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WO2006064871A1 - Composition de matiere grasse solide contenant de l’acide nucleique porteur de donnees - Google Patents

Composition de matiere grasse solide contenant de l’acide nucleique porteur de donnees Download PDF

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Publication number
WO2006064871A1
WO2006064871A1 PCT/JP2005/023039 JP2005023039W WO2006064871A1 WO 2006064871 A1 WO2006064871 A1 WO 2006064871A1 JP 2005023039 W JP2005023039 W JP 2005023039W WO 2006064871 A1 WO2006064871 A1 WO 2006064871A1
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WO
WIPO (PCT)
Prior art keywords
nucleic acid
information
solid fat
fat composition
information nucleic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP2005/023039
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English (en)
Japanese (ja)
Inventor
Hiroshi Yokoyama
Masahiko Yamanaka
Kentarou Watanabe
Tsunehiko Higuchi
Shigeki Hirabayashi
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nissan Motor Co Ltd
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Nissan Motor Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nissan Motor Co Ltd filed Critical Nissan Motor Co Ltd
Publication of WO2006064871A1 publication Critical patent/WO2006064871A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11C—FATTY ACIDS FROM FATS, OILS OR WAXES; CANDLES; FATS, OILS OR FATTY ACIDS BY CHEMICAL MODIFICATION OF FATS, OILS, OR FATTY ACIDS OBTAINED THEREFROM
    • C11C3/00—Fats, oils, or fatty acids by chemical modification of fats, oils, or fatty acids obtained therefrom

Definitions

  • the present invention relates to an information nucleic acid-containing solid fat composition, and more particularly to a solid fat composition containing an information nucleic acid that can be used for individual authentication.
  • DNA is an information biomolecule originally possessed by all living organisms and including all genetic information in each organism. Many of them correspond to the amino acid sequences of many proteins. In other words, deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC) and deoxythymine (dT) forces are bound with a certain direction through S-phosphate bond, and the number of bases If n is n, there are 4 n types of DNA of that length. Thus, for example, there can be about 4.3 billion distinct DNAs with only 16 bases. At present, any DNA sequence with several tens of base sequences can be synthesized arbitrarily. In addition, if the amount of DNA exceeds a certain level, the sequence can be automatically determined by an automatic sequence reader (sequencer).
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2004-159502
  • Patent Document 1 basically relates to a method of mixing DNA and a water non-solvent, and as a method for confirming the authenticity of a product, the presence or absence of amplification of ribonucleic acid by a PCR method. By confirming the above, it is only shown that the target product containing ribonucleic acid is identified.
  • the present invention has been made in view of the above-described problems of the prior art, and the purpose of the invention is to specifically identify the source / history product.
  • An object of the present invention is to provide a solid fat composition containing an information nucleic acid.
  • the information nucleic acid-containing solid fat composition of the present invention is a solid fat composition containing an information nucleic acid having a site having an arbitrary and known base sequence.
  • the target product can be individually authenticated by determining the base arrangement 1J of the information nucleic acid contained in a trace amount.
  • solid fat means that whose viscosity exceeds lOO (mPa's) at a temperature of 30 ° C.
  • the information nucleic acid-containing solid fat composition of the present invention as described above is a solid fat composition containing an information nucleic acid having a site having an arbitrary and known base sequence.
  • an ester-based or paraffin-based one that is not particularly limited can be used.
  • ester-based oil and fat components include higher fatty acid and glycerin triesters and higher fatty acid and higher alcohol monoesters.
  • Paraffin-based fat and oil components include, for example, 16 to 40 carbon atoms, particularly carbon number. Mention may be made of 20-30 paraffins.
  • Such a solid oil composition can be used for, for example, wax, cosmetics, sarcophagus, and the like, and can contain information nucleic acids that are difficult to remove, and thus can be specifically and specifically authenticated.
  • the information nucleic acid means DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and derivatives thereof, which may be natural type or artificial type, but are included in a solid oil composition that is used in harsh environments. In view of this, it is preferable to use an artificial mold that is structurally stable. In the artificial type, a sequence that does not exist in the natural type can be formed.
  • the fact that the base sequence site is arbitrary indicates that it can be selected at random as long as it is a detectable base sequence, and that the base sequence site is known is used for individual authentication. It shows that the base sequence to be used is known beforehand.
  • the content of the information nucleic acid used is preferably from 0.5 to 500 / ig, preferably from 50 to: 100, and more preferably from 100 to 100 g of the composition. ,.
  • the information nucleic acid may be detected from the solid fat composition, and the base coordination IJ may not be determined. If it exceeds 500 / g, the information nucleic acid may be identified. The impact is not good, but the cost is high.
  • the size of the information nucleic acid is preferably such that the number of bases in the whole nucleic acid is 200 or less. If it exceeds 200, unreacted sites are generated little by little at the synthesis stage, and the content of those lacking bases easily increases. More preferably, it is about 100 bases.
  • thymines are not adjacent to each other in the above base sequence. As a result, dimerization of thymine can be suppressed.
  • the information nucleic acid is derived from a protective group from the viewpoint of improving stability when used in combination with a compound that reacts with a ⁇ H group or when used in a harsh environment. It is preferable that Specifically, the hydroxyl group in one or both of the 5 and 3 groups is derivatized using a phosphate ester group, an acylol group, an alkoxycarbonyl group, a benzyl group, a substituted benzynole group and an aryl group. be able to.
  • Fig. 1 (A) shows natural DNA, and (B) shows DNA derivatized at the 5 'position.
  • the hydroxyl group at the 5 ′ position is preferable to derivatize the hydroxyl group at the 5 ′ position with piotin or a fluorescent molecule.
  • vithion when vithion is used, it is easily adsorbed selectively on a column to which a protein called avidin is bound.
  • avidin a protein called avidin is bound.
  • a fluorescent molecule such as fluorescein
  • the nucleic acid itself becomes fluorescent, so that it can be detected with high sensitivity and purification becomes easy. In this way, individual authentication becomes extremely easy if the convenience of isolation and purification is enhanced.
  • the product extracted with water can be easily separated by passing it through a column of a carrier coated with gold (Au).
  • Au gold
  • two standing hydroxyl groups can be derivatized with the above protecting groups from the viewpoint of improving stability.
  • the above base sequence site is a site used for amplification of the information nucleic acid It is preferable that As a powerful method for amplifying information nucleic acid, polymerase chain reaction (PCR) that can be amplified synergistically can be appropriately employed.
  • PCR polymerase chain reaction
  • thermostable DNA polymerase even if the amount of information nucleic acid is extremely small, it can be amplified extremely. For example, temperature control is performed in the presence of a base (primer) complementary to several tens of bases of DNA. When the thermostable DNA polymerase is allowed to act, the original DNA can be doubled. For example, if this is repeated 30 times, it can be amplified several hundred million times.
  • Such amplification makes it possible to obtain a sufficient amount of DNA to determine its sequence even from a very small amount of sample, and by extension, the “identity” of the product that contained it from the information corresponding to the sequence. Will be revealed.
  • the sites used for the amplification have primer corresponding sites necessary for polymerase chain reaction (PCR) at both ends. That is, as informational nucleic acid Although it is possible to use one that does not have a primer-corresponding site, it is possible to identify in a shorter time by providing a primer-corresponding site.
  • PCR polymerase chain reaction
  • the lower limit of the number of bases is preferably 5. More preferably, it is 10 or more. If the number of bases is less than 5, the number of distinguishable nucleic acids decreases, and it takes time to identify many products.
  • the upper limit for the number of bases is preferably 100. This is because when the number of bases exceeds 100, the ratio of by-products lacking a base at any position increases, and purification takes time and tends to be difficult in some cases.
  • RNA When RNA is used as the information nucleic acid, DNA complementary to the sequence can be obtained using reverse transcriptase, and PCR can be performed using this DNA.
  • the information nucleic acid further has an authentication information part in addition to the base sequence part, so that individual authentication can be performed by setting more detailed information.
  • an authentication information part in addition to the base sequence part, so that individual authentication can be performed by setting more detailed information.
  • FIG. 2 in the case of information DNA having primer-corresponding sites at both ends, a sequence of m bases is placed in the center (B to B), and the sequence information of this portion is used as authentication information.
  • sequences (X to X, P to P) complementary to 1 (el) and n primers are ligated, respectively.
  • the presence of this part is the first time PCR
  • the information DNA can be used as an information element as a single-stranded DNA or a double-stranded DNA complexed with a complementary sequence of DNA.
  • This primer-corresponding site IJ can be devised so that the binding of complementary sequences is as stable as possible and amplification by the PCR method proceeds smoothly.
  • each of X to X, B to B, and P to P is deoxy.
  • dA adenosine
  • dG deoxyguanosine
  • dC deoxycytosine
  • dT deoxythymine
  • the solution of the information nucleic acid extracted from the solid fat composition force, the PCR buffer solution, the sterilization Mix distilled water, at least one primer, 2,3_dioxynucleoside triphosphate (dNTP) and polymerase, (1) heat at 92-95 °° for 2-5 minutes, then ( 2a) 92-95. (2b) 20-50 for 30-60 seconds at C. 30-60 seconds at C, (2c) 70-80. In C 30 ⁇ : 120 ⁇ 50 times, after 20 ⁇ 50 times of the heat cycle, (3) 70 ⁇ 80. Heat treatment is preferably performed for 1 to 10 minutes. It is preferable to use two types of primers from the viewpoint of increasing the arbitraryness of the base sequence of the information nucleic acid.
  • dNTP 2,3_dioxynucleoside triphosphate
  • (2b) 30 seconds at 40 ° C is particularly preferred. If it is shorter than 30 seconds at 20 ° C, it will be difficult to bind the primer and DNA, and if it is longer than 60 seconds at 50 ° C, the enzyme will be deactivated. Also, in (2c), 30 seconds at 72 ° C Is particularly preferred. If it is shorter than 30 seconds at 70 ° C, the elongation becomes insufficient, and if it is longer than 120 seconds at 80 ° C, the enzyme is inactivated.
  • the repetition of the heating cycles (2a) to (2c) is because the amplification factor decreases if 30 times is less than 20 times, which is particularly preferable, and if it exceeds 50 times, time is wasted.
  • FIG. 3 shows a flow chart of an embodiment of a method for detecting the information nucleic acid contained in the information nucleic acid-containing solid fat composition.
  • S1 information DNA is extracted from the solid fat composition.
  • S2 concentrate by lyophilization.
  • S3 add two types of primers and polymerase.
  • S4 DNA is amplified by repeating PCR.
  • S5 the remaining excess primer is degraded by single-stranded DNA cleavage enzyme.
  • S6 double-stranded information DNA is purified by gel filtration.
  • S7 the sequencer determines the sequence.
  • the solid fat composition is mixed with a small amount of water, for example, when the information DNA is chemically bound when it is supported on the fine particles, Hydrolysis Etc., it can be extracted efficiently.
  • it may be concentrated using, for example, a centrifugal evaporator.
  • Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Vent polymerase, Pfu polymerase, Bca BEST polymerase, KOD DNA polymerase, etc. can be used as the single-stranded DNA cleavage enzyme.
  • the target DNA may be amplified between S6 and S7 by repeating the same operations as in S3 and 4.
  • the sequence determination may be performed by a mass spectrometer, or may be combined with the sequence determination by a sequencer.
  • the base sequence it is desirable to compare the information nucleic acid data extracted from the product with the information nucleic acid database including at least the information nucleic acid data. This is because the time required for product certification can be significantly reduced by comparing with a database of information nucleic acids obtained in advance.
  • Examples of data stored in such a database include the electrophoresis time and the distance traveled by gel filtration (this is sufficient if the information nucleic acid itself is allowed to flow into the control lane).
  • the contained information nucleic acid is supported on fine particles.
  • “supporting” means that the information nucleic acid is in close contact with the fine particles so that they can move together with the fine particles, and the information nucleic acids are attached to the surfaces of the fine particles. Even if it is firmly fixed, it may adhere to the surface of the fine particles or in the recesses with a strength sufficient to withstand use.
  • nucleic acid Since the nucleic acid is water-soluble, it cannot be said that there is no possibility that it will flow out of the product together with water even if it is incorporated in the product.
  • the fine particles are not particularly limited as long as they can carry an information nucleic acid, but in addition to silica and zinc oxide, titanium oxide, molybdenum oxide, tungsten oxide, nor titanate and the like are suitable. Can be used. In addition, the dispersibility at the time of mixing with a solid fat composition can be improved by silica-coating the surface.
  • the content of the fine particles is 0.5 with respect to the total amount of the composition. It is preferably ⁇ 50%, more preferably 0.5 to 5%.
  • the content of fine particles is less than 0.5%, the amount of fine particles in the sampled solid fat composition may be small, and the detection accuracy may be reduced. On the other hand, if it exceeds 50%, the applicability of wax is reduced and the skin may be damaged in the case of cosmetics.
  • the average particle size is preferably 0.01 to 5 ⁇ m, more preferably 0.02 to: l x m.
  • the coating film may be damaged in the case of wax. In the case of cosmetics, there is a possibility of damaging the skin.
  • the information nucleic acid-carrying fine particles are formed by, for example, dissolving the information nucleic acid as it is in a suspension obtained by adding sterile distilled water to the fine particles, or by dissolving a part or all of the information nucleic acid in sterile distilled water. It can be obtained by adding as a solution, desirably adding a specific solvent, and drying.
  • the information nucleic acid can be reliably supported on the surface of the fine particles by drying after being held in a state, and the information nucleic acid is supported on the fine particles by using such information nucleic acid-supported fine particles.
  • the information nucleic acid can be reliably added to the solid fat composition and stably dispersed as compared with the case of introducing the information nucleic acid alone.
  • the suspension includes alcohol (eg, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, etc.), ester (eg, ethyl acetate, butyl acetate, Propyl acetate, etc.), ketones (eg, acetone, dimethyl ketone, methyl ethyl ketone, jetyl ketone, etc.) and aromatic solvents ( It is desirable to further add a solvent such as toluene, hexane, cyclohexane, xylene, etc. This improves the dispersibility of the fine particles in the suspension, and after the addition of the information nucleating acid. The volatilization of the water and solvent will be promoted.
  • alcohol eg, methanol, ethanol, propanol, butanol, pentanol, hexanol, hept
  • solvents are not limited to only one type, and two or more types of solvents can be used in combination. These solvents may be added at the same time as the information nucleic acid or after the information nucleic acid is added.
  • the volume ratio of sterilized distilled water Z alcohol is in the range of 1 to 99, that is, a solvent other than alcohol, that is, an ester.
  • the volume ratio of sterile distilled water Z solvent is preferably in the range of 1 to 75.
  • Informational DNA represented by SEQ ID NO: 1 (including a verifiable DNA represented by SEQ ID NO: 2) was synthesized by sequentially binding nucleosides by the phosphoramidite method.
  • an information DNA represented by SEQ ID NO: 1 was used as the information nucleic acid.
  • Zinc oxide with an average particle diameter of 0.02 xm (ZS-032 manufactured by Showa Denko KK) coated with silica was used as fine particles for the carrier, and 30% ethanol by volume ratio was added to 4 g of the zinc oxide. 15 mL of sterilized distilled water contained was added and stirred to obtain a zinc oxide suspension.
  • the fully dried lump fine powder was put in a mortar and crushed to obtain information nucleic acid-carrying fine particles as the original fine powder.
  • fine particles for the carrier zinc oxide with an average particle size of 0.02 xm (ZS _032 manufactured by Showa Denko KK) coated with silica coating, aluminum oxide with an average particle size of 5 ⁇ m coated with silica coating (Co., Ltd.) Using Micron's AX10-32) or silica-coated ano-reminium oxide with an average particle diameter of 20 ⁇ m (Micron's AX116), the content of the information nucleic acid carried on the microparticles is adjusted, or The information-nucleated acid-containing solid fat composition (wax) of this example was prepared by adjusting the content of the information-containing nucleic acid-carrying fine particles. Tables 1 and 2 show the specifications of the information nucleic acid-containing solid oil composition (wax) in each of the above examples.
  • Zinc phosphate-treated 70111111 150111111 0.8mmt dull steel plate with cationic electrodeposition paint (Nippon Paint Co., Ltd., trade name "Power Top U600M”) to have a dry film thickness of 20 zm
  • a gray paint made by NOF Corporation, trade name “HIEIPICO No. 500” was applied thereon.
  • the coating was applied to a dry film thickness of 30 xm and baked at 140 ° C for 30 minutes to form an intermediate coating layer and a base coat layer.
  • Talia paint (made by Nippon Paint Co., Ltd., trade name “Super Parrak O-130 GN3”) was applied so that the dry film thickness was 30 zm, and 3 ° C at 140 ° C.
  • a talya layer was formed by baking for 0 minutes.
  • test pieces in each of the above examples were washed with a detergent (manufactured by Taiho Kogyo Co., Ltd., Clean View) to remove the wax component, and then the degree of scratches on the coating film surface was visually confirmed.
  • a detergent manufactured by Taiho Kogyo Co., Ltd., Clean View
  • the results obtained are also shown in Tables 1 and 2. In the table, “o” indicates “no flaw” and “ ⁇ ” indicates “slightly flawed”.
  • the information nucleic acid-containing solid fat composition of the present invention can identify the information nucleic acid.
  • Example 18 has a smaller number of PCR amplifications than the information-containing nucleic acid-containing solid fat composition of Examples 13 to 17: It can be seen that the information nucleic acid can be identified.
  • the information-containing nucleic acid-containing solid fat composition of Examples 1 to 17 can form a wax coating without damaging the coating surface.
  • FIG. 1 is a structural formula showing natural DNA and DNA in which the 5 ′ position is derivatized.
  • FIG. 2 is a schematic diagram showing an information nucleic acid having primer-corresponding sites at both ends at the authentication information site.
  • FIG. 3 is a flowchart showing one embodiment of a method for detecting an information nucleic acid contained in an information nucleic acid-containing solid fat composition.

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  • Chemical & Material Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Detergent Compositions (AREA)
  • Fats And Perfumes (AREA)
  • Cosmetics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention concerne une composition de matière grasse solide contenant un acide nucléique porteur de données, ledit acide nucléique porteur de données permettant la détermination individuelle et spécifique de l’origine et de l’historique du produit. Ledit acide nucléique porteur de données contenu par ladite composition de matière grasse solide comprend un site portant une séquence de base arbitraire et connue, ledit acide nucléique porteur de donnés étant véhiculé par de fines particules ; de plus, la teneur dudit acide nucléique porteur de données dans ladite composition de matière grasse solide s'inscrit dans la plage de 0,5 à 500 µg pour 100 g de la composition.
PCT/JP2005/023039 2004-12-15 2005-12-15 Composition de matiere grasse solide contenant de l’acide nucleique porteur de donnees Ceased WO2006064871A1 (fr)

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JP2004-362147 2004-12-15
JP2004362147A JP2006169338A (ja) 2004-12-15 2004-12-15 情報化核酸含有固形油脂組成物

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10355494A1 (de) 2003-11-27 2005-07-07 Webasto Ag System und Verfahren zum Umsetzen von Brennstoff und Oxidationsmittel zu Reformat

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0707790D0 (en) * 2007-04-23 2007-05-30 Oxford Ancestors Ltd Nucleic acid-containing media

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63503242A (ja) * 1986-04-09 1988-11-24 バイオタル・リミテツド 真偽を立証したい物品の標識
WO2004035831A1 (fr) * 2002-10-16 2004-04-29 Bioneer Corporation Procede permettant d'identifier un vehicule et marqueur d'oligonucleotide utilise pour ce faire

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS63503242A (ja) * 1986-04-09 1988-11-24 バイオタル・リミテツド 真偽を立証したい物品の標識
WO2004035831A1 (fr) * 2002-10-16 2004-04-29 Bioneer Corporation Procede permettant d'identifier un vehicule et marqueur d'oligonucleotide utilise pour ce faire

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
MAHLER ET AL: "DNA-labeled clay: A sensitive new method for tracing particle transport", GEOLOGY, vol. 26, no. 9, 1998, pages 831 - 834, XP002996483 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10355494A1 (de) 2003-11-27 2005-07-07 Webasto Ag System und Verfahren zum Umsetzen von Brennstoff und Oxidationsmittel zu Reformat

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