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WO2012046859A1 - Support d'information d'identification pour identifier le sujet à identifier et son utilisation - Google Patents

Support d'information d'identification pour identifier le sujet à identifier et son utilisation 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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WO
WIPO (PCT)
Prior art keywords
information holding
identification
barrier
identification information
solid phase
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Ceased
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PCT/JP2011/073269
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English (en)
Japanese (ja)
Inventor
孝介 丹羽
廣田　寿一
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NGK Insulators Ltd
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NGK Insulators Ltd
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Priority to JP2012537783A priority Critical patent/JPWO2012046859A1/ja
Publication of WO2012046859A1 publication Critical patent/WO2012046859A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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.

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Abstract

Cette invention concerne un support d'information d'identification capable de porter une information d'identification de manière stable et de maintenir une bonne capacité d'identification. Le support d'information d'identification selon la présente invention comprend : un support solide ; une ou plusieurs zones portant l'information et contenant un ou plusieurs fragments d'ADN immobilisés sur le support solide, lesdits fragments d'ADN ayant, à titre de données d'identification, une séquence de bases à des fins d'identification qui a été préalablement liée au sujet à identifier ; et une ou plusieurs barrières entourant lesdites zones portant l'information.
PCT/JP2011/073269 2010-10-07 2011-10-07 Support d'information d'identification pour identifier le sujet à identifier et son utilisation Ceased WO2012046859A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2013000047A (ja) * 2011-06-15 2013-01-07 Univ Of Tokyo Ｄｎａインクを用いたオンサイト物品認証システム
EP2594639A4 (fr) * 2010-07-16 2014-02-05 Ngk Insulators Ltd Support qui porte des informations d'identification pour l'identification d'un sujet d'identification et son utilisation

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Publication number Priority date Publication date Assignee Title
JP2002357604A (ja) * 2001-04-17 2002-12-13 Nisshinbo Ind Inc 反応容器及びこれを用いる生物学的に活性な物質の分析方法
WO2004092376A1 (fr) * 2003-04-11 2004-10-28 Olympus Corporation Methode de designation de sequences normalement orthogonales, methode de production d'acides nucleiques se presentant normalement sous forme de sequences orthogonales et acides nucleiques ainsi obtenus
WO2005012574A2 (fr) * 2003-04-29 2005-02-10 Genvault Corporation Code a barres biologique
WO2005111212A2 (fr) * 2004-04-29 2005-11-24 Genvault Corporation Code-barres biologique
JP2006167558A (ja) * 2004-12-15 2006-06-29 Nissan Motor Co Ltd 補修塗膜及び補修塗装方法
WO2006101229A1 (fr) * 2005-03-25 2006-09-28 Ngk Insulators, Ltd. Matrice de sondes et son procédé de fabrication

Patent Citations (6)

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Publication number Priority date Publication date Assignee Title
JP2002357604A (ja) * 2001-04-17 2002-12-13 Nisshinbo Ind Inc 反応容器及びこれを用いる生物学的に活性な物質の分析方法
WO2004092376A1 (fr) * 2003-04-11 2004-10-28 Olympus Corporation Methode de designation de sequences normalement orthogonales, methode de production d'acides nucleiques se presentant normalement sous forme de sequences orthogonales et acides nucleiques ainsi obtenus
WO2005012574A2 (fr) * 2003-04-29 2005-02-10 Genvault Corporation Code a barres biologique
WO2005111212A2 (fr) * 2004-04-29 2005-11-24 Genvault Corporation Code-barres biologique
JP2006167558A (ja) * 2004-12-15 2006-06-29 Nissan Motor Co Ltd 補修塗膜及び補修塗装方法
WO2006101229A1 (fr) * 2005-03-25 2006-09-28 Ngk Insulators, Ltd. Matrice de sondes et son procédé de fabrication

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2594639A4 (fr) * 2010-07-16 2014-02-05 Ngk Insulators Ltd Support qui porte des informations d'identification pour l'identification d'un sujet d'identification et son utilisation
JP2013000047A (ja) * 2011-06-15 2013-01-07 Univ Of Tokyo Ｄｎａインクを用いたオンサイト物品認証システム

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