WO2012046859A1 - Identifying information carrier for identifying subject to be identified and utilization of same - Google Patents

Identifying information carrier for identifying subject to be identified and utilization of same Download PDF

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Publication number
WO2012046859A1
WO2012046859A1 PCT/JP2011/073269 JP2011073269W WO2012046859A1 WO 2012046859 A1 WO2012046859 A1 WO 2012046859A1 JP 2011073269 W JP2011073269 W JP 2011073269W WO 2012046859 A1 WO2012046859 A1 WO 2012046859A1
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WIPO (PCT)
Prior art keywords
information holding
identification
barrier
identification information
solid phase
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PCT/JP2011/073269
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French (fr)
Japanese (ja)
Inventor
孝介 丹羽
廣田　寿一
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日本碍子株式会社
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Priority to JP2012537783A priority Critical patent/JPWO2012046859A1/en
Publication of WO2012046859A1 publication Critical patent/WO2012046859A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips

Definitions

  • the present invention relates to a holding body that holds identification information for identifying an object and its use, and more particularly to a holding body that holds identification information containing DNA and its use.
  • Such distribution and movement monitoring and management of various identification objects are usually performed by providing identification information specific to the identification object or associated in advance and identifying the identification information in a timely manner.
  • identification information specific to the identification object or associated in advance
  • identifying the identification information in a timely manner.
  • a technique for avoiding counterfeiting or falsification by preparing a DNA ink using the DNA base sequence, printing it on the identification target using this DNA ink, and detecting the DNA base sequence in a timely manner. It has been proposed (Patent Document 1).
  • the identification information holding body holding the identification information is exposed to various environments.
  • the inventors of the present invention have a great influence on the accuracy of identification and proof by the amount and state of DNA to be held, and the identification information holding body holds DNA as identification information stably until the time of proof. I found out that is important. That is, it is important to stably hold DNA until the identification information holding body is applied to an article to be identified (for example, after the identification information holding body itself is manufactured and distributed). It is also important to maintain the DNA stably after applying the identification information holding body to the article to be identified until the time of certification through the distribution channel of the article.
  • DNA may be damaged by ultraviolet light having a wavelength of about 200 to 300 nm.
  • the DNA when a frictional external force is applied during the distribution or a change in temperature or humidity occurs, the DNA may be detached from the identification target and may be cleaved on the identification target. In such a case, it is difficult to accurately detect the assigned DNA, and the discrimination ability may be reduced.
  • an object of the present invention is to provide an identification information holding body that can stably hold identification information and maintain good discrimination ability, and use thereof.
  • the present inventors have found an effective configuration for stably holding DNA used as identification information for identifying an identification target on the solid phase carrier, and completed the present invention. According to the present invention, the following means are provided.
  • a solid support One or more information holding regions in which one or two or more DNAs having identification base sequences previously associated with identification objects as identification information are held on the solid phase carrier; A barrier surrounding one or more of the information holding areas; A holding body for identification information.
  • a structure comprising a plurality of identification information holding bodies of the present invention integrally.
  • a method for producing the identification information holding body of the present invention Immobilizing the one or two or more DNAs on the solid phase carrier to form one or more of the information holding regions; Fixing a barrier member serving as a barrier surrounding one or more of the information holding regions on the solid phase carrier;
  • a manufacturing method comprising:
  • An identification method of an identification object Contacting the information holding region of the identification information holding body of the present invention given to the identification target with one or more probes having a base sequence complementary to the identification base sequence; Detecting a hybridization product of the identification base sequence and the probe; A method comprising:
  • FIG. 1 It is a figure which shows an example of the identification information holding body of this invention. It is a figure which shows another example of the identification information holding body of this invention. It is a figure which shows the example in which identification information comprises the pattern. It is a figure which shows an example of a structure of the identification information holding body of this invention, and a usage pattern. It is a figure which shows an example of a structure of the identification information holding body of this invention, and a usage pattern. It is a figure which shows the spot pattern in an Example. It is a figure which shows an example of the holding body used in an Example. It is a figure which shows another example of the holding body used in an Example. It is a figure which shows another example of the holding body used in an Example. It is a figure which shows the evaluation result of Experiment 1. It is a figure which shows the evaluation result of the experiment 2. FIG. It is a figure which shows the evaluation result of the experiment 3. FIG. It is a figure which shows the evaluation result of the experiment 4. FIG.
  • the identification information holding body of the present invention since detachment and decomposition of DNA serving as identification information are suppressed and stably held on the solid phase carrier, the identification accuracy, accuracy and reproducibility are excellent. A holder having good discrimination ability is provided. In addition, since DNA as identification information is held on the solid phase carrier in this way, rapid and simple detection is possible. In addition, when a discrimination base sequence selected from the base sequences represented by SEQ ID NOs: 1 to 100 and its complementary sequence is used, a highly specific discrimination ability is ensured and the discrimination target is highly selective. And can be identified quickly.
  • FIGS. 1 and FIG. 2 An example of the identification information holding body of the present invention (hereinafter also simply referred to as the present holding body) is shown in FIGS.
  • the holder has a barrier surrounding the information holding region on the surface of the solid phase carrier holding identification information.
  • a barrier may protect DNA or the like held as identification information in the information holding area from friction or impact from the outside in a state where the identification information holding body itself or an identification target to which the identification information is applied is in circulation. Therefore, the identification information can be stably held on the solid phase carrier. For this reason, identification and proof of an identification object can be performed correctly.
  • the identification information can be protected even if the identification information holding body is fixed so that the information holding area faces the outside of the article to be identified. Furthermore, by having a barrier, the information holding area can be provided to the article side without exposing the information holding area to the outside of the article by fixing the information holding area so as to be removable from the article at the time of identification.
  • the identification information can be stably held during the distribution after the identification information holding body itself is manufactured.
  • the identification information is more reliably protected. it can.
  • the barrier may not necessarily be configured as a part of the solid phase carrier, and may be a barrier member that is fixed to the solid phase carrier via an adhesive or an adhesive.
  • the barrier member may comprise such an immobilization layer and a plastic barrier layer.
  • the barrier also functions as a mark for clarifying the region where the hybridization solution containing the probe is dropped when the probe is supplied to the information holding region to detect hybridization. It can also function as a container for reliably supplying a predetermined amount to the holding region. As a result, the identification target can be accurately identified and proved.
  • a plurality of identification information holding bodies preferably 10 or more, more preferably 20 or more information holding areas are formed on a single solid phase carrier, and these information holding areas are partitioned by a barrier.
  • the identification information holding body can be efficiently manufactured by manufacturing the structure as described above.
  • the barrier may be an index for individually identifying a plurality of information holding areas. Therefore, the identification information body having one or two or more information holding areas can be separated using the barrier as an index to obtain the final identification information holding body.
  • the barrier in the identification information holding body or structure of the present invention can be configured using, for example, a separator disclosed in International Publication No. WO2006 / 101229 (PCT / JP2006 / 306134) as a barrier member.
  • a separator it is preferable to use a material selected from the group consisting of polycarbonate, polyolefin (PET, PE), polyamide, polyimide, acrylic resin, and fluorides and halides thereof.
  • PET polyolefin
  • PE polyamide
  • polyimide polyimide
  • acrylic resin and fluorides and halides thereof.
  • solid phase carrier in the identification information holding body or structure of the present invention is described in, for example, JP-A-2006-184016, JP-A-2006-71309 and Nucleic Acid Research, 2007, vol.35, No1.e3.
  • the substrate can be used.
  • the DNA can be fixed to the solid phase carrier with sufficient strength, and the DNA can be protected from an impact from the outside, including during distribution.
  • This holding body is a holding body that holds identification information for identifying an identification target, and has, as identification information, a DNA having an identification base sequence that is associated with the identification target in advance.
  • the identification object identified by the holder is not particularly limited, and various distribution articles and media can be mentioned.
  • Distribution articles include all articles that are distributed commercially or non-commercially. Examples include various industrial products, parts (including intermediate products), marine products, agricultural products, arts, books, banknotes, securities, and the like.
  • various certificates are also included.
  • This holder can take various forms.
  • identification information may be held for carriers of various shapes such as films, sheets, and substrates.
  • the holder is fixed to the identification object by chemical means such as adhesion or other physical means.
  • the holder may be a part of the identification target.
  • identification information may be held in a part of the identification target.
  • This holder can take a form in which identification information is held on a carrier.
  • the carrier shape include films, flat plates, particles, molded articles (beads, strips, wells or strips of multiwell plates, tubes, meshes, foams, membranes, paper, needles, fibers, plates, slides, and cells. Culture vessels, etc.) and latex.
  • the region to which the identification information is applied is flat.
  • DNA or the like can be immobilized by physical adsorption or chemical bonding, and normal hybridization conditions If it can endure, it will not be restrict
  • such a carrier material examples include plastics, inorganic polymers, metals, natural polymers, and ceramics.
  • the plastic is not particularly limited as long as it can immobilize biomolecules by ultraviolet irradiation, and specific examples include thermoplastic resins, thermosetting resins, and copolymers. .
  • thermoplastic resin examples include ionomers (styrene-based, olefin-based), polynorbornene, polyacetal, polyarylate, polyether ether ketone, polyethylene oxide, polyoxymethylene, polyethylene terephthalate, polycarbonate, polystyrene, polysulfone, Polyparamethylstyrene, polyallylamine, polyphenylene ether, polyphenylene sulfide, polybutadiene, polybutylene terephthalate, polypropylene, polymethylpentene, polyethersulfone, polyphenylene sulfide, polyoxybenzoyl, polyoxyethylene, cellulose acetate, polydimethylsiloxane, polyisobutylene , Cellulose triacetate, poly-p-phenylene terephthalamide, poly Soprene, polyacrylonitrile, polymethylpentene, chlorine plastic (polyvinyl chloride, polychlorinated ethylene
  • Thermosetting resins include epoxy, polyxylene, polyguanamine, polydiallyl phthalate, polyvinyl ester, polyphenol, unsaturated polyester, polyfuran, polyimide, polyurethane, polymaleic acid, melamine, urea, alkyd, benzoguanamine, polycyanate, polycyanate.
  • An isocyanate etc. are mentioned.
  • the copolymer includes isobutylene maleic anhydride copolymer, acrylonitrile acrylate styrene copolymer, acrylonitrile EPDM styrene copolymer, acrylonitrile styrene copolymer, acrylonitrile butadiene styrene copolymer, butadiene styrene methyl methacrylate copolymer.
  • Ethylene vinyl chloride copolymer ethylene vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, acrylonitrile-butadiene styrene copolymer, polyether ether ketone copolymer, fluorinated ethylene polypropylene copolymer, tetrafluoroethylene
  • Ethylene vinyl chloride copolymer ethylene vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, acrylonitrile-butadiene styrene copolymer, polyether ether ketone copolymer, fluorinated ethylene polypropylene copolymer, tetrafluoroethylene
  • perfluoroalkyl vinyl ether copolymers and tetrafluoroethylene ethylene copolymers.
  • polycarbonate particularly preferred are polycarbonate, polymethyl methacrylate, acrylonitrile butadiene styrene copolymer, polyethylene, polyethylene terephthalate, polyphenol, polystyrene, polyacrylonitrile, polyvinyl chloride, aramid and the like.
  • dyes, color formers, plasticizers, pigments, polymerization inhibitors, surface modifiers, stabilizers, adhesion-imparting agents, thermosetting agents, dispersants, UV degradation inhibitors, etc. may be added to the synthetic resin as necessary.
  • the synthetic resin may be laminated with different types of the synthetic resins in order to maintain the shape, or may be a single synthetic resin.
  • the polymer alloy which mixed 2 or more types of the said synthetic resin may be sufficient.
  • fibers such as vein fibers, fruit fibers, animal hair fibers, cocoon fibers, feather fibers, chitin, chitosan and asbestos (asbestos) may be mixed with the synthetic resin.
  • the inorganic polymer include glass, crystal, carbon, silica gel, and graphite.
  • Specific examples of the metal include gold, platinum, silver, copper, iron, aluminum, a magnet, and a paramagnet.
  • natural polymers include polyamino acids, cellulose, chitin, chitosan, alginic acid, and derivatives thereof.
  • Specific examples of the ceramic include apatite, alumina, silica, silicon carbide, silicon nitride, and boron carbide.
  • the DNA or the like may be directly immobilized on the carrier or the like, but an immobilization phase for immobilization may be added to the carrier or the like.
  • an immobilized phase as long as it is supported on the carrier or the like, it may be supported simply using physical adhesiveness, or may be chemically supported via a covalent bond or the like. Good.
  • the said fixed phase may be carry
  • the immobilized phase include small organic molecules in addition to the materials described above as materials for the carrier and the like. Specific examples of the organic low molecule include a carbodiimide group-containing compound, an isocyanate group-containing compound, a nitrogen iperit group-containing compound, an aldehyde group-containing compound, and an amino group-containing compound.
  • the immobilized phase is preferably supported as a film on a carrier or the like.
  • a known method such as spraying, dipping, brushing, stamping, vapor deposition, or coating using a film coater can be used.
  • a carbodiimide group (resin) over the entire surface of a glass carrier or the like
  • a solution obtained by dissolving an amino-substituted organoalkoxysilane such as 3-aminopropyltriethoxysilane in a suitable solvent After immersing the carrier or the like under a temperature of about 70 to 80 ° C. for about 2 to 3 hours, the carrier is taken out, washed with water, and further heated and dried at about 100 to 120 ° C. for about 4 to 5 hours.
  • the substrate After drying, the substrate is immersed in a suitable solvent, carbodiimide resin is added, and the mixture is stirred for about 12 hours at a temperature of about 30 to 170 ° C. and washed.
  • the amino group of the 3-aminopropyltriethoxysilane may be reacted with a functional group other than the nucleic acid binding group of the nitrogen iperit group using an appropriate solvent to introduce the nitrogen iperit group onto the surface of a glass carrier or the like. it can.
  • plastic carriers mentioned above already have the above functional group on the surface of the carrier, etc., and in this case, without introducing the functional group on the surface of the carrier, etc. Can also be used for the production of carriers and the like. Further, even such a plastic carrier can be used for the production of the carrier by further introducing a functional group.
  • a known photopolymerization initiator can be mixed with the above-mentioned carrier or the like and the material of the immobilized phase.
  • a photopolymerization initiator By mixing a photopolymerization initiator, the reactivity at the time of immobilizing nucleic acid by irradiation with electromagnetic waves such as ultraviolet rays can be improved.
  • the identification information means information for identifying an identification target.
  • the identification information is included in one or more DNAs.
  • DNA includes other compounds that can form base pairs in the same manner as DNA.
  • compounds having other known main chain structures such as BNA and PNA and having a base as a side chain are included.
  • the identification information may be included in a pattern formed by providing these DNAs on a carrier. The pattern will be described later.
  • DNA has a polymer structure of deoxyribonucleotide having adenine (A), thymine (T), cytosine (C), and guanine (G) as bases, as well as other main chain structures, It means a compound (polymer) having A, T, C and G as a base.
  • the identification information is associated with one identification object, and the identification information is composed of one or two or more identification base sequences provided in one or two or more DNAs. Therefore, one identification object may be identified by one DNA, or may be identified by two or more DNAs.
  • 1 DNA has 1 identifying base sequence.
  • the identification base sequence is associated in advance with one identification target. With this association, the identification target is identified by the identification information.
  • the identification base sequence may or may not be unique to the identification target.
  • the case where the identification target is unique is, for example, the case where the identification target has a unique base sequence or mutation on the genome, and the unique base sequence itself is used as the identification base sequence.
  • the identification base sequence may be naturally derived or artificially designed.
  • an artificially designed base sequence is used as a sequence for identification, for example, a set is formed in which there is no mishybridization with each other in advance and can be reliably hybridized and detected under common hybridization conditions.
  • identification target detection by hybridization can be performed quickly and with high accuracy.
  • Examples of such artificial base sequences include base sequences represented by SEQ ID NOs: 1 to 100 and complementary base sequences thereof. All of these base sequences have the same base length and a melting temperature (Tm) of 40 ° C. or higher and 80 ° C. or lower, preferably 50 ° C. or higher and 70 ° C. or lower. Obtainable. It is preferable that the melting temperatures of two or more artificial base sequences used simultaneously are as close as possible.
  • the melting temperature calculated by the GC% method, the Wallace method, the method based on Current Protocols in Molecular Molecular Biology (developed by Shujunsha's Bio Experiment Illustrated 3 practically increased PCR p.25), etc. can be adopted.
  • the calculation is preferably performed by the Nearest-Neighbor method which can take into account the range of melting temperature and the base sequence concentration in the present invention.
  • the melting temperature by the Nearest-Neighbor method is, for example, software with Visual OMP (Tomy Digital Biology Co., Ltd.) or software provided by the Japan Genetic Research Institute (http://www.ngrl.co.jp/). (OligoCalculator; http://www.ngrl.co.jp/tool/ngrl#tool.html).
  • Such an identification sequence in an artificial base sequence is also referred to as an orthonormalized sequence, for example, a continuous match length for a DNA sequence of a predetermined base length obtained from a random number, melting temperature prediction by Nearest-Neighbor method, Hamming distance, Designed by performing secondary structure prediction calculations.
  • the orthonormalized sequence is a base sequence of nucleic acid having a uniform melting temperature, that is, a sequence designed so that the melting temperature is within a certain range, and the nucleic acid itself is intramolecular. It means a base sequence that does not form a stable hybrid other than a base sequence that is structured in the above and does not inhibit hybridization with a complementary sequence.
  • a sequence included in one orthonormalized sequence group hardly reacts between sequences other than the desired combination and within a self-sequence, or does not generate a reaction. Further, when the orthonormalized sequence is amplified in PCR, the amount of nucleic acid corresponding to the initial amount of the nucleic acid having the orthonormalized sequence is quantitatively affected without being affected by the problems such as the above-mentioned cross-hybridization. Has the property of being amplified.
  • the orthonormalized array as described above is described in detail in H. Yoshida and A.Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vl.54, 9-20 (2000) and Japanese Patent Application No. 2003-108126. Are listed. Orthonormalized sequences can be designed using the methods described in these references.
  • the identification information may be composed of one or more identification base sequences. Since the identification information is constituted by two or more DNAs each having a different base sequence for identification, identification with higher accuracy can be made possible. Further, even if a certain number of artificial base sequences are used, by combining them, it is possible to identify more identification objects than the certain number.
  • the identification base sequence preferably has a thymine base ratio smaller than the thymine-rich base sequence described later. That is, the identification base sequence preferably has a T (thymine) content (the number of thymine bases in the identification base sequence / the total number of bases in the identification base sequence ⁇ 100) of less than 50%. This is because, if thymine is present in the identification base sequence, it tends to deteriorate with respect to the amount of immobilization and / or reaction with the probe, as well as irradiation with ultraviolet rays after immobilization.
  • the thymine ratio is not particularly limited, but is preferably 40% or less, more preferably 30% or less, and even more preferably 20% or less. More preferably, it is 10% or less, more preferably 5% or less, and still more preferably 1% or less. Most preferably, it is 0%.
  • DNA may have a thymine rich base sequence together with one base sequence for identification.
  • thymine is considered to be involved in chemical bonding of DNA to a carrier, and having a thymine-rich base sequence allows DNA to be easily and firmly bound to a carrier.
  • a thymine-rich base sequence means that a continuous region containing the thymine specified by two thymines (T) selected in the base sequence of DNA has a higher thymine content than other regions.
  • T thymine
  • the content of T (thymine) is 50% or more. More preferably, it is 60% or more, more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, and still more preferably. Is 98% or more, most preferably 100%.
  • the thymine-rich base sequence includes a single thymine with no interval.
  • the tin-rich base sequence preferably contains two or more thymines in succession. More preferably, it consists only of continuous thymine.
  • the thymine is preferably 3 or 4 or more, more preferably 5 or 6 or more, and still more preferably 7 or 8 or more.
  • the base sequence rich in thymine extends to almost the whole or the whole of DNA is mentioned.
  • the base sequence may be in any part of the DNA, but is preferably not part of the identification base sequence. That is, the thymine-rich base sequence is preferably provided separately from the identification base sequence.
  • the thymine-rich base sequence may be provided including the 5 ′ end and / or the 3 ′ end, or not including the end, but provided on the 5 ′ end side and / or the 3 ′ end side. May be.
  • identification information consisting of one or more DNAs is held on the carrier, whereby the identification base sequence in the DNA functions as identification information.
  • the identification information is preferably held on a carrier or the like with an appropriate pattern (two-dimensional form). That is, the pattern is not particularly limited, but may be a combination of symbols, letters, numbers, barcodes, images, and the like. Such a pattern is preferably visible.
  • the identification information may be configured as a plurality of dot-like bodies containing DNA.
  • Each dot-like body is composed of one or more DNAs.
  • the DNA constituting the dot-like body is composed of the one DNA.
  • each dot-like body may consist of only one DNA or may consist of two or more DNAs. Alternatively, both dot-like bodies may be included.
  • a plurality of dot-like bodies may be aggregated to form a visible pattern (characters, symbols, figures, combinations thereof, etc.).
  • the pattern By configuring the pattern with the dot-like body, the required amount of DNA can be reduced and the DNA concentration in the dot can be increased, so that the detection sensitivity is increased, and detection with high accuracy can be performed easily and quickly.
  • One pattern is usually configured to specify one identification target.
  • the one pattern can be composed of a dot-like body composed of one or two or more DNAs.
  • One identification target may be provided with one pattern, or two or more types may be provided.
  • Two or more types of patterns (in FIG. 3, ⁇ and ⁇ ) can be simultaneously provided on one carrier or identification target.
  • the overlapping positions may be arranged so that the individual dot-like bodies belonging to the respective patterns do not overlap, or may be arranged overlappingly.
  • the place where a pattern overlaps may be comprised only by the dot-like body which belongs to any one pattern.
  • the dot-like bodies belonging to both patterns may not be arranged at the places where the patterns overlap. This is because the existence of the pattern itself can be confirmed even in such a state.
  • two or more types of patterns may be provided to identify one identification target, or may be provided to identify individual identification targets.
  • a technique for forming a dot-like body containing DNA on a carrier or the like for example, a technique such as a known DNA microarray can be applied. That is, a liquid in which DNA is dispersed or dissolved may be spotted on a carrier by a known method such as an ink jet method or a pin method.
  • the area on the carrier on which one or two or more DNAs associated with the identification target are thus formed can constitute an information holding area.
  • This holder has a barrier on the surface of the carrier that surrounds one or more information holding areas.
  • the barrier is provided with a predetermined height from the carrier surface.
  • the barrier may be a continuous barrier that continuously surrounds the information holding area, or a plurality of barriers that intermittently (discontinuously) surround the information holding area are arranged. It may be.
  • the barrier has a height of 10 ⁇ m or more, preferably 50 ⁇ m or more, more preferably 100 ⁇ m or more from the information holding region (the surface of the solid phase carrier). When the thickness is at least 10 ⁇ m, the identification information can be protected from physical failure. Further, the barrier has a height of 500 ⁇ m or less, more preferably 300 ⁇ m or less, and still more preferably 200 ⁇ m or less from the information holding region (the surface of the solid phase carrier). This is because if the height is 500 ⁇ m or less, the handling property of the identification information holding body is not affected.
  • the barrier is preferably formed so that the information holding area can be visually identified. This is because the information holding area can be easily visually recognized, the probe solution can be easily supplied, and the determination of the hybridization result is facilitated. In this case, it is possible to adopt a method of coloring the thickness of the barrier or the inside or outside of the barrier.
  • the barrier may be a part of the solid phase carrier, or may be a barrier member integrated on the solid phase carrier separately from the solid phase carrier.
  • the barrier member may be integrated with the solid phase carrier with an adhesive or an adhesive.
  • the barrier preferably has a hydrophobic surface.
  • the probe solution supplied in the barrier that is, in the information holding region, can be stably held so as not to flow out.
  • the hydrophobic surface is formed by a hydrophobic layer when the barrier is a barrier member including an adhesive layer or an adhesive layer and a hydrophobic layer in this order from the solid phase carrier side. it can.
  • the barrier can be provided with a surface layer part including an adhesive or an adhesive. By providing such a surface layer portion, other members can be fixed via the surface layer portion.
  • the barrier can be provided with the surface layer part on the uppermost layer of the barrier (the highest part far from the solid support).
  • the cover which covers the upper part of an information holding area
  • the cover effectively protects the information holding area from failure. That is, it is possible to suppress detachment due to friction of DNA and decomposition due to ultraviolet irradiation until the time of use.
  • the cover may be a film or sheet. Further, the cover may be formed on the identification information area by being applied to the barrier.
  • the cavity can be used as a cavity for hybridization.
  • the cavity can be provided with an opening through which a probe solution for hybridization can be injected.
  • the opening can be provided in a timely manner. For example, you may provide the weak part which an opening is formed by the press from the outside.
  • the cover may be provided so that it can be easily peeled off from the barrier.
  • such a barrier includes a first adhesive layer, a hydrophobic (water-repellent) layer, and a second adhesive layer from the solid support side, and 1 or 2 on the solid support.
  • You may be comprised with the barrier member which is a laminated body provided with the pattern which encloses and partitions the above information holding area
  • the first pressure-sensitive adhesive layer has stronger adhesiveness than the second pressure-sensitive adhesive, so that the barrier member can be firmly fixed to the solid phase carrier, while the cover is interposed via the second pressure-sensitive adhesive layer. Can be fixed easily.
  • a pressure-sensitive adhesive layer can be formed on the surface of the solid support that is not the information holding area of the solid support, and the permanent support can be fixed to the identification flaw through the pressure-sensitive adhesive layer.
  • Such a barrier member also includes, for example, a first pressure-sensitive adhesive layer, a first hydrophobic layer, a second pressure-sensitive adhesive layer, and a second hydrophobic layer as shown in FIG. And a third pressure-sensitive adhesive layer.
  • the third pressure-sensitive adhesive layer can be provided with a release layer. At this time, it is preferable that the first pressure-sensitive adhesive layer is stronger than the second pressure-sensitive adhesive layer, and the third pressure-sensitive adhesive layer is also stronger than the second pressure-sensitive adhesive layer. Is preferred.
  • the release layer also functions as a cover that protects the information holding area.
  • the third pressure-sensitive adhesive layer can function as an immobilization layer to the holding body by sticking and fixing the third pressure-sensitive adhesive layer exposed by peeling the release layer to the identification target.
  • providing the barrier surrounding the information holding region on the solid phase carrier may be in a state where the carrier surface for the identification information region is formed lower than the surrounding carrier surface.
  • the carrier when it includes a recess having a bottom surface, it may be an information holding area that holds identification information in the recess.
  • the concave portion can also be used as a cavity for hybridization.
  • a cavity for hybridization is provided by a barrier or the like, so that the contact process can be easily performed in the identification of the identification target described later.
  • two or more patterns can be provided in the information holding region, and the two or more patterns are obtained from one or more DNAs having a base sequence for identification that is not common between the patterns. It can be composed of a dot-like body. Further, the two or more types of patterns may be arranged so that at least a part thereof overlaps.
  • the carrier When the holder is provided with identification information on a solid phase carrier and should be separately immobilized on an identification target, the carrier preferably includes an immobilization means for the identification target.
  • the immobilization means include an adhesive layer and an adhesive layer.
  • Such immobilization means is effective when the holder is in the form of a film or sheet.
  • the immobilization means by latching, fitting, etc. can be provided in addition to the adhesive layer and the adhesive layer.
  • the immobilization means is preferably capable of separating the holder from the identification target. By making it separable, the identification target to be described later can be easily identified.
  • the structure of the present invention can be provided with a plurality of the holding bodies integrally. Such a structure is convenient because a plurality of the holders can be supplied at once. It is preferable that the plurality of holders are arranged in a predetermined pattern. For example, it is provided in a pattern as shown in FIGS. Such a pattern is suitable for individual separation.
  • the plurality of holders are preferably provided with a continuous or single solid support as a solid support common to the plurality of holders. This is because it is convenient to manufacture and handle a plurality.
  • the structure is formed so that a plurality of book holders can be separated. This is because it can be easily separated into individual book holders. It is preferable that the structure has a fragile portion capable of separating a plurality of the present holding bodies. By having the fragile portion, the holder can be easily separated with a small force.
  • the fragile portion is preferably formed so as to partition a plurality of book holders.
  • the thickness between the adjacent main holders is 20 ⁇ m to 1500 ⁇ m, preferably 40 ⁇ m to 1000 ⁇ m, more preferably 50 to 600 ⁇ m.
  • the plurality of holders can be separated from each other by using barriers provided by the respective holders, and the barriers can be used as an index.
  • the plurality of book holders can include a continuous or single barrier as a barrier common to the plurality of book holders.
  • the plurality of holders can be provided with a continuous or single immobilization layer containing an adhesive or a pressure-sensitive adhesive on the front or back surface of the structure.
  • an immobilization layer may be provided with respect to a plurality of barriers (the uppermost part) of the book holder, and the information holding region of the solid phase carrier is formed. It can also be provided on the surface opposite to the surface to be applied.
  • the method for producing the holder includes a step of fixing one or more DNAs on a solid phase carrier to form one or more information holding regions, and a barrier surrounding the one or more information holding regions A barrier member to be fixed on the solid phase carrier.
  • this manufacturing method is a method for manufacturing the present holding body, it can also be carried out as a method for manufacturing the present structure including a plurality of the present holding bodies at the same time.
  • the barrier member can have an opening pattern (plan view) corresponding to one or more information holding regions as shown in FIGS. 1, 2, 4 and 5.
  • the barrier member is provided with an adhesive layer or an adhesive, and is fixed to the solid phase carrier, and is laminated on the fixed layer. Barrier layer.
  • the barrier member can also comprise a further immobilization layer containing an adhesive or adhesive that is laminated to the barrier layer.
  • the solid phase carrier may be provided with an immobilization layer containing an adhesive or an adhesive on the surface opposite to the surface forming the information holding region of the solid phase carrier.
  • a step of providing a cover for covering the upper part of the one or more information holding regions via the barrier member may be provided.
  • DNA that may have a thymine-rich base sequence in addition to the identification base sequence can be supplied to the surface of the solid phase carrier, and both can be contacted and immobilized.
  • the DNA is usually supplied in a form contained in water or a buffer so that the activity of the DNA immobilized in both contact reactions is maintained.
  • the immobilization method is not particularly limited, and a known method can be adopted.
  • a known compound such as DNA and a carbodiimide resin, nitrogen ipellite, polyamino acid, nitrocellulose, or the like is chemically or physically bound. In this state, the mixture and the carrier may be brought into contact with each other and fixed, and the fixation at this time may be performed by irradiating an electromagnetic wave as described later.
  • DNA which is identification information when DNA which is identification information has a thymine-rich base sequence, it can be immobilized by irradiating an electromagnetic wave during or after contact between the DNA and the solid phase carrier.
  • a well-known photoinitiator can also be mixed in water or a buffer.
  • the electromagnetic wave used for immobilization is preferably ultraviolet light having a wavelength of 220 nm to 380 nm.
  • it may be an ultraviolet ray having a broad waveform including a wavelength of 280 nm.
  • Irradiation dose is preferably 10 ⁇ 5000mJ / cm 2, more preferably 100 ⁇ 2000mJ / cm 2.
  • the DNA solution can be dried after spotting and before ultraviolet irradiation.
  • a drying method of a nucleic acid solution you may dry naturally and you may heat and dry.
  • the heating temperature is usually 30 to 100 ° C., preferably 35 to 45 ° C.
  • a known technique can be used without any particular limitation to supply a small amount of DNA, usually, water or buffer containing DNA in a dotted manner to a carrier or the like.
  • This manufacturing method may further include a blocking step.
  • the holding body can be further blocked by bringing an excess amount of bovine serum albumin (BSA), casein, salmon sperm DNA or the like into contact with a carrier or the like as necessary.
  • BSA bovine serum albumin
  • the method for producing an identifiable article according to the present invention can include a step of preparing the holding body and a step of fixing the holding body to an article to be identified.
  • the means for fixing the holding body to the article in the fixing step is not particularly limited. As already described, it may be fixed by means such as an adhesive or a pressure-sensitive adhesive, or may be fixed by locking means like a tag.
  • the form to be fixed is not particularly limited.
  • the identification information holding body may be fixed to the article such that the information holding area is directed to the article side or the identification information is directed to the outside of the article.
  • Identification method Next, a method for identifying an identification target using the holder will be described. A step of bringing one or more probes having a base sequence complementary to the identification base sequence into contact with the information holding region of the holder provided to the identification target; a high level of the identification base sequence and the probe; Detecting a hybridization product. According to this identification method, since a good discrimination capability is ensured in the holder, the identification target can be identified with good accuracy.
  • the identification step can be applied directly to methods such as management, monitoring, authentication, identification, and tracking of identification targets.
  • the contact step is a step of bringing one or more probes having a base sequence complementary to the identification base sequence included in the identification information into contact with the identification information on the holder attached to the identification target It can be.
  • a hybridization product with the probe can be formed. That is, the purpose of this contact step is to allow hybridization between the identification base sequence in the DNA and the probe.
  • the probe only a probe corresponding to the identification base sequence previously assigned to the identification target may be supplied, or a probe that is universally applicable to many identification targets may be supplied.
  • the probe may be complementary to the identification base sequence to the extent that it can form a hybridized product, but is preferably completely complementary.
  • the discriminating base sequence is selected from the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary base sequences
  • the probe has the base sequence represented by SEQ ID NOs: 1 to 100 and its complementary bases Selected from the sequence.
  • the probe is preferably labeled for subsequent detection.
  • a conventionally known one can be appropriately selected and used. It may be various dyes such as a fluorescent substance that emits a fluorescent signal when excited by itself, or may be a substance that emits various signals in combination with the second component by an enzyme reaction or an antigen-antibody reaction.
  • a fluorescent labeling substance such as Cy3, Alexa555, Cy5, Alexa647 can be used.
  • biotin and streptavidin HPR may be combined for detection by color development such as by treatment with a substrate.
  • the probe may include a probe specific to the identification target, but may be a probe set that is universally configured to be applicable to many identification targets. Since probes having the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary base sequences have no mishybridization with each other, universal holders using these base sequences as identification sequences are universal. It can be a set applicable to.
  • the conditions for the contact process are not particularly limited.
  • a normal hybridization medium can be used.
  • An appropriate temperature can be set.
  • a temperature of 30 ° C. or higher and 80 ° C. or lower, preferably 30 ° C. or higher and 40 ° C. or lower can be adopted. More preferably, it is 35 degreeC or more and 40 degrees C or less.
  • time is 1 second or more and 1 hour or less.
  • the contact step is 30 ° C. or higher and 40 ° C. or lower, more preferably 35 ° C. or higher and 40 ° C. or lower, and preferably 1 second or longer and 5 minutes or shorter, more preferably 1 second or longer and 1 minute or shorter. it can.
  • the holder may be separated from the identification target.
  • the contact step can be performed in a state separated from the identification target. Further, if possible, a contact process may be performed on the identification target. For example, there is a case where a hybridization cavity is provided on the holder.
  • DNA as identification information is immobilized on a carrier or the like, the hybridization product is retained on the carrier or the like even if an excess probe is washed.
  • the detection step may be a step of detecting a hybridization product between the identification base sequence in the identification information and the probe. By detecting such a hybridization product, the identification target can be identified.
  • the method for detecting the hybridized product in the detection step is not particularly limited.
  • the label may be detected.
  • the double strand may be detected by an electrical detection method or the like.
  • the identification target is identified. That is, since the identity of the identification target is determined, it can be determined that it has not been tampered with, has not been replaced, or has not been damaged. Further, when the hybridized product is not detected, it is determined that the identification target does not exist or the identification target is not identical. That is, it is determined whether the identification target is lost, altered, or damaged.
  • identification information when one or more DNAs form a visible pattern, the identification target can be easily identified by visually recognizing the pattern, and the identification target is identified.
  • the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary sequences are used, highly selective hybridization products are generated, so that rapid and highly accurate discrimination is possible. Further, even when two or more DNAs, that is, two or more identification base sequences are associated with one identification target, identification with high accuracy is possible.
  • the time required for the detection process is not particularly limited, but can be 1 second or more and 1 hour or less.
  • a general detection temperature 50 Hybridization and detection are possible at 40 ° C. or lower (for example, about 37 ° C.) compared to (° C.-70 ° C.), so that the detection process can be accelerated. More preferably, they are 1 second or more and 5 minutes or less, More preferably, they are 1 second or more and 1 minute or less.
  • the contact process and the detection process can be easily and quickly performed on the holder having a good identification ability. Therefore, identification with high accuracy is possible.
  • Such an identification method also has the same effect in a management method, a monitoring method, an authentication method, and the like related to the distribution and storage of various articles.
  • the identification kit of the identification target disclosed in the present specification can include the holder and one or more probes having a base sequence complementary to the identification base sequence. According to such a kit, identification by this holding body can be implemented. For the holder and the probe in the identification kit, the various aspects described above can be applied as they are.
  • the following advantageous effects can be obtained.
  • (1) By providing a barrier surrounding the information holding region on the solid phase carrier and using this barrier as a hybridization cavity, it becomes easy to drip the hybridization solution, and even if bubbles are generated on the information holding region. The bubbles can be quickly eliminated. Moreover, the amount of the hybridization solution can be reduced by the cavity formed by the barrier. Furthermore, because of such an open cavity, it is possible to perform a quick cleaning only by flowing a cleaning liquid, and it is possible to speed up the entire process. (2) By providing a barrier, it is possible to suppress contact with an external element and rubbing to the information holding area in the detection step, and detection with good reproducibility is possible.
  • oligonucleotide identification base sequence there are three types of sequences: one with a low proportion of thymine (T) base, one with a high proportion of thymine base, and one without any thymine base, Oligonucleotides having a sequence in which polyT (thymine-rich base sequence) was connected to the 5 ′ end of these sequences were synthesized. These oligonucleotides were spotted and fixed on a resin substrate.
  • T thymine
  • the hybridization reaction with the probe before and after the ultraviolet treatment was performed, and the intensities of the fluorescence signals were compared.
  • the ultraviolet irradiation treatment was performed by continuously irradiating ultraviolet light with a wavelength of about 200 to 300 nm on the carrier on which the oligonucleotide was fixed for a long time.
  • Oligonucleotides are Oligo1-1, 2-1, 3-1 (SEQ ID NO: 101 to 103) having 10, 2 and 0 thymine bases in the identification base sequence, and 5 ′ of these.
  • aqueous solution of the synthetic oligonucleotide was prepared as follows.
  • Aqueous solution for spot using additive 1 SSC system 6 ⁇ SSC (thinned 20 ⁇ SSC from Invitrogen) and 100 pmol / ⁇ l of oligonucleotide mixed in equal amounts (each final concentration 50 pmol / ⁇ l, 3 ⁇ SSC)
  • B Aqueous solution for spot using additive 2: PBS system 2 ⁇ PBS (thin diluted 10 ⁇ PBS from Invitrogen) and 100 pmol / ⁇ l of oligonucleotide mixed in equal amounts (each final concentration 50 pmol / ⁇ l, 1 ⁇ PBS)
  • the synthetic oligonucleotide was immobilized on a carrier (substrate) by the following procedure. That is, the spotted substrate was set in a UV irradiation apparatus (Spectroline XL-1500 UV Crosslinker) and irradiated with ultraviolet light at 600 mJ / cm 2 . Next, the substrate set on the slide rack is shaken up and down 50 times in a 3-5% BSA aqueous solution, then shaken up and down 10 times in sterilized water, and then drained by centrifugation (1000 rpm ⁇ 2 minutes). did.
  • a UV irradiation apparatus Spectroline XL-1500 UV Crosslinker
  • Probe mixture (2.5nM, each) * 1.5 ⁇ l Hybri Solution ( ⁇ 2) * 9.0 ⁇ l milliQ water 7.5 ⁇ l total 18.0 ⁇ l * Probe mixture composition (2.5nM, each) Probe 1 (100nM) 10 ⁇ l Probe 2 (100nM) 10 ⁇ l Probe 3 (100nM) 10 ⁇ l TE (pH 8.0) 370 ⁇ l 400 ⁇ l * Hybri Solution composition ( ⁇ 2) 20 x SSC 2.0 ml 10% SDS 0.8 ml 100% Formamide 12.0 ml 100 mM EDTA 0.8 ml milliQ 24.4 ml 40.0 ml
  • reaction of probe DNA solution with substrate The prepared reaction probe DNA solution was heated at 90 ° C. for 1 minute using GeneAmp PCR System 9700 from Applied Biosystems, and then heated at 80 ° C. for 1 minute using a heat block (TAITEC DTU-N). Each 9 ⁇ l of the probe solution was applied to the spot area on the substrate, and the reaction was performed by leaving it at 37 ° C. for 60 minutes using a comfort / plus thermoblock Slide (eppendorf) to prevent drying.
  • TITEC DTU-N a heat block
  • the washing solution was transferred to a glass staining vat.
  • the substrate after the reaction with the probe DNA solution was immersed and shaken up and down for 5 minutes.
  • the substrate was transferred to a glass staining vat containing sterilized water and shaken up and down for 1 minute. Subsequently, it was centrifuged and dried at 2000 rpm for 1 minute to remove moisture remaining on the substrate.
  • each substrate obtained by irradiating with ultraviolet light was reacted with the probe DNA in the same manner as the method performed in (2), and the excess probe on the substrate was washed.
  • the method for preparing an aqueous solution for oligonucleotide spots in the PBS system had a slightly larger ratio of reduction in the fluorescence intensity of the spot due to the irradiation of additional ultraviolet light than the SSC system, and it was desirable to prepare the aqueous solution for spots in the SSC system.
  • an identification information holding body including a barrier member was produced and evaluated.
  • the evaluation was performed according to the following procedure.
  • (1) Synthetic DNA probe for detection of sample DNA is supported on a carrier (film (Example) / two types of film and slide substrate (glass and plastic) (for control)), and the synthetic DNA probe is retained by immobilizing spots and probes.
  • the spotted carrier is immersed in a blocking solution designated by the manufacturer for 5 minutes at room temperature, then immersed in boiling water for 2 minutes, further immersed in sterilized water and quenched for 2 minutes, and then airgun. According to draining.
  • an aqueous solution in which a synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) in which the 5 ′ end is modified with an amino group is dissolved in each of a commercially available plastic substrate and a commercially available glass substrate is a capture probe.
  • spotting was performed at NGK Corporation using a GENESHOT (registered trademark) spotter.
  • the synthetic DNA was immobilized by washing the spotted carrier with 2 ⁇ SSC / 0.2% SDS for 15 minutes and then with 95 ° C. ⁇ 2 ⁇ SSC / 0.2% SDS for 5 minutes. After performing sterilized water washing (shaking up and down 10 times) three times, the liquid was removed by centrifugation (1000 rpm ⁇ 3 minutes).
  • the synthetic oligo DNA sequences used for spotting in the preparation of the probe holder and the control probe holder are two sequences (from the sequence table shown in the following table, which are already known to have high sample reactivity) ( D1-001, D1-100) (SEQ ID NOS: 1 and 100) were selected and used.
  • the spot pattern of each probe was carried out as shown in FIG. 6, and three types were used: a spot where only D1-001 was immobilized, a spot where only D1-100 was immobilized, and a spot where both were immobilized. .
  • barrier member (with / without a cover) composed of a laminate on a carrier, which is capable of holding a sample solution, on the various DNA probe holders prepared in (1), as shown in FIGS.
  • Three types of barrier members (I to III, I have no cover, II and III have a cover) were attached.
  • the barrier members II and III were both attached with the barrier member during storage, and the cover (weakly adhesive Lintec film) was peeled off to react with the sample DNA to expose the probe on the carrier.
  • a reaction solution using each of the two types of sample DNAs was prepared, and the reaction was performed on a synthetic DNA probe holding carrier (film and two types of control substrates). The detailed procedure is described below. Furthermore, the sample DNA reaction was carried out with no barrier member attached to the film and the two control substrates. Since the leakage of the following sample DNA solution for reaction cannot be prevented with respect to the film having no barrier member and the two kinds of substrates as the control probe holder, the reaction was carried out with the liquid volume increased to 50 ⁇ l.
  • Hybri Solution composition ( ⁇ 2) 20 x SSC 2.0 ml 10% SDS 0.8 ml 100% Formamide 12.0 ml 100 mM EDTA 0.8 ml milliQ 24.4 ml 40.0 ml
  • reaction of sample DNA solution to holder The sample solution (rD1-001 only, rD1-100 only and mixed) prepared as described above was heated at 90 ° C. for 1 minute using an Applied Biosystems GeneAmp PCR System 9700, and then heat block (TAITEC Using DTU-N) and heated at 80 ° C. for 1 minute. Next, 9 ⁇ l each of the above sample solution is dropped on the spot area on each holder, and then the reaction is carried out by allowing to stand at 37 ° C. for 60 minutes in a comfort / plus thermoblock Slide (eppendorf) to prevent drying. went.
  • the cleaning solution prepared as described above was transferred to a glass staining vat, the substrate after completion of the sample DNA solution reaction was immersed, and shaken up and down for 5 minutes. Further, the substrate was transferred to a glass staining vat containing sterilized water, shaken up and down for 1 minute, and then centrifugally dried at 2000 rpm for 1 minute to remove water remaining on the substrate.
  • the probe holder was cut into each pattern unit and reacted with sample DNA.
  • the cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.
  • the probe holder was cut into each pattern unit and reacted with sample DNA.
  • the cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.
  • the probe holder was cut into each pattern unit and reacted with sample DNA.
  • the cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.

Abstract

Provided is an identifying information carrier whereby identifying information can be stably carried and good identification ability can be maintained. The identifying information carrier according to the present invention comprises: a solid support; one or more information-carrying areas wherein one or more DNAs are carried on the solid support, said DNA having, as identifying data, a base sequence for identification that has been preliminarily linked to a subject to be identified; and one or more barriers surrounding the information-carrying areas.

Description

識別対象を識別するための識別情報の保持体及びその利用Retainer of identification information for identifying identification object and use thereof
　本発明は、対象物を識別するための識別情報を保持する保持体及びその利用に関し、特に、ＤＮＡを含有する識別情報を保持する保持体及びその利用に関する。 The present invention relates to a holding body that holds identification information for identifying an object and its use, and more particularly to a holding body that holds identification information containing DNA and its use.
　近年、製品、部品（中間製品含む）、水産物、農産物、銀行券、証券等の各種の流通物品を品質、安全性、改ざん防止、真偽判定の観点から、管理することが要請されるようになってきている。 In recent years, it has been requested that various distribution items such as products, parts (including intermediate products), marine products, agricultural products, banknotes, securities, etc. be managed from the viewpoints of quality, safety, tampering prevention, and authenticity judgment. It has become to.
　こうした各種の識別対象の流通や移動の監視・管理は、通常、識別対象に固有のあるいは予め関連付けられた識別情報を付与しておき、その識別情報を、適時に識別することによって行われる。例えば、ＤＮＡの塩基配列を利用してＤＮＡインキを作製し、このＤＮＡインキを用いて識別対象に印刷し、適時に、ＤＮＡの塩基配列を検出することで、偽造や改ざん等を回避する技術も提案されている(特許文献１)。 Such distribution and movement monitoring and management of various identification objects are usually performed by providing identification information specific to the identification object or associated in advance and identifying the identification information in a timely manner. For example, a technique for avoiding counterfeiting or falsification by preparing a DNA ink using the DNA base sequence, printing it on the identification target using this DNA ink, and detecting the DNA base sequence in a timely manner. It has been proposed (Patent Document 1).
特開2008-187991号公報JP 2008-187991 A
　流通や移動を通じて、識別情報を保持した識別情報保持体は、様々な環境に曝されることになる。本発明者らは、識別及び証明の精度は、保持されるＤＮＡの量や状態に大きく影響されるものであり、識別情報保持体は、いかに安定して識別情報たるＤＮＡを証明時まで保持するかが重要であることを見出すに至った。すなわち、識別情報保持体を識別対象たる物品に適用するまでの間（例えば、識別情報保持体自体の製造後から流通時）、ＤＮＡを安定的に保持することが重要である。また、一旦、識別対象たる物品に識別情報保持体を適用後、その物品の流通経路を通じて証明時までＤＮＡを安定して維持することも重要である。 Through the distribution and movement, the identification information holding body holding the identification information is exposed to various environments. The inventors of the present invention have a great influence on the accuracy of identification and proof by the amount and state of DNA to be held, and the identification information holding body holds DNA as identification information stably until the time of proof. I found out that is important. That is, it is important to stably hold DNA until the identification information holding body is applied to an article to be identified (for example, after the identification information holding body itself is manufactured and distributed). It is also important to maintain the DNA stably after applying the identification information holding body to the article to be identified until the time of certification through the distribution channel of the article.
　また、ＤＮＡは、波長200～300nm程度の紫外光で損傷が生じることがある。また、流通中に、摩擦的な外力がかかったり、温度や湿度の変化など生じると、ＤＮＡが識別対象から脱離されるほか、識別対象上において切断される可能性もある。このような場合には、付与されたＤＮＡを正確に検出することが困難になり、識別能が低下する恐れがある。また、精度よく識別対象を識別するには、その検出が簡易にかつ迅速に実行できることも重要である。 In addition, DNA may be damaged by ultraviolet light having a wavelength of about 200 to 300 nm. In addition, when a frictional external force is applied during the distribution or a change in temperature or humidity occurs, the DNA may be detached from the identification target and may be cleaved on the identification target. In such a case, it is difficult to accurately detect the assigned DNA, and the discrimination ability may be reduced. In addition, in order to identify the identification target with high accuracy, it is also important that the detection can be performed easily and quickly.
　そこで、本発明は、安定して識別情報を保持して良好な識別能を維持できる識別情報の保持体及びその利用等を提供することを目的とする。 Therefore, an object of the present invention is to provide an identification information holding body that can stably hold identification information and maintain good discrimination ability, and use thereof.
　本発明者らは、識別対象を識別するための識別情報として用いるＤＮＡをその固相担体上において安定的に保持させる等のために効果的な構成を見出し、本発明を完成した。本発明によれば、以下の手段が提供される。 The present inventors have found an effective configuration for stably holding DNA used as identification information for identifying an identification target on the solid phase carrier, and completed the present invention. According to the present invention, the following means are provided.
Ｉ．固相担体と、
　識別対象に予め関連付けられた識別用塩基配列を識別情報として有する１又は２以上のＤＮＡが前記固相担体上に保持された１又は２以上の情報保持領域と、
　１又は２以上の前記情報保持領域を包囲する障壁と、
を備える、識別情報の保持体。 I. A solid support;
One or more information holding regions in which one or two or more DNAs having identification base sequences previously associated with identification objects as identification information are held on the solid phase carrier;
A barrier surrounding one or more of the information holding areas;
A holding body for identification information.
II．本発明の識別情報保持体を複数個一体に備える、構造体。 II. A structure comprising a plurality of identification information holding bodies of the present invention integrally.
III．本発明の識別情報保持体の製造方法であって、
　前記固相担体上に、前記１又は２以上のＤＮＡを固定して１又は２以上の前記情報保持領域を形成する工程と、
　１又は２以上の前記情報保持領域を包囲する障壁となる障壁部材を前記固相担体上に固定する工程と、
を備える、製造方法。 III. A method for producing the identification information holding body of the present invention,
Immobilizing the one or two or more DNAs on the solid phase carrier to form one or more of the information holding regions;
Fixing a barrier member serving as a barrier surrounding one or more of the information holding regions on the solid phase carrier;
A manufacturing method comprising:
IV．本発明の識別情報保持体を準備する工程と、
　前記識別情報保持体を識別対象とする物品に固定化する工程と、
を備える、識別可能な物品の製造方法。 IV. Preparing the identification information holding body of the present invention;
Immobilizing the identification information holding body on an article to be identified;
A method for producing an identifiable article.
V．識別対象の識別方法であって、
　前記識別対象に付与された本発明の識別情報保持体の前記情報保持領域に対して、前記識別用塩基配列と相補的な塩基配列を有する１又は２以上のプローブを接触させる工程と、
　前記識別用塩基配列と前記プローブとのハイブリダイゼーション産物を検出する工程と、
を備える、方法。 V. An identification method of an identification object,
Contacting the information holding region of the identification information holding body of the present invention given to the identification target with one or more probes having a base sequence complementary to the identification base sequence;
Detecting a hybridization product of the identification base sequence and the probe;
A method comprising:
本発明の識別情報保持体の一例を示す図である。It is a figure which shows an example of the identification information holding body of this invention. 本発明の識別情報保持体の他の一例を示す図である。It is a figure which shows another example of the identification information holding body of this invention. 識別情報がパターンを構成している例を示す図である。It is a figure which shows the example in which identification information comprises the pattern. 本発明の識別情報保持体の構成、使用形態の一例を示す図である。It is a figure which shows an example of a structure of the identification information holding body of this invention, and a usage pattern. 本発明の識別情報保持体の構成、使用形態の一例を示す図である。It is a figure which shows an example of a structure of the identification information holding body of this invention, and a usage pattern. 実施例におけるスポットパターンを示す図である。It is a figure which shows the spot pattern in an Example. 実施例で用いる保持体の一例を示す図である。It is a figure which shows an example of the holding body used in an Example. 実施例で用いる保持体の他の一例を示す図である。It is a figure which shows another example of the holding body used in an Example. 実施例で用いる保持体の他の一例を示す図である。It is a figure which shows another example of the holding body used in an Example. 実験１の評価結果を示す図である。It is a figure which shows the evaluation result of Experiment 1. 実験２の評価結果を示す図である。It is a figure which shows the evaluation result of the experiment 2. FIG. 実験３の評価結果を示す図である。It is a figure which shows the evaluation result of the experiment 3. FIG. 実験４の評価結果を示す図である。It is a figure which shows the evaluation result of the experiment 4. FIG.
　本発明の識別情報保持体によれば、識別情報たるＤＮＡの脱離や分解が抑制され固相担体上に安定して保持されているため、識別の正確性、精度及び再現性に優れた、識別能が良好な保持体が提供される。また、このように識別情報たるＤＮＡが固相担体上に保持されることで、迅速でかつ簡易な検出も可能となる。また、配列番号１～１００で表される塩基配列及びその相補的配列から選択される識別用塩基配列を用いている場合には、特異性の高い識別能が確保され、識別対象を高い選択性で、迅速に識別することができる。以下、本明細書の開示の実施形態について詳細に説明する。 According to the identification information holding body of the present invention, since detachment and decomposition of DNA serving as identification information are suppressed and stably held on the solid phase carrier, the identification accuracy, accuracy and reproducibility are excellent. A holder having good discrimination ability is provided. In addition, since DNA as identification information is held on the solid phase carrier in this way, rapid and simple detection is possible. In addition, when a discrimination base sequence selected from the base sequences represented by SEQ ID NOs: 1 to 100 and its complementary sequence is used, a highly specific discrimination ability is ensured and the discrimination target is highly selective. And can be identified quickly. Hereinafter, embodiments of the present disclosure will be described in detail.
　本発明の識別情報保持体（以下、単に、本保持体ともいう。）の一例を図１及び図２に示す。図１及び図２に示すように、本保持体は、情報保持領域を包囲する障壁を、識別情報を保持する固相担体表面に有する。障壁が、情報保持領域に識別情報として保持されるＤＮＡ等を、識別情報保持体自体あるいはこれが適用された識別対象が流通等におかれた状態において、外部からの摩擦や衝撃から保護することができるため、識別情報を安定して固相担体上に保持できる。このため、識別対象の識別及び証明を正確に行うことができる。 An example of the identification information holding body of the present invention (hereinafter also simply referred to as the present holding body) is shown in FIGS. As shown in FIG. 1 and FIG. 2, the holder has a barrier surrounding the information holding region on the surface of the solid phase carrier holding identification information. A barrier may protect DNA or the like held as identification information in the information holding area from friction or impact from the outside in a state where the identification information holding body itself or an identification target to which the identification information is applied is in circulation. Therefore, the identification information can be stably held on the solid phase carrier. For this reason, identification and proof of an identification object can be performed correctly.
　また、障壁があれば、情報保持領域を識別対象たる物品の外方に向けるようにして、識別情報保持体を固定化しても、識別情報を保護することができる。さらに、障壁を有することで、情報保持領域を物品側に向けて、識別時には物品から脱着可能に固定化するようにして、情報保持領域を物品外方に曝すことなく付与することもできる。 Also, if there is a barrier, the identification information can be protected even if the identification information holding body is fixed so that the information holding area faces the outside of the article to be identified. Furthermore, by having a barrier, the information holding area can be provided to the article side without exposing the information holding area to the outside of the article by fixing the information holding area so as to be removable from the article at the time of identification.
　さらに、障壁を介して情報保持領域の上方を覆うカバーを備えることで、識別情報保持体自体の製造後から流通時において識別情報を安定して保持できる。また、こうしたカバーを備えることで、情報保持領域を物品に向けて物品に適用された場合であってもあるいは物品外方に向けて適用された場合であっても、識別情報をより確実に保護できる。 Furthermore, by providing a cover that covers the upper portion of the information holding area through a barrier, the identification information can be stably held during the distribution after the identification information holding body itself is manufactured. In addition, by providing such a cover, even when the information holding area is applied to the article toward the article or applied to the outside of the article, the identification information is more reliably protected. it can.
　障壁は、必ずしも固相担体の一部として構成されなくてもよく、接着剤や粘着剤を介して固相担体に対して固定化される障壁部材であってもよい。障壁部材は、こうした固定化層とプラスチック製の障壁層を備えることができる。 The barrier may not necessarily be configured as a part of the solid phase carrier, and may be a barrier member that is fixed to the solid phase carrier via an adhesive or an adhesive. The barrier member may comprise such an immobilization layer and a plastic barrier layer.
　障壁は、また、情報保持領域に対してプローブを供給してハイブリダイズを検出するとき、プローブを含むハイブリダイズ溶液を滴下する領域を明確化するための目印として機能するとともに、ハイブリダイズ溶液を情報保持領域に対して確実に所定量を供給するための容器としても機能することができる。この結果、識別対象の識別及び証明を正確に行うことができる。 The barrier also functions as a mark for clarifying the region where the hybridization solution containing the probe is dropped when the probe is supplied to the information holding region to detect hybridization. It can also function as a container for reliably supplying a predetermined amount to the holding region. As a result, the identification target can be accurately identified and proved.
　識別情報保持体は、複数個、好ましくは、１０個以上、より好ましくは２０個以上の情報保持領域を単一の固相担体上に形成して、これらの情報保持領域を障壁で区画することで、一挙に効率的に製造することができる。すなわち、上記した構造体として製造することで、効率的に識別情報保持体を製造できる。
　このとき、障壁は、複数の情報保持領域を個別に識別する指標とすることもできる。したがって、障壁を指標として、１又は２以上の情報保持領域を有する識別情報体を分離して、最終的な識別情報保持体を取得できる。 A plurality of identification information holding bodies, preferably 10 or more, more preferably 20 or more information holding areas are formed on a single solid phase carrier, and these information holding areas are partitioned by a barrier. Thus, it can be manufactured efficiently at a stroke. That is, the identification information holding body can be efficiently manufactured by manufacturing the structure as described above.
At this time, the barrier may be an index for individually identifying a plurality of information holding areas. Therefore, the identification information body having one or two or more information holding areas can be separated using the barrier as an index to obtain the final identification information holding body.
　本願発明の識別情報保持体又は構造体における障壁は、例えば、国際公開第WO2006/101229号（PCT/JP2006/306134）に開示されるセパレーターを障壁部材とし用いて構成することができる。こうしたセパレーターとしては、ポリカーボネート、ポリオレフィン（PET、PE）、ポリアミド、ポリイミド、アクリル樹脂、これらのフッ化物及びハロゲン化物からなる群から選択される材料を用いることが好ましい。また、接着剤又は粘着剤を含む固定化層を備えていることが好ましい。 The barrier in the identification information holding body or structure of the present invention can be configured using, for example, a separator disclosed in International Publication No. WO2006 / 101229 (PCT / JP2006 / 306134) as a barrier member. As such a separator, it is preferable to use a material selected from the group consisting of polycarbonate, polyolefin (PET, PE), polyamide, polyimide, acrylic resin, and fluorides and halides thereof. Moreover, it is preferable to provide the fixed layer containing an adhesive agent or an adhesive.
　また、本願発明の識別情報保持体又は構造体における固相担体は、例えば、特開2006-184016号公報、特開2006-71309号公報及びNucleic Acid Research,2007, vol.35, No1.e3 記載の基板を用いることができる。 Further, the solid phase carrier in the identification information holding body or structure of the present invention is described in, for example, JP-A-2006-184016, JP-A-2006-71309 and Nucleic Acid Research, 2007, vol.35, No1.e3. The substrate can be used.
　また、こうした固相担体に対するＤＮＡの結合には、固相担体表面の活性エステル基とＤＮＡの５’末端及び３’末端に結合したアミノ基との反応によるアミド構造（共有結合）の形成を用いることができる。こうした結合方法によると、十分に強力にＤＮＡを固相担体に固定でき、流通時を含んでＤＮＡを外部からの衝撃から保護することができる。 In addition, for the binding of DNA to such a solid phase carrier, formation of an amide structure (covalent bond) by reaction between an active ester group on the surface of the solid phase carrier and an amino group bound to the 5 ′ end and 3 ′ end of DNA is used. be able to. According to such a binding method, the DNA can be fixed to the solid phase carrier with sufficient strength, and the DNA can be protected from an impact from the outside, including during distribution.
　以下、本発明の実施形態について詳細に説明する。 Hereinafter, embodiments of the present invention will be described in detail.
（識別情報保持体）
　本保持体は、識別対象を識別するため識別情報を保持する保持体であって、識別対象に予め関連付けられた識別用塩基配列を有するＤＮＡを識別情報として有する。 (Identification information holder)
This holding body is a holding body that holds identification information for identifying an identification target, and has, as identification information, a DNA having an identification base sequence that is associated with the identification target in advance.
（識別対象）
　本保持体によって識別される識別対象は、特に限定されないで、各種の流通物品や媒体が挙げられる。流通物品としては、商業的にあるいは非商業的に流通する全ての物品が対象となる。例えば、各種工業製品、部品（中間製品含む）、水産物、農産物、芸術品、書籍などのほか、銀行券、証券などが挙げられる。また、個人を識別するためのＩＤカードのほか、各種証明書等も含まれる。 (Identification target)
The identification object identified by the holder is not particularly limited, and various distribution articles and media can be mentioned. Distribution articles include all articles that are distributed commercially or non-commercially. Examples include various industrial products, parts (including intermediate products), marine products, agricultural products, arts, books, banknotes, securities, and the like. In addition to an ID card for identifying an individual, various certificates are also included.
　本保持体は、各種形態を採ることができる。例えば、フィルム、シート、基板などの各種形状の担体に対して識別情報を保持するものであってもよい。この場合、本保持体は、識別対象に対して、接着などの化学的手段やそのほかの物理的手段により、固定化される。また、本保持体が、識別対象の一部であってもよい。例えば、識別対象の一部に識別情報が保持されていてもよい。 本 This holder can take various forms. For example, identification information may be held for carriers of various shapes such as films, sheets, and substrates. In this case, the holder is fixed to the identification object by chemical means such as adhesion or other physical means. Further, the holder may be a part of the identification target. For example, identification information may be held in a part of the identification target.
　本保持体は、担体に識別情報が保持された形態を採ることができる。担体の形状としては、例えば、フィルム、平板、粒子、成形品（ビーズ、ストリップ、マルチウェルプレートのウェルまたはストリップ、チューブ、メッシュ、連続発泡フォーム、膜、紙、針、ファイバー、プレート、スライド及び細胞培養容器等）、ラテックス等を挙げることができる。また、それらの大きさについては、特に制限は無い。検出を考慮すると、識別情報を付与する領域が平坦状であることが好ましい。 This holder can take a form in which identification information is held on a carrier. Examples of the carrier shape include films, flat plates, particles, molded articles (beads, strips, wells or strips of multiwell plates, tubes, meshes, foams, membranes, paper, needles, fibers, plates, slides, and cells. Culture vessels, etc.) and latex. Moreover, there is no restriction | limiting in particular about those magnitude | sizes. In consideration of detection, it is preferable that the region to which the identification information is applied is flat.
　ＤＮＡ等である識別情報が保持される担体又は識別対象の部位（担体等）を構成する材料としては、物理的吸着又は化学結合によってＤＮＡ等を固定化することができ、通常のハイブリダイゼーションの条件に耐えうるものであれば特に制限されない。具体的には、核酸の固定及びハイブリダイゼーション等に用いる溶剤に不溶であり、かつ常温若しくはその付近の温度範囲内（例えば０～１００℃）で固体又はゲル状であるものが挙げられる。 As a material constituting a carrier for holding identification information such as DNA or a site to be identified (carrier etc.), DNA or the like can be immobilized by physical adsorption or chemical bonding, and normal hybridization conditions If it can endure, it will not be restrict | limited. Specific examples include those that are insoluble in a solvent used for immobilization of nucleic acid, hybridization, and the like, and are solid or gel at room temperature or in the vicinity of a temperature range (for example, 0 to 100 ° C.).
　このような担体等の材質としては、具体的には、プラスチック、無機高分子、金属、天然高分子、セラミック等が挙げられる。上記プラスチックとして具体的には、紫外線照射により生体分子を固定化することができるものであれば特に制限されず、具体的には、熱可塑性樹脂、熱硬化性樹脂及び共重合体等が挙げられる。さらに具体的には、熱可塑性樹脂としては、アイオノマー（スチレン系、オレフィン系）、ポリノルボルネン、ポリアセタール、ポリアリレート、ポリエーテルエーテルケトン、ポリエチレンオキサイド、ポリオキシメチレン、ポリエチレンテレフタレート、ポリカーボネート、ポリスチレン、ポリスルホン、ポリパラメチルスチレン、ポリアリルアミン、ポリフェニレンエーテル、ポリフェニレンサルファイド、ポリブタジエン、ポリブチレンテレフタレート、ポリプロピレン、ポリメチルペンテン、ポリエーテルスルホン、ポリフェニレンスルフィド、ポリオキシベンゾイル、ポリオキシエチレン、酢酸セルロース、ポリジメチルシロキサン、ポリイソブチレン、セルローストリアセテート、ポリ－ｐ－フェニレンテレフタラミド、ポリイソプレン、ポリアクリロニトリル、ポリメチルペンテン、塩素プラスティック（ポリ塩化ビニル、ポリ塩化エチレン、塩素化ポリプロピレン、ポリ塩化ビニリデン）、フッ素プラスチック（テトラフルオロエチレン、ポリクロロトリフルオロエチレン、ポリフッ化ビニリデン）、ポリアミド（ナイロン6、ナイロン66）、ポリアミドイミド、ポリイミド（熱可塑性ポリイミド、ポリエーテルイミド）、ポリエチレンプラスティック（塩素化、高密度、低密度）、ポリビニルプラスティック（ポリ塩化ビニル、ポリ酢酸ビニル、ポリパラビニルフェノール、ポリビニルアルコール、ポリビニルエーテル、ポリビニルブチラール、ポリビニルホルマール）、液晶ポリマー（ポリエステル系液晶高分子）、アクリレートプラスティック（アミノポリアクリルアミド、ポリアクリル酸メチル、ポリメチルメタクリレート、エチルポリメタクリレート、ブチルポリメタクリレート）、熱可塑性エラストマー（スチレン系、オレフィン系、ウレタン系、ポリエステル系、ポリアミド系、1，2－ポリブタジエン系、塩化ビニル系、フッ素系、ポリアイオノマー系、塩素化ポリエチレン系、シリコーン系）等が挙げられる。 Specific examples of such a carrier material include plastics, inorganic polymers, metals, natural polymers, and ceramics. Specifically, the plastic is not particularly limited as long as it can immobilize biomolecules by ultraviolet irradiation, and specific examples include thermoplastic resins, thermosetting resins, and copolymers. . More specifically, examples of the thermoplastic resin include ionomers (styrene-based, olefin-based), polynorbornene, polyacetal, polyarylate, polyether ether ketone, polyethylene oxide, polyoxymethylene, polyethylene terephthalate, polycarbonate, polystyrene, polysulfone, Polyparamethylstyrene, polyallylamine, polyphenylene ether, polyphenylene sulfide, polybutadiene, polybutylene terephthalate, polypropylene, polymethylpentene, polyethersulfone, polyphenylene sulfide, polyoxybenzoyl, polyoxyethylene, cellulose acetate, polydimethylsiloxane, polyisobutylene , Cellulose triacetate, poly-p-phenylene terephthalamide, poly Soprene, polyacrylonitrile, polymethylpentene, chlorine plastic (polyvinyl chloride, polychlorinated ethylene, chlorinated polypropylene, polyvinylidene chloride), fluoroplastic (tetrafluoroethylene, polychlorotrifluoroethylene, polyvinylidene fluoride), polyamide (nylon) 6, nylon 66), polyamideimide, polyimide (thermoplastic polyimide, polyetherimide), polyethylene plastic (chlorinated, high density, low density), polyvinyl plastic (polyvinyl chloride, polyvinyl acetate, polyparavinylphenol, polyvinyl) Alcohol, polyvinyl ether, polyvinyl butyral, polyvinyl formal), liquid crystal polymer (polyester-based liquid crystal polymer), acrylate plastic (aminopoly) Kurylamide, polymethyl acrylate, polymethyl methacrylate, ethyl polymethacrylate, butyl polymethacrylate), thermoplastic elastomer (styrene, olefin, urethane, polyester, polyamide, 1,2-polybutadiene, vinyl chloride, Fluorine, polyionomer, chlorinated polyethylene, and silicone).
　また、熱硬化性樹脂としては、エポキシ、ポリキシレン、ポリグアナミン、ポリジアリルフタレート、ポリビニルエステル、ポリフェノール、不飽和ポリエステル、ポリフラン、ポリイミド、ポリウレタン、ポリマレイン酸、メラミン、ユリア、アルキド、ベンゾグアナミン、ポリシアナート、ポリイソシアナート等が挙げられる。 Thermosetting resins include epoxy, polyxylene, polyguanamine, polydiallyl phthalate, polyvinyl ester, polyphenol, unsaturated polyester, polyfuran, polyimide, polyurethane, polymaleic acid, melamine, urea, alkyd, benzoguanamine, polycyanate, polycyanate. An isocyanate etc. are mentioned.
　さらに、共重合体としては、イソブチレン無水マレイン酸共重合体、アクリロニトリルアクリレートスチレン共重合体、アクリロニトリルEPDMスチレン共重合体、アクリロニトリルスチレン共重合体、アクリロニトリルブタジエンスチレン共重合体、ブタジエンスチレンメチルメタクリレート共重合体、エチレン塩化ビニル共重合体、エチレン酢酸ビニル共重合体、エチレン－エチルアクリレート共重合体、アクリロニトリル－ブタジエンスチレン共重合体、ポリエーテルエーテルケトン共重合体、フッ化エチレンポリプロピレン共重合体、テトラフルオロエチレンパーフロロアルキルビニルエーテル共重合体、テトラフルオロエチレンエチレン共重合体等が挙げられる。 Further, the copolymer includes isobutylene maleic anhydride copolymer, acrylonitrile acrylate styrene copolymer, acrylonitrile EPDM styrene copolymer, acrylonitrile styrene copolymer, acrylonitrile butadiene styrene copolymer, butadiene styrene methyl methacrylate copolymer. , Ethylene vinyl chloride copolymer, ethylene vinyl acetate copolymer, ethylene-ethyl acrylate copolymer, acrylonitrile-butadiene styrene copolymer, polyether ether ketone copolymer, fluorinated ethylene polypropylene copolymer, tetrafluoroethylene Examples include perfluoroalkyl vinyl ether copolymers and tetrafluoroethylene ethylene copolymers.
　上記の合成樹脂のうち、特に好ましいものとしては、ポリカーボネート、ポリメチルメタクリレート、アクリロニトリルブタジエンスチレン共重合体、ポリエチレン、ポリエチレンテレフタレート、ポリフェノール、ポリスチレン、ポリアクリロニトリル、ポリ塩化ビニル、アラミド等が挙げられる。 Among the above synthetic resins, particularly preferred are polycarbonate, polymethyl methacrylate, acrylonitrile butadiene styrene copolymer, polyethylene, polyethylene terephthalate, polyphenol, polystyrene, polyacrylonitrile, polyvinyl chloride, aramid and the like.
　また、上記合成樹脂に、染料、発色剤、可塑剤、顔料、重合禁止剤、表面改質剤、安定剤、密着性付与剤、熱硬化剤、分散剤、紫外線劣化防止剤等を必要に応じて添加した合成樹脂を用いることができる。さらに、前記合成樹脂は、形状を保持するために異なる種類の前記合成樹脂が積層しても良く、単一合成樹脂であっても良い。また、前記合成樹脂を２種類以上混合したポリマーアロイであっても良い。さらに、上記合成樹脂に、葉脈繊維、果実繊維、獣毛繊維、繭繊維、羽毛繊維、キチン、キトサン、石綿（アスベスト）等の線維を混合してもよい。 In addition, dyes, color formers, plasticizers, pigments, polymerization inhibitors, surface modifiers, stabilizers, adhesion-imparting agents, thermosetting agents, dispersants, UV degradation inhibitors, etc., may be added to the synthetic resin as necessary. Can be used. Furthermore, the synthetic resin may be laminated with different types of the synthetic resins in order to maintain the shape, or may be a single synthetic resin. Moreover, the polymer alloy which mixed 2 or more types of the said synthetic resin may be sufficient. Furthermore, fibers such as vein fibers, fruit fibers, animal hair fibers, cocoon fibers, feather fibers, chitin, chitosan and asbestos (asbestos) may be mixed with the synthetic resin.
　無機高分子として具体的には、ガラス、水晶、カーボン、シリカゲル及びグラファイト等が挙げられる。金属として具体的には、金、白金、銀、銅、鉄、アルミニウム、磁石、パラマグネット等が挙げられる。天然高分子としては、ポリアミノ酸、セルロース、キチン、キトサン、アルギン酸及びそれらの誘導体が挙げられる。セラミックとして具体的には、アパタイト、アルミナ、シリカ、炭化ケイ素、窒化ケイ素及び炭化ホウ素等が挙げられる。 Specific examples of the inorganic polymer include glass, crystal, carbon, silica gel, and graphite. Specific examples of the metal include gold, platinum, silver, copper, iron, aluminum, a magnet, and a paramagnet. Examples of natural polymers include polyamino acids, cellulose, chitin, chitosan, alginic acid, and derivatives thereof. Specific examples of the ceramic include apatite, alumina, silica, silicon carbide, silicon nitride, and boron carbide.
　上記担体等には、直接ＤＮＡ等を固定化してもよいが、さらに、担体等に対して固定化のための固定化相を付与してもよい。こうした固定化相としては、上記担体等上に担持される限り、単に物理的な接着性を利用して担持されていてもよく、また、化学的に共有結合等を介して担持されていてもよい。また、上記固定化相は、必要に応じ、担体等上の全面において担持されても、また、その一部において担持されてもよい。固定化相としては、上記した担体等の材料として説明した材料の他、有機低分子が挙げられる。上記有機低分子として具体的には、カルボジイミド基含有化合物、イソシアネート基含有化合物、窒素イペリット基含有化合物、アルデヒド基含有化合物、アミノ基含有化合物等が挙げられる。 The DNA or the like may be directly immobilized on the carrier or the like, but an immobilization phase for immobilization may be added to the carrier or the like. As such an immobilized phase, as long as it is supported on the carrier or the like, it may be supported simply using physical adhesiveness, or may be chemically supported via a covalent bond or the like. Good. Moreover, the said fixed phase may be carry | supported on the whole surface on a support | carrier etc. as needed, and may be carry | supported in a part. Examples of the immobilized phase include small organic molecules in addition to the materials described above as materials for the carrier and the like. Specific examples of the organic low molecule include a carbodiimide group-containing compound, an isocyanate group-containing compound, a nitrogen iperit group-containing compound, an aldehyde group-containing compound, and an amino group-containing compound.
　固定化相は、担体等上に皮膜として担持されることが好ましい。担体等上に固定化相を皮膜で担持させる方法としては、スプレー、浸漬、ブラッシング、スタンプ、蒸着、フィルムコータを用いたコーティング等の公知の方法を用いることができる。 The immobilized phase is preferably supported as a film on a carrier or the like. As a method for supporting the immobilized phase as a film on a carrier or the like, a known method such as spraying, dipping, brushing, stamping, vapor deposition, or coating using a film coater can be used.
　例えば、ガラス製担体等の表面全体にカルボジイミド基（樹脂）を導入する方法については、まず、３－アミノプロピルトリエトキシシラン等のアミノ置換オルガノアルコキシシランを適当な溶媒に溶解して得られた溶液に７０～８０℃程度の温度条件下で担体等を概ね２～３時間程度浸漬した後、これを取り出して水洗し、さらに、１００～１２０℃程度で約４～５時間加熱乾燥する。乾燥後、適当な溶媒中に浸し、カルボジイミド樹脂を加え３０～１７０℃程度の温度条件下で１２時間程度攪拌し、洗浄すればよい。また、上記３－アミノプロピルトリエトキシシランのアミノ基と窒素イペリット基の核酸結合基以外の官能基を適当な溶媒を用いて反応させ、ガラス製担体等の表面に窒素イペリット基を導入することもできる。 For example, for a method of introducing a carbodiimide group (resin) over the entire surface of a glass carrier or the like, first, a solution obtained by dissolving an amino-substituted organoalkoxysilane such as 3-aminopropyltriethoxysilane in a suitable solvent Then, after immersing the carrier or the like under a temperature of about 70 to 80 ° C. for about 2 to 3 hours, the carrier is taken out, washed with water, and further heated and dried at about 100 to 120 ° C. for about 4 to 5 hours. After drying, the substrate is immersed in a suitable solvent, carbodiimide resin is added, and the mixture is stirred for about 12 hours at a temperature of about 30 to 170 ° C. and washed. In addition, the amino group of the 3-aminopropyltriethoxysilane may be reacted with a functional group other than the nucleic acid binding group of the nitrogen iperit group using an appropriate solvent to introduce the nitrogen iperit group onto the surface of a glass carrier or the like. it can.
　また、ガラス製担体等にアミノ基以外場合や、担体等がガラス以外の材料からなる場合においても、上記担体の説明で挙げた各種材料表面に種々の官能基を導入することは、従来より一般的に行われていることであり、その方法も公知であるので、このような公知の方法を用いて担体等の表面への官能基の導入を行うことができる。 In addition, when a glass carrier or the like other than an amino group is used, or when the carrier or the like is made of a material other than glass, it is conventionally common to introduce various functional groups on the surfaces of various materials mentioned in the description of the carrier Since this method is known and its method is also known, it is possible to introduce a functional group onto the surface of a carrier or the like using such a known method.
　さらに、上記で挙げたプラスチック製担体等の中には、担体等表面に既に上記のような官能基を有するものも有り、この場合には担体等表面に官能基を導入することなしに、これをそのまま担体等の製造に用いることも可能である。また、このようなプラスチック製担体等であってもさらに官能基を導入して上記担体等の製造に用いることも可能である。 Furthermore, some of the plastic carriers mentioned above already have the above functional group on the surface of the carrier, etc., and in this case, without introducing the functional group on the surface of the carrier, etc. Can also be used for the production of carriers and the like. Further, even such a plastic carrier can be used for the production of the carrier by further introducing a functional group.
　また、上記担体等や固定化相の材料に公知の光重合開始剤を混合することもできる。光重合開始剤を混合することによって、紫外線等の電磁波の照射による核酸の固定化の際の反応性が向上し得る。 Also, a known photopolymerization initiator can be mixed with the above-mentioned carrier or the like and the material of the immobilized phase. By mixing a photopolymerization initiator, the reactivity at the time of immobilizing nucleic acid by irradiation with electromagnetic waves such as ultraviolet rays can be improved.
（識別情報）
　本明細書において、識別情報とは、識別対象を識別するための情報を意味する。識別情報は、１又は２以上のＤＮＡに含まれている。なお、ＤＮＡには、ＤＮＡと同様に塩基対を形成しうる他の化合物が含まれる。例えば、ＢＮＡ、ＰＮＡ等の公知の他の主鎖構造を有し、塩基を側鎖として有しうる化合物が包含される。さらには、識別情報は、これらのＤＮＡが担体上に付与されて構成するパターンに含まれていてもよい。パターンについては後述する。 (Identification information)
In this specification, the identification information means information for identifying an identification target. The identification information is included in one or more DNAs. DNA includes other compounds that can form base pairs in the same manner as DNA. For example, compounds having other known main chain structures such as BNA and PNA and having a base as a side chain are included. Furthermore, the identification information may be included in a pattern formed by providing these DNAs on a carrier. The pattern will be described later.
　本明細書において、ＤＮＡとは、アデニン（Ａ）、チミン（Ｔ）、シトシン（Ｃ）及びグアニン（Ｇ）をそれぞれ塩基として有するデオキシリボヌクレオチドの重合体のほか、他の主鎖構造を有し、Ａ、Ｔ、Ｃ及びＧを塩基として有する化合物（重合体）を意味する。 In this specification, DNA has a polymer structure of deoxyribonucleotide having adenine (A), thymine (T), cytosine (C), and guanine (G) as bases, as well as other main chain structures, It means a compound (polymer) having A, T, C and G as a base.
　識別情報は、一つの識別対象に関連付けえられており、また、識別情報は、１又は２以上のＤＮＡ中に備えられる１又は２以上の識別用塩基配列によって構成されている。したがって、一つの識別対象は、１のＤＮＡによって識別されるようになっていてもよいし、２以上のＤＮＡによって識別されるようになっていてもよい。 The identification information is associated with one identification object, and the identification information is composed of one or two or more identification base sequences provided in one or two or more DNAs. Therefore, one identification object may be identified by one DNA, or may be identified by two or more DNAs.
　１のＤＮＡは、１の識別用塩基配列を有している。識別用塩基配列は、１の識別対象に予め関連付けられている。この関連付けによって、識別対象が識別情報によって識別されることになる。識別用塩基配列は、識別対象に固有のものであってもよいし、そうでなくてもよい。 1 DNA has 1 identifying base sequence. The identification base sequence is associated in advance with one identification target. With this association, the identification target is identified by the identification information. The identification base sequence may or may not be unique to the identification target.
　識別対象に固有である場合とは、例えば、識別対象がゲノム上の特有の塩基配列や変異を有するとき、当該特有の塩基配列自体を識別用塩基配列とする場合が挙げられる。 The case where the identification target is unique is, for example, the case where the identification target has a unique base sequence or mutation on the genome, and the unique base sequence itself is used as the identification base sequence.
　識別用塩基配列は、天然由来であってもよいが、人工的に設計されたものであってもよい。人工的に設計された塩基配列を識別用配列として用いる場合とは、例えば、予め相互にミスハイブリダイゼーションがなく、そして、共通のハイブリダイゼーション条件で確実にハイブリダイズし検出できるような集合を構成する人工的塩基配列を用いることで、ハイブリダイゼーションによる、識別対象の検出を迅速かつ精度の高いものとすることができる。 The identification base sequence may be naturally derived or artificially designed. When an artificially designed base sequence is used as a sequence for identification, for example, a set is formed in which there is no mishybridization with each other in advance and can be reliably hybridized and detected under common hybridization conditions. By using an artificial base sequence, identification target detection by hybridization can be performed quickly and with high accuracy.
　こうした人工的塩基配列としては、例えば、配列番号１～１００で表される塩基配列及びその相補的な塩基配列が挙げられる。これらの塩基配列は全て同一塩基長であり、融解温度（Ｔｍ）が４０℃以上８０℃以下、好ましくは５０℃以上７０℃以下であって、同一条件でのハイブリダイズにおいて均質なハイブリダイズ結果が得ることができる。同時に使用する２種類以上の人工的塩基配列の融解温度は、できるだけ近いことが好ましい。 Examples of such artificial base sequences include base sequences represented by SEQ ID NOs: 1 to 100 and complementary base sequences thereof. All of these base sequences have the same base length and a melting temperature (Tm) of 40 ° C. or higher and 80 ° C. or lower, preferably 50 ° C. or higher and 70 ° C. or lower. Obtainable. It is preferable that the melting temperatures of two or more artificial base sequences used simultaneously are as close as possible.
　なお、融解温度は、ＧＣ％法、Wallace法、Current Protocols in Molecular Biologyに準拠した方法（秀潤社刊バイオ実験イラストレイテッド３ 本当に増えるＰＣＲｐ．２５に記載）等により算出したものを採用できるが、本発明における融解温度の範囲および塩基配列濃度の影響を加味できるNearest-Neighbor法によって算出されることが好ましい。Nearest-Neighbor法による融解温度は、例えば、Visual OMP（トミーデジタルバイオロジー株式会社製）とのソフトウエアや日本遺伝子研究所（http://www.ngrl.co.jp/）が提供するソフトウエア（OligoCalculator;http://www.ngrl.co.jp/tool/ngrl#tool.html）により容易に取得できる。 The melting temperature calculated by the GC% method, the Wallace method, the method based on Current Protocols in Molecular Molecular Biology (developed by Shujunsha's Bio Experiment Illustrated 3 practically increased PCR p.25), etc. can be adopted. The calculation is preferably performed by the Nearest-Neighbor method which can take into account the range of melting temperature and the base sequence concentration in the present invention. The melting temperature by the Nearest-Neighbor method is, for example, software with Visual OMP (Tomy Digital Biology Co., Ltd.) or software provided by the Japan Genetic Research Institute (http://www.ngrl.co.jp/). (OligoCalculator; http://www.ngrl.co.jp/tool/ngrl#tool.html).
　このような人工的塩基配列における識別配列は、正規直交化配列ともいい、たとえば乱数から得られた所定塩基長のＤＮＡ配列に対して連続一致長、Nearest-Neighbor法による融解温度予測、ハミング距離、二次構造予測の計算を行うことにより設計される。正規直交化配列は、核酸の塩基配列であって、その融解温度が均一であるもの、即ち融解温度が一定範囲内に揃うように設計された配列であって、核酸自身が分子内（intramolecular）で構造化して、相補的な配列とのハイブリッド形成を阻害することのない配列であり、尚且つこれに相補的な塩基配列以外とは安定したハイブリッドを形成しない塩基配列を意味する。１つの正規直交化配列群に含まれる配列は、所望の組み合わせ以外の配列間および自己配列内において反応が生じ難いか、または反応が生じない。また、正規直交化配列は、ＰＣＲにおいて増幅させると、たとえば上述のクロスハイブリダイズのような問題に影響されずに、当該正規直交化配列を有する核酸の初期量に応じた量の核酸が定量的に増幅される性質を有している。上記のような正規直交化配列は、H.Yoshida and A.Suyama,"Solution to 3-SAT by breadth first search",DIMACS Vl.54, 9-20(2000)および特願２００３－１０８１２６に詳細が記載されている。これらの文献に記載の方法を使用して正規直交化配列を設計することができる。 Such an identification sequence in an artificial base sequence is also referred to as an orthonormalized sequence, for example, a continuous match length for a DNA sequence of a predetermined base length obtained from a random number, melting temperature prediction by Nearest-Neighbor method, Hamming distance, Designed by performing secondary structure prediction calculations. The orthonormalized sequence is a base sequence of nucleic acid having a uniform melting temperature, that is, a sequence designed so that the melting temperature is within a certain range, and the nucleic acid itself is intramolecular. It means a base sequence that does not form a stable hybrid other than a base sequence that is structured in the above and does not inhibit hybridization with a complementary sequence. A sequence included in one orthonormalized sequence group hardly reacts between sequences other than the desired combination and within a self-sequence, or does not generate a reaction. Further, when the orthonormalized sequence is amplified in PCR, the amount of nucleic acid corresponding to the initial amount of the nucleic acid having the orthonormalized sequence is quantitatively affected without being affected by the problems such as the above-mentioned cross-hybridization. Has the property of being amplified. The orthonormalized array as described above is described in detail in H. Yoshida and A.Suyama, “Solution to 3-SAT by breadth first search”, DIMACS Vl.54, 9-20 (2000) and Japanese Patent Application No. 2003-108126. Are listed. Orthonormalized sequences can be designed using the methods described in these references.
　識別情報は、１又は２以上の識別用塩基配列によって構成されていてもよい。異なる識別用塩基配列をそれぞれ有する２以上のＤＮＡによって識別情報が構成されることで、より確度の高い識別が可能とすることができる。また、一定数の人工的塩基配列を用いても、それらを組み合わせることで、当該一定数よりも多い識別対象を識別することができる。 The identification information may be composed of one or more identification base sequences. Since the identification information is constituted by two or more DNAs each having a different base sequence for identification, identification with higher accuracy can be made possible. Further, even if a certain number of artificial base sequences are used, by combining them, it is possible to identify more identification objects than the certain number.
　識別用塩基配列は、後述するチミンリッチな塩基配列よりもチミン塩基比率が小さいことが好ましい。すなわち、識別用塩基配列は、Ｔ（チミン）の含有量（識別用塩基配列中のチミン塩基数／識別用塩基配列の全塩基数×１００）が５０％未満であることが好ましい。識別用塩基配列中にチミンが存在すると、固定化量及び／又はプローブとの反応量のほか、固定化後の紫外線照射等に対して劣化しやすくなるからである。所望の固定化量及び／又は反応量、劣化抑制が得られれば、チミン比率は特に限定しないが、好ましくは、４０％以下であり、より好ましくは３０％以下であり、さらに好ましくは２０％以下であり、一層好ましくは１０％以下であり、より一層好ましくは５％以下であり、さらに一層好ましくは、１％以下である。最も好ましくは、０％である。 The identification base sequence preferably has a thymine base ratio smaller than the thymine-rich base sequence described later. That is, the identification base sequence preferably has a T (thymine) content (the number of thymine bases in the identification base sequence / the total number of bases in the identification base sequence × 100) of less than 50%. This is because, if thymine is present in the identification base sequence, it tends to deteriorate with respect to the amount of immobilization and / or reaction with the probe, as well as irradiation with ultraviolet rays after immobilization. As long as the desired immobilization amount and / or reaction amount and deterioration suppression are obtained, the thymine ratio is not particularly limited, but is preferably 40% or less, more preferably 30% or less, and even more preferably 20% or less. More preferably, it is 10% or less, more preferably 5% or less, and still more preferably 1% or less. Most preferably, it is 0%.
　ＤＮＡは、一つの識別用塩基配列とともに、チミンリッチな塩基配列を有していてもよい。チミンは、後述するように、ＤＮＡの保持体への化学結合に関与していると考えられ、チミンリッチな塩基配列を有することで簡易にかつ強固に担体にＤＮＡを結合させることができる。 DNA may have a thymine rich base sequence together with one base sequence for identification. As will be described later, thymine is considered to be involved in chemical bonding of DNA to a carrier, and having a thymine-rich base sequence allows DNA to be easily and firmly bound to a carrier.
　チミンリッチな塩基配列とは、ＤＮＡの塩基配列において選択される２つのチミン（Ｔ）で特定される当該チミンを両端に含む一続きの領域が、他の領域よりもチミンの含有量が高いことを意味している。より好ましくは、Ｔ（チミン）の含有量（チミン塩基数／チミンリッチな塩基配列の全塩基数×１００）が５０％以上である。より好ましくは６０％以上であり、さらに好ましくは７０％以上であり、一層好ましくは８０％以上であり、より一層好ましくは９０％以上であり、さらに一層好ましくは９５％以上であり、さらにまた好ましくは９８％以上であり、最も好ましくは１００％である。したがって、チミンリッチな塩基配列は、一つのチミンが連続することなく間隔をおいて含まれていることが好ましい。チンリッチな塩基配列は、好ましくは、２以上のチミンが連続してチミンを含む。そして、より好ましくは連続するチミンのみからなる。チミンは、３又は４以上連続していることが好ましく、より好ましくは、５又は６以上連続していることが好ましく、さらに好ましくは、７又は８以上連続している。なお、チミンリッチな塩基配列がＤＮＡのほぼ全体又は全体に及んでいる場合が挙げられる。 A thymine-rich base sequence means that a continuous region containing the thymine specified by two thymines (T) selected in the base sequence of DNA has a higher thymine content than other regions. I mean. More preferably, the content of T (thymine) (the number of thymine bases / the total number of bases of a thymine-rich base sequence × 100) is 50% or more. More preferably, it is 60% or more, more preferably 70% or more, still more preferably 80% or more, still more preferably 90% or more, still more preferably 95% or more, and still more preferably. Is 98% or more, most preferably 100%. Therefore, it is preferable that the thymine-rich base sequence includes a single thymine with no interval. The tin-rich base sequence preferably contains two or more thymines in succession. More preferably, it consists only of continuous thymine. The thymine is preferably 3 or 4 or more, more preferably 5 or 6 or more, and still more preferably 7 or 8 or more. In addition, the case where the base sequence rich in thymine extends to almost the whole or the whole of DNA is mentioned.
　チミンリッチな塩基配列を備える場合、当該塩基配列は、ＤＮＡのいずれの部位にあってもよいが、識別用塩基配列の一部でないことが好ましい。すなわち、チミンリッチな塩基配列は、識別用塩基配列とは別個に備えられることが好ましい。また、チミンリッチな塩基配列は、５’末端及び／又は３'末端を含んで備えられていてもよいし、当該末端を含まないが、５’末端側及び／又は３’末端側に備えられていてもよい。また、中央部にあってもよい。好ましくは、５’末端及び／又は３'末端を含んで備えられる。 In the case of providing a thymine-rich base sequence, the base sequence may be in any part of the DNA, but is preferably not part of the identification base sequence. That is, the thymine-rich base sequence is preferably provided separately from the identification base sequence. In addition, the thymine-rich base sequence may be provided including the 5 ′ end and / or the 3 ′ end, or not including the end, but provided on the 5 ′ end side and / or the 3 ′ end side. May be. Moreover, you may exist in a center part. Preferably, it comprises a 5 'end and / or a 3' end.
　こうした１又は２以上のＤＮＡからなる識別情報が担体に保持されることで、ＤＮＡ中の識別用塩基配列が識別情報として機能することとなる。識別情報は、好ましくは、適当なパターン（二次元形態）を伴って担体上等に保持される。すなわち、パターンとしては、特に限定されないが、例えば、記号、文字、数字、バーコード、画像のほか、これらの組み合わせであってもよい。こうしたパターンは目視可能であることが好ましい。 Such identification information consisting of one or more DNAs is held on the carrier, whereby the identification base sequence in the DNA functions as identification information. The identification information is preferably held on a carrier or the like with an appropriate pattern (two-dimensional form). That is, the pattern is not particularly limited, but may be a combination of symbols, letters, numbers, barcodes, images, and the like. Such a pattern is preferably visible.
　図３に示すように、識別情報は、ＤＮＡを含む、複数のドット状体として構成されていてもよい。個々のドット状体は、１又は２以上のＤＮＡから構成されている。一つの識別対象を識別するために一つのＤＮＡを用いる場合には、ドット状体を構成するＤＮＡは、当該一つのＤＮＡで構成されている。また、一つの識別対象を識別するために、２以上のＤＮＡを用いる場合には、個々のドット状体は、一つのＤＮＡのみからなっていてもよいし、２以上のＤＮＡからなっていてもよいし、この両者のドット状体を含んでいてもよい。 As shown in FIG. 3, the identification information may be configured as a plurality of dot-like bodies containing DNA. Each dot-like body is composed of one or more DNAs. When one DNA is used to identify one identification target, the DNA constituting the dot-like body is composed of the one DNA. Further, when two or more DNAs are used to identify one identification target, each dot-like body may consist of only one DNA or may consist of two or more DNAs. Alternatively, both dot-like bodies may be included.
　図３に示すように、複数のドット状体が集合して目視可能なパターン（文字、記号、図形、これらの組み合わせ等）を構成していてもよい。ドット状体でパターンを構成することで、必要なＤＮＡ量を低減できるとともに、ドット内のＤＮＡ濃度を高めることができるため、検出感度を高め、簡易かつ迅速に確度の高い検出が可能となる。一つのパターンは、通常、一つの識別対象の特定のために構成される。当該一つのパターンは、１又は２以上のＤＮＡからなるドット状体で構成されうる。なお、一つの識別対象に対して、一つのパターンを備えていてもよいし、２種以上を備えていてもよい。 As shown in FIG. 3, a plurality of dot-like bodies may be aggregated to form a visible pattern (characters, symbols, figures, combinations thereof, etc.). By configuring the pattern with the dot-like body, the required amount of DNA can be reduced and the DNA concentration in the dot can be increased, so that the detection sensitivity is increased, and detection with high accuracy can be performed easily and quickly. One pattern is usually configured to specify one identification target. The one pattern can be composed of a dot-like body composed of one or two or more DNAs. One identification target may be provided with one pattern, or two or more types may be provided.
　２種以上のパターン（図３においては、○と×である。）を、同時に一つの担体又は識別対象上に備えることもできる。この場合、２種以上のパターンをコンパクトに保持するためには、２種以上のパターンの少なくとも一部が重なるように配置されることが好ましい。重なって配置される箇所は、それぞれのパターンに属する個々のドット状体が重ならないようにして配置されていてもよいし、重複して配置されていてもよい。また、パターンが重なる箇所は、いずれか一方のパターンに属するドット状体のみで構成されていてもよい。さらには、パターンが重なる箇所は、双方のパターンに属するドット状体がいずれも配置されていないとしてもよい。こうした状態であっても、パターン自体の存在は確認できるからである。 Two or more types of patterns (in FIG. 3, ◯ and ×) can be simultaneously provided on one carrier or identification target. In this case, in order to hold two or more types of patterns in a compact manner, it is preferable that at least a part of the two or more types of patterns overlap each other. The overlapping positions may be arranged so that the individual dot-like bodies belonging to the respective patterns do not overlap, or may be arranged overlappingly. Moreover, the place where a pattern overlaps may be comprised only by the dot-like body which belongs to any one pattern. Further, the dot-like bodies belonging to both patterns may not be arranged at the places where the patterns overlap. This is because the existence of the pattern itself can be confirmed even in such a state.
　なお、２種以上のパターンは、一つの識別対象を識別するために備えられていてもよいし、それぞれ個別の識別対象を識別するために備えられていてもよい。いずれにしても、前記パターン間で共通しない前記識別用塩基配列を有する１又は２以上のＤＮＡからなるドット状体で構成されていることが好ましい。 Note that two or more types of patterns may be provided to identify one identification target, or may be provided to identify individual identification targets. In any case, it is preferable to be composed of a dot-like body made of one or more DNAs having the identification base sequence that is not common among the patterns.
　なお、担体上等にＤＮＡを含むドット状体を形成する技術は、たとえば、公知のＤＮＡマイクロアレイなどの技術を適用することができる。すなわち、ＤＮＡを分散又は溶解した液体を、インクジェット方式あるいはピン方式など、公知の手法で担体上にスポットすればよい。 In addition, as a technique for forming a dot-like body containing DNA on a carrier or the like, for example, a technique such as a known DNA microarray can be applied. That is, a liquid in which DNA is dispersed or dissolved may be spotted on a carrier by a known method such as an ink jet method or a pin method.
　こうして形成される、識別対象に関連付けられる１又は２以上のＤＮＡが固定化された担体上の領域は、情報保持領域を構成することができる。識別対象に対して１つでもよいし２つ以上でもよい。また、一つの担体上に１つであってもよいし２つ以上であってもよい。 The area on the carrier on which one or two or more DNAs associated with the identification target are thus formed can constitute an information holding area. There may be one or two or more for the identification target. Moreover, one may be sufficient on one support | carrier and two or more may be sufficient.
　本保持体は、１又は２以上の情報保持領域を包囲する障壁を担体表面に備えている。障壁は、担体表面から所定の高さをもって備えられている。また、障壁は、情報保持領域の周囲を連続的に包囲する連続する障壁であってもよいし、情報保持領域の周囲を断続的（不連続的に）に包囲する複数個の障壁が配列されたものであってもよい。 This holder has a barrier on the surface of the carrier that surrounds one or more information holding areas. The barrier is provided with a predetermined height from the carrier surface. The barrier may be a continuous barrier that continuously surrounds the information holding area, or a plurality of barriers that intermittently (discontinuously) surround the information holding area are arranged. It may be.
　障壁は、情報保持領域（固相担体の表面）から１０μｍ以上、好ましくは５０μｍ以上、より好ましくは１００μｍ以上の高さを有する。少なくとも１０μｍであると、物理的な障害から識別情報を保護することができる。また、障壁は、情報保持領域（固相担体の表面）から５００μｍ以下、より好ましくは３００μｍ以下、さらに好ましくは２００μｍ以下の高さを有する。高くても５００μｍ以下であれば、識別情報保持体の取り扱い性等に影響を及ぼさないからである。 The barrier has a height of 10 μm or more, preferably 50 μm or more, more preferably 100 μm or more from the information holding region (the surface of the solid phase carrier). When the thickness is at least 10 μm, the identification information can be protected from physical failure. Further, the barrier has a height of 500 μm or less, more preferably 300 μm or less, and still more preferably 200 μm or less from the information holding region (the surface of the solid phase carrier). This is because if the height is 500 μm or less, the handling property of the identification information holding body is not affected.
　障壁は、情報保持領域を目視で識別可能に形成されていることが好ましい。こうすることで、情報保持領域を容易に視認でき、プローブ溶液を供給しやすく、また、ハイブリダイゼーション結果の判定も容易になるからである。この場合、障壁の厚みや障壁の内部や外部に着色等するなどする手法を採用できる。 The barrier is preferably formed so that the information holding area can be visually identified. This is because the information holding area can be easily visually recognized, the probe solution can be easily supplied, and the determination of the hybridization result is facilitated. In this case, it is possible to adopt a method of coloring the thickness of the barrier or the inside or outside of the barrier.
　障壁は、固相担体の一部であってもよいし、固相担体とは別個に、固相担体上に一体化された障壁部材であってもよい。障壁が障壁部材からなる場合、障壁部材は、粘着剤又は接着剤で固相担体に一体化されていてもよい。 The barrier may be a part of the solid phase carrier, or may be a barrier member integrated on the solid phase carrier separately from the solid phase carrier. When the barrier is composed of a barrier member, the barrier member may be integrated with the solid phase carrier with an adhesive or an adhesive.
　障壁は、疎水性表面を有していることが好ましい。疎水性表面を有していることで、障壁内部、すなわち、情報保持領域内に供給されたプローブ溶液を外部に流出しないように安定して保持することができる。疎水性表面は、例えば、図１及び図２に示すように、障壁が、固相担体側から粘着剤層又は接着剤層、疎水性層の順で備える障壁部材としてときの疎水性層により形成できる。 The barrier preferably has a hydrophobic surface. By having the hydrophobic surface, the probe solution supplied in the barrier, that is, in the information holding region, can be stably held so as not to flow out. For example, as shown in FIGS. 1 and 2, the hydrophobic surface is formed by a hydrophobic layer when the barrier is a barrier member including an adhesive layer or an adhesive layer and a hydrophobic layer in this order from the solid phase carrier side. it can.
　障壁は、粘着剤又は接着剤を含む表層部を備えることができる。こうした表層部を備えることで、表層部を介して他の部材を固定化できる。 The barrier can be provided with a surface layer part including an adhesive or an adhesive. By providing such a surface layer portion, other members can be fixed via the surface layer portion.
　障壁は、表層部を障壁の最上層（最も固相担体からはなれた高い部位）に備えることができる。こうした表層部を備えるとき、表層部を介して識別対象に本保持体を固定化可能となる。また、こうした表層部を備えるとき、表層部を介して情報保持領域の上方を覆うカバーを固定化させて備えることができる。カバーは、効果的に情報保持領域を障害から保護する。すなわち、使用時までの間、ＤＮＡの摩擦による脱離や紫外線照射による分解を抑制することができる。カバーは、フィルム状体、シート状体であってもよい。また、カバーが、障壁に対して付与されることで識別情報領域上にキャビティを形成するものであってもよい。さらに、このようなキャビティを形成するカバーの場合、当該キャビティをハイブリダイゼーションのためのキャビティとして用いることも可能となる。この場合には、キャビティにハイブリダイゼーションのためのプローブ溶液などを注入可能な開口を備えることができる。あるいは、適時に当該開口を形成可能に設けることができる。例えば、外部から押圧により、開口が形成されるような脆弱部を設けてあってもよい。カバーを障壁から容易に剥離できるように設けられていてもよい。 The barrier can be provided with the surface layer part on the uppermost layer of the barrier (the highest part far from the solid support). When such a surface layer part is provided, it becomes possible to fix this holding body to the identification target via the surface layer part. Moreover, when providing such a surface layer part, the cover which covers the upper part of an information holding area | region through a surface layer part can be fixed and provided. The cover effectively protects the information holding area from failure. That is, it is possible to suppress detachment due to friction of DNA and decomposition due to ultraviolet irradiation until the time of use. The cover may be a film or sheet. Further, the cover may be formed on the identification information area by being applied to the barrier. Further, in the case of a cover forming such a cavity, the cavity can be used as a cavity for hybridization. In this case, the cavity can be provided with an opening through which a probe solution for hybridization can be injected. Alternatively, the opening can be provided in a timely manner. For example, you may provide the weak part which an opening is formed by the press from the outside. The cover may be provided so that it can be easily peeled off from the barrier.
　こうした障壁は、例えば、図４に示すように、固相担体側から、第１の粘着性層、疎水性（撥水性）層及び第２粘着性層を備え、固相担体上の１又は２以上の情報保持領域を包囲し区画するパターンを備える積層体である障壁部材で構成されていてもよい。第１の粘着剤層は、第２の粘着剤よりも強い粘着性を有していることで、障壁部材を固相担体に強固に固定できる一方、第２の粘着剤層を介して、カバーを剥離容易に固定することができる。この場合、本保持体の固相担体の情報保持領域でない面に粘着剤層を形成して、当該粘着剤層を介して本保持体を識別致傷に固定化できる。 For example, as shown in FIG. 4, such a barrier includes a first adhesive layer, a hydrophobic (water-repellent) layer, and a second adhesive layer from the solid support side, and 1 or 2 on the solid support. You may be comprised with the barrier member which is a laminated body provided with the pattern which encloses and partitions the above information holding area | region. The first pressure-sensitive adhesive layer has stronger adhesiveness than the second pressure-sensitive adhesive, so that the barrier member can be firmly fixed to the solid phase carrier, while the cover is interposed via the second pressure-sensitive adhesive layer. Can be fixed easily. In this case, a pressure-sensitive adhesive layer can be formed on the surface of the solid support that is not the information holding area of the solid support, and the permanent support can be fixed to the identification flaw through the pressure-sensitive adhesive layer.
　こうした、障壁部材は、また、例えば、図５に示すように、固相担体側から、第１の粘着剤層、第１の疎水性層、第２の粘着剤層、第２の疎水性層及び第３の粘着剤層を備えることができる。第３の粘着剤層には、剥離層を備えることができる。このとき、第１の粘着剤層は第２の粘着剤層よりも強い粘着性であることが好ましく、また、第３の粘着剤層も第２の粘着剤層よりも強い粘着性であることが好ましい。剥離層は、情報保持領域を保護するカバーとしても機能する。剥離層を剥離して露出される第３の粘着剤層を識別対象に密着させ固定することで、第３の粘着剤層は、保持体への固定化層として機能することができる。 Such a barrier member also includes, for example, a first pressure-sensitive adhesive layer, a first hydrophobic layer, a second pressure-sensitive adhesive layer, and a second hydrophobic layer as shown in FIG. And a third pressure-sensitive adhesive layer. The third pressure-sensitive adhesive layer can be provided with a release layer. At this time, it is preferable that the first pressure-sensitive adhesive layer is stronger than the second pressure-sensitive adhesive layer, and the third pressure-sensitive adhesive layer is also stronger than the second pressure-sensitive adhesive layer. Is preferred. The release layer also functions as a cover that protects the information holding area. The third pressure-sensitive adhesive layer can function as an immobilization layer to the holding body by sticking and fixing the third pressure-sensitive adhesive layer exposed by peeling the release layer to the identification target.
　なお、固相担体上の情報保持領域を包囲する障壁を設けることは、識別情報領域のための担体表面がその周囲の担体表面よりも低く形成されているような状態であってもよい。例えば、担体が、底面を有する凹部を備える場合、当該凹部に識別情報を保持する情報保持領域としてもよい。また、当該凹部は、ハイブリダイゼーションのためのキャビティとしても用いることができる。 It should be noted that providing the barrier surrounding the information holding region on the solid phase carrier may be in a state where the carrier surface for the identification information region is formed lower than the surrounding carrier surface. For example, when the carrier includes a recess having a bottom surface, it may be an information holding area that holds identification information in the recess. The concave portion can also be used as a cavity for hybridization.
　本保持体において、障壁等によってハイブリダイゼーションのためのキャビティを備えることで、後述する識別対象の識別において、容易に接触工程を実施できる。 In this holder, a cavity for hybridization is provided by a barrier or the like, so that the contact process can be easily performed in the identification of the identification target described later.
　情報保持領域内には、図３に示すように、パターンを２種以上備えることができ、当該２種以上のパターンは、パターン間で共通しない識別用塩基配列を有する１又は２以上のＤＮＡからなるドット状体で構成されることができる。さらに、２種以上のパターンは、少なくともその一部が重なって配置されていてもよい。 As shown in FIG. 3, two or more patterns can be provided in the information holding region, and the two or more patterns are obtained from one or more DNAs having a base sequence for identification that is not common between the patterns. It can be composed of a dot-like body. Further, the two or more types of patterns may be arranged so that at least a part thereof overlaps.
　本保持体が、固相担体上に識別情報を備えて、別途、識別対象に固定化されるべきものである場合、担体には、識別対象に対する固定化手段を備えていることが好ましい。例えば、当該固定化手段は、接着層、粘着層などが挙げられる。こうした固定化手段は、本保持体がフィルム状、シート状の場合に有効である。また、本保持体がタグ形態を有する場合、固定化手段としては、接着層や粘着層のほか、係止、嵌合等による固定化手段を備えることができる。固定化手段は、識別対象から本保持体を分離可能なものであることが好ましい。分離可能とすることで、後述する識別対象の識別を容易に実施できる。 When the holder is provided with identification information on a solid phase carrier and should be separately immobilized on an identification target, the carrier preferably includes an immobilization means for the identification target. For example, examples of the immobilization means include an adhesive layer and an adhesive layer. Such immobilization means is effective when the holder is in the form of a film or sheet. Moreover, when this holding body has a tag form, as an immobilization means, the immobilization means by latching, fitting, etc. can be provided in addition to the adhesive layer and the adhesive layer. The immobilization means is preferably capable of separating the holder from the identification target. By making it separable, the identification target to be described later can be easily identified.
（構造体）
　本発明の構造体は、本保持体を複数個一体に備えることができる。こうした構造体は、複数個の本保持体を一挙に供給できるため都合がよい。複数個の本保持体は、所定パターンで配列して備えることが好ましい。例えば、図１及び図２に示すようなパターンで備えられる。こうしたパターンであると、個々に分離するのに適している。複数個の本保持体は、連続する又は単一の固相担体を複数個の本保持体に共通する固相担体として備えることが好ましい。複数個を一括して製造し取り扱うのに都合がよいからである。 (Structure)
The structure of the present invention can be provided with a plurality of the holding bodies integrally. Such a structure is convenient because a plurality of the holders can be supplied at once. It is preferable that the plurality of holders are arranged in a predetermined pattern. For example, it is provided in a pattern as shown in FIGS. Such a pattern is suitable for individual separation. The plurality of holders are preferably provided with a continuous or single solid support as a solid support common to the plurality of holders. This is because it is convenient to manufacture and handle a plurality.
　本構造体は、複数個の本保持体を分離可能に形成されていることが好ましい。個々の本保持体に容易に分離できるからである。構造体は、複数個の本保持体を分離可能な脆弱部を有していることが好ましい。脆弱部を有していることで、小さい力で容易に本保持体を分離できる。脆弱部は、複数個の本保持体を区画するように形成されていることが好ましい。 It is preferable that the structure is formed so that a plurality of book holders can be separated. This is because it can be easily separated into individual book holders. It is preferable that the structure has a fragile portion capable of separating a plurality of the present holding bodies. By having the fragile portion, the holder can be easily separated with a small force. The fragile portion is preferably formed so as to partition a plurality of book holders.
　また、構造体において、隣接する本保持体間の厚みは、２０μｍ～１５００μｍの厚み、好ましくは４０μｍ～１０００μｍの厚み、さらに好ましくは５０～６００μｍの厚みを有する。 Further, in the structure, the thickness between the adjacent main holders is 20 μm to 1500 μm, preferably 40 μm to 1000 μm, more preferably 50 to 600 μm.
　複数個の本保持体は、それぞれが備える障壁によって区画され、障壁を指標として相互に分離可能とすることができる。また、複数個の本保持体は、連続する又は単一の障壁を複数個の本保持体に共通する障壁として備えることができる。 The plurality of holders can be separated from each other by using barriers provided by the respective holders, and the barriers can be used as an index. In addition, the plurality of book holders can include a continuous or single barrier as a barrier common to the plurality of book holders.
　図４及び図５に示すように、複数個の本保持体は、接着剤又は粘着剤を含有する連続する又は単一の固定化層を構造体の表面又は裏面に備えることができる。また、図４及び図５に示すように、こうした固定化層は、複数個の本保持体の障壁（その最上部）に対して備えていてもよいし固相担体の前記情報保持領域が形成される面とは反対側の面に備えることもできる。 As shown in FIGS. 4 and 5, the plurality of holders can be provided with a continuous or single immobilization layer containing an adhesive or a pressure-sensitive adhesive on the front or back surface of the structure. Further, as shown in FIGS. 4 and 5, such an immobilization layer may be provided with respect to a plurality of barriers (the uppermost part) of the book holder, and the information holding region of the solid phase carrier is formed. It can also be provided on the surface opposite to the surface to be applied.
（本保持体の製造方法）
　本保持体の製造方法は、固相担体上に、１又は２以上のＤＮＡを固定して１又は２以上の情報保持領域を形成する工程と、１又は２以上の情報保持領域を包囲する障壁となる障壁部材を前記固相担体上に固定する工程と、を備えることができる。本製造方法は、本保持体を製造する方法であるが、同時に、複数個の本保持体を備える本構造体を製造する方法としても実施することができる。 (Method for manufacturing the holder)
The method for producing the holder includes a step of fixing one or more DNAs on a solid phase carrier to form one or more information holding regions, and a barrier surrounding the one or more information holding regions A barrier member to be fixed on the solid phase carrier. Although this manufacturing method is a method for manufacturing the present holding body, it can also be carried out as a method for manufacturing the present structure including a plurality of the present holding bodies at the same time.
　障壁部材は、図１、図２、図４及び図５に示すように、１又は２以上の情報保持領域に対応する開口パターン（平面視）を有することができる。また、図１、図２，図４及び図５に示すように、障壁部材は、接着剤又は粘着剤を備えて固相担体に固定化するための固定化層と、この固定化層に積層される障壁層とを備えることができる。障壁部材は、障壁層に積層される、粘着剤又は接着剤を含有するさらに別の固定化層を備えることもできる。固相担体は、固相担体の情報保持領域を形成する面と反対側の面には、粘着剤又は接着剤を含有する固定化層を備えることもできる。 The barrier member can have an opening pattern (plan view) corresponding to one or more information holding regions as shown in FIGS. 1, 2, 4 and 5. In addition, as shown in FIGS. 1, 2, 4 and 5, the barrier member is provided with an adhesive layer or an adhesive, and is fixed to the solid phase carrier, and is laminated on the fixed layer. Barrier layer. The barrier member can also comprise a further immobilization layer containing an adhesive or adhesive that is laminated to the barrier layer. The solid phase carrier may be provided with an immobilization layer containing an adhesive or an adhesive on the surface opposite to the surface forming the information holding region of the solid phase carrier.
　さらに、障壁部材を介して、１又は２以上の前記情報保持領域の上方を覆うカバーを付与する工程を備えることもできる。 Furthermore, a step of providing a cover for covering the upper part of the one or more information holding regions via the barrier member may be provided.
　情報保持領域の形成工程では、固相担体の表面に、識別用塩基配列のほか、チミンリッチな塩基配列を有していてもよいＤＮＡを供給して両者を接触させて固定化することができる。固定化では、例えば、両者の接触反応において固定化されるＤＮＡの活性が維持されるように、通常、ＤＮＡは水またはバッファー中に含まれる形で供給される。固定化の手法は特に限定されないで、公知の方法を採用することができ、ＤＮＡとカルボジイミド樹脂、窒素イペリット、ポリアミノ酸、ニトロセルロール等の公知の化合物を化学的に結合又は物理的に結合した状態で、これら混合物と担体を接触させ固定させてもよく、また、このときの固定は後述するように電磁波を照射して行ってもよい。 In the information holding region forming step, DNA that may have a thymine-rich base sequence in addition to the identification base sequence can be supplied to the surface of the solid phase carrier, and both can be contacted and immobilized. In the immobilization, for example, the DNA is usually supplied in a form contained in water or a buffer so that the activity of the DNA immobilized in both contact reactions is maintained. The immobilization method is not particularly limited, and a known method can be adopted. A known compound such as DNA and a carbodiimide resin, nitrogen ipellite, polyamino acid, nitrocellulose, or the like is chemically or physically bound. In this state, the mixture and the carrier may be brought into contact with each other and fixed, and the fixation at this time may be performed by irradiating an electromagnetic wave as described later.
　また、識別情報たるＤＮＡがチミンリッチな塩基配列を有している場合には、ＤＮＡと固相担体との接触中又は接触後に電磁波を照射することによって固定化することもできる。また、水またはバッファー中に公知の光重合開始剤を混合することもできる。固定化に用いる電磁波としては、２２０ｎｍ～３８０ｎｍの波長の紫外線が好ましい。なかでも、２８０nmの波長を含む紫外線を照射することが好ましい。具体的には、波長280nmを含むブロードな波形を有する紫外線であっても良い。照射量は、１０～５０００ｍＪ/cm2が好ましく、１００～２０００ｍＪ/cm2がさらに好ましい。好ましくは、200mJ/cm2以上である。また、ＤＮＡ溶液をスポット後紫外線照射前に乾燥させることができる。なお、核酸溶液の乾燥方法としては、自然に乾燥させてもよく、加熱して乾燥させてもよい。加熱する場合の温度は、通常３０～１００℃、好ましくは３５～４５℃である。 Moreover, when DNA which is identification information has a thymine-rich base sequence, it can be immobilized by irradiating an electromagnetic wave during or after contact between the DNA and the solid phase carrier. Moreover, a well-known photoinitiator can also be mixed in water or a buffer. The electromagnetic wave used for immobilization is preferably ultraviolet light having a wavelength of 220 nm to 380 nm. Among these, it is preferable to irradiate ultraviolet rays including a wavelength of 280 nm. Specifically, it may be an ultraviolet ray having a broad waveform including a wavelength of 280 nm. Irradiation dose is preferably 10 ~ 5000mJ / cm 2, more preferably 100 ~ 2000mJ / cm 2. Preferably, it is 200 mJ / cm 2 or more. Alternatively, the DNA solution can be dried after spotting and before ultraviolet irradiation. In addition, as a drying method of a nucleic acid solution, you may dry naturally and you may heat and dry. The heating temperature is usually 30 to 100 ° C., preferably 35 to 45 ° C.
　本明細書において微量のＤＮＡを、通常は、ＤＮＡを含有する水またはバッファーを、担体等に点状に供給するには、公知の手法を特に限定することなく用いることができる。 In the present specification, a known technique can be used without any particular limitation to supply a small amount of DNA, usually, water or buffer containing DNA in a dotted manner to a carrier or the like.
　本製造方法は、さらに、ブロッキング工程を備えていてもよい。本保持体は、さらに、必要に応じて過剰量のウシ血清アルブミン（ＢＳＡ）、カゼイン、サケ***ＤＮＡ等を担体等に接触させ、ブロッキングを施すこともできる。 This manufacturing method may further include a blocking step. The holding body can be further blocked by bringing an excess amount of bovine serum albumin (BSA), casein, salmon sperm DNA or the like into contact with a carrier or the like as necessary.
（識別可能な物品の製造方法）
　本発明の識別可能な物品の製造方法は、本保持体を準備する工程と、本保持体を識別対象とする物品に固定化する工程と、を備えることができる。固定化工程における、本保持体の物品への固定化手段は特に限定されない。既に説明したように、接着剤や粘着剤等の手段で固定されてもよいし、タグのように係止手段により固定化されるものであってもよい。固定化される形態も特に限定されない。情報保持領域が物品側に指向するように又は識別情報が物品の外方を指向するように、識別情報保持体を物品に固定化してもよい。 (Method for manufacturing identifiable article)
The method for producing an identifiable article according to the present invention can include a step of preparing the holding body and a step of fixing the holding body to an article to be identified. The means for fixing the holding body to the article in the fixing step is not particularly limited. As already described, it may be fixed by means such as an adhesive or a pressure-sensitive adhesive, or may be fixed by locking means like a tag. The form to be fixed is not particularly limited. The identification information holding body may be fixed to the article such that the information holding area is directed to the article side or the identification information is directed to the outside of the article.
（識別方法）
　次に、本保持体を用いて識別対象を識別する方法について説明する。識別対象に付与された本保持体の情報保持領域に対して、識別用塩基配列と相補的な塩基配列を有する１又は２以上のプローブを接触させる工程と、識別用塩基配列とプローブとのハイブリダイゼーション産物を検出する工程と、を備えることができる。本識別方法によれば、本保持体において良好な識別能が確保されているため、良好な確度で識別対象を識別できる。そして、当該識別工程は、別途、識別対象の管理、監視、認証、同定、追跡等の方法にそのまま適用することができる。 (Identification method)
Next, a method for identifying an identification target using the holder will be described. A step of bringing one or more probes having a base sequence complementary to the identification base sequence into contact with the information holding region of the holder provided to the identification target; a high level of the identification base sequence and the probe; Detecting a hybridization product. According to this identification method, since a good discrimination capability is ensured in the holder, the identification target can be identified with good accuracy. The identification step can be applied directly to methods such as management, monitoring, authentication, identification, and tracking of identification targets.
（接触工程）
　接触工程は、識別対象に付与された本保持体に上の識別情報に対して、この識別情報に含まれる識別用塩基配列と相補的な塩基配列を有する１又は２以上のプローブを接触させる工程とすることができる。接触工程において、識別対象が予め関連付けられた識別用塩基配列を備えているとき、プローブとハイブリダイズ産物を形成することができる。すなわち、本接触工程は、ＤＮＡ中の識別用塩基配列とプローブとのハイブリダイゼーションを行わせることを目的としている。プローブは、識別対象に予め付与した識別用塩基配列に対応するプローブだけを供給してもよいし、ユニバーサルに多くの識別対象に適用可能に組成したプローブを供給してもよい。 (Contact process)
The contact step is a step of bringing one or more probes having a base sequence complementary to the identification base sequence included in the identification information into contact with the identification information on the holder attached to the identification target It can be. In the contacting step, when the identification target has an identification base sequence associated in advance, a hybridization product with the probe can be formed. That is, the purpose of this contact step is to allow hybridization between the identification base sequence in the DNA and the probe. As the probe, only a probe corresponding to the identification base sequence previously assigned to the identification target may be supplied, or a probe that is universally applicable to many identification targets may be supplied.
　プローブは、識別用塩基配列とハイブリダイズ産物を形成できる程度に相補的であればよいが、好ましくは、完全に相補的である。識別用塩基配列が、配列番号１～１００で表される塩基配列及びその相補的な塩基配列から選択されるとき、プローブは、配列番号１～１００で表される塩基配列及びその相補的な塩基配列から選択される。 The probe may be complementary to the identification base sequence to the extent that it can form a hybridized product, but is preferably completely complementary. When the discriminating base sequence is selected from the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary base sequences, the probe has the base sequence represented by SEQ ID NOs: 1 to 100 and its complementary bases Selected from the sequence.
　プローブは、その後の検出のために、標識されていることが好ましい。標識としては従来公知のものを適宜選択して用いることができる。それ自体励起されると蛍光シグナルを発する蛍光物質などの各種色素であってもよいし、さらに酵素反応や抗原抗体反応により第２成分と組み合わせて各種シグナルを発する物質であってもよい。典型的には、Ｃｙ３、Alexa555、Ｃｙ５、Alexa647等の蛍光標識物質を用いることができる。また、ビオチンとストレプトアビイジンＨＰＲとを組み合わせて基質による処理等による発色による検出を用いてもよい。 The probe is preferably labeled for subsequent detection. As the label, a conventionally known one can be appropriately selected and used. It may be various dyes such as a fluorescent substance that emits a fluorescent signal when excited by itself, or may be a substance that emits various signals in combination with the second component by an enzyme reaction or an antigen-antibody reaction. Typically, a fluorescent labeling substance such as Cy3, Alexa555, Cy5, Alexa647 can be used. Alternatively, biotin and streptavidin HPR may be combined for detection by color development such as by treatment with a substrate.
　プローブは、識別対象に固有のプローブを含んでいてもよいが、ユニバーサルに多くの識別対象に適用可能に組成したプローブセットであってもよい。配列番号１～１００で表される塩基配列及びその相補的塩基配列を有するプローブであれば、相互にミスハイブリダイゼーションがないため、こうした塩基配列を識別用配列として用いた本保持体であればユニバーサルに適用可能なセットとすることができる。 The probe may include a probe specific to the identification target, but may be a probe set that is universally configured to be applicable to many identification targets. Since probes having the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary base sequences have no mishybridization with each other, universal holders using these base sequences as identification sequences are universal. It can be a set applicable to.
　接触工程の条件は特に限定しない。通常のハイブリダイズ媒体を用いることができる。適度な温度に設定することができる。例えば、配列番号１～１００で表される塩基配列又は当該塩基配列に相補的な配列などの高度に選択的な識別用塩基配列を利用する場合には、配列番号１～１００で表される塩基配列又は当該塩基配列に相補的な塩基配列を利用する場合には、３０℃以上８０℃以下、好ましくは３０℃以上４０℃以下の温度を採用できる。より好ましくは、３５℃以上４０℃以下である。また、時間は、1秒以上１時間以下であることが好ましい。より好ましくは、1秒以上５分以下であり、さらに好ましくは１秒以上１分以下である。好ましくは、３０℃以上４０℃以下、より好ましくは、３５℃以上４０℃以下で、かつ、好ましくは１秒以上５分以下、より好ましくは、１秒以上１分以下の接触工程とすることができる。 The conditions for the contact process are not particularly limited. A normal hybridization medium can be used. An appropriate temperature can be set. For example, when a highly selective identification base sequence such as the base sequence represented by SEQ ID NO: 1 to 100 or a sequence complementary to the base sequence is used, the base represented by SEQ ID NO: 1 to 100 When a sequence or a base sequence complementary to the base sequence is used, a temperature of 30 ° C. or higher and 80 ° C. or lower, preferably 30 ° C. or higher and 40 ° C. or lower can be adopted. More preferably, it is 35 degreeC or more and 40 degrees C or less. Moreover, it is preferable that time is 1 second or more and 1 hour or less. More preferably, they are 1 second or more and 5 minutes or less, More preferably, they are 1 second or more and 1 minute or less. Preferably, the contact step is 30 ° C. or higher and 40 ° C. or lower, more preferably 35 ° C. or higher and 40 ° C. or lower, and preferably 1 second or longer and 5 minutes or shorter, more preferably 1 second or longer and 1 minute or shorter. it can.
　なお、接触工程に際しては、本保持体を識別対象から分離してもよい。分離可能な固定化手段により本保持体が識別対象に固定化されている場合には、識別対象とから分離した状態で接触工程を実施できる。また、可能な場合には、識別対象上で接触工程を実施してもよい。例えば、本保持体上に、ハイブリダイゼーション用のキャビティを備える場合が挙げられる。 In the contact process, the holder may be separated from the identification target. When the holder is fixed to the identification target by the separable fixing means, the contact step can be performed in a state separated from the identification target. Further, if possible, a contact process may be performed on the identification target. For example, there is a case where a hybridization cavity is provided on the holder.
　後段の検出工程に先立って、過剰のプローブを洗浄除去することが好ましい。識別情報たるＤＮＡは担体等に固定化されているため、余分なプローブを洗浄しても、ハイブリダイゼーション産物は、担体等上に保持される。 It is preferable to wash away excess probe prior to the subsequent detection step. Since DNA as identification information is immobilized on a carrier or the like, the hybridization product is retained on the carrier or the like even if an excess probe is washed.
（検出工程）
　検出工程は、前記識別情報中の識別用塩基配列と前記プローブとのハイブリダイゼーション産物を検出する工程とすることができる。こうしたハイブリダイゼーション産物を検出することにより、識別対象を識別できる。検出工程におけるハイブリダイズ産物の検出方法は特に限定されない。連結分子が標識を有する場合には、その標識を検出すればよい。また、電気的な検出方法などにより、二重鎖を検出してもよい。 (Detection process)
The detection step may be a step of detecting a hybridization product between the identification base sequence in the identification information and the probe. By detecting such a hybridization product, the identification target can be identified. The method for detecting the hybridized product in the detection step is not particularly limited. When the linking molecule has a label, the label may be detected. Alternatively, the double strand may be detected by an electrical detection method or the like.
　ハイブリダイズ産物が検出されたとき、識別対象が同定される。すなわち、識別対象の同一性が判定されるため、改ざんされていないこと、置換されていないこと、損なわれていないことなどが判定できる。また、ハイブリダイズ産物が検出されないとき、識別対象の不存在ないし識別対象の非同一と判定される。すなわち、識別対象は失われたか、改ざんされたか、損なわれたかなどと判定される。 When the hybridized product is detected, the identification target is identified. That is, since the identity of the identification target is determined, it can be determined that it has not been tampered with, has not been replaced, or has not been damaged. Further, when the hybridized product is not detected, it is determined that the identification target does not exist or the identification target is not identical. That is, it is determined whether the identification target is lost, altered, or damaged.
　識別情報として、１又は２以上のＤＮＡが目視可能なパターンを形成している場合には、そのパターンを目視で視認することにより容易に識別対象の識別が可能となり、識別対象が同定される。また、配列番号１～１００で表される塩基配列及びその相補的配列を利用する場合には、高度に選択的にハイブリダイズ産物が生成されるため、迅速に確度の高い識別が可能である。さらに、一つの識別対象に対して、２以上のＤＮＡ、すなわち、２以上の識別用塩基配列が関連付けられている場合にも、確度の高い識別が可能となる。 As identification information, when one or more DNAs form a visible pattern, the identification target can be easily identified by visually recognizing the pattern, and the identification target is identified. In addition, when the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary sequences are used, highly selective hybridization products are generated, so that rapid and highly accurate discrimination is possible. Further, even when two or more DNAs, that is, two or more identification base sequences are associated with one identification target, identification with high accuracy is possible.
　検出工程に要する時間は、特に限定しないが、1秒以上１時間以下とすることできる。本方法においては、例えば、配列番号１～１００で表される塩基配列及びその相補的配列から選択されるような高度に選択的な識別用塩基配列を用いることで、一般的な検出温度(５０℃～７０℃)と比べ４０℃以下（例えば３７℃程度）でハイブリダイズ及び検出が可能であるため、検出工程の迅速化も可能となっている。より好ましくは1秒以上５分以下であり、さらに好ましくは１秒以上１分以下である。 The time required for the detection process is not particularly limited, but can be 1 second or more and 1 hour or less. In this method, for example, by using a highly selective identification base sequence selected from the base sequences represented by SEQ ID NOs: 1 to 100 and their complementary sequences, a general detection temperature (50 Hybridization and detection are possible at 40 ° C. or lower (for example, about 37 ° C.) compared to (° C.-70 ° C.), so that the detection process can be accelerated. More preferably, they are 1 second or more and 5 minutes or less, More preferably, they are 1 second or more and 1 minute or less.
　以上説明したように、本識別方法によれば、良好な識別能を有する本保持体に対して、簡易にかつ迅速に接触工程及び検出工程を実施できる。したがって、確度の高い識別が可能となっている。また、こうした識別方法は、各種物品の流通、保管に関する、管理方法、監視方法、認証方法等においても同様の効果を奏する。 As described above, according to the identification method, the contact process and the detection process can be easily and quickly performed on the holder having a good identification ability. Therefore, identification with high accuracy is possible. Such an identification method also has the same effect in a management method, a monitoring method, an authentication method, and the like related to the distribution and storage of various articles.
（識別キット）
　本明細書に開示される識別対象の識別キットは、本保持体と、識別用塩基配列と相補的な塩基配列を有する１又は２以上のプローブと、を備えることができる。こうしたキットによれば、本保持体による識別を実施することができる。本識別キットにおける本保持体及びプローブについては、既に説明したこれらについての各種態様をそのまま適用することができる。 (Identification kit)
The identification kit of the identification target disclosed in the present specification can include the holder and one or more probes having a base sequence complementary to the identification base sequence. According to such a kit, identification by this holding body can be implemented. For the holder and the probe in the identification kit, the various aspects described above can be applied as they are.
　以上説明した本発明によれば、以下の有利な作用を奏することもできる。
（１）固相担体上に情報保持領域を包囲する障壁を設け、この障壁をハイブリダイゼーションのキャビティとして用いることで、ハイブリダイゼーション溶液の滴下が容易となり、情報保持領域上において気泡が発生してもその気泡を速やかに解消させることができる。また、障壁によるキャビティによって、ハイブリダイゼーション溶液の量を低減させることができる。さらに、こうした開放的キャビティであるため、洗浄液を流すだけの迅速な洗浄が可能となり、工程全体を迅速化することができる。
（２）障壁を備えることで、検出工程においての情報保持領域への外部要素との接触やこすれの発生を抑制し、再現性のよい検出が可能となる。
（３）固相担体表面に、DNA末端のアミノ基と共有結合の形成が可能な官能基を形成させることで、末端アミノ基修飾DNAを固相担体に共有結合するようにしたことで、低濃度のサンプル液であっても検出が可能となる。 According to the present invention described above, the following advantageous effects can be obtained.
(1) By providing a barrier surrounding the information holding region on the solid phase carrier and using this barrier as a hybridization cavity, it becomes easy to drip the hybridization solution, and even if bubbles are generated on the information holding region. The bubbles can be quickly eliminated. Moreover, the amount of the hybridization solution can be reduced by the cavity formed by the barrier. Furthermore, because of such an open cavity, it is possible to perform a quick cleaning only by flowing a cleaning liquid, and it is possible to speed up the entire process.
(2) By providing a barrier, it is possible to suppress contact with an external element and rubbing to the information holding area in the detection step, and detection with good reproducibility is possible.
(3) By forming a functional group capable of forming a covalent bond with the amino group at the end of the DNA on the surface of the solid phase carrier, the terminal amino group-modified DNA is covalently bound to the solid phase carrier. Even a sample solution with a concentration can be detected.
　以下、本発明を具現化した実施例について説明するが、本明細書の開示は、以下の実施例に限定されるものではない。 Hereinafter, examples embodying the present invention will be described, but the disclosure of the present specification is not limited to the following examples.
　本実施例では、２３ｍｅｒの人工設計オリゴヌクレオチドの識別用塩基配列のうち、チミン（Ｔ）塩基の割合が少ないもの、チミン塩基の割合が多いもの、チミン塩基が全くないものの３通りの配列と、これら配列の５’末端にｐｏｌｙＴ（チミンリッチな塩基配列）をつないだ配列からなるオリゴヌクレオチドを合成した。これらのオリゴヌクレオチドを樹脂基板にスポットし、固定した。スポット固定後の紫外線照射による識別対象の識別能（検出能）への影響を評価するため、紫外線処理前後でのプローブとのハイブリダイズ反応を行い、蛍光シグナルの強度を比較した。なお、紫外線照射処理は、オリゴヌクレオチドが固定された担体上に２００－３００ｎｍ程度の紫外光を長時間連続照射することにより行った。 In this example, among the 23mer artificially designed oligonucleotide identification base sequence, there are three types of sequences: one with a low proportion of thymine (T) base, one with a high proportion of thymine base, and one without any thymine base, Oligonucleotides having a sequence in which polyT (thymine-rich base sequence) was connected to the 5 ′ end of these sequences were synthesized. These oligonucleotides were spotted and fixed on a resin substrate. In order to evaluate the influence of the ultraviolet irradiation after spot fixation on the discrimination ability (detection ability) of the discrimination target, the hybridization reaction with the probe before and after the ultraviolet treatment was performed, and the intensities of the fluorescence signals were compared. The ultraviolet irradiation treatment was performed by continuously irradiating ultraviolet light with a wavelength of about 200 to 300 nm on the carrier on which the oligonucleotide was fixed for a long time.
（１）オリゴヌクレオチドの固定化体の作製
　熱可塑性 HYPERLINK "http://ja.wikipedia.org/wiki/%E5%90%88%E6%88%90%E6%A8%B9%E8%84%82" \o "合成樹脂" プラスチックの一種であるポリカーボネート基板（２５ｍｍ±０．０５ｍｍ×７６ｍｍ±０．０５ｍｍ×）上に、あらかじめ調製した合成オリゴヌクレオチド（ＤＮＡ）（日本遺伝子研究所社製：表１参照）を溶かした水溶液を日本ガイシ株式会社製ＧＥＮＥＳＨＯＴスポッターを用いてスポットした。オリゴヌクレオチドは、識別用塩基配列中のチミン塩基数が１０個、２個及び０個のＯｌｉｇｏ１－１、２－１、３－１の３種（配列番号１０１～１０３）と、これらの５’末端にそれぞれＴが１２個、１０個及び１０個のＰｏｌｙＴを結合させたＯｌｉｇｏ１－２、２－２、３－２の３種（配列番号１０４～１０６）とした。 (1) Preparation of immobilized oligonucleotides Thermoplastic HYPERLINK "http://en.wikipedia.org/wiki/%E5%90%88%E6%88%90%E6%A8%B9%E8%84% 82 "\ o" Synthetic Resin "Synthetic oligonucleotide (DNA) prepared in advance on a polycarbonate substrate (25 mm ± 0.05 mm × 76 mm ± 0.05 mm ×) which is a kind of plastic 1) was spotted using a GENESHOT spotter manufactured by NGK Corporation. Oligonucleotides are Oligo1-1, 2-1, 3-1 (SEQ ID NO: 101 to 103) having 10, 2 and 0 thymine bases in the identification base sequence, and 5 ′ of these. Three types of oligos 1-2, 2-2, and 3-2 (SEQ ID NOs: 104 to 106) each having 12, 10 and 10 PolyTs bonded to the ends were used.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　なお、合成オリゴヌクレオチドの水溶液は以下のようにして調製した。
（ａ）添加剤１を用いたスポット用水溶液：ＳＳＣ系
　６×ＳＳＣ（ｉｎｖｉｔｒｏｇｅｎ社２０×ＳＳＣを薄めたもの）と１００ｐｍｏｌ／μｌのオリゴヌクレオチドをそれぞれを等量混合したもの（各終濃度５０ｐｍｏｌ／μｌ，３×ＳＳＣ）
（ｂ）添加剤２を用いたスポット用水溶液：ＰＢＳ系
　２×ＰＢＳ（ｉｎｖｉｔｒｏｇｅｎ社１０×ＰＢＳを薄めたもの）と１００ｐｍｏｌ／μｌのオリゴヌクレオチドをそれぞれを等量混合したもの（各終濃度５０ｐｍｏｌ／μｌ，１×ＰＢＳ） In addition, the aqueous solution of the synthetic oligonucleotide was prepared as follows.
(A) Aqueous solution for spot using additive 1: SSC system 6 × SSC (thinned 20 × SSC from Invitrogen) and 100 pmol / μl of oligonucleotide mixed in equal amounts (each final concentration 50 pmol / μl, 3 × SSC)
(B) Aqueous solution for spot using additive 2: PBS system 2 × PBS (thin diluted 10 × PBS from Invitrogen) and 100 pmol / μl of oligonucleotide mixed in equal amounts (each final concentration 50 pmol / μl, 1 × PBS)
スポットの後、以下の手順で合成オリゴヌクレオチドの担体（基板）への固定を行った。すなわち、スポット済み基板をＵＶ照射装置（Ｓｐｅｃｔｒｏｌｉｎｅ社ＸＬ－１５００ＵＶ Ｃｒｏｓｓｌｉｎｋｅｒ）にセットし、６００ｍＪ／ｃｍ2で紫外線光を照射した。次いで、スライドラックにセットした基板を３～５％ＢＳＡ水溶液中にて５０回上下振とう後、さらに、滅菌水中にて１０回上下振とうし、その後、遠心（１０００ｒｐｍ×２分）により液切りした。 After the spot, the synthetic oligonucleotide was immobilized on a carrier (substrate) by the following procedure. That is, the spotted substrate was set in a UV irradiation apparatus (Spectroline XL-1500 UV Crosslinker) and irradiated with ultraviolet light at 600 mJ / cm 2 . Next, the substrate set on the slide rack is shaken up and down 50 times in a 3-5% BSA aqueous solution, then shaken up and down 10 times in sterilized water, and then drained by centrifugation (1000 rpm × 2 minutes). did.
（２）プローブＤＮＡとの反応
　（１）で作製した合成ＤＮＡ保持担体上のオリゴヌクレオチドを検出するためのプローブとして、５’末端をＣｙ３で修飾された合成ＤＮＡ（日本遺伝子研究所社製：以下３種の配列参照、配列番号１０７～１０９）を使用した。 (2) Reaction with probe DNA As a probe for detecting the oligonucleotide on the synthetic DNA holding carrier prepared in (1), a synthetic DNA whose 5 ′ end is modified with Cy3 (manufactured by Nippon Genetic Institute, Inc .: Three sequence references, SEQ ID NOs 107-109) were used.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　まず初めに、上記３種プローブを用いた反応溶液を以下の組成で調製し、合成オリゴヌクレオチド保持担体（基板）への反応を行った。詳細な手順を以下に説明する。 First, a reaction solution using the above three kinds of probes was prepared with the following composition, and reacted to a synthetic oligonucleotide holding carrier (substrate). A detailed procedure will be described below.
（プローブDNA溶液の調製）
Probe mixture(2.5nM, each) *                 1.5 μｌ
Hybri Solution(×2)*                         9.0 μｌ
milliQ水                                     7.5 μｌ
total                                        18.0 μｌ

* Probe mixture組成（2.5nM, each）
Probe 1(100nM)                               10 μｌ
Probe 2(100nM)                               10 μｌ
Probe 3(100nM)                               10 μｌ
TE(ｐH8.0)                                   370 μｌ
                                             400 μｌ

*Hybri Solution組成（×2）
20×SSC                                      2.0 mｌ
10％SDS                                      0.8 ml
100％ Formamide                              12.0 ml
100 mM EDTA                                  0.8 ml
milliQ                                       24.4 mｌ
                                             40.0 mｌ (Preparation of probe DNA solution)
Probe mixture (2.5nM, each) * 1.5 μl
Hybri Solution (× 2) * 9.0 μl
milliQ water 7.5 μl
total 18.0 μl

* Probe mixture composition (2.5nM, each)
Probe 1 (100nM) 10 μl
Probe 2 (100nM) 10 μl
Probe 3 (100nM) 10 μl
TE (pH 8.0) 370 μl
400 μl

* Hybri Solution composition (× 2)
20 x SSC 2.0 ml
10% SDS 0.8 ml
100% Formamide 12.0 ml
100 mM EDTA 0.8 ml
milliQ 24.4 ml
40.0 ml
（基板とのプローブDNA溶液の反応）
　調製した反応プローブＤＮＡ溶液を、Applied Biosystems社のGeneAmp PCR System 9700を使用し、９０℃で１分加熱した後、ヒートブロック（ＴＡＩＴＥＣ社ＤＴＵ－Ｎ）を使用し８０℃で１分加熱した。上記プローブ溶液を各９μｌずつ、基板上のスポットエリアにかけ、乾燥防止のためコンフォート／プラス用サーモブロックＳｌｉｄｅ（eppendorf社）を使用し、３７℃で６０分間静置することにより反応を行った。 (Reaction of probe DNA solution with substrate)
The prepared reaction probe DNA solution was heated at 90 ° C. for 1 minute using GeneAmp PCR System 9700 from Applied Biosystems, and then heated at 80 ° C. for 1 minute using a heat block (TAITEC DTU-N). Each 9 μl of the probe solution was applied to the spot area on the substrate, and the reaction was performed by leaving it at 37 ° C. for 60 minutes using a comfort / plus thermoblock Slide (eppendorf) to prevent drying.
（反応後基板洗浄液の調製）
Milli Q                                      188.0 mｌ
20×SSC                                      10.0 ml
10％SDS                                      2.0 mｌ
         total                               200.0 mｌ (Preparation of substrate cleaning solution after reaction)
Milli Q 188.0 ml
20 x SSC 10.0 ml
10% SDS 2.0 ml
total 200.0 ml
（基板上の余剰プローブの洗浄）
　上記洗浄液をガラス染色バットに移した。プローブＤＮＡ溶液との反応終了後の基板を浸漬し５分間上下振とうした。滅菌水を入れたガラス染色バットに基板を移し、１分間上下振とうした。次いで、２０００ｒｐｍで１分間遠心乾燥し、基板上に残った水分を除去した。 (Cleaning of excess probe on the substrate)
The washing solution was transferred to a glass staining vat. The substrate after the reaction with the probe DNA solution was immersed and shaken up and down for 5 minutes. The substrate was transferred to a glass staining vat containing sterilized water and shaken up and down for 1 minute. Subsequently, it was centrifuged and dried at 2000 rpm for 1 minute to remove moisture remaining on the substrate.
（３）（１）で作製された担体に追加で紫外光照射された基板の作製及びその基板へのプローブＤＮＡを用いた反応
　（１）にて作製済みの基板をＵＶ照射装置（Spectroline社XL-1500UV Crosslinker）にセットし、追加で紫外光を照射(それぞれ６００ｍＪ/ｃｍ2及び１２００ｍＪ/ｃｍ2)した基板を作製した。 (3) Preparation of a substrate additionally irradiated with ultraviolet light on the carrier prepared in (1) and reaction using the probe DNA to the substrate The substrate prepared in (1) is subjected to UV irradiation equipment (Spectroline XL -1500UV Crosslinker) to set to produce a substrate with the ultraviolet light is irradiated (respectively 600 mJ / cm 2 and 1200 mJ / cm 2) in addition.
　追加で紫外光を照射して得た各基板に対し、（２）で実施した方法と同様の方法でプローブDNAとの反応を行い、基板上の余剰プローブを洗浄した。 Further, each substrate obtained by irradiating with ultraviolet light was reacted with the probe DNA in the same manner as the method performed in (2), and the excess probe on the substrate was washed.
（４）データ解析
（スキャナーによる蛍光検出）
（２）及び（３）において得た反応済み各基板について、Appleied Precision社ArrayWoRxを使用して適宜、露光時間を調節し、基板表面の蛍光画像を取得した。さらにGenePix Proを使用し、得られた画像の蛍光シグナルの数値化を行った。（２）で得られた基板上の各スポットの蛍光強度について結果を比較した。その結果を以下の表に示す。 (4) Data analysis (fluorescence detection with a scanner)
For each of the reacted substrates obtained in (2) and (3), an exposure time was appropriately adjusted using ArrayWoRx manufactured by Appleied Precision, and a fluorescence image of the substrate surface was obtained. Furthermore, using GenePix Pro, the fluorescence signal of the obtained image was digitized. The results were compared for the fluorescence intensity of each spot on the substrate obtained in (2). The results are shown in the following table.
Figure JPOXMLDOC01-appb-T000003
Figure JPOXMLDOC01-appb-T000003
　表３に示すように、オリゴヌクレオチド用水溶液の調製方法（ＳＳＣ系、ＰＢＳ系）に関係なく、オリゴヌクレオチドの識別用塩基配列中のチミン塩基数が少なくなるにつれ（Ｏｌｉｇｏ１－１、２－１、３－１およびＯｌｉｇｏ１－２、２－２、３－２の順）、プローブ反応量が向上する結果を確認した。また、オリゴヌクレオチドの末端のｐｏｌｙＴ結合の有無によるプローブ反応量への影響を確認したところ、ｐｏｌｙＴを結合したオリゴヌクレオチド（Ｏｌｉｇｏ１－２、２－２、３－２）において、結合していないオリゴヌクレオチド（Ｏｌｉｇｏ１－１、２－１、３－１）と比べてプローブ反応量が数倍から１０倍程度に向上する結果を確認した。 As shown in Table 3, regardless of the method for preparing the aqueous solution for oligonucleotides (SSC system, PBS system), as the number of thymine bases in the nucleotide sequence for oligonucleotide identification decreases (Oligo 1-1, 2-1, 3-1 and Oligo1-2, 2-2, 3-2 in this order), and the result that the probe reaction amount was improved was confirmed. In addition, when the influence of the presence or absence of polyT binding at the end of the oligonucleotide on the amount of probe reaction was confirmed, in oligonucleotides bound to polyT (Oligo1-2, 2-2, 3-2), oligonucleotides that were not bound Compared to (Oligo 1-1, 2-1, 3-1), the probe reaction amount was improved from several times to about 10 times.
　また、上記（３）により得られた基板上の各スポットの蛍光強度についての結果（ＳＳＣ系）を比較した。上記（２）の結果も加味した結果を以下の表に示す。 Also, the results (SSC system) for the fluorescence intensity of each spot on the substrate obtained by (3) above were compared. The following table shows the results including the results of (2) above.
Figure JPOXMLDOC01-appb-T000004
Figure JPOXMLDOC01-appb-T000004
　表４に示すように、オリゴヌクレオチド水溶液の調製方法がＳＳＣ系においては、Ｏｌｉｇｏ３－２（識別用塩基配列中のチミン数０、ＰｏｌｙＴ有り）において追加紫外光照射によるスポットの蛍光強度低減割合を従来の数十分の１から約１／３程度までに抑制できることを確認した。 As shown in Table 4, when the oligonucleotide aqueous solution preparation method is the SSC system, the fluorescence intensity reduction rate of the spot due to irradiation with additional ultraviolet light in Oligo 3-2 (zero thymine in identification base sequence, with PolyT) is conventionally It was confirmed that it was possible to suppress the tens of 1 to about 1/3.
　オリゴヌクレオチドの識別用塩基配列中のチミン塩基数が少なく、かつオリゴヌクレオチドの末端にｐｏｌｙＴが結合した配列において、基板上に固定されたオリゴヌクレオチドの紫外線による影響を小さくすることが可能である。 It is possible to reduce the influence of the oligonucleotide fixed on the substrate due to ultraviolet rays in a sequence in which the number of thymine bases in the oligonucleotide identification base sequence is small and polyT is bonded to the end of the oligonucleotide.
　また、オリゴヌクレオチドスポット用水溶液調製方法がＰＢＳ系においては、ＳＳＣ系と比べ、追加紫外光照射によるスポットの蛍光強度低減割合がやや大きく、ＳＳＣ系でのスポット用水溶液調製が望ましいことがわかった。 Further, it was found that the method for preparing an aqueous solution for oligonucleotide spots in the PBS system had a slightly larger ratio of reduction in the fluorescence intensity of the spot due to the irradiation of additional ultraviolet light than the SSC system, and it was desirable to prepare the aqueous solution for spots in the SSC system.
　本実施例では、障壁部材を備える識別情報保持体を作製し、その評価を行った。評価は以下の手順で行った。
（１）サンプルDNA検出用合成DNAプローブを担体上（フィルム（実施例）/フィルム及びスライド基材２種（ガラス及びプラスチック）（対照用））にスポットならびにプローブの固定化による合成DNAプローブが保持された担体の作製の実施
（２）サンプル液の保持が可能なに障壁部材（カバーあり/なし）のプローブ保持体（フィルム）の担体上への貼付の実施
（３）作製したプローブ保持体を用いた評価
実験１：作製された担体へのサンプルDNAを用いた反応(スポット配置に応じた反応パターンの確認)の実施
実験２：作製したプローブ保持体へのサンプルDNAを用いた反応（経時変化の影響評価）
実験３：作製したプローブ保持体に紫外光照射した後のサンプルDNAを用いた反応(紫外線の影響評価)の実施
実験４：作製したプローブ保持体上のプローブの物理的な摩擦（こすれ）を生じさせた後のサンプルDNAを用いた反応の実施
（４）実験１～４の反応結果のスキャナーによる検出及びデータ解析 In this example, an identification information holding body including a barrier member was produced and evaluated. The evaluation was performed according to the following procedure.
(1) Synthetic DNA probe for detection of sample DNA is supported on a carrier (film (Example) / two types of film and slide substrate (glass and plastic) (for control)), and the synthetic DNA probe is retained by immobilizing spots and probes. (2) Attaching the barrier member (with / without cover) to the probe holder (film) on the carrier so that the sample liquid can be held (3) The prepared probe holder Evaluation experiment 1 used: Reaction using sample DNA to the prepared carrier (reaction pattern confirmation according to spot arrangement) Experiment 2: Reaction using sample DNA to the prepared probe holder (change over time) Impact assessment)
Experiment 3: Implementation of reaction using UV-irradiated sample DNA (evaluation of influence of ultraviolet rays) on the prepared probe holder Experiment 4: Physical friction (rubbing) of the probe on the prepared probe holder occurs (4) Detection and data analysis of the reaction results of Experiments 1 to 4 using a scanner
（１）サンプルDNA検出用合成DNAプローブを担体上にスポット・固定することによる合成DNAプローブ（識別情報）が保持された保持体の作製
　プローブ保持体の固相担体として、住友ベークライト株式会社製DNA共有結合用フィルムに、5'末端をアミノ基で修飾した合成オリゴDNA（株式会社日本遺伝子研究所製）を溶かした水溶液をキャプチャープローブとし、日本ガイシ株式会社にてGENESHOT（登録商標）スポッターを用いてスポットした。なお、プローブの固定化は、スポット後の担体を、メーカー指定ブロッキング液に、室温で５分浸漬後、沸騰水中に２分浸漬し、さらに、滅菌水中に浸漬して２分間急冷後、エアーガンで液切りを行うことによった。 (1) Preparation of a holder holding a synthetic DNA probe (identification information) by spot-fixing a synthetic DNA probe for detection of sample DNA on a carrier As a solid carrier of the probe holder, DNA manufactured by Sumitomo Bakelite Co., Ltd. An aqueous solution prepared by dissolving a synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) with a 5 'end modified with an amino group on a covalent bond film is used as a capture probe, and GENESHOT (registered trademark) spotter is installed at NGK Spotted. In order to fix the probe, the spotted carrier is immersed in a blocking solution designated by the manufacturer for 5 minutes at room temperature, then immersed in boiling water for 2 minutes, further immersed in sterilized water and quenched for 2 minutes, and then airgun. According to draining.
　なお、対照用プローブ保持体として、市販のプラスチック基板、市販のガラス基板のそれぞれに、5'末端をアミノ基で修飾した合成オリゴDNA（株式会社日本遺伝子研究所製）を溶かした水溶液をキャプチャープローブとし、日本ガイシ株式会社にてGENESHOT（登録商標）スポッターを用いてスポットを実施した。対照用プローブ保持体においては、合成ＤＮＡの固定化は、スポット後の担体を、2×SSC/0.2％SDSで１５分洗浄後、95℃ 2×SSC/0.2％SDSで５分洗浄し、さらに、滅菌水洗浄（10回上下振とう）を３回行った後、遠心（1000rpm×3分)により液切りを行うことによった。 In addition, as a probe holder for the control, an aqueous solution in which a synthetic oligo DNA (manufactured by Nippon Genetic Laboratory Co., Ltd.) in which the 5 ′ end is modified with an amino group is dissolved in each of a commercially available plastic substrate and a commercially available glass substrate is a capture probe. And spotting was performed at NGK Corporation using a GENESHOT (registered trademark) spotter. In the control probe holder, the synthetic DNA was immobilized by washing the spotted carrier with 2 × SSC / 0.2% SDS for 15 minutes and then with 95 ° C. × 2 × SSC / 0.2% SDS for 5 minutes. After performing sterilized water washing (shaking up and down 10 times) three times, the liquid was removed by centrifugation (1000 rpm × 3 minutes).
　プローブ保持体及び対照用プローブ保持体の作製に際し、スポットするのに用いた合成オリゴDNA配列は、サンプル反応性の高いことが既に分かっている以下の表に記載の配列表の中から２配列（Ｄ１－００１，Ｄ１－１００）（配列番号１及び１００）を選定し使用した。 The synthetic oligo DNA sequences used for spotting in the preparation of the probe holder and the control probe holder are two sequences (from the sequence table shown in the following table, which are already known to have high sample reactivity) ( D1-001, D1-100) (SEQ ID NOS: 1 and 100) were selected and used.
Figure JPOXMLDOC01-appb-T000005
Figure JPOXMLDOC01-appb-T000005
　各プローブのスポットパターンは図６に示すように行い、Ｄ１－００１のみが固定化されたスポットと、Ｄ１－１００のみが固定化されたスポットと、双方が固定化されたスポットの３種とした。 The spot pattern of each probe was carried out as shown in FIG. 6, and three types were used: a spot where only D1-001 was immobilized, a spot where only D1-100 was immobilized, and a spot where both were immobilized. .
（２）積層体で構成されたサンプル液の保持が可能な障壁部材（カバーあり/なし）の担体上への貼付
　（１）で作製した各種DNAプローブ保持体上に図７～図９に示す三種類の障壁部材（I～III,Iはカバーなし、II及びIIIはカバーあり）を貼り付けた。なお、障壁
部材II及びIIIは、ともに保管時は障壁部材を貼付したままとし、サンプルＤＮＡとの反応時にカバー（弱粘着性のリンテック製フィルム）をはがして担体上のプローブを露出させた。 (2) Affixing a barrier member (with / without a cover) composed of a laminate on a carrier, which is capable of holding a sample solution, on the various DNA probe holders prepared in (1), as shown in FIGS. Three types of barrier members (I to III, I have no cover, II and III have a cover) were attached. The barrier members II and III were both attached with the barrier member during storage, and the cover (weakly adhesive Lintec film) was peeled off to react with the sample DNA to expose the probe on the carrier.
（３）各種評価
（実験１）
　障壁部材を貼付したプローブ保持体及び障壁部材を貼り付けていないプローブ保持体および対照用プローブ保持体に対するサンプルDNAの反応(スポット配置に応じた反応パターンの確認
　サンプルDNAとして、5’末端をCy3で修飾した合成DNA(日本遺伝子研究所社：以下に示す２種配列参照)を使用して反応を実施した。
Figure JPOXMLDOC01-appb-T000006
 (3) Various evaluations (Experiment 1)
Reaction of sample DNA to probe holder with barrier member attached and probe holder without barrier member and control probe holder (confirmation of reaction pattern according to spot arrangement) The reaction was carried out using the modified synthetic DNA (Nippon Genetics Laboratories Co., Ltd .: see the following two sequences).
Figure JPOXMLDOC01-appb-T000006
　まず、上記２種サンプルＤＮＡをそれぞれ用いた反応溶液を調製し、合成DNAプローブ保持担体（フィルムおよび対照用基板２種）への反応を行った。詳細な手順を以下に記載する。さらに、フィルム及び対照用基板２種に対しては、障壁部材を貼付せずの状態でサンプルDNAの反応を実施した。対照用プローブ保持体である障壁部材なしのフィルムおよび基板2種に対しては、下記反応用サンプルDNA溶液の漏出を防ぐことができないため、液量を50μｌと増やして反応を実施した。 First, a reaction solution using each of the two types of sample DNAs was prepared, and the reaction was performed on a synthetic DNA probe holding carrier (film and two types of control substrates). The detailed procedure is described below. Furthermore, the sample DNA reaction was carried out with no barrier member attached to the film and the two control substrates. Since the leakage of the following sample DNA solution for reaction cannot be prevented with respect to the film having no barrier member and the two kinds of substrates as the control probe holder, the reaction was carried out with the liquid volume increased to 50 μl.
（反応用サンプルDNA溶液の調製）
サンプル液(2.5nM, each)                      1.5 μｌ
Hybri Solution(×2)*                         9.0 μｌ
milliQ水                                     7.5 μｌ
total                                        18.0 μｌ (Preparation of sample DNA solution for reaction)
Sample solution (2.5 nM, each) 1.5 μl
Hybri Solution (× 2) * 9.0 μl
milliQ water 7.5 μl
total 18.0 μl
　上記組成において、サンプル液を混液とするときは、以下の組成のサンプル液を用いる。
*Sample mixture組成（2.5nM, each）
ｒD1-001(100nM)                              10 μｌ
ｒD1-100(100nM)                              10 μｌ
TE(ｐH8.0)                                   380 μｌ
　                                           400 μｌ In the above composition, when a sample liquid is used as a mixed liquid, a sample liquid having the following composition is used.
* Sample mixture composition (2.5nM, each)
rD1-001 (100nM) 10 μl
rD1-100 (100nM) 10 μl
TE (pH 8.0) 380 μl
400 μl
*Hybri Solution組成（×2）
20×SSC                                      2.0 mｌ
10％SDS                                      0.8 ml
100％ Formamide                              12.0 ml
100 mM EDTA                                  0.8 ml
milliQ                                       24.4 mｌ
                                             40.0 mｌ * Hybri Solution composition (× 2)
20 x SSC 2.0 ml
10% SDS 0.8 ml
100% Formamide 12.0 ml
100 mM EDTA 0.8 ml
milliQ 24.4 ml
40.0 ml
（保持体へのサンプルDNA溶液の反応）
　上記のようにして調製したサンプル溶液（ｒＤ１－００１のみ、ｒＤ１－１００のみ、及び混合）を、Applied Biosystems社のGeneAmp PCR System 9700を使用し、90℃で1分加熱した後、ヒートブロック(TAITEC社DTU-N）を使用し80℃で1分加熱した。次いで、上記サンプル溶液を各9μｌずつ、各保持体上のスポットエリアに滴下後、乾燥防止のためコンフォート／プラス用サーモブロックＳｌｉｄｅ（eppendorf社）で、37℃で60分間静置することにより反応を行った。 (Reaction of sample DNA solution to holder)
The sample solution (rD1-001 only, rD1-100 only and mixed) prepared as described above was heated at 90 ° C. for 1 minute using an Applied Biosystems GeneAmp PCR System 9700, and then heat block (TAITEC Using DTU-N) and heated at 80 ° C. for 1 minute. Next, 9 μl each of the above sample solution is dropped on the spot area on each holder, and then the reaction is carried out by allowing to stand at 37 ° C. for 60 minutes in a comfort / plus thermoblock Slide (eppendorf) to prevent drying. went.
（反応後基板洗浄液の調製）
Milli Q                                      188.0 mｌ
20×SSC                                      10.0 ml
10％SDS                                      2.0 mｌ
         total                               200.0 mｌ (Preparation of substrate cleaning solution after reaction)
Milli Q 188.0 ml
20 x SSC 10.0 ml
10% SDS 2.0 ml
total 200.0 ml
（保持体上の余剰サンプルの洗浄）
　上記のように調製した洗浄液をガラス染色バットに移し、サンプルDNA溶液反応終了後の基板を浸漬し、5分間上下振とうした。さらに、滅菌水を入れたガラス染色バットに基板を移し、1分間上下振とう後、2000rpmで1分間遠心乾燥し、基板上に残った水分を除去した。 (Washing excess sample on the holder)
The cleaning solution prepared as described above was transferred to a glass staining vat, the substrate after completion of the sample DNA solution reaction was immersed, and shaken up and down for 5 minutes. Further, the substrate was transferred to a glass staining vat containing sterilized water, shaken up and down for 1 minute, and then centrifugally dried at 2000 rpm for 1 minute to remove water remaining on the substrate.
（実験２）プローブ保持体における経時変化の影響評価
　障壁部材を貼付したプローブ保持体及び対照用プローブ保持体につき、プローブを固定化から以下の表に示す一定期間経過後にそれぞれサンプルＤＮＡ（Ｄ１－０１とＤ１－１００の混合液）との反応を実施した。なお、プローブ固定化からの時間変化評価のために、プローブ保持体及び対照用プローブ保持体は、常温で透明な袋に収納して保管し、その後、サンプルＤＮＡとの反応を行った。混合液の調製は以下の通りとした。 (Experiment 2) Evaluation of influence of change over time in probe holder Each of the sample holder (D1-01) after the probe was immobilized and the fixed period of time shown in the following table passed after the probe was fixed on the probe holder and the control probe holder. And a mixture of D1-100). In addition, for the time change evaluation after probe immobilization, the probe holder and the control probe holder were stored and stored in a transparent bag at room temperature, and then reacted with sample DNA. The mixture was prepared as follows.
Figure JPOXMLDOC01-appb-T000007
Figure JPOXMLDOC01-appb-T000007
　プローブ保持体については、各パターン単位に切断した上でサンプルDNAとの反応を行った。なお切断は反応直前に実施した。切断にはカッター、はさみ、超音波カッターのいずれも用いることが可能であった。サンプルＤＮＡ（Ｄ１－０１とＤ１－１００の混合液）との反応、洗浄は、実験１と同様に行った。 The probe holder was cut into each pattern unit and reacted with sample DNA. The cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.
（実験３）プローブ保持体に対する紫外光照射の影響評価
　障壁部材を貼付したプローブ保持体及び対照用プローブ保持体に対して、作製直後にUV照射装置（Spectroline社XL-1500UV Crosslinker）にセットし、以下に示す線量で紫外光を照射した。紫外線照射後、サンプルＤＮＡとの反応を行った。 (Experiment 3) Evaluation of the influence of ultraviolet light irradiation on the probe holder For the probe holder and the control probe holder to which the barrier member was attached, set the UV irradiation apparatus (Spectroline XL-1500UV Crosslinker) immediately after production, Ultraviolet light was irradiated at the dose shown below. After UV irradiation, reaction with sample DNA was performed.
Figure JPOXMLDOC01-appb-T000008
Figure JPOXMLDOC01-appb-T000008
　プローブ保持体については、各パターン単位に切断した上でサンプルDNAとの反応を行った。なお切断は反応直前に実施した。切断にはカッター、はさみ、超音波カッターのいずれも用いることが可能であった。サンプルＤＮＡ（Ｄ１－０１とＤ１－１００の混合液）との反応、洗浄は、実験１と同様に行った。 The probe holder was cut into each pattern unit and reacted with sample DNA. The cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.
（実験４）プローブ保持体への物理的な摩擦（こすれ）の影響評価
　障壁部材を貼付したプローブ保持体及び対照用プローブ保持体に対して、作製直後に以下の表に示す条件で固相担体上のスポット面に意図的に摩擦（こすれ）を生じさせた。 (Experiment 4) Evaluation of influence of physical friction (rubbing) on probe holder The solid phase carrier was immediately subjected to the conditions shown in the following table for the probe holder to which the barrier member was attached and the control probe holder. Friction (rubbing) was intentionally generated on the upper spot surface.
Figure JPOXMLDOC01-appb-T000009
Figure JPOXMLDOC01-appb-T000009
　プローブ保持体については、各パターン単位に切断した上でサンプルDNAとの反応を行った。なお切断は反応直前に実施した。切断にはカッター、はさみ、超音波カッターのいずれも用いることが可能であった。サンプルＤＮＡ（Ｄ１－０１とＤ１－１００の混合液）との反応、洗浄は、実験１と同様に行った。 The probe holder was cut into each pattern unit and reacted with sample DNA. The cleavage was performed immediately before the reaction. Any of a cutter, scissors, and an ultrasonic cutter could be used for cutting. Reaction with sample DNA (mixture of D1-01 and D1-100) and washing were carried out in the same manner as in Experiment 1.
（４）スキャナーによる蛍光検出及びデータ解析
　上記実験１～４において得た反応済みの各プローブ保持体について、moleculardevice社GenePix4000Bを使用して適宜、laser powerおよびPMTを調節し、保持体表面の蛍光画像を取得した。さらにGene Pix Proを使用し、得られた画像の蛍光シグナルの数値化を行った。 (4) Fluorescence detection and data analysis by scanner For each of the reacted probe holders obtained in Experiments 1 to 4 above, the laser power and PMT were adjusted as appropriate using moleculardevice GenePix4000B, and the fluorescence image of the holder surface was obtained. Acquired. Furthermore, using Gene Pix Pro, the fluorescence signal of the obtained image was digitized.
（実験１の評価結果）
　評価の結果を図１０に示す。図１０に示すように、サンプルＤＮＡとしてｒＤ１－００１のみ、ｒＤ１－１００のみ、及びこれらの双方を保持体に対して供給したとき、それぞれサンプルＤＮＡに対し狙いとする蛍光画像が得られた。すなわち、ｒＤ１－００１のみを供給したときには、「Ｎ」が提示され、ｒＤ１－１００のみを供給したときには、「Ｙ」が提示され、両者を供給したときには、「Ｎ」と「Ｙ」との重複した画像が提示された。また、二つのプローブを一つのスポットに固定したエリアにおいても顕著なシグナルの低下といった問題は起こらず、目視での認識に問題ない結果であることが分かった。 (Evaluation result of Experiment 1)
The evaluation results are shown in FIG. As shown in FIG. 10, when only rD1-001, only rD1-100, and both of them were supplied to the holder as sample DNA, a target fluorescence image was obtained for each sample DNA. That is, “N” is presented when only rD1-001 is supplied, “Y” is presented when only rD1-100 is supplied, and “N” and “Y” overlap when both are supplied. Image was presented. In addition, it was found that there was no problem of significant signal decrease even in an area where two probes were fixed to one spot, and there was no problem with visual recognition.
（実験２の評価結果）
　評価の結果を以下の表及び図１１に示す。なお、以下の説明において、プローブ保持体に対して、障壁部材Ｉ～IIIをそれぞれ貼付したものを、保持体I～IIIと標記する。 (Evaluation result of Experiment 2)
The evaluation results are shown in the following table and FIG. In the following description, those obtained by attaching the barrier members I to III to the probe holder are denoted as holders I to III, respectively.
　表９及び図１１に示すように、保持体Iに対しては作製からの時間が経
過するにつれ、作製直後の蛍光強度からの蛍光強度の低下が確認された。一方、保持体II、IIIにおいては作製からの時間経過に対する蛍光強度の低下は起こらず、長期保存安定性の高い保持体構造であることが分かった。一方で、障壁部材I～IIIを貼り付けることにより、
使用するサンプル量が半分以下（50μｌ→18μｌ）に低減することができた。 As shown in Table 9 and FIG. 11, for the holder I, it was confirmed that the fluorescence intensity decreased from the fluorescence intensity immediately after the production as the time from the production passed. On the other hand, in the holders II and III, it was found that the fluorescence intensity did not decrease with the passage of time from the production, and the holder structure had high long-term storage stability. On the other hand, by attaching the barrier members I to III,
The amount of sample used could be reduced to less than half (50 μl → 18 μl).
Figure JPOXMLDOC01-appb-T000010
Figure JPOXMLDOC01-appb-T000010
（実験３の評価結果）
　評価の結果を以下の表及び図１２に示す。表１０及び図１２に示すように、保持体Iに対しては紫外線照射線量が増大するにつれ、作製直後の蛍光強度か
らの蛍光強度の顕著な低下が確認された。一方、保持体II、IIIにおいては作製からの時間経
過に対する蛍光強度の低下は起こらず、紫外線の影響の小さな保持体構造であることが分かった。 (Evaluation result of Experiment 3)
The results of the evaluation are shown in the following table and FIG. As shown in Table 10 and FIG. 12, with respect to the holder I, it was confirmed that the fluorescence intensity significantly decreased from the fluorescence intensity immediately after the production as the ultraviolet irradiation dose increased. On the other hand, in the holders II and III, it was found that the fluorescence intensity did not decrease with the lapse of time from the production, and the holder structure was less affected by ultraviolet rays.
Figure JPOXMLDOC01-appb-T000011
Figure JPOXMLDOC01-appb-T000011
（実験４の評価結果）
　評価の結果を以下の表及び図１３に示す。表１１及び図１３に示すように、保持体II及びIIIに比較して保持体Iに対しては、摩擦(こすれ)に対してプ
ローブの保持を継続することは困難であることが確認された。一方、保持体II及びIIIにおいて
はこすれに対して安定な構造であることが確認できた。また、保持体なしの場合に比べ保持体が貼り付けられている場合の方がプローブの損傷が抑えられることも確認できた。 (Evaluation result of Experiment 4)
The results of the evaluation are shown in the following table and FIG. As shown in Table 11 and FIG. 13, it was confirmed that it is difficult for the holding body I to keep holding the probe against friction (rubbing) compared to the holding bodies II and III. . On the other hand, it was confirmed that the holders II and III have a stable structure against rubbing. It was also confirmed that the damage to the probe was suppressed when the holder was affixed compared to the case without the holder.
Figure JPOXMLDOC01-appb-T000012
Figure JPOXMLDOC01-appb-T000012
　以上の結果から、障壁部材に対してカバーを備えることで、長期安定性、紫外線照射、さらにはこすれに対して本来のプローブの性能を大幅に低下することなく使用できることが確認できた。また、保持体IIとIIIとでは、障壁の高さがより高い保持体IIIがより効果高い傾向があった。 From the above results, it was confirmed that by providing a cover for the barrier member, it was possible to use the probe without significantly reducing the performance of the original probe against long-term stability, ultraviolet irradiation, and rubbing. Further, in the holding bodies II and III, the holding body III having a higher barrier height tended to be more effective.
配列番号１～１０９：合成オリゴヌクレオチド SEQ ID NOs: 1 to 109: Synthetic oligonucleotide

Claims (37)

  1. 　固相担体と、
    　識別対象に予め関連付けられた識別用塩基配列を識別情報として有する１又は２以上のＤＮＡが前記固相担体上に保持された１又は２以上の情報保持領域と、
    　１又は２以上の前記情報保持領域を包囲する障壁と、
    を備える、識別情報の保持体。     A solid support;
    One or more information holding regions in which one or two or more DNAs having identification base sequences previously associated with identification objects as identification information are held on the solid phase carrier;
    A barrier surrounding one or more of the information holding areas;
    A holding body for identification information.
  2. 　前記障壁は、前記固相担体の一部である、又は、前記固相担体上に一体化された障壁部材である、請求項１に記載の保持体。 The holding body according to claim 1, wherein the barrier is a part of the solid phase carrier or a barrier member integrated on the solid phase carrier.
  3. 　前記障壁部材は、粘着剤又は接着剤で前記固相担体に一体化されている、請求項２に記載の保持体。 The holding body according to claim 2, wherein the barrier member is integrated with the solid phase carrier with an adhesive or an adhesive.
  4. 　前記障壁は、疎水性表面を有している、請求項１～３のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 3, wherein the barrier has a hydrophobic surface.
  5. 　前記障壁は、前記情報保持領域の周囲を連続的又は不連続的に包囲する、請求項１～４のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 4, wherein the barrier surrounds the information holding area continuously or discontinuously.
  6. 　前記障壁は、前記情報保持領域から１０μｍ以上、好ましくは５０μｍ以上、より好ましくは１００μｍ以上の高さを有する、請求項１～５のいずれかに記載の保持体。 The holder according to any one of claims 1 to 5, wherein the barrier has a height of 10 µm or more, preferably 50 µm or more, more preferably 100 µm or more from the information holding region.
  7. 　前記障壁は、前記情報保持領域から５００μｍ以下、より好ましくは３００μｍ以下、さらに好ましくは２００μｍ以下の高さを有する、請求項１～６に記載の保持体。 The holder according to any one of claims 1 to 6, wherein the barrier has a height of 500 µm or less, more preferably 300 µm or less, and further preferably 200 µm or less from the information holding region.
  8. 　前記障壁は、前記情報保持領域を目視で識別可能に形成されている、請求項１～７のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 7, wherein the barrier is formed so that the information holding area can be visually identified.
  9. 　前記障壁は、粘着剤又は接着剤を含む表層部を備えている、請求項１～８のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 8, wherein the barrier includes a surface layer portion containing an adhesive or an adhesive.
  10. 　前記表層部を介して前記識別対象に固定化可能である、請求項９に記載の保持体。 The holder according to claim 9, which can be fixed to the identification target via the surface layer portion.
  11. 　前記障壁を介して前記情報保持領域の上方を覆うカバーを備える、請求項１～１０のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 10, further comprising a cover that covers an upper portion of the information holding area through the barrier.
  12. 　前記識別用塩基配列は、添付する配列表に記載の配列番号１～１００及びその相補配列から選択される、請求項１～１１のいずれかに記載の保持体。 The holder according to any one of claims 1 to 11, wherein the base sequence for identification is selected from SEQ ID NOs: 1 to 100 shown in the attached sequence listing and complementary sequences thereof.
  13. 　前記１又は２以上のＤＮＡは前記固相担体に化学結合により結合されている、請求項１～１２のいずれかに記載の保持体。 The holder according to any one of claims 1 to 12, wherein the one or more DNAs are bonded to the solid phase carrier by chemical bonds.
  14. 　前記１又は２以上のＤＮＡを含む、複数個のドット状体が、目視で識別可能なパターンを構成する、請求項１～１３のいずれかに記載の保持体。 The holding body according to any one of claims 1 to 13, wherein a plurality of dot-like bodies containing the one or more DNAs constitute a visually distinguishable pattern.
  15. 　前記保情報持領域内に、前記パターンを２種以上備える、請求項１４に記載の保持体。 The holding body according to claim 14, comprising two or more types of the patterns in the holding information holding area.
  16. 　２種以上の前記パターンは、前記パターン間で共通しない前記識別用塩基配列を有する１又は２以上のＤＮＡからなるドット状体で構成されている、請求項１５に記載の保持体。 The holding body according to claim 15, wherein the two or more types of patterns are composed of dot-like bodies made of one or more DNAs having the identification base sequence that is not common between the patterns.
  17. 　２種以上の前記パターンは、少なくともその一部が重なって配置されている、請求項１５又は１６に記載の保持体。 The holder according to claim 15 or 16, wherein at least a part of the two or more patterns are arranged to overlap each other.
  18. 　請求項１～１７のいずれかに記載の識別情報保持体を複数個一体に備える、構造体。 A structure comprising a plurality of the identification information holding bodies according to any one of claims 1 to 17 integrally.
  19. 　複数個の前記識別情報保持体を、所定パターンで配列して備える、請求項１８に記載の構造体。 The structure according to claim 18, comprising a plurality of the identification information holding bodies arranged in a predetermined pattern.
  20. 　複数個の前記識別情報保持体は、部分的に連続する又は単一の固相担体を前記固相担体として備える、請求項１８又は１９に記載の構造体。 The structure according to claim 18 or 19, wherein the plurality of identification information holding bodies include a partially continuous or single solid phase carrier as the solid phase carrier.
  21. 　前記構造体は、複数個の前記識別情報保持体を分離可能に形成されている、請求項１８～２０のいずれかに記載の構造体。 The structure according to any one of claims 18 to 20, wherein the structure is formed so that a plurality of the identification information holding bodies can be separated.
  22. 　前記構造体は、複数個の前記識別情報保持体を分離可能な脆弱部を有する、請求項１８～２１のいずれかに記載の構造体。 The structure according to any one of claims 18 to 21, wherein the structure has a fragile portion capable of separating a plurality of the identification information holding bodies.
  23. 　複数個の前記識別情報保持体を、各識別情報体間で分離可能に前記構造体の当該識別情報保持体の間が２０μｍ～１５００μｍの厚み、好ましくは４０μｍ～１０００μｍの厚み、さらに好ましくは５０～６００μｍの厚みを有する、請求項１８～２２のいずれかに記載の構造体。 A plurality of the identification information holding bodies are separable between the identification information bodies, the thickness between the identification information holding bodies of the structure is 20 μm to 1500 μm, preferably 40 μm to 1000 μm, more preferably 50 to The structure according to any one of claims 18 to 22, having a thickness of 600 袖 m.
  24. 　複数個の前記識別情報保持体は、前記障壁によって区画され、前記障壁を指標として相互に分離可能である、請求項１８～２３のいずれかに記載の構造体。 The structure according to any one of claims 18 to 23, wherein the plurality of identification information holding bodies are partitioned by the barrier and are separable from each other using the barrier as an index.
  25. 　複数個の前記識別情報保持体は、連続する又は単一の障壁を前記障壁として備える、請求項１８～２４のいずれかに記載の構造体。 The structure according to any one of claims 18 to 24, wherein the plurality of identification information holding bodies include a continuous or single barrier as the barrier.
  26. 　複数個の前記識別情報保持体は、接着剤又は粘着剤を含有する連続する又は単一の固定化層を前記構造体の表面又は裏面に備える、請求項１８～２５のいずれかに記載の構造体。 The structure according to any one of claims 18 to 25, wherein the plurality of identification information holding bodies include a continuous or single fixing layer containing an adhesive or a pressure-sensitive adhesive on the front surface or the back surface of the structure. body.
  27. 　前記固定化層を複数個の前記識別情報保持体の障壁に対して備える、請求項２６に記載の構造体。 27. The structure according to claim 26, wherein the fixing layer is provided against a plurality of barriers of the identification information holding body.
  28. 　前記固定化層を複数個の前記識別情報保持体の前記固相担体の前記情報保持領域が形成される面とは反対側の面に備える、請求項２６又は２７に記載の構造体。 28. The structure according to claim 26 or 27, wherein the immobilization layer is provided on a surface opposite to a surface on which the information holding region of the solid-phase carrier of the plurality of identification information holding bodies is formed.
  29. 　請求項１～１７のいずれかに記載の識別情報保持体の製造方法であって、
    　前記固相担体上に、前記１又は２以上のＤＮＡを固定して１又は２以上の前記情報保持領域を形成する工程と、
    　１又は２以上の前記情報保持領域を包囲する障壁となる障壁部材を前記固相担体上に固定する工程と、
    を備える、製造方法。     A method for producing an identification information holding body according to any one of claims 1 to 17,
    Immobilizing the one or two or more DNAs on the solid phase carrier to form one or more of the information holding regions;
    Fixing a barrier member serving as a barrier surrounding one or more of the information holding regions on the solid phase carrier;
    A manufacturing method comprising:
  30. 　前記障壁部材は、１又は２以上の前記情報保持領域に対応する開口パターンを有する、請求項２９に記載の製造方法。 The manufacturing method according to claim 29, wherein the barrier member has an opening pattern corresponding to one or more of the information holding regions.
  31. 　前記障壁部材は、接着剤又は粘着剤を備えて前記固相担体に固定化するための第１の固定化層と、前記固定化層に積層される障壁層とを備える、請求項２９又は３０に記載の製造方法。 The barrier member includes a first immobilization layer for immobilizing the solid phase carrier with an adhesive or a pressure-sensitive adhesive, and a barrier layer laminated on the immobilization layer. The manufacturing method as described in.
  32. 　前記障壁部材は、前記障壁層に積層される、粘着剤又は接着剤を含有する第２の固定化層を備える、請求項２９～３１のいずれかに記載の製造方法。 The manufacturing method according to any one of claims 29 to 31, wherein the barrier member includes a second fixing layer containing an adhesive or an adhesive, which is laminated on the barrier layer.
  33. 　さらに、前記障壁部材を介して、１又は２以上の前記情報保持領域の上方を覆うカバーを付与する工程と、備える、請求項２９～３２のいずれかに記載の方法。 The method according to any one of claims 29 to 32, further comprising a step of providing a cover that covers the upper part of the one or more information holding regions via the barrier member.
  34. 　前記固相担体は、前記固相担体の前記情報保持領域を形成する面と反対側の面には、粘着剤又は接着剤を含有する固定化層を備える、請求項２９～３３のいずれかに記載の方法。 The solid phase carrier is provided with an immobilization layer containing an adhesive or an adhesive on a surface of the solid phase carrier opposite to a surface forming the information holding region. The method described.
  35. 　請求項１～１７のいずれかに記載の識別情報保持体を準備する工程と、
    　前記識別情報保持体を識別対象とする物品に固定化する工程と、
    を備える、識別可能な物品の製造方法。     Preparing the identification information holding body according to any one of claims 1 to 17,
    Immobilizing the identification information holding body on an article to be identified;
    A method for producing an identifiable article.
  36. 　前記固定化工程は、前記情報保持領域が前記物品側に指向する又は前記識別情報が前記物品の外方を指向するように、前記識別情報保持体が前記物品に固定化する工程である、請求項３５に記載の方法。 The fixing step is a step in which the identification information holding body is fixed to the article such that the information holding area is directed to the article side or the identification information is directed to the outside of the article. Item 36. The method according to Item 35.
  37. 　識別対象の識別方法であって、
    　前記識別対象に付与された請求項１～１７のいずれかに記載の識別情報保持体の前記情報保持領域に対して、前記識別用塩基配列と相補的な塩基配列を有する１又は２以上のプローブを接触させる工程と、
    　前記識別用塩基配列と前記プローブとのハイブリダイゼーション産物を検出する工程と、
    を備える、方法。     An identification method of an identification object,
    The one or more probes having a base sequence complementary to the identification base sequence for the information holding region of the identification information holding body according to any one of claims 1 to 17 attached to the identification target A step of contacting
    Detecting a hybridization product of the identification base sequence and the probe;
    A method comprising:
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