JP4689475B2 - Nucleic acid immobilization molded body and nucleic acid immobilization method - Google Patents

Description

  The present invention relates to a nucleic acid immobilization molded article used for a nucleic acid microarray in which nucleic acids (DNA fragments and oligonucleotides) useful for analysis of gene expression, mutation, polymorphism, etc., that is, nucleic acid probes are aligned and immobilized on a solid phase surface. And a nucleic acid immobilization method of immobilizing a nucleic acid probe on the nucleic acid immobilization molded article.

In recent years, with the progress of genome sequencing, the entire base sequence of the genome of each organism is being clarified, and a technique for effectively utilizing this result is desired.
Nucleic acid microarray is a technology that can analyze multiple genes simultaneously, and is developed not only as a base sequencing method but also as a method for efficiently examining gene expression levels and polymorphisms. Biological classification such as tailor-made medicine and fungi Application to identification of diseases and diagnosis of diseases has been developed.

  Nucleic acid microarrays that can be used for tailor-made medicine, as well as biological classification or variant identification, are generally sufficient to qualitatively detect specific base sequences. Furthermore, considering the risk of nosocomial infection, an inexpensive disposable one such as a test paper is required.

  On the other hand, the base material used for the nucleic acid microarray is generally mainly glass or silicon. When nucleic acid probes are immobilized on the surface of these base materials, the surface of the base material is modified with chemical functional groups on the surface by using, for example, a silane coupling reaction, while the ends of the nucleic acid probes are used. And a method of modifying the functional group that can be covalently bonded to the chemical functional group and immobilizing it by forming a covalent bond. Patent Document 1 discloses a combination of an amino group and an epoxy group, a thiol group and a maleimide group, and the like. There is also a technique for synthesizing a nucleic acid probe directly on a fixed surface using a lithography technique or the like.

  However, when a substrate such as glass is used, an operator must handle the substrate carefully. If you get hurt by a piece of glass, there is a risk of nosocomial infection. Moreover, the above-mentioned various nucleic acid probe immobilization methods on a substrate such as glass require manufacturing costs.

  On the other hand, attempts have been made to immobilize nucleic acid probes on inexpensive plastic substrates. In these plastic substrates, the substrate surface is hydrophobic (water repellent), and it is difficult to immobilize a nucleic acid probe that is a hydrophilic polymer.

  In view of such circumstances, a technique is also disclosed in which a functional group is generated on the surface of a plastic substrate by oxidizing it, and a nucleic acid probe is immobilized by covalent bonding in the same manner as described above (Patent Document 2). ).

  However, these various nucleic acid probe immobilization methods still require manufacturing costs, and still cannot provide inexpensive nucleic acid microarrays suitable for tailor-made medicine.

JP 2000-270896 A JP 2005-010004 A

  An object of the present invention is to provide a molded article for nucleic acid immobilization capable of immobilizing a nucleic acid probe at a low cost and effectively in producing a nucleic acid microarray, and to provide an effective method for immobilizing a nucleic acid probe. is there.

  In order to solve the above-mentioned problems, the present inventors have conducted extensive research, and as a result, a molded article for nucleic acid immobilization having a structure in which a base material is a plastic layer and includes at least a hydrophilic coat layer on the surface thereof, The inventors have found that a nucleic acid probe can be effectively immobilized on a molded article for nucleic acid immobilization by devising the contact angle of the hydrophilic coat layer and the thickness of the hydrophilic coat layer, and the present invention has been completed.

That is, this invention consists of the following.
1. On the surface of the plastic substrate, at least a nucleic acid fixing molded hydrophilic polymeric resin is applied, the center line average surface roughness Ra of the substrate is at 100nm or 1000nm or less, the hydrophilic high A molded article for nucleic acid immobilization, wherein the hydrophilic coating layer formed by applying a molecular resin has a thickness of 5% to 70 % of the center line average surface roughness Ra of the substrate.
2. 2. The molded article for nucleic acid immobilization according to item 1, wherein a contact angle of the hydrophilic coating layer is 0 ° or more and 60 ° or less.
3. 3. The molded article for nucleic acid fixation according to item 1 or 2, wherein the plastic is any one selected from polystyrene, polycarbonate, polypropylene, polyamide, and acrylic resin.
4). The hydrophilic polymer resin is mainly composed of one or more selected from siloxane resins, polyvinyl alcohol, ethylene vinyl alcohol, polyvinyl acetate, polyethylene glycol, polyester resins, acrylic resins, and melanin resins. 4. The molded article for nucleic acid immobilization according to any one of items 1 to 3, wherein
5. 5. Nucleic acid to a nucleic acid immobilization molded article, wherein a nucleic acid probe-containing aqueous solution containing a nucleic acid probe is dropped on the nucleic acid immobilization molded article according to any one of items 1 to 4 and dried. Probe immobilization method.
6). 6. The method for immobilizing a nucleic acid probe according to item 5, wherein polythymine is added in advance to the nucleic acid probe.
7). 7. The method for immobilizing a nucleic acid probe according to 5 or 6 above, wherein ultraviolet rays are irradiated during the drying.

  The molded article for nucleic acid immobilization according to the present invention uses a plastic layer as a base material and has a hydrophilic coat layer laminated on the surface thereof, so that it is economical, convenient for handling, and efficient nucleic acid probe. Can be fixed, and there is no risk of nosocomial infections. According to the nucleic acid immobilization method using the molded article for nucleic acid immobilization of the present invention, the nucleic acid probe can be effectively immobilized.

  In the present invention, the “molded body for nucleic acid immobilization” refers to one for immobilizing a nucleic acid probe in a nucleic acid microarray generally used in the field of gene analysis. The molded article for nucleic acid immobilization of the present invention has a configuration in which a plastic layer is used as a base material and at least a hydrophilic coat layer is laminated on the surface thereof.

Examples of the shape of the molded body include a plate material, a container, a film, and a tube. Among them, a rectangular film or a plate-like material is preferable from the viewpoint of easy handling, but is not limited thereto. Further, the size of the molded body is not particularly limited, but a surface to be detected needs a certain area from the viewpoint of facilitating detection. For example, if the molded body is a rectangular film or plate material, the area of the surface, from the viewpoint it is easy to handle, about 40～1000Mm ^2, preferably about 60~300mm ^2.

  The manufacturing method of the said molded object can be selected according to a condition by those skilled in the art. For example, extrusion molding, injection molding, melt molding, compression molding and the like can be mentioned. From the viewpoint of production cost and ease, extrusion molding is preferable, but the present invention is not limited thereto.

  In the present invention, a hydrophobic general-purpose plastic that is widely used in industry can be used as the plastic used as the substrate. Specific examples include polyethylene, polystyrene, polycarbonate, polypropylene, polyamide, phenol resin, epoxy resin, polycarbodiimide resin, polyvinyl chloride, polyvinylidene fluoride, polyvinyl fluoride, polyimide, and acrylic resin. Of these, polystyrene, polycarbonate, polypropylene, polyamide and acrylic resin are preferred from the viewpoint of being inexpensive, relatively strong and easy to handle, but the present invention is not limited thereto.

  In the present invention, the “centerline surface average roughness” is defined by JIS surface roughness JIS-B-0601 and refers to a parameter that can be represented by Ra. In the present invention, the method for measuring the surface average roughness Ra can be determined using, for example, an atomic force microscope, but is not limited thereto. The plastic used for the base material of the molded body of the present invention can be selected from those having an average surface roughness of 100 to 1000 nm, preferably 100 to 900 nm.

  In the present invention, the “contact angle” means an angle formed by a contact portion between the liquid droplet and the solid surface, although a liquid droplet is formed when the liquid touches the solid surface. In other words, it refers to the angle between the tangential plane and the liquid / solid surface at the liquid surface and liquid / solid contact points. The contact angle can generally be expressed between 0 and 180 °. As the droplets, water droplets are generally used, and as the angle approaches 0 °, the water droplets extend flat on the solid coating surface, that is, show a hydrophilic state, and the solid surface can be said to be hydrophilic. . On the other hand, as the angle approaches 180 °, the water droplets are completely repelled from the fixed surface and show a state of being in contact with the painted solid surface in a shape similar to a sphere, and the solid surface can be said to be hydrophobic. The contact angle of the hydrophilic coat layer of the present invention is 0 to 60 °, preferably 0 to 40 °, more preferably 0 to 30 ° from the viewpoint of improving the nucleic acid immobilization rate. This is because it becomes difficult to fix the hydrophilic nucleic acid probe when the angle exceeds 60 °.

Various natural hydrophilic polymer substances and synthetic water-soluble polymer substances can be used as the hydrophilic material for forming the hydrophilic coat layer satisfying the above conditions.
Examples of natural hydrophilic polymer materials include starches such as carboxymethyl starch and dialdehyde starch; celluloses such as carboxymethyl cellulose and hydroxyethyl cellulose; polysaccharides such as tannin, lignin, alginic acid, gum arabic heparin, chitin, and chitosan. It can be illustrated.

  Synthetic hydrophilic polymer materials include: siloxane-based, tetraethoxysilane; vinyl alcohol-based, polyvinyl alcohol; polyalkylene oxide-based, polyethylene oxide; polyalkylene glycol-based, polyethylene glycol: acrylic acid-based, polyacrylic Acid soda; maleic anhydride as a methyl vinyl ether maleic anhydride copolymer, methyl vinyl ether maleic anhydride soda, methyl vinyl ether maleic anhydride ammonium salt, maleic anhydride ethyl ester copolymer; phthalic acid as polyhydroxyethyl Phthalate ester; water-soluble polyester, polydimethylolpropionate ester; acrylamide type, polyacrylamide hydrolyzate, polyacrylamid Quaternaries, further, polyvinylpyrrolidone, polyethyleneimine, polyethylene sulfonate, and water-soluble nylon and the like.

  As the hydrophilic material for forming the hydrophilic coating layer of the present invention, among the above hydrophilic materials, in particular, from the viewpoint of processability of the coating liquid, tetraethoxysilane, polyvinyl alcohol, polyvinyl acetate, polyethylene glycol, Polyester resins, acrylic resins or melanin resins are preferred. In order to sufficiently satisfy the contact angle, these resin materials may be mixed or copolymerized. Further, it may be possible to prevent the contact angle from being lowered to some extent by performing a plasma treatment using argon, oxygen, nitrogen alone or a mixed gas after the resin coating.

  The hydrophilic coating layer of the present invention can contain an inorganic additive as long as the above contact angle condition is satisfied. Examples of such inorganic additives include spherical particles, fibers, and flakes. From the viewpoint of dispersibility of inorganic additives, spherical particles are preferable. Further, the particle size of the spherical particles can be appropriately selected while taking the above-mentioned blending amount so that the surface roughness of the molded body to be blended is within the range described later, but the inorganic additive dispersibility in the resin In view of the above, 0.01 to 0.10 μm is preferable, and 0.02 to 0.08 μm is more preferable.

  Examples of the inorganic additive include titanium oxide, titanium dioxide, silica, diatomaceous earth, alumina, iron oxide, zinc oxide, magnesium oxide, aluminum hydroxide, magnesium hydroxide, and calcium carbonate. From the group consisting of these, At least one selected may be contained as a subcomponent of the hydrophilic coat layer. Among these, silica is preferable from the viewpoint of hydrophilicity, but the present invention is not limited to this.

  The blending amount of the inorganic additive is appropriately set according to the main component of the hydrophilic coat layer in which the inorganic additive is blended and the kind of the inorganic additive. From the viewpoint of dispersibility of the inorganic additive, for example, The amount is 10 parts by weight or less, preferably 1 part by weight or less, based on 100 parts by weight of the main component.

  In the present invention, the thickness of the hydrophilic coat layer can be 5 to 90%, preferably 20 to 80%, more preferably 30 to 70% of the surface average roughness of the plastic layer. If it is higher than 90%, the surface on which the nucleic acid probe is immobilized becomes smooth, and it becomes difficult to immobilize the nucleic acid probe. On the other hand, if it is lower than 5%, the hydrophilicity of the surface on which the nucleic acid probe is immobilized cannot be sufficiently obtained, and the wettability between the surface to be immobilized and the aqueous solution of nucleic acid is not improved, so the nucleic acid probe is difficult to immobilize. It is. In general, when a nucleic acid probe, which is a hydrophilic polymer, is immobilized on the surface of a hydrophobic plastic layer, a person skilled in the art considers only the affinity between the surface to be immobilized and the nucleic acid probe, and the plastic layer is hydrophilic. It is conceivable to form a thick coat layer and to immobilize the nucleic acid probe on the hydrophilic coat layer. However, when the hydrophilic coat layer is formed thick, the nucleic acid probe is not immobilized as expected.

  The method for forming the hydrophilic coating layer on the plastic layer can be appropriately selected by those skilled in the art in consideration of the affinity between the plastic layer and the hydrophilic coating layer, and is not particularly limited. , Dip coating method, Langmuir-Blodgett method, roll coating method, micro gravure method and die method, etc., and roll coating method or micro gravure method is preferable from the viewpoint of production cost, but the present invention is limited to this. It is not a thing.

  The solvent of the hydrophilic material used for the coating can be appropriately selected by those skilled in the art in consideration of the coating method, the solubility of the hydrophilic material, the dispersibility of the accessory components, and the affinity between the solvent and the plastic layer. Although not limited, for example, when the true solvent component of the hydrophilic material is alcoholic, ethanol, isopropyl alcohol, or the like is used.

  The present invention also extends to a method for immobilizing a nucleic acid probe on the molded article for nucleic acid immobilization of the present invention. The nucleic acid probe can be immobilized by a spotting method. Specifically, it can be carried out by dropping a nucleic acid-containing aqueous solution containing a nucleic acid probe prepared in advance onto the molded article for nucleic acid fixation of the present invention and drying it. The dropping of the nucleic acid-containing aqueous solution can be performed using a spotter device provided in the nucleic acid microarray manufacturing apparatus, or can be discharged by an ink jet method. After dropping the nucleic acid-containing aqueous solution, the nucleic acid-containing aqueous solution can be dried by an ordinary method, and the nucleic acid probe can be immobilized on the nucleic acid-immobilized molded body. Moreover, ultraviolet irradiation can also be given at the time of drying.

  As a nucleic acid probe to be immobilized, DNA, RNA, PNA (peptide nucleic acid) or the like can be used. Among these, single-stranded DNA is selected from the viewpoint of production cost and frequency of use, and is generally referred to as a DNA probe. As a method for preparing DNA, for example, synthesized DNA (oligonucleotide) or cDNA reversely transcribed from mRNA may be used. The oligonucleotide can be synthesized using a commercially available automatic nucleic acid synthesizer or the like. Examples include nucleic acid synthesis or partial synthesis modification, cDNA that is complementary DNA using mRNA as a template, PCR products, and the like. In the nucleic acid of the present invention, a functional group may be added as a reaction site on the substrate, for example, polythymine may be added.

  The nucleotide sequence of the nucleic acid probe and the like can be appropriately designed by those skilled in the art depending on the purpose of use and is not particularly limited. For example, when a synthetic oligonucleotide probe is used, the length of the probe can be designed to be about 10 to 30 bases, preferably about 12 to 26 bases.

  Furthermore, in order to improve the immobilization rate of the nucleic acid probe to the base material, a method of adding about 50 to 300 bases of an irrelevant base sequence that does not affect the hybridization reaction to the end of the nucleic acid probe can be used. Examples of the irrelevant base sequence include polyadenine, polyguanine, and polythymine. Among them, polythymine is preferable from the viewpoint of improving the immobilization rate of the nucleic acid probe. This addition of polythymine can be appropriately carried out by those skilled in the art using, for example, terminal transferase.

  Hereinafter, in order to deepen the understanding of the present invention, examples will be shown and described, but the present invention is not limited to these examples.

Example 1
A white polyethylene terephthalate film (Lumirror E20, Toray Industries, Inc.) having an average surface roughness of 0.19 μm is referred to as a base material (hereinafter referred to as “base material A”). The hydrophilic polymer resin (Fressera R manufactured by Matsushita Electric Works Co., Ltd.) is added to one side of the film by a gravure coater at a solid content concentration of 0.1%. The solution is diluted with isopropyl alcohol until the coating thickness is 0.1 μm (about 53% of the average surface roughness), and the hydrophilic coating layer is laminated, thereby fixing the nucleic acid according to the present invention. A molded product was obtained. Moreover, when the pure water contact angle on the hydrophilic coat layer surface side was measured, it was 18 °.

(Example 2)
A molded article for nucleic acid immobilization according to the present invention having a hydrophilic coating layer in the same manner as in Example 1 except that a white polyethylene terephthalate film having a surface roughness of 0.13 μm (Toyobo Co., Ltd., Chrisper K1211) was used as a substrate. Got. Further, when the pure water contact angle on the hydrophilic coat layer side was measured, it was 19 °.

(Example 3)
As the hydrophilic polymer resin for forming the hydrophilic coating layer, polyvinyl alcohol (Nippon Synthetic Chemical Co., Ltd. Goseifamer Z100) with 1% colloidal silica particle size of 20 nm was used. The substrate A was hydrophilic in the same manner as in Example 1, except that the pure water contact angle was adjusted to 60 ° or less after coating and drying, and then 0.1 μm thick coating was applied. A molded article for nucleic acid immobilization having a constitution having an adhesive coat layer was obtained. Further, when the pure water contact angle on the hydrophilic coat layer surface side was measured, it was 18 °.

Example 4
The nucleic acid immobilization molded body obtained in Example 1 was further subjected to oxygen plasma treatment on the surface of the hydrophilic coat layer to obtain a nucleic acid immobilization molded body. Moreover, when the pure water contact angle on the hydrophilic coat layer surface side was measured, it was 12 °.

(Comparative Example 1)
It was produced in the same manner as in Example 1 except that the hydrophilic coating layer had a thickness of about 0.3 μm (about 150% of the average surface roughness) with respect to the substrate A to obtain a molded article for nucleic acid immobilization.

(Comparative Example 2)
A smooth polyethylene terephthalate film (Toyobo A4100) having a surface average roughness of 0.07 μm was used as the plastic layer, and a hydrophilic coating layer (same as in Examples 1 and 2) was applied thereon with a gravure coater. A molded article for nucleic acid fixation was obtained.

(Comparative Example 3)
Without performing hydrophilic coating on the base material A, the base material A was directly used as a molded article for nucleic acid fixation.

(Comparative Example 4)
The substrate A was not subjected to hydrophilic coating, and was directly subjected to oxygen plasma treatment to obtain a molded article for nucleic acid immobilization.

(Experimental example 1) Determination of the amount of nucleic acid immobilized by production and detection of nucleic acid microarrays Nucleic acid probes were immobilized on the nucleic acid immobilization molded bodies of Examples 1 to 4 and Comparative Examples 1 to 4, and the detection was visually determined. Thus, whether or not the nucleic acid was successfully immobilized was examined.

1) Preparation of nucleic acid probe As the nucleic acid probe, the nucleic acid probe represented by SEQ ID NO: 1 (provided by Sigma Genosys Japan) was used. The nucleic acid probe of SEQ ID NO: 1 in the sequence listing is an estrogen receptor allele (Xba I polymorphism) present in the intron region between the first exon and the second exon, and the sequence that is not cleaved by the restriction enzyme Xba I ( x-type) can be detected. Polythymine was added to the nucleic acid probe using a terminal transferase (New England Biolab) at the 5 ′ end. Specifically, 4 μl of terminal transferase (20 units / μl), 10 μl of thymidine triphosphate (10 pmol / μl), 4 μl of the nucleic acid probe of SEQ ID NO: 1 (50 mM), 5 μl of NEBuffer 4 attached to the product, and product After preparing 50 μl of purified water containing 5 μl of the attached cacodylate buffer solution, reacting at 37 ° C. for 4 hours, and adding 50 μl of 20 × SSC buffer solution attached to the product, polythymine-added nucleic acid probe (about 300 bp) 2 pmol / μl was obtained respectively.

Sequence number 1: gtggtctaga gttggg 16

2) Immobilization of nucleic acid probe and production of nucleic acid microarray Next, the nucleic acid probe of SEQ ID NO: 1 to which polythymine was added was diluted with 10 × SSC buffer attached to the product so as to be 1.0 pmol / μl. The nucleic acid microarray was manufactured by applying 0.5 μL each to the molded article for nucleic acid immobilization of 1-4 and Comparative Examples 1-4 and irradiating them with 312 nm ultraviolet rays for 2 minutes.

3) Amplification of specimen genomic DNA Reverse the xx-type specimen genomic DNA having the forward primer having the base sequence shown in SEQ ID NO: 2 and the base sequence shown in 3 and having biotin bound to the 5 ′ end. A base sequence containing the Xba I polymorphism was amplified by a PCR method using a primer and Taq DNA polymerase (Roche Diagnostics). PCR reaction conditions were 94 ° C. and 30 seconds for the denaturation process, 55 ° C. and 20 seconds for the annealing process, 72 ° C. and 20 seconds for the chain extension process, and amplification was performed by performing this process for 30 cycles.

Sequence number 2: gttccaaatg tcccagccgt 20
Sequence number 3: cctgcaccag aatatgttac c 21

4) Nucleic acid detection and confirmation of nucleic acid immobilization amount To 20 μL of the amplified DNA solution, a solution (20 μL) of sodium hydroxide (5 M) and ethylenediaminetetraacetic acid (0.05 M) was added, stirred well, and allowed to stand for 5 minutes. The amplified DNA was denatured into single strands. In this sample solution, a solution (1 mL) of sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium citrate (1.0 w / v%) and the nucleic acid microarray were added. Each was added and reacted by shaking for 30 minutes at a reaction temperature of 45 ° C. Thereafter, alkaline phosphatase labeled streptavidin is added, and nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) are added to bind to each probe on the detection strip. The alkaline phosphatase bound to the prepared sample was developed.

  The above results are summarized in the following table. Examples 1 to 4 all had good color development, while Comparative Examples 1 to 4 were difficult to visually confirm color development. That is, the usefulness of the molded article for nucleic acid fixation of the present invention was suggested.

5) Nucleic acid detection and confirmation of nucleic acid fixation

  The molded article for immobilizing nucleic acid of the present invention uses a plastic layer as a base material, and thus is economical, convenient for handling, and has no risk of nosocomial infection. Moreover, according to the nucleic acid immobilization method using the nucleic acid immobilization molded body of the present invention, nucleic acids can be immobilized effectively, and the cost for industrial production is not required.

Claims (7)

On the surface of the plastic substrate, at least a nucleic acid fixing molded hydrophilic polymeric resin is applied, the center line average surface roughness Ra of the substrate is at 100nm or 1000nm or less, the hydrophilic high A molded article for nucleic acid immobilization, wherein the hydrophilic coating layer formed by applying a molecular resin has a thickness of 5% to 70 % of the center line average surface roughness Ra of the substrate. The molded article for nucleic acid immobilization according to claim 1, wherein a contact angle of the hydrophilic coat layer is 0 ° or more and 60 ° or less. The nucleic acid immobilization molded article according to claim 1 or 2, wherein the plastic is any one selected from polystyrene, polycarbonate, polypropylene, polyamide, and acrylic resin. The hydrophilic polymer resin is mainly composed of one or more selected from siloxane resins, polyvinyl alcohol, ethylene vinyl alcohol, polyvinyl acetate, polyethylene glycol, polyester resins, acrylic resins, and melanin resins. The molded article for nucleic acid fixation according to any one of claims 1 to 3, wherein A nucleic acid probe-containing aqueous solution containing a nucleic acid probe is dropped on the nucleic acid-immobilized molded article according to any one of claims 1 to 4, and dried, to the nucleic acid-immobilized molded article. Nucleic acid probe immobilization method. 6. The nucleic acid probe immobilization method according to claim 5, wherein polythymine is added in advance to the nucleic acid probe. The method for immobilizing a nucleic acid probe according to claim 5 or 6, wherein ultraviolet rays are irradiated during the drying.