JP2006169337A - Opaque resin composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an opaque resin composition containing an informative nucleic acid that can individually and concretely specify the origin and history of a particular product. <P>SOLUTION: The opaque resin composition contains an informative nucleic acid having a moiety having an arbitrary and known base sequence. The opaque resin composition is embodied by one in which the informative nucleic acid is carried by microparticles or one in which the content of the informative nucleic acid is 0.5 to 500 μg per 100 g of the composition. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to an opaque resin composition, and more particularly to an opaque resin composition containing an information nucleic acid that can be used for individual authentication.

  Conventionally, for individual authentication, a license plate, watermark printing such as banknotes, an IC chip and a photo of a credit card are used. However, these individual authentication means have a drawback that they can be removed from the product by peeling, cutting, erasing or the like. For this reason, it has been expected to develop authentication information that cannot be removed from the product, that is, does not disappear.

On the other hand, 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. That is, deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), and deoxythymine (dT) are bonded with a certain direction through a phosphate ester bond, and the number of bases is n. Thus, there are ⁴ⁿ types of DNA having that length. Thus, for example, there can be about 4.3 billion types of distinguishable DNAs with only 16 types of bases. At present, any DNA having a sequence of several tens of bases can be arbitrarily synthesized. In addition, if the amount of DNA exceeds a certain level, the sequence can be automatically determined by an automatic sequence reader (sequencer).

From such a background, it has been proposed to use a forgery-preventing label in which DNA is contained in a water-insoluble medium in a product to clarify the authenticity of the product by using the presence or absence of the DNA as a clue. (See Patent Document 1).
JP 2004-159502 A

  However, the technique described in Patent Document 1 basically relates to a method of mixing DNA and a water non-solvent, and the authenticity of a product is confirmed by the presence or absence of amplification of ribonucleic acid by PCR. Thus, it has only been shown to identify target products containing ribonucleic acid. In addition, authenticity verification data that uses the presence / absence of DNA as a test index is data related to individual authentication that uses DNA sequence as a test index and enables authentication for each product even for the same type of product. Is not disclosed.

  On the other hand, in traffic accidents, there is a demand for specifying a target vehicle from a paint film piece, a glass piece, a plastic piece, etc. left at the accident site.

  The present invention has been made in view of such problems of the prior art, and the object of the present invention is to provide an informatized nucleic acid capable of individually and specifically identifying the source / history product. It is in providing the opaque resin composition containing.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have recognized the nucleic acid typified by DNA as one piece of authentication information, and later detected the information nucleic acid in the product. As a result, the present invention has been completed.

  That is, the opaque resin composition of the present invention is an opaque resin composition containing an information nucleic acid having a site having an arbitrary and known base sequence.

  According to the present invention, even for mass-produced products such as industrial products, the target product can be individually authenticated by determining the base sequence of the information nucleic acid contained in a trace amount.

  Hereinafter, the opaque resin composition of the present invention will be described in detail. In the present specification and claims, “%” indicates a mass percentage unless otherwise specified. “Opaque” means that the light transmittance is less than 50%.

  As described above, the opaque resin composition of the present invention is an opaque resin composition containing an information nucleic acid having a site having an arbitrary and known base sequence. As the opaque resin, a conventionally known one can be used, and specifically, a material selected from polypropylene (PP), polycarbonate (PC), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC) and the like. A mixture of suitable pigments can be used. Since such an opaque resin composition contains an information nucleic acid that is difficult to remove, it can be individually and specifically authenticated.

  Here, information nucleic acid refers to DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and derivatives thereof, which may be natural or artificial, but are considered to be included in opaque resin compositions where the usage environment is severe. Then, 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.

  Moreover, in the information nucleic acid, that the base sequence site is arbitrary indicates that it can be randomly selected 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 is known beforehand.

  The information nucleic acid used in the present invention is not particularly limited, but the content of the information nucleic acid is preferably 0.5 to 500 μg, preferably 50 to 100 μg, relative to 100 g of the composition. It is more preferable. If the amount is less than 0.5 μg, there is a possibility that the information nucleic acid cannot be detected from the opaque resin composition and the base sequence cannot be determined. If the amount exceeds 500 μg, the color is changed when moisture penetrates into the opaque resin composition. There is a high possibility that consistency will be reduced.

  In the present invention, the size of the information nucleic acid is preferably such that the number of bases in the whole nucleic acid is 200 or less. When it exceeds 200, unreacted sites are generated little by little at the stage of synthesis, and the content of those lacking a base tends to increase. More preferably, it is about 100 bases.

  Furthermore, it is preferable that thymines are not adjacent to each other in the above base sequence. Thereby, it can suppress that thymine dimerizes.

  In the present invention, the information nucleic acid is preferably derivatized with a protecting group from the viewpoint of improving stability when used in combination with a compound that reacts with an OH group or when used in a severe environment. . Specifically, a hydroxyl group at one or both of the 5′-position and the 3′-position is derivatized with a phosphate ester group, an acyl group, an alkoxycarbonyl group, a benzyl group, a substituted benzyl group, an allyl group, or the like. can do. FIG. 1A shows natural DNA, and FIG. 1B shows DNA derivatized at the 5 ′ position.

  Furthermore, from the viewpoint of enhancing the convenience of isolation and purification, it is preferable to derivatize the hydroxyl group at the 5 ′ position with biotin or a fluorescent molecule. Specifically, when vithion is used, it is easily adsorbed selectively on a column to which a protein called avidin is bound. On the other hand, when a fluorescent molecule such as fluorescein is used, the nucleic acid itself becomes fluorescent, so that it can be detected with high sensitivity and purification is facilitated. Thus, when the convenience of isolation and purification is increased, individual authentication becomes extremely easy.

  Furthermore, for example, when the 5′-position is substituted with sulfur, it can be easily separated by passing the column extracted with water through a carrier column coated with gold (Au). When RNA is used as the information nucleic acid, the 2′-position hydroxyl group can be derivatized with the above-described protecting group from the viewpoint of improving stability.

  In addition, in the case of performing individual authentication by extracting an information nucleic acid, the base sequence portion may be a portion used for amplification of the information nucleic acid from the viewpoint of efficiently detecting even a small content. preferable. As a method for amplifying such information nucleic acid, polymerase chain reaction (PCR) that can be amplified synergistically can be appropriately employed.

  In the PCR method, even if the amount of information nucleic acid is extremely small, it can be amplified extremely. For example, while controlling the temperature in the presence of a base (primer) complementary to several tens of bases of DNA, When the sex 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. As a result, the “identity” of the product that contained it from the information corresponding to the sequence can be obtained. Will be revealed.

  At this time, it is preferable that the sites used for the amplification have primer-corresponding sites necessary for polymerase chain reaction (PCR) at both ends. That is, as the information nucleic acid, a nucleic acid that does not have a primer-corresponding site can be used, but by providing the primer-corresponding site, it becomes possible to identify in a shorter time.

  For such a primer corresponding site, the lower limit of the number of bases is preferably 5. More preferably, it is 10 or more. When the number of bases is less than 5, the number of distinguishable nucleic acids decreases, and it takes time to identify many products. On the other hand, the upper limit of 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 becomes high, so that purification takes time and tends to be difficult in some cases.

  In addition, 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.

  Further, the information nucleic acid preferably further has an authentication information site in addition to the base sequence site, whereby individual authentication can be performed with more detailed information setting.

For example, as shown in FIG. 2, in the case of information-oriented DNA having primer-corresponding sites at both ends, a sequence with m bases is placed in the center (B ₁ -B _m ), and the sequence information of this part is authenticated. Correspond to information. At both ends, sequences (X _{1 to} X ₁ , P _{1 to} P _n ) complementary to l (el) and n primers, respectively, are linked. The presence of this part makes it possible to adopt the PCR method for the first time.

  The information DNA can be used as an information element of this single-stranded DNA or a double-stranded DNA complexed with a complementary sequence of DNA. The sequence of the primer corresponding site can be devised so that the binding of complementary sequences is as stable as possible and amplification by the PCR method proceeds smoothly.

In FIG. 2, each of X _{1 to} X ₁ , B _{1 to} B _m , and P _{1 to} P _n is deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), or deoxythymine (dT). And any combination thereof.

  As a preferable example of the PCR method, in determining the base sequence of the information nucleic acid contained in the opaque resin composition, a solution of the information nucleic acid extracted from the opaque resin composition, a PCR buffer, sterile distilled water, Mix one primer, 2,3-dideoxynucleoside triphosphate (dNTP) and polymerase, (1) heat at 92-95 ° C. for 2-5 minutes, then (2a) 30-92 at 92-95 ° C. The heating cycle of (2b) at 20 to 50 ° C. for 30 to 60 seconds, (2c) at 70 to 80 ° C. for 30 to 120 seconds is repeated 20 to 50 times, and then (3) at 70 to 80 ° C. It is preferable to heat-process for 1 to 10 minutes. In addition, it is preferable to use two types of primers from the viewpoint of enhancing the arbitraryness of the base sequence of the information nucleic acid.

  In (1), 5 minutes at 94 ° C. is particularly preferable. If it is shorter than 2 minutes at 92 ° C., it will be difficult to separate the DNA into two differences, and if it is longer than 5 minutes at 95 ° C., the enzyme will be inactivated. Note that this is not necessary when the information nucleic acid contained in the product is a single strand.

  Further, in (2a), 30 seconds at 94 ° C. is particularly preferable. This is because the amplification factor decreases when the temperature is shorter than 30 seconds at 92 ° C., and the enzyme is deactivated when the temperature is longer than 60 seconds at 95 ° C.

  Further, in (2b), 30 seconds at 40 ° C. is particularly preferable. This is because, when it is shorter than 30 seconds at 20 ° C., it becomes difficult to bind the primer and DNA, and when it is longer than 60 seconds at 50 ° C., the enzyme is deactivated.

  Further, in (2c), 30 seconds at 72 ° C. is particularly preferable. This is because the elongation becomes insufficient when the temperature is shorter than 30 seconds at 70 ° C., and the enzyme is deactivated when the temperature is longer than 120 seconds at 80 ° C.

  Further, in (3), 7 minutes at 72 ° C. is particularly preferable. This is because if the temperature is shorter than 1 minute at 70 ° C., the elongation becomes insufficient, and if it is longer than 10 minutes at 80 ° C., time is wasted.

  Furthermore, the repetition of the heating cycles (2a) to (2c) is particularly preferably 30 times, and if it is less than 20 times, the amplification factor decreases, and if it is more than 50 times, time is wasted.

  FIG. 3 shows a flowchart of an embodiment of a method for detecting an information nucleic acid contained in an opaque resin composition containing the information nucleic acid. As shown in the figure, information DNA is extracted from the opaque resin composition in S1. In S2, it is concentrated by lyophilization. In S3, two kinds of primers and a polymerase are added. In S4, DNA is amplified by repeating PCR. In S5, the remaining excess primer is degraded with a single-stranded DNA cleavage enzyme. In S6, the double-stranded information DNA is purified by gel filtration. In S7, sequencing is performed by a sequencer.

  Here, in S1, for example, the opaque resin composition may be powdered and mixed with a small amount of water. For example, when the information DNA is chemically bonded to the fine particles, hydrolysis may be performed. Can be extracted more efficiently. Moreover, in S2, you may concentrate using a centrifugal evaporator, for example. Further, in S5, for example, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Vent polymerase, Pfu polymerase, Bca BEST polymerase, KOD DNA polymerase and the like can be used as the single-stranded DNA cleaving enzyme. Further, the target DNA may be amplified between S6 and S7 by repeating the same operations as in S3 and S4. In S7, sequencing may be performed by a mass spectrometer, or may be combined with sequencing by a sequencer.

  In determining the base sequence, it is desirable to compare the data of the information nucleic acid extracted from the product with the information nucleic acid database including at least the data of the information nucleic acid. This is because the time required for product authentication can be greatly reduced by comparing with a database of information nucleic acids obtained in advance.

  Examples of data stored in such a database include electrophoresis time and movement distance when gel filtration is performed (this is sufficient if the information nucleic acid itself is allowed to flow in the control lane).

  In the present invention, the contained information nucleic acid is preferably carried on fine particles.

  Here, “supported” in the present invention means that the information nucleic acid is in close contact with the fine particles so as to be able to move together with the fine particles, and the information nucleic acid is attached to the surfaces of the fine particles. Even if it is firmly fixed, it may be adhered to the surface of the fine particles or in the recesses with a strength sufficient to withstand use. 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. In addition to silica and zinc oxide, titanium oxide, molybdenum oxide, tungsten oxide, barium titanate, and the like can be suitably used. . In addition, the dispersibility at the time of mixing with a resin composition can be improved by silica-coating the surface.

  In the present invention, the content of the fine particles is preferably 0.5 to 50%, more preferably 0.5 to 2% with respect to the composition. When the content of fine particles is less than 0.5%, the amount of fine particles in the sampled opaque resin composition may be small, and the detection accuracy may be reduced. Moreover, when it exceeds 50%, color consistency may fall.

  Furthermore, the size of the fine particles is preferably 0.01 to 40 μm, more preferably 0.02 to 0.1 μm. When the average particle size of the microparticles is less than 0.01 μm, the detection accuracy of the information nucleic acid decreases, and when it exceeds 40 μm, the dispersibility in a solvent such as sterilized distilled water or alcohol tends to decrease. .

  Furthermore, the amount of the information nucleic acid supported on the fine particles is preferably 0.5 to 500 μg, more preferably 10 to 100 μg, based on 100 g of the fine particles. When the amount of the information nucleic acid supported is less than 0.5 μg per 100 g of fine particles, the information nucleic acid cannot be stably added to the opaque resin composition, and the detection accuracy is lowered, and the information nucleic acid exceeds 500 μg. However, there is a tendency that the information nucleic acid cannot be stably supported on the fine particles, and a free information nucleic acid is generated and difficult to disperse.

  Such information-containing nucleic acid-carrying fine particles are added, for example, to a suspension obtained by adding sterilized distilled water to fine particles, or as a solution obtained by dissolving a part or all of the information nucleic acid in sterilized distilled water as it is. Desirably, a specific solvent can be added and dried.

  A solution in which an information nucleic acid having a site having an arbitrary and known base sequence is dissolved directly in a suspension of fine particles such as silica and zinc oxide, or a part or all of the information nucleic acid is dissolved in sterilized distilled water. After being added in the state, the information nucleic acid can be reliably supported on the surface of the fine particles by drying, and the information nucleic acid is supported on the fine particles by using such information nucleic acid-supported fine particles. By introducing it into the opaque resin composition, it becomes possible to reliably add the information nucleic acid to the opaque resin composition and stably disperse it compared to the case where it is introduced in the state of the information nucleic acid alone. Moreover, it is possible to prevent outflow of information nucleic acid due to moisture, and it is extremely excellent that individual authentication of the origin and history of products using the base sequence of information nucleic acid as a detection index is possible over a long period of time. Fruit is brought about.

  In addition, the suspension includes alcohol (eg, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, etc.), ester (eg, ethyl acetate, butyl acetate, propyl acetate, etc.), ketone ( For example, acetone, dimethyl ketone, methyl ethyl ketone, diethyl ketone, etc.) and aromatic solvents (toluene, hexane, cyclohexane, xylene, etc.) are preferably added to improve the dispersibility of fine particles in suspension. At the same time, volatilization of moisture and solvent after the addition of the information nucleic acid is promoted.

  In addition, these solvents are not limited to only 1 type, It is also possible to use 2 or more types of solvents together. These solvents may be added at the same time as the information nucleic acid or after the information nucleic acid is added.

  Further, as the amount of the solvent added, in the case of alcohol, the volume ratio of sterilized distilled water / alcohol is preferably in the range of 1 to 99. In the case of a solvent other than alcohol, that is, ester, ketone, aromatic solvent In this case, the volume ratio of sterile distilled water / solvent is preferably in the range of 1 to 75. That is, if the addition amount of the solvent is too small, the above-mentioned effect due to the addition of the solvent cannot be obtained sufficiently, and conversely, even if it is too much, the water tends to remain without volatilization due to a decrease in compatibility with water. There is a tendency to become insufficient.

  Hereinafter, the present invention will be described in more detail with reference to some examples, but the present invention is not limited to these examples.

1. Preparation of Polypropylene Containing Informational DNA with Primer Corresponding Site 10 g of fine particles having the following different particle sizes and immobilized informational DNA with primer corresponding site were mixed with 1000 g of polypropylene (TX2028B43 manufactured by Nippon Polypro). .

2. Fine particles used (1) Average particle size 0.02 μm: ZS-032 (Zinc oxide) manufactured by Showa Denko K.K.
(2) Average particle size of 40 μm: AW40-74 (aluminum oxide) manufactured by Micron Corporation
(3) AW50-74 (aluminum oxide) manufactured by Micron Corporation with an average particle size of 60 μm

3. Molding of polypropylene 1010 g of polypropylene containing 1 information DNA, 200 g of talc (JA-48R manufactured by Asada Flour Milling), and 40 g of additive (TZ-100 manufactured by Titanium Industry) may be used in a super mixer (manufactured by Zhouen Seisakusho) The mixture was mixed, and an extruder (manufactured by Kurimoto Tekko Co., Ltd.) was heated to 210 ° C. and extruded to obtain pellets. The pellets were set at a mold temperature of 30 to 40 ° C. and a cylinder temperature of 210 ° C., and injection molded in 40 to 90 seconds to produce an opaque resin composition (PP plate: 150 mm long, 70 mm wide, 5 mm thick).

  The obtained opaque resin compositions of Examples 1 to 22 were evaluated for DNA detection and color consistency as follows.

<Detection of information DNA>
(1) A test piece, which is a PP plate containing information DNA, was finely cut with a cutter.
(2) 5 mL of sterilized distilled water was added to the test strip and stirred with a magnetic stirrer to extract DNA into the aqueous layer.
(3) The test strip and the aqueous layer were separated using a centrifuge, and the aqueous layer was concentrated using a centrifugal evaporator.
(4) DNA solution (5 μL) eluted and collected, PCR buffer (5 μL), Taq polymerase (0.25 μL), sterile distilled water (24.75 μL), 5 μM primer 1 (5 μL), 5 μM primer 2 (5 μL) , And 2 mM dNTP (5 μL).
・ Primer 1 ... 5'-TGCACGCACCGTGTACTC-3 '
・ Primer 2 ... 5'-CCGACCAACGTGTCCACT-3 '
(5) After heating at 94 ° C. for 5 minutes, [94 ° C. for 30 seconds → 40 ° C. for 30 seconds → 72 ° C. for 30 seconds] was repeated 30 times.
(6) After being treated at 72 ° C for 7 minutes, it was stored at 4 ° C.
(7) Using a single-stranded DNA cleaving enzyme (S1 nuclease), the excess primer was decomposed, and the target double-stranded information DNA was purified by gel filtration.
(8) One kind of primer (primer 1: 5′-TGCACGCACCGTGTACTC-3 ′) and fluorescently labeled 2,3-dideoxynucleoside triphosphate were mixed with the purified information DNA.
(9) The same operations as in the above steps (4) to (6) were performed.
(10) After gel filtration purification, it was subjected to sequencing by using an automatic sequencer.
As shown in Tables 1 and 2, when these steps were performed and detection of informational DNA was attempted, it was found that identification of the informational DNA was easy, and that further PCR treatment was necessary. It was.

<Evaluation of color consistency>
Except not including the information DNA, the difference from the color of the opaque resin composition equivalent to those in Examples 1-22 above was visually evaluated. As shown in Tables 1 and 2, the equivalents were evaluated as ○, Different ones (with whiteness) were marked with Δ, and large ones (with strong whiteness) were marked with ×.

  As is clear from Tables 1 and 2, the opaque resin compositions of Examples 1 to 12 can all be distinguished well from the information DNA, and exhibit good color consistency. In other words, the target color consistency can be obtained with the same workability as normal molding without being limited to the narrow molding conditions specified in particular, and the opaque resin composition can be identified.

  On the other hand, since the opaque resin compositions of Examples 13 to 16 have a content of information DNA that is less than the preferred range of the present invention, the result is that the discriminability of the information DNA is reduced. In the opaque resin composition of No. 22, any one of the content of information DNA, the particle size of fine particles, and the amount used deviates from the preferred range of the present invention. At least one of these declines occurred.

This is a structural formula showing natural DNA and DNA derived from this 5 'position. It is the schematic which shows the information-ized nucleic acid which has a primer corresponding | compatible site | part at both ends in an authentication information site | part. It is a flowchart which shows one Embodiment of the detection method of the information-ized nucleic acid contained in an opaque resin composition.

Claims (5)

  An opaque resin composition comprising an information nucleic acid having a site having an arbitrary and known base sequence.   The opaque resin composition according to claim 1, wherein the content of the information nucleic acid is 0.5 to 500 μg with respect to 100 g of the composition.   3. The opaque resin composition according to claim 1, wherein the information nucleic acid is supported on fine particles.   4. The opaque resin composition according to claim 3, wherein the average particle size of the fine particles is 0.01 to 40 [mu] m.   The opaque resin composition according to claim 3 or 4, wherein the content of the fine particles is 0.5 to 50% with respect to the composition.

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