easily judged visually, whereas Comparative Example 1 DNA microarrays made from PP substrates that did not contain any titanium dioxide produced almost no color. This indicates that inclusion of an inorganic filler in the plastic substrate can efficiently fix the nucleic acid to the plastic substrate. Example 2

A DNA microarray was produced in the same manner as in Example 1 except that a polyethylene terephthalate (PET) substrate was used instead of the polypropylene substrate in Example 1.

Comparative Example 2

In Example 2, a DNA microarray was produced in the same manner as in Example 2 except that no inorganic filler was contained.

Experimental example 2: Inspection using DN A microarray 2

Similar to Experimental Example 1 except that DNA microarrays manufactured from PET substrates of Example 2 and Comparative Example 2 were used instead of DNA microarrays manufactured from PP substrates of Example 1 and Comparative Example 1. Thus, the nucleic acid was detected.

Figure 2 shows the results of color development. The DNA microarray prepared from the PET substrate containing the inorganic filler of Example 2 can be easily judged visually, while the comparative example

The DNA microarray made from the PET substrate that does not contain 2 inorganic fillers hardly developed color.

Example 3

A DNA microarray was produced in the same manner as in Example 1 except that a polycarbonate (PC) substrate was used instead of the PP substrate in Example 1. Figure 3 shows the color development.

Example 4

Samples for surface roughness measurement and X-ray photoelectron spectroscopy (XP S) were prepared. Specifically, in Example 3, the substrate itself was used without immobilizing the nucleic acid.

Experimental example 3: Measurement of surface roughness of substrate for DNA microarray

Before and after ion beam irradiation on the polycarbonate substrate surface used in Example 4 The surface roughness Ra was measured using a laser microscope (manufactured by Keyence Corporation). Specifically, the average surface roughness in the observation field was measured using the analysis software attached to the product. As a result, it was about 0.0 before the ion beam irradiation, but about

0.15 111, and it was confirmed that the surface roughness Ra increased.

Example 5

Samples for X-ray photoelectron spectroscopy (XPS) were prepared. Specifically, a nucleic acid microarray substrate was produced in the same manner as in Example 4 except that in Example 4, the number of ion beam irradiations was set to 2. In this example, since the purpose is to prepare a sample for X-ray photoelectron spectroscopy (XPS), no nucleic acid is immobilized. The ion irradiation interval was set to 30 seconds.

Example 6

In Example 5, a nucleic acid microarray substrate was produced in the same manner as in Example 5 except that the number of ion beam irradiations was three.

Example 7

In Example 5, a nucleic acid microarray substrate was produced in the same manner as in Example 5 except that the number of ion beam irradiations was changed to 4.

Comparative Example 4

In Example 4, a nucleic acid microarray substrate was produced in the same manner as in Example 4 except that the substrate did not contain an inorganic filler.

Example 4: Surface analysis of a DNA microarray substrate

The elemental composition ratios on the polycarbonate substrate surfaces of Examples 4 to 7 and Comparative Example 4 were measured using an X-ray photoelectron spectrometer (XPS, ULVAC-FAI). Specifically, A 1 した ο; line was used as the X-ray source, and measurement was performed using a charged neutralization gun at approximately 25 ° C and under reduced pressure (approximately 10 to ¹⁵ Pa or less) (analysis area: Diameter 8 0 0 / m). The results are shown in Table 1. Since titanium was confirmed in the region 5 nm deep from the surface, it was confirmed that titanium dioxide was contained in at least the surface portion of the polycarbonate substrate. In addition, by increasing the number of ion beam irradiation, It was confirmed that the composition ratio of titanium increased. This is considered to be the reason why the nucleic acid is more immobilized by increasing the number of ion beam irradiations.

table 1

Industrial applicability.

According to the present invention, a nucleic acid microarray and a substrate for nucleic acid microarray can be provided at low cost. Further, the base material can fix the nucleic acid so as to be strong and suitable for hybridization. According to the method for producing a nucleic acid microarray of the present invention, the nucleic acid microarray can be produced inexpensively and efficiently. According to the detection method using the nucleic acid microarray of the present invention, DNA expression analysis and genotyping can be performed at low cost and with high sensitivity, compared with the conventional method. In addition, according to the detection method of the present invention, by introducing a specific label into the nucleic acid hybrid, nucleic acid can be detected and determined with a so-called bedside without using a large-scale detection apparatus.

.

This application is based on Japanese patent application No. 2 0 0 5 — 1 0 5 3 9 2 (filing date: March 3 1st, 2000), the contents of which are described in this specification. All are included.

Claims

The scope of the claims

1. A nucleic acid microarray in which nucleic acid is immobilized on a plastic substrate, and at least a surface portion of the substrate is made of an oxide-based filler, a hydroxide-based filler, a carbonate-based filler, and a sulfate-based filler. A nucleic acid microarray containing an inorganic filler selected from the group consisting of:

2. The nucleic acid microphone mouth array according to claim 1, wherein the inorganic filler is titanium dioxide.

3. The nucleic acid microarray according to claim 1 or 2, wherein the plastic is polypropylene, polymethyl methacrylate, polycarbonate, or polyethylene terephthalate.

4. A nucleic acid microarray in which a nucleic acid is immobilized on a plasma-treated or ion-beam-treated plastic substrate, and an oxide-based, hydroxide-based, A nucleic acid microarray containing an inorganic filler selected from the group consisting of a salt-based filler and a sulfate-based filler.

5. The nucleic acid microarray according to claim 4, wherein the inorganic filler is titanium dioxide.

6. The nucleic acid microarray according to claim 4 or 5, wherein the plastic is polypropylene, polymethyl methacrylate, polycarbonate, or polyethylene terephthalate.

7. The nucleic acid microarray according to any one of claims 4 to 6, wherein the substrate has a surface roughness R a (J I S B 060 1-1 9 94) of 0.06 to 0.5;

8. Plasma treatment or ion beam treatment of a plastic substrate containing an inorganic filler at least on the surface portion; and

A method for producing a nucleic acid microarray comprising:

9. The plasma processing step is performed in an inert gas atmosphere, and the ion beam processing step is performed using an inert gas as an ionized gas. The manufacturing method according to claim 8.

10. The method according to claim 9, wherein the inert gas is nitrogen gas.

1 1. The production method according to any one of claims 8 to 10, wherein the nucleic acid has a length of 50 bases or more.

1 2. The production method according to any one of claims 8 to 11, wherein the step of fixing the nucleic acid is performed by UV irradiation.

1 3. Nucleic acid microarray substrate made of plastic, consisting of an oxide-based filler, a hydroxide-based filler, a carbonate-based filler and a sulfate-based filler on at least the surface of the substrate. A substrate containing an inorganic filler selected from the group.

14. The substrate according to claim 13, wherein the inorganic filler is titanium dioxide.

1 5. The substrate according to claim 13 or 14, wherein the substrate surface is further subjected to plasma treatment or ion beam treatment.

1 6. The substrate according to claim 15, wherein the substrate has a surface roughness R a (JI S B 06 01— 1 9 94) force S 0.06 to 0.5 / X m.

1 7. A step of contacting the nucleic acid microarray according to any one of claims 1 to 7 or the nucleic acid microarray obtained by the production method according to any one of claims 8 to 12 and a sample containing the nucleic acid to be analyzed. , and

Detecting the nucleic acid hybrid formed by the contacting step

A method for detecting a nucleic acid having a specific base sequence.

1 8. The detection method according to claim 17, wherein the nucleic acid hybrid is labeled.

1 9. The detection method according to claim 18, wherein the label is alkaline phosphatase.