JP2000270879A - Nucleic acid-immobilized gel-holding fiber, fiber array and flakes thereof - Google Patents

Abstract

(57) Abstract: A nucleic acid-immobilized gel-holding fiber, a nucleic acid-immobilized gel-holding fiber array, and a slice thereof. Effect A thin section having a fiber cross section of a nucleic acid-immobilized gel-holding fiber array in which nucleic acids are arbitrarily arranged at high density and accurately can be efficiently obtained with good reproducibility. Using this slice, the type and amount of nucleic acid in the specimen can be examined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a polymer material on which nucleic acids are immobilized. More specifically, the present invention relates to a nucleic acid-immobilized gel holding fiber in which a nucleic acid-immobilized gel is held on a fiber surface, a nucleic acid-immobilized gel-holding fiber array, and a thin slice thereof.

[0002]

2. Description of the Related Art In recent years, genome projects for various organisms have been promoted, and a large number of genes including human genes and their base sequences have been rapidly identified. The function of a gene whose sequence has been clarified can be examined by various methods. As one of the promising methods, gene expression analysis using the clarified nucleotide sequence information is known. For example, methods using various nucleic acid: nucleic acid hybridization reactions and various PCR reactions, such as Northern hybridizations, have been developed, and the relationship between various genes and their biological function expression should be examined by the method. Can be. However, these methods have limitations on the number of genes that can be applied. Therefore, in view of the entire complex reaction system consisting of a large number of genes at the individual level, which is being revealed through today's genome project, comprehensive and systematic analysis of genes by the above method is necessary. It is difficult. Recently, a DNA microarray method (DNA
A new analytical method or methodology called the chip method has been developed and attracted attention.

[0003] These methods are basically the same as conventional methods in that they are nucleic acid detection and quantification methods based on a nucleic acid: nucleic acid hybridization reaction, but they are mounted on a flat substrate called a microarray or chip. There is a great feature that a large number of DNA fragments are arranged and fixed at a high density. As a specific use of the microarray method, for example, a sample obtained by labeling an expression gene or the like of a cell to be studied with a fluorescent dye or the like is hybridized on a flat base piece, and nucleic acids (DN
A or RNA), a label is labeled with a fluorescent dye or the like, and then read at high speed by a high-resolution analyzer. Thus, the amount of each gene in the sample can be quickly estimated. In other words, the essence of this new method is basically the integration of the miniaturization of the reaction sample and the technology to sequence and align the reaction sample in a form that can be analyzed and quantified in a reproducible manner in a large, rapid and systematic manner. It is understood that there is.

[0004] As a technique for immobilizing nucleic acids on a substrate, similar to the Northern method described above, other than a method of immobilizing nucleic acids on a nylon sheet or the like at a high density, a technique for immobilizing nucleic acids on a substrate such as glass is used to further increase the density. And immobilized by coating with polylysine or the like, or a method of directly solid-phase synthesizing short-chain nucleic acids on a substrate such as silicon.

[0005] However, for example, the method of spotting and immobilizing nucleic acids on a substrate, such as glass, which has been chemically or physically modified on a solid surface [Science 270, 467-470 (1995)], uses a spot density rather than a sheet method. Although excellent in spot density and the amount of nucleic acid that can be fixed per spot, the direct synthesis method on a silicon substrate (US Patent 5,445,934, US Patent
t 5,774,305), and it is pointed out that it is difficult to reproduce. On the other hand, a method of regularly solid-phase synthesizing various kinds of short-chain nucleic acids in situ using a photolithography technique on a substrate such as silicon has been studied in terms of the number of nucleic acid species that can be synthesized per unit area (spot density) and Although the amount of immobilization per spot (the amount of synthesis) and the reproducibility are said to be superior to the spotting method, the types of compounds that can be immobilized are limited to relatively short-chain nucleic acids that can be controlled by photolithography. further,
Due to the expensive manufacturing equipment and the multi-stage manufacturing process, it is difficult to greatly reduce the cost per chip. In addition, as a technique for solid-phase synthesis of a nucleic acid on a small carrier to form a library, a method using minute beads is known.
This method is considered to be capable of synthesizing many kinds of long-chain nucleic acids at a lower cost than the chip method, and also capable of immobilizing longer-chain nucleic acids than cDNA and the like. However, unlike the chip method, it is difficult to produce a compound in which specified compounds are aligned with high reproducibility based on specified sequence criteria.

[0006]

Under these circumstances, the nucleic acid can be immobilized at a predetermined concentration regardless of the chain length, and can be arranged in a measurable form at a high density with good reproducibility. The establishment of a new systematic methodology that can be adapted is strongly required for genetic analysis, which is expected to increase in the future, and is a problem to be solved by the present invention.

[0007] Specifically, the problem to be solved by the present invention is that nucleic acid immobilization is more difficult than a method for producing a nucleic acid sequence by spotting or dispensing a small amount on a two-dimensional carrier such as a nylon sheet or a glass substrate. An array having a high amount and capable of increasing the density of nucleic acid molecular species arranged per unit area and suitable for mass production, that is, a two-dimensional (planar) array having nucleic acids immobilized thereon (immobilized nucleic acid Dimensional array). In addition, the problem to be solved by the present invention can be applied to long-chain nucleic acids including cDNA, compared to a method for producing a high-density oligonucleic acid sequence by a combination of photolithography and solid-phase synthesis on a silicon substrate,
It is an establishment of a method for producing an immobilized nucleic acid two-dimensional array having a lower production cost. Therefore, an object of the present invention is to provide a nucleic acid-immobilized gel-holding fiber, a nucleic acid-immobilized gel-holding fiber array, and a slice thereof.

[0008]

Means for Solving the Problems The inventors of the present invention have made intensive studies to solve the above-mentioned problems, and as a result, a conventional method in which a nucleic acid alignment process and an immobilization process are performed on the same two-dimensional carrier. By revising the idea of the method, the nucleic acid immobilization process is performed independently on the fiber as a one-dimensional structure (on one fiber), and various fiber shaping technologies are introduced into the alignment process. By producing a fiber bundle as a three-dimensional structure, and through a process of sectioning the resulting fiber bundle, it was found that an immobilized nucleic acid two-dimensional high-density array could be produced, and the present invention was completed. . That is, the present invention
This is a nucleic acid-immobilized gel holding fiber in which a gel in which a nucleic acid is immobilized on the surface of a fiber is held.

Further, the present invention is a nucleic acid-immobilized gel holding fiber array comprising the bundle of nucleic acid-immobilized gel holding fibers. Examples of the array include those in which the fibers in the array are regularly arrayed, and those that include 100 or more fibers per cm ² . In addition, examples of the type of nucleic acid contained in the nucleic acid-immobilized gel include different types in all or a part of each fiber. Furthermore, the present invention is a thin section of the nucleic acid-immobilized gel-holding fiber array, having a cut surface crossing a fiber axis of the nucleic acid-immobilized gel-holding fiber array.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
The present invention relates to a fiber holding a gel on which a nucleic acid is immobilized (hereinafter, also referred to as “nucleic acid-immobilized gel-holding fiber”). In the present invention, nucleic acids to be immobilized on a gel include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Is mentioned. The nucleic acid used in the present invention may be a commercially available nucleic acid or a nucleic acid obtained from a living cell or the like.

[0011] The preparation of DNA or RNA from living cells
For a known method, for example, DNA extraction, the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (19
76)), and for extraction of RNA,
oro et al. (Favaloro et al., Methods Enzymol. 65:
718 (1980)). The nucleic acid to be immobilized further includes a linear or circular plasmid DN.
A or chromosomal DNA, a DNA fragment obtained by cleaving them with a restriction enzyme or chemically, DNA synthesized by an enzyme or the like in a test tube, or a chemically synthesized oligonucleotide can also be used.

The type of the gel that can be used in the present invention is not particularly limited. For example, acrylamide, N, N-
Dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N
One or more monomers such as acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, (meth) acrylic acid, allyldextrin, and methylenebis (meth) acrylamide, polyethylene glycol A gel obtained by copolymerizing a polyfunctional monomer with (meth) acrylate or the like can be used. Other gels that can be used in the present invention include, for example, gels of agarose, alginic acid, dextran, polyvinyl alcohol, polyethylene glycol, and the like, and gels obtained by cross-linking these.

In the present invention, the nucleic acid may be immobilized on the gel as it is, or a derivative obtained by chemically modifying the nucleic acid, or a denatured nucleic acid may be immobilized if necessary.
The nucleic acid may be immobilized on the gel by physically encapsulating the gel or by directly binding to the gel components.The nucleic acid is once shared with a carrier such as a polymer or inorganic particles. The carrier may be bound by a bond or a non-covalent bond, and the carrier may be immobilized on a gel.

For example, a vinyl group can be introduced into the terminal group of a nucleic acid (WO98 / 39351) and copolymerized with a gel component such as acrylamide. Copolymerization includes a method of copolymerizing with a monomer, a polyfunctional monomer and a polymerization initiator, and a method of copolymerizing with a monomer and a polymerization initiator and then gelling with a crosslinking agent. Alternatively, agarose can be imidcarbonated by the cyanogen bromide method, and then gelled after binding to the amino group of the terminally aminated nucleic acid. At this time, a mixed gel of agarose immobilized with nucleic acid and another gel (for example, acrylamide gel) may be used.

As another method, there is a method in which a nucleic acid is bound to a carrier such as a polymer particle or an inorganic particle, and the particle is entrapped and immobilized in the above-mentioned gel. For example, agarose beads on which nucleic acids are immobilized can be obtained by reacting biotinylated nucleic acids with avidinated agarose beads (eg, avidinated agarose manufactured by Sigma). The nucleic acid-immobilized agarose beads can be comprehensively immobilized on an acrylamide gel or the like. Further, in the case of binding to a gel or a carrier, the binding can be carried out using a crosslinking agent such as glutaraldehyde or 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC).

As a general chemical modification of nucleic acid, amination, biotinylation, digoxigenation and the like are known, [Current Protocols In Molecular Biology, Ed .;
Frederick M. Ausubel et al. (1990), De-isotope experiment protocol (1) DIG hybridization (Shujunsha)], and in the present invention, any of these modification methods can be adopted. As an example, introduction of an amino group into a nucleic acid will be described.

The position of the bond between the aliphatic hydrocarbon chain having an amino group and the single-stranded nucleic acid is not particularly limited, and is not limited to the 5'-terminus or 3'-terminus of the nucleic acid but also in the nucleic acid chain (for example, phosphorous). Acid diester binding site or base site). This single-stranded nucleic acid derivative is disclosed in
No. 4,667,025, U.S. Pat. No. 4,789,737 and the like. In addition to this method, for example, using a commercially available reagent for introducing an amino group [for example, Aminolink II (trade name); PE Biosystems Japan, Amino Modifiers (trade name); It can be prepared according to a well-known method (Nucleic Acids Res., 11 (18), 6513- (1983)) for introducing an aliphatic hydrocarbon chain having an amino group into the 5 ′ terminal phosphoric acid. In the present invention, the fibers that can be used as the nucleic acid-immobilized gel-holding fibers include synthetic fibers, semi-synthetic fibers, regenerated fibers, chemical fibers such as inorganic fibers, and natural fibers.

As typical examples of synthetic fibers, nylon 6,
Nylon 66, various polyamide-based fibers such as aromatic polyamide, polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and other polyester-based fibers, polyacrylonitrile and other acrylic-based fibers, polyethylene and polypropylene and the like. Various fibers of polyolefin, various fibers of polyvinyl alcohol, various fibers of polyvinylidene chloride, various fibers of polyvinyl chloride, various fibers of polyurethane, phenolic fiber, fluorine based on polyvinylidene fluoride, polytetrafluoroethylene, etc. Fibers and various kinds of polyalkylene paraoxybenzoate fibers are exemplified. Fibers other than those for clothing, for example, optical fibers mainly composed of a transparent amorphous polymer such as polymethyl methacrylate or polystyrene can also be used.

Typical examples of semi-synthetic fibers include various cellulose-based fibers made from diacetate, triacetate, chitin, chitosan and the like, and various protein-based fibers called promix. Typical examples of the regenerated fiber include various cellulosic regenerated fibers (rayon, cupra, polynosic, etc.) obtained by a viscose method, a copper-ammonia method, or an organic solvent method. Representative examples of inorganic fibers include glass fibers,
And carbon fiber.

Representative examples of natural fibers include vegetable fibers such as cotton, flax, ramie, jute, animal fibers such as wool and silk, and mineral fibers such as asbestos. The form of the fiber used in the present invention is not particularly limited. Also,
It may be a monofilament or a multifilament. Further, a spun yarn obtained by spinning short fibers may be used. When multifilament or spun yarn fibers are used, voids between single fibers can be used for holding the nucleic acid-immobilized gel. The fiber used in the present invention may be used as it is in an untreated state, but if necessary,
The fiber may be a fiber into which a reactive functional group is introduced, or may be a fiber which has been subjected to a plasma treatment or a radiation treatment such as γ-ray or electron beam.

The method for producing the nucleic acid-immobilized gel-holding fiber is not particularly limited, but various chemical or physical interactions between the fiber and the gel, that is, the functional groups of the fiber And a chemical or physical interaction between the gel and the components constituting the gel.
For example, a method in which a fiber is immersed in a liquid in which a monomer, a polyfunctional monomer, an initiator, and a nucleic acid or a nucleic acid having a vinyl group at a terminal are mixed, and polymerization and gelation are performed. Also, monomers,
A method of immersing the fiber in a liquid obtained by mixing a nucleic acid bonded to a carrier such as a polyfunctional monomer, an initiator and polymer particles or inorganic particles, and polymerizing and gelling the fiber may be used.

In addition, a method in which a fiber is immersed in a liquid obtained by mixing a monomer, an initiator and a nucleic acid having a vinyl group at a terminal with a cross-linking agent to form a gel, and the monomer is polymerized with an initiator A method in which fibers are immersed and gelled in a liquid obtained by mixing particles in which a nucleic acid or a carrier such as nucleic acid or polymer particles or inorganic particles is bonded to a carrier, such as a crosslinking agent and a nucleic acid, or agarose or the like in which nucleic acids are immobilized by heating and dissolving the fibers. And a method of cooling and gelling.

The nucleic acid-immobilized gel holding fiber obtained by the above method can be subjected to an appropriate treatment as long as the gel is not broken. For example, the immobilized nucleic acid is denatured by performing a heat treatment, an alkali treatment, a surfactant treatment, or the like. Alternatively, when using nucleic acids obtained from raw materials such as cells and cells, unnecessary cell components and the like are removed. Then, the nucleic acid-immobilized gel holding fiber after the treatment can be used as a material for detecting a nucleic acid.
Note that these processes may be performed separately or simultaneously. In addition, it may be appropriately performed before the sample containing the nucleic acid is held on the fiber.

The nucleic acid-immobilized gel holding fiber prepared as described above can be used as a basic unit constituting the nucleic acid-immobilized gel holding fiber array of the present invention. The nucleic acid-immobilized gel-holding fibers can be bundled and then adhered to form a nucleic acid-immobilized gel-holding fiber array. On this occasion,
By regularly arranging the nucleic acid-immobilized gel-holding fibers and bonding them with a resin adhesive or the like, for example, a nucleic acid-immobilized gel-holding fiber array in which the nucleic acid-immobilized gel-holding fibers are regularly and regularly arranged in a matrix is obtained. be able to. Although the shape of the nucleic acid-immobilized gel-holding fiber array is not particularly limited, it is usually formed in a square or rectangular shape by regularly arranging the nucleic acid-immobilized gel-holding fibers.

The term "regularly" means that the nucleic acid-immobilized gel-holding fibers contained in a frame of a fixed size are arranged in order so that the number thereof is constant. For example, diameter 1
mm of nucleic acid-immobilized gel-holding fibers in bundles
mm and a width of 10 mm, it is necessary to arrange the square within the square frame (1 cm ² ).
The number of nucleic acid-immobilized gel holding fibers included in the side is set to 10,
The ten nucleic acid-immobilized gel-holding fibers are bundled in a single row to form a one-layer sheet, and the sheets are stacked so that the number of layers is ten. As a result, a total of 100 nucleic acid-immobilized gel-holding fibers can be arranged, that is, 10 vertically and 10 horizontally. However,
The method of regularly arranging the nucleic acid-immobilized gel holding fibers is as follows.
However, the present invention is not limited to the sheet stacking as described above.

In this case, it is desirable to arrange the positions of the nucleic acid-immobilized gel holding fibers on which the specific nucleic acids are immobilized in a predetermined state, but it is not always necessary to arrange them in such a manner. The reason is that at the stage when the array is formed, it is not known where the nucleic acid-immobilized gel holding fiber on which the specific nucleic acid is immobilized is located, but after cutting the cross section of the array, a hybridization technique or the like is used once. By determining the arrangement position of the nucleic acid in the cross section using
This is because the position of the nucleic acid-immobilized gel holding fiber on which the specific nucleic acid is immobilized can be confirmed. Therefore, using this technique, once the positions of a plurality of types of nucleic acids arranged in a slice are determined, the slices obtained from the same sequence are all located at the same position. The positional arrangement of the nucleic acids in all the obtained slices is known.

In the present invention, the number of the nucleic acid-immobilized gel holding fibers to be bundled is 100 or more, preferably 1,0.
The number is from 00 to 10,000,000, and can be appropriately set according to the purpose. However, the density of the nucleic acid-immobilized gel holding fiber in the array is 100 to 1.0 per cm ² .
It is preferable to prepare it so that the number becomes 00,000.
Then, in order to arrange the nucleic acid-immobilized gel holding fibers to obtain a thin slice of the array in which the nucleic acids are immobilized at a high density,
The thickness of the nucleic acid-immobilized gel holding fiber is preferably thin. In a preferred embodiment of the present invention, one nucleic acid-immobilized gel holding fiber needs to have a thickness of 1 mm or less. In the case of a monofilament, for example, a commercially available fishing line has a thickness of 50 to 900 μm. Further, according to the recent spinning technology, monofilaments of 1 dtex (in the case of polyethylene terephthalate, the diameter becomes about 14 μm) can be produced, and further fine fibers (ultrafine fibers or ultrafine fibers) can be produced (diameter 1). 〜１０10 μm).

When the nucleic acid-immobilized gel holding fiber is a monofilament having a diameter of 50 μm, 200 fibers can be arranged per 1 cm. Therefore, the number of fibers that can be arranged in a 1 cm ² square is 40,000. .
Therefore, in this case, up to 40,000 kinds of nucleic acids can be immobilized per 1 cm ² . On the other hand, in the case of multifilament, 83dtex / 36 filament and 8dtex
A 2dtex / 45 filament or the like can be used as it is.

The type of nucleic acid immobilized on the gel can be different for each fiber in the array, and any number of fibers from the same nucleic acid immobilized fiber can be used. It is also possible to select fibers, bundle the selected fibers, and arrange them appropriately. That is, according to the present invention, the type of immobilized nucleic acid and the order of the sequence can be arbitrarily set according to the purpose.

In the present invention, the nucleic acid-immobilized gel-holding fiber array cross section is cut by cutting the above-described nucleic acid-immobilized gel-holding fiber array in a direction crossing the fiber axis, preferably in a direction perpendicular to the fiber axis. Can be obtained. As a cutting method at this time, for example, a method of cutting a thin section from the array using a microtome and the like can be mentioned. The thickness of the flake can be arbitrarily adjusted, but is usually 1 to 5,000 μm, preferably 10 to 2,000 μm.

In the obtained slice having a cross section of the nucleic acid-immobilized gel-holding fiber array, nucleic acids corresponding to the number of the nucleic acid-immobilized gel-holding fibers constituting the array are present. With respect to the number of nucleic acids per cross-sectional area of a flake, by appropriately selecting the outer diameter of the nucleic acid-immobilized gel-holding fiber to be used, the method for preparing an array, and the like, 100 or more, more preferably 1,000 or more per 1 cm ^{2 of} flake cross-sectional area It is also possible to prepare a slice on which the nucleic acid is immobilized.

These slices can be used for detecting a nucleic acid having a specific base sequence in a sample by reacting with the sample using the immobilized nucleic acid as a probe and performing hybridization. The probe referred to in the present invention refers to a nucleic acid having a nucleotide sequence complementary to the nucleotide sequence of a gene to be detected in a narrow sense. That is, a thin section having a cross section of the nucleic acid-immobilized gel-holding fiber array of the present invention is reacted with a sample to perform hybridization, thereby forming a hybrid with a probe and a nucleic acid present in a sample complementary to the sample, and detecting the hybrid. By doing so, it is possible to detect a nucleic acid in a sample having a target base sequence. In the broad sense, the probe in the present invention refers to a nucleic acid capable of specifically binding to a protein, a low-molecular compound, or the like existing in a specimen. Therefore, these thin sections can be used not only for detecting nucleic acids that form hybrids with immobilized nucleic acids (probes), but also for proteins or small molecules that specifically bind to the immobilized nucleic acids. Use for detecting various samples such as compounds (for example, biological components) can be mentioned.

Known means can be used to detect nucleic acids that form hybrids with the immobilized nucleic acids and various biological components that specifically bind to the immobilized nucleic acids.
For example, a labeled substance such as a fluorescent substance, a luminescent substance, or a radioisotope is allowed to act on a nucleic acid, protein, low-molecular compound, or the like in a sample, and the labeled substance can be detected.
Regarding the type of these labels and the method of introducing the labels, etc.,
There is no particular limitation, and various conventionally known means can be used.

[0034]

The present invention will be described in more detail with reference to the following examples. However, the technical scope of the present invention is not limited by these examples. Reference Example 1 Preparation of Oligonucleotide and Oligonucleotide Having Amino Group or Biotin at 5 ′ Terminal: The following oligonucleotides (probe A and probe B) were synthesized. Probe A: GCGATCGAAACCTTGCTGTACGAGCGAGGGCTC (SEQ ID NO: 1) Probe B: GATGAGGTGGAGGTCAGGGTTTGGGACAGCAG (SEQ ID NO: 2)

Oligonucleotide synthesis was performed using a DNA / RNA synthesizer (model 39) of PE Biosystems Inc.
4), and in the final step of DNA synthesis, aminolink II (trade name) (Applied Biosystems) was used to add NH to the 5 'end of each oligonucleotide.
A probe aminated by introducing ₂ (CH ₂ ) _6- and a biotinylated probe were prepared using biotin amidite. These were used after deprotection and purification by a general method.

Example 1 Preparation of Nucleic Acid-Immobilized Gel-Retaining Fiber An aqueous solution containing the oligonucleotide having a biotin group at the 5 'end obtained in Reference Example 1 and having the following composition was prepared. Acrylamide 3.7 parts by weight Methylenebisacrylamide 0.3 parts by weight 2,2'-azobis (2-amidinopropane) dihydrochloride 0.1 parts by weight Biotinylated oligonucleotide (probe A or probe B) 0.005 parts by weight Avidinated agarose (6%) suspension 1.0 part by weight of suspension

After immersing a cotton yarn (washed with methyl ethyl ketone beforehand and dried) in which two spun yarns of 25 tex are immersed in the solution, the solution is transferred to a sealed glass container whose inside is saturated with steam, and is heated at 80 ° C. for 4 hours. The polymerization reaction was performed by leaving it to stand. As a result, there was obtained a fiber holding a gel in which an oligonucleotide (probe A or probe B) was immobilized via a biotin-avidin bond.

Example 2 Preparation of Nucleic Acid-Immobilized Gel-Retaining Fiber Array 20 fibers holding the gel on which the probe A obtained in Example 1 was immobilized were brought into close contact with each other in a Teflon plate shape without overlapping. And fixed at both ends. To this, a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd. Coronate 4403, Nipporan 4223) was thinly applied, and after the polyurethane resin was sufficiently hardened, this was peeled off from the Teflon plate, and the nucleic acid on which the probe A was immobilized was removed. A sheet in which the immobilized gel holding fibers were arranged in a row was obtained. On the other hand, with respect to the nucleic acid-immobilized gel holding fiber to which the probe B was immobilized, a sheet-like material was obtained by the same operation. Next, 20 sheets of these sheets are laminated so as to have the arrangement shown in FIG. 1 (3) and adhered using the above-mentioned adhesive. Was obtained.

Example 3 Preparation of Thin Section of Nucleic Acid-Immobilized Gel-Retaining Fiber Array: Example 2
By cutting out the nucleic acid-immobilized gel holding fiber array obtained in the above to a thickness of 100 μm using a microtome in a direction perpendicular to the fiber axis, a total of 400 fiber cross sections of 20 lengths and widths are regularly squared. A slice of the arranged nucleic acid-immobilized gel-holding fiber array was obtained (FIG. 1 (4)).

Reference Example 2 Labeling of sample nucleic acid: As a model of the sample nucleic acid, oligonucleotides (C, C) complementary to a part of the oligonucleotide (probe A, probe B) synthesized in Reference Example 1 were used.
D) was synthesized. Oligonucleotide C: GAGCCCTCGCTCGTACAGCAAGGTTTC
G (SEQ ID NO: 3) Oligonucleotide D: CTGCTGTCCCAAACCCTGACCTCCACC
(SEQ ID NO: 4) The 5 ′ end of each of these oligonucleotides was ligated to the 5 ′ end of each oligonucleotide using aminolink II (trade name) (PE Biosystems Japan) in the same manner as in Reference Example 4. After the introduction of ₂ (CH ₂ ) ₆ −, it was labeled with digoxigenin (DIG: Digoxigenin, Roche Diagnostics Co., Ltd.) as follows.

Each of the terminally aminated oligonucleotides was dissolved in 100 mM borate buffer (pH 8.5) to a final concentration of 2 mM. Equivalent amount of Digoxigenin-3-O-methylcarb
onyl-ε-aminocapronic acid-N-hydroxy-succinimide e
ster (26 mg / ml dimethylformamide solution) was added, and the mixture was allowed to stand at room temperature overnight. Adjust the volume to 100 μl, add 2 μl glycogen (Roche Diagnostics, Inc.),
10 μl of 3M sodium acetate (pH 5.2) and 300 μl of cold ethanol were added, and the precipitate was collected by centrifugation at 15,000 rpm for 15 minutes. 500 μl of 70% ethanol was added to the precipitate, and the precipitate was collected again at the bottom of the tube by centrifugation at 15,000 rpm for 5 minutes. The precipitate was air-dried and dissolved in 100 μl of 10 mM Tris-HCl (pH 7.5), 1 mM EDTA. The DIG-labeled oligonucleotide thus obtained was used as a model of the sample nucleic acid.

Reference Example 3 Hybridization: A thin piece of the nucleic acid-immobilized gel-retaining fiber array prepared in Example 3 was placed in a hybridization bag, and a hybridization solution having the following composition was poured into the bag and pre-pressed at 45 ° C. for 30 minutes. Hybridization was performed. The DIG-labeled DNA obtained in Reference Example 2 was added, and hybridization was performed at 45 ° C. for 15 hours.

[0043]

Reference Example 4 Detection: After completion of hybridization, a slice of the nucleic acid-immobilized gel-retaining fiber array was warmed in advance.
transfer to 0.1 ml of 0.1 x SSC, 0.1% SDS solution and shake
Washing for 0 minutes was performed three times at 45 ° C. DIG buffer 1 was added, and SDS was removed while shaking at room temperature. After repeating this again, DIG buffer 2 was added and shaken for 1 hour.
After removing the buffer, 1 / 10,000 anti-DI was added to DIG buffer 2.
An antigen-antibody reaction was carried out by adding 10 ml of a solution to which a G alkaline phosphatase-labeled antibody solution was added and shaking slowly for 30 minutes. Next, the plate was washed by shaking twice with DIG buffer 1 containing 0.2% Tween 20 for 15 minutes, and then immersed in DIG buffer 3 for 3 minutes. After removing DIG buffer 3, 3 ml of DIG buffer containing AMPPD was added and equilibrated for 10 minutes.

After draining the water, the mixture was transferred to a new hybridization bag and kept at 37 ° C. for 1 hour, and then sandwiched together with the X-ray film in a binder for X-ray film to expose the film. As a result, it was confirmed that the oligonucleotide C was bound to the place where the probe A was placed, and the oligonucleotide D was bound to the place where the probe B was placed. DIG buffer 1: 0.1 M maleic acid, 0.15 M sodium chloride
(pH 7.5) DIG buffer 2: DIG buffer 1 with 0.5% blocking reagent added DIG buffer 3: 0.1 M Tris-HCl (pH 9.5), 0.1 M sodium chloride, 0.05 M magnesium chloride Blocking reagent: Anti-DIG alkaline phosphatase-labeled antibody solution and AMPPD are reagents in a DIG Detection kit (Roche Diagnostics Inc.).

[0046]

Industrial Applicability According to the present invention, a nucleic acid-fixed gel holding fiber, a nucleic acid-fixed gel holding fiber array, and a slice thereof are provided. ADVANTAGE OF THE INVENTION According to this invention, the slice which has the fiber cross section of the nucleic acid fixed gel holding | maintenance fiber array in which the nucleic acid was arrange | positioned arbitrarily at high density and accurately can be obtained efficiently with good reproducibility. Using this slice, the type and amount of nucleic acid in the specimen can be examined. Advantages and usefulness of the present invention compared to the conventional method include, for example, that the immobilization process is not performed on a two-dimensional plane, but is performed separately and independently on fibers as a one-dimensional structure. Quantitative fixation of nucleic acids has become possible, and various fiber shaping technologies or fabric production technologies have been introduced into the alignment process, enabling higher densities. In order to produce the desired two-dimensional array from a fiber bundle as a three-dimensional structure, a thinning process that was not available in the conventional method was newly introduced. This eliminates the need for operation and enables mass production through continuous sectioning.

[0047]

[Sequence List] SEQUENCE LISTING <110> MITSUBISHI RAYON CO., LTD. <120> A FIBER HOLDING NUCLEIC ACID-FIXED GEL, AN ARRAY OF THE FIBERS AND A SLICE OF THE ARRAY <130> P99-0167 <140> <141 > <160> 4 <170> PatentIn Ver. 2.0 <210> 1 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 1 gcgatcgaaa ccttgctgta cgagcgaggg ctc 33 <210> 2 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 2 gatgaggtgg aggtcagggt ttgggacagc ag 32 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 3 gagccctcgc tcgtacagca aggtttcg 28 <210> 4 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Synthetic DNA <400> 4 ctgctgtccc aaaccctgac ctccacc 27

[0048]

[Sequence List Free Text]

 SEQ ID NO: 1: Synthetic DNA SEQ ID NO: 2: Synthetic DNA SEQ ID NO: 3: Synthetic DNA SEQ ID NO: 4: Synthetic DNA

[Brief description of the drawings]

FIG. 1 is a schematic view of a nucleic acid-immobilized gel-holding fiber, a nucleic acid-immobilized gel-holding fiber array, and a slice thereof. (1)
Is a nucleic acid-immobilized gel holding fiber having probe A immobilized thereon,
(2) is a nucleic acid-immobilized gel holding fiber on which probe B is immobilized, (3) is a nucleic acid-immobilized gel holding fiber array composed of these two nucleic acid-immobilized gel holding fibers, and (4) is a nucleic acid-immobilized gel holding fiber. 1 shows a cross section of a fiber array cut in a direction perpendicular to a fiber axis.

 ──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Wataru Fujii 10-1 Ogurocho, Tsurumi-ku, Yokohama-shi, Kanagawa Prefecture Inside the Chemical Development Laboratory, Mitsubishi Rayon Co., Ltd. (72) Inventor Takashi Akita 20 Miyukicho, Otake-shi, Hiroshima Prefecture No. 1 Mitsubishi Rayon Co., Ltd. Central Research Laboratory (72) Inventor Kei Murase 20-1 Miyuki-cho, Otake City, Hiroshima Prefecture Mitsubishi Rayon Co., Ltd. Central Technology Research Center (72) Inventor Chiho Ito Miyuki Otake City, Hiroshima Prefecture No. 20-1 in the Mitsubishi Rayon Co., Ltd. Central Research Laboratory F-term (reference) 4B024 AA20 BA80 CA09 HA11 4B029 AA21 BB20 CC05 4B063 QA01 QA13 QQ42 QR32 QR55 QS34 4L033 AB02 AC15 BA45 BA76 CA01

Claims (6)

[Claims] 1. A nucleic acid-immobilized gel holding fiber in which a nucleic acid-immobilized gel is held on the surface of a fiber. 2. A nucleic acid-immobilized gel-holding fiber array comprising the bundle of nucleic acid-immobilized gel-holding fibers according to claim 1. 3. The nucleic acid-immobilized gel holding fiber array according to claim 2, wherein the fibers in the fiber array are regularly arranged. 4. The nucleic acid-immobilized gel-holding fiber array according to claim 2, wherein the fiber bundle contains 100 or more fibers per 1 cm ² . 5. The nucleic acid-immobilized gel-holding fiber array according to any one of claims 2 to 4, wherein the kind of the nucleic acid is different in all or a part of each fiber. 6. A thin section of the nucleic acid-immobilized gel-holding fiber array, having a cut surface crossing a fiber axis of the fiber array according to any one of claims 2 to 5.