JP2008222966A - Dna-containing ink composition - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an ink containing DNA, which is hardly decomposed by external stimulation such as ultraviolet rays, heat, acids and alkali, and to provide a method for simply analyzing DNA in an ink composition. <P>SOLUTION: The ink composition containing DNA and having water resistance of a prescribed degree or higher, is prepared. Furthermore, the DNA in a printed matter prepared by using an ink composition like this is rapidly extracted and analyzed by using water or a water solution. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a DNA-containing ink composition that can be used in describing or printing characters, images, and various codes. More specifically, for example, the present invention relates to a DNA-containing ink composition that is used for printing securities, cards and the like, and is suitable for personal authentication and authenticity verification using vermilion and various coloring tools, and a simple DNA extraction method using the same.

  At present, personal authentication such as criminal investigation and parent / child appraisal is carried out using a unique part with high discrimination power using DNA. In recent years, in addition to such applications, a method for authenticating authenticity has also been considered, in which checks, banknotes, various certificates, cards, and the like are printed and counterfeited with ink containing DNA. For example, it is disclosed that a thin film pattern is formed using a personal identification DNA ink in which a DNA molecule and a cationic surfactant are mixed, and the formed pattern is used as personal identification information (see Patent Document 1). ). In this method, a complicated thin film pattern formed by developing DNA molecules in a thin film shape is optically detected as identification information, and the unique biological information of the DNA molecules is not used.

  There is also disclosed a method for identifying the authenticity of a stamp or handwriting by using the base sequence of a specific DNA fragment whose base sequence is known in advance as an index (for example, Patent Document 2). On the other hand, plastic resin microcapsules or gel materials with DNA encapsulated in DNA or ultra-fine plastic resin surfaces with DNA bound are prepared in advance and mixed with ink. Some have been made (Patent Documents 3 and 4).

  The techniques using the DNA-containing ink as described above are all different from conventional authentication systems in that DNA is used, but the DNA in the printed ink is light, ultraviolet light, heat, acid, It is often decomposed by an external stimulus such as alkali. Therefore, when DNA is directly contained in ink, the amount of DNA decreases immediately after printing, making detection difficult. In addition, according to the method of encapsulating in microcapsules or the like and supporting the particles on a carrier such as particles, although it is resistant to external stimuli, for example, it takes 1 day or more to detect DNA in the ink after printing. This is the current situation.

JP 2002-167530 A JP-A-2005-247900 JP 2004-175922 A JP 2004-331832 A

  Thus, there is a problem that DNA is easily decomposed by the action of ultraviolet rays, heat, acid, alkali, etc. When mixed with ink and used for producing printed matter, there is a possibility that it is not in an identifiable state at the time of analysis. Furthermore, recovery of DNA after printing requires multiple steps and is difficult. Despite the necessity of establishing a DNA ink composition and its analysis method that can maintain a good discriminating ability of DNA after printing and analyze it quickly and easily, it is necessary to ensure DNA. Ink compositions and methods for detecting them have not been found. Therefore, this solution has been desired.

  As a result of intensive studies to provide a DNA-containing ink composition that can withstand external stimuli such as ultraviolet rays, heat, acids, and alkalis, and a simple DNA detection method, the present inventors are strong against external stimuli and are capable of printing. Thereafter, an ink composition from which DNA can be easily extracted was found, a detection method using the ink composition was established, and the present invention was completed.

  That is, the present invention provides a water-resistant ink composition containing DNA.

  The water-resistant ink composition may be a mixture of water-resistant ink and DNA.

  The water-resistant ink composition can be a composition containing water, a colorant, a resin, and DNA.

  The resin may be at least one selected from the group consisting of urethane resins, acrylic resins, and epoxy resins.

The content of the DNA may be 3 × ^{10 −10} μg or more and 20 μg or less per 1 μl of the ink composition.

  The present invention also provides a method for extracting DNA from a print on a medium, the method comprising a step of bringing a print produced using any of the ink compositions described above into contact with water or an aqueous solution.

  The medium can be paper, plastic resin, or fabric.

  The present invention is also a method for determining the authenticity of a print on a medium, comprising the step of bringing a print produced using the ink composition into contact with water or an aqueous solution; and contacting the print with water or an aqueous solution. And a step of analyzing the DNA in the prepared DNA-containing aqueous solution.

  The medium can be paper, plastic resin, or fabric.

  The ink composition of the present invention is resistant to external stimuli such as ultraviolet rays, heat, acids, alkalis, etc., and can keep the rate of DNA degradation low even after standing for a long time after printing. Further, DNA in the ink composition can be easily extracted in a short time with water or an aqueous solution. Furthermore, if the ink composition of the present invention is used, DNA can be recovered from the same printed material several times. Therefore, using the ink composition of the present invention, for example, it is possible to establish a very good authentication system for printed matter.

  Hereinafter, the present invention will be described in detail.

  The term “water-resistant ink composition” of the present invention refers to an ink composition that does not fall into the category of conventional water-based inks and oil-based inks and can be expressed as “water-resistant”. In the present specification, the term “water-resistant ink composition” includes a colorant such as DNA, dye or pigment, a resin for dye or pigment fixing material and water, and appropriately contains a preservative, a water-soluble organic solvent, a surface active agent. And other additives such as pH buffering agent, and satisfy the following conditions.

  That is, normal temperature (25 ° C.) water can be contained (mixed) in an amount of 5% by weight or more based on the ink composition, and in normal temperature (25 ° C.) water (tap water, distilled water, deionized water, etc.). When the printed material is taken out after being immersed for 48 hours, rubbed with a 100% polyester cloth, and the ink is peeled off, the ink composition does not penetrate into the polyester cloth and no bleeding of the printed material is observed. When the ink easily penetrates into the polyester cloth or the printed matter oozes, it becomes a “water-based ink composition”.

  The DNA contained in the ink composition of the present invention is not limited, and for example, any of natural genes derived from human genes, animal or microbial genes, or synthetic DNAs can be used, single-stranded or double-stranded. Any of the chains can be used. The molecular weight is not limited, and it may be a DNA fragment of short chain of about 10 bp to medium long chain of 200 bp or more. Usually, a DNA fragment of about 20 bp to 100 bp is easy to use, and can be appropriately selected from those according to the purpose. In the case of using naturally-derived DNA, a gene region or an intergenic region such as a microsatellite region can be used in the base sequence of the total DNA of the cell. When synthetic DNA is used, for example, desired DNA can be synthesized by a commercially available DNA synthesizer and purified. The desired DNA may be preferably used in the present invention even if it has the same or similar sequence as the naturally-occurring DNA, or a completely different sequence.

The content of the DNA fragment of the present invention is 3 × ^{10 −10} μg or more and 20 μg or less, preferably 1 ng to 1 μg, more preferably 10 ng to 5000 ng per 1 μl of the ink composition.

  The water-resistant ink composition of the present invention is not limited, but can be prepared by mixing DNA with a commercially available water-resistant ink. Alternatively, a colorant such as DNA, dye or pigment, a resin for dye or pigment fixing material, water, and other additives such as preservatives, water-soluble organic solvents, surfactants, pH buffering agents, etc. Ink composition.

  Here, the term “water-resistant ink” of the present invention refers to a water-resistant ink that does not fall into the category of conventional water-based inks and oil-based inks and can be displayed as “water-resistant”. Preferably, the term “water-resistant ink” in the present specification includes a colorant, a resin for dye or pigment fixing material and water, and appropriately contains preservatives, water-soluble organic solvents, surfactants, pH buffering agents, and the like. This refers to ink containing other additives. The “water-resistant ink” in this specification satisfies the following parameters.

  In other words, it can contain (mix) 5% by weight or more of water at room temperature (25 ° C) with respect to the ink, and the printed matter can be contained in water (tap water, distilled water, deionized water, etc.) at room temperature (25 ° C). When the ink is removed after being immersed for 48 hours, rubbed with a 100% polyester cloth, and the ink is peeled off, the ink does not penetrate into the polyester cloth, and the printed material is not observed to spread. If the ink easily penetrates into the polyester cloth or the printed matter oozes, it becomes “water-based ink”.

    Although not particularly limited, the “water-resistant ink” includes, for example, a non-volatile component of 44.0 to 46.0 (Wt%), a viscosity of 75 to 700 (CP / 25 ° C.), and a pH of 7.5 to 9.0 ( 25 ° C.). The viscosity at this time is measured by using a BH type rotational viscometer manufactured by TOKIMEC Co., Ltd., and measuring the viscosity of the composition whose temperature is adjusted to 25 ° C. with a rotating rotor at 20 rpm.

  As the colorant contained in the water-resistant ink composition of the present invention, a dye or a pigment can be used. Transparent ink can also be used by selecting the type of pigment.

  The dye may be used by directly dissolving as a component of the ink, or may be used by being supported on or contained in inorganic or organic fine particles.

  As a dye contained in the water-resistant ink composition of the present invention, it can be used by mixing with other colorants as required. As the water-soluble dye to be used, those which are classified into acid dyes, direct dyes, basic dyes, reactive dyes and food dyes in the color index and which have excellent water resistance and light resistance are used.

  The colorant contained in the water-resistant ink composition of the present invention may be an organic pigment or an inorganic pigment. Organic pigments include azo, phthalocyanine, anthraquinone, dioxazine, indigo, thioindigo, perylene, isoindolenone, aniline black, azomethine, rhodamine B lake pigment, carbon black Examples of inorganic pigments include iron oxide, titanium oxide, calcium carbonate barium sulfate, aluminum hydroxide, barium yellow, bitumen, cadmium red, chrome yellow, and metal powder.

  The resin for the dye or pigment fixing material used in the present invention is not particularly limited as long as it is a resin that can be usually used in the production of water-resistant ink, but is preferably a urethane resin, an acrylic resin, or an epoxy resin. . The resin content is preferably 3% to 70% by weight, more preferably 5% to 50% by weight of the total ink composition.

  As the composition of the DNA ink composition of the present invention, a surfactant can be used, and the wettability to the recording paper can be improved by the surfactant. Preferred surfactants include polyoxyethylene alkyl ether acetates, dialkyl sulfosuccinates, polyoxyethylene alkyl ethers, polyoxyethylene alkyl phenyl ethers, polyoxyethylene polyoxypropylene block copolymers, and acetylene glycol surfactants. Agents and the like.

  Furthermore, by using lithium ions, quaternary ammonium, or quaternary phosphonium as a counter ion of the surfactant of the present invention, the surfactant exhibits excellent dissolution stability.

  Furthermore, a penetrant can also be used as a component of the DNA ink composition of the present invention for the purpose of adjusting the surface tension.

  As the other pH adjusting agent, any substance can be used as long as it can adjust the pH appropriately without adversely affecting the prepared ink. Examples thereof include amines such as diethylamine and triethanolamine, hydroxides of alkali metal elements such as lithium hydroxide and sodium hydroxide, ammonium hydroxide, quaternary ammonium hydroxide, and quaternary phosphonium hydroxide. Products, alkali metal carbonates such as lithium carbonate, sodium carbonate and potassium carbonate.

  Further, conventionally known antiseptic / antifungal agents, rust preventives and other additives can be added to the DNA ink composition of the present invention. For example, as a preservative and antifungal agent, sodium dehydroacetate, sodium sorbate, sodium 2-pyridinethiol-1-oxide, sodium benzoate, sodium pentachlorophenol, isothiazoline and the like can be used in the present invention.

  Examples of the rust preventive include acidic sulfite, sodium thiosulfate, ammonium thiodiglycolate, diisopropyl ammonium nitrite, pentaerythritol tetranitrate, dicyclohexyl ammonium nitrite and the like.

  In addition, a water-soluble ultraviolet absorber, a water-soluble infrared absorber, and an antifoaming agent can be added depending on the purpose.

  Examples of the antifoaming agent include oils and fats, fatty acids, fatty acid esters, alcohols, phosphate esters, amines, amides, metal soaps, sulfate esters, and silicones. These can be used alone or in combination of two or more. Among these, a silicone-based antifoaming agent having a siloxane bond is preferable from the viewpoint of easy control of the negative pressure of the sponge absorbent in the ink tank. In particular, a polysiloxane antifoaming agent to which polyethylene oxide and / or polypropylene oxide is added is effective.

  Furthermore, the ink composition of the present invention may contain alcohols. Examples of the alcohol include methanol, ethanol, isopropanol and the like, and the content ratio may be any as long as it is in the range of 0% by weight to 70% by weight of the entire ink composition.

  By appropriately printing the DNA-containing ink composition of the present invention thus prepared on a medium, it is possible to create an identifiable document, printed matter, and the like. Here, “printing” includes not only a mode of printing using ink, but also a mode of applying ink to a medium or handwriting characters on a medium with ink. Examples of the medium at this time include paper, plastic resin, and fabric, but are not limited, and any medium that can be used for ordinary printed materials can be used. The printed matter thus obtained can stably hold DNA over a long period of time even when handled in the same manner as before. Moreover, recovery is easy and quick detection is possible.

  That is, for example, in the present invention, DNA can be easily extracted or analyzed by bringing the print on the medium thus obtained into contact with water or an aqueous solution. Here, the aqueous solution refers to various buffer solutions, PCR solutions, DNA hybridization solutions or equivalents in which components that do not affect the resin components contained in the ink are added to water.

  The contact with water or an aqueous solution can be carried out by dropping a water droplet directly on the print and leaving it for a short time such as 1 second or more and 1 hour or less, and leaving it for 10 seconds to 1 minute for simpler operation. As long as the contact with water or an aqueous solution is a time of 48 hours or less, a longer time can be set as required.

  After standing, water or an aqueous solution can be collected, and the contained DNA can be amplified by PCR or the like and analyzed in a short time. It is also possible to hybridize the collected solution directly to the probe.

  Alternatively, the printed portion can be cut out together with a medium such as paper and directly added to the PCR solution or the like to perform PCR.

  In printing using the ink composition containing the DNA of the present invention, the authenticity of the printing can be judged using the discriminating power of DNA. In this method, a print made using the ink composition of the present invention is brought into contact with an aqueous solution such as water or a buffer, the resulting aqueous DNA-containing solution is recovered, and the DNA in the aqueous solution is analyzed. included.

  The analysis is not limited, but after amplifying DNA by the PCR method, confirming the size of the DNA fragment by agarose gel electrophoresis or determining the base sequence to confirm that the DNA is a predetermined DNA It can be done by the method. Alternatively, it is also preferable to detect by using a hybridization method using a labeled probe more simply. Specifically, a synthetic DNA having a sequence complementary to a part of the target DNA is prepared in advance, hybridization is performed under a certain condition, and the presence or absence of double strand formation is determined by, for example, a colloidal gold solution. It can also be identified by the presence or absence of discoloration, and can be identified by a fluorescent reagent such as Cyber Green or Cyber Safe.

  The PCR method itself is well known, and kits and devices for that purpose are also commercially available. Therefore, it can be easily carried out by synthesizing the forward side primer and reverse side primer to be used. Furthermore, an identification probe is also well known and can be easily implemented if it is appropriately synthesized according to the sequence of the target DNA. The specific method is also specifically described in the following examples.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these are not intended to limit the technical scope of the present invention. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

  The DNA used in the following Examples and Comparative Examples was obtained by purifying and desalting 85 Mer (ATTAACCCTCACTAAAGGGATCAATAAAACAAAACAAAACGCGCGGCTCACGGGCGCCTAGGAGTGCCCTATAGTGAGTCGTATT) shown in SEQ ID NO: 1 in the Sequence Listing. All PCR was performed using Bioline Bio Taq DNA polymerase under the conditions recommended by Bioline.

(Example 1)
Preparation of water-resistant ink composition Water-resistant ink (clear: obtained from Osaka Sealing Printing Co., Ltd.) and DNA solution (10 μg / μl, 1 μg / μl, 100 ng / μl, 10 ng / μl) at a DNA concentration of TE (10 Water resistant to a final DNA concentration of 5 μg / μl, 500 ng / μl, 50 ng / μl, 5 ng / μl by mixing 1: 1 with Tris-HCl, 1 mM EDTA, pH 8.8) Each ink composition was obtained.

(Example 2)
Preparation of water-resistant ink composition TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.8) at the DNA concentration of water-resistant ink (clear: obtained from Osaka Sealing Printing Co., Ltd.) and DNA solution (10 μg / μl, 1 μg / μl) )) Were mixed at a ratio of 99: 1 to obtain water-resistant ink compositions having final DNA concentrations of 100 ng / μl and 10 ng / μl, respectively.

(Example 3)
A water-resistant ink composition was obtained in the same manner as in Example 1 using black (comics black ink water-resistant BLACK 4 manufactured by DELETER) as the water-resistant ink.
(Example 4)
A water-resistant ink composition was obtained in the same manner as in Example 2 using black (commercial black ink water-resistant BLACK 4 manufactured by DELETER) as the water-resistant ink.

(Comparative Example 1)
Preparation of ink composition containing oil-based ink and DNA Oil-based transparent ink (Sakata ink Oil-based transparent ink brown Oil-based transparent ink white) 3 g of DNA solution ((10 μg DNA / μl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.8)) 10 μl was mixed to obtain an oil-based ink composition having a final DNA concentration of 33 μg / g.

(Comparative Example 2)
Preparation of ink composition containing oil-based ink and DNA 200 μl of toluene was added to 10 mg of the oil-based ink composition of Comparative Example 1 and mixed to obtain an oil-based DNA ink composition having a final DNA concentration of 1.15 ng / μl.
(Comparative Example 3)
Oil-based transparent ink (Sakata ink Oil-based transparent ink Brown Oil-based transparent ink white) Add 200 μl of toluene, mix well, and mix with DNA solution ((1 μg DNA / μl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.8)) ) 0.33 μl and final DNA concentration is 1.15 ng
An oil-based ink composition having a concentration of / μl was obtained.

(Comparative Example 4)
Preparation of Ink Composition Containing Aqueous Ink and DNA DNA solution (10 μg / μl, 1 μg / μl, 100 ng / μl, 10 ng / μl) of DNA in stock aqueous black ink (black ink for comics manufactured by DELETER) TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.8)) was mixed at a ratio of 1: 1, and the final DNA concentrations were 5 μg / μl, 500 ng / μl, 50 ng / μl, 5 ng A water-based ink composition having a concentration of / μl was obtained.
(Comparative Example 5)
Use black (commercial black ink BLACK3 for comics made by DELETER) as water-based ink, mix with DNA solution (10 μg / μl, TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.8)) at 99: 1, and finally A water-resistant ink composition having a DNA concentration of 100 ng / μl was obtained.

The ink types and DNA contents in each Example and each Comparative Example are as shown in Table 1.

  Using the ink compositions obtained in Examples and Comparative Examples, DNA recovery rates were compared from printed materials, DNA extraction time and recovery rates were evaluated, UV resistance analysis, and multiple DNA recovery evaluations were performed.

(Comparison of DNA recovery rate)
First, each ink composition was applied to paper (copy paper just) using a brush and dried well in the dark at room temperature overnight. However, in the case of the oil-based ink composition of Comparative Example 1, since the viscosity of the ink was very high, even if it was applied, it did not adapt well to the paper and it was difficult to perform the operation itself.

Next, DNA was recovered from the obtained DNA ink print and attempted to be purified. First, 1 cm ² of the applied portion was cut out with a disposable razor, and the sections were collected while cutting into smaller pieces.

  To this, 500 μl of water was added, and further 400 μl of toluene and 100 μl of methyl isobutyl ketone were added and mixed well. Placed at room temperature for 30 minutes, and separated into an organic layer and an aqueous layer for 5 minutes, the aqueous layer portion (lower layer) was collected and purified by phenol / chloroform extraction and ethanol precipitation. The operation was performed at room temperature. It was confirmed by PCR whether or not the target DNA was contained in the recovered material.

  As a result, in the case of Example 1 using the durable ink composition, about 25% to 30% of DNA was recovered by the operation at room temperature compared with the original DNA weight. In the case of Examples 2 and 3, DNA recovery was sometimes confirmed, but sometimes recovery was not confirmed (Comparative Example 2: 1 out of 4 times Comparative Example 3: 1 out of 2 times). Furthermore, for the purpose of improving the recovery method of Comparative Examples 2 and 3, recovery was performed using a commercially available thinner (Asahi Pen Paint Thin Solution S), but there was no significant change in the results, and the reliability of the recovery itself was poor. It was. It is considered that the ink using the oil-based ink and the organic solvent cannot be dissolved uniformly due to the difference in polarity, and the DNA concentration in the ink is uneven.

  Next, without using an organic solvent, add 500 μl of water to the cut sections, and leave them at room temperature for 30 minutes. Then, purify the solution by phenol / chloroform extraction and ethanol precipitation, and measure the amount of DNA contained. The amount recovered was checked. When recovered with water alone, in the case of Example 1, about 35% to 40% of DNA was recovered in the operation at room temperature compared to the original DNA weight. Comparative Examples 2 and 3 In the case of, recovery of DNA could not be confirmed.

また、実施例2、4では、最終DNA濃度100 ng/μlの調製物0.2 μlを4mm²にスポットし、60℃16時間で乾燥させ、１０分室温に置き、冷ましたスポットに10μlの水を置き、10秒静置、ピペッティングをスポット上で行い、一度水を回収し20秒待ち、同じ水をもう一度スポット上にのせ、ピペッティングを行い、この水に含まれるDNA量を測定した。実施例2では5回調べ、平均値を算出したところ36.8 ％のDNAが回収された。この回収液1μlを使用してPCRを行ったところ、実施例２，４ともに5回調べたうち全てで増幅は観察された。比較例５で同様の実験を行ったところ、PCRによる増幅は観察されなかった。 (Evaluation of DNA extraction time and recovery rate)
Next, the recovery with water was examined by changing the extraction time. For the ink composition of Example 1, the cases where the extraction time was 2 minutes, 5 minutes, 10 minutes, 20 minutes, and 30 minutes (4 times for 30 minutes, 2 times for others) were calculated, and the average value was calculated. . The results are shown in Table 2.

In Examples 2 and 4, 0.2 μl of a preparation with a final DNA concentration of 100 ng / μl was spotted on 4 mm ² , dried at 60 ° C. for 16 hours, placed at room temperature for 10 minutes, and 10 μl of water was added to the cooled spot. The sample was allowed to stand for 10 seconds, and pipetting was performed on the spot. Water was collected once, waited for 20 seconds, the same water was placed on the spot again, pipetting was performed, and the amount of DNA contained in the water was measured. In Example 2, it was examined 5 times, and the average value was calculated. As a result, 36.8% of DNA was recovered. When PCR was carried out using 1 μl of this recovered solution, amplification was observed in all of the five examinations in Examples 2 and 4. When a similar experiment was performed in Comparative Example 5, amplification by PCR was not observed.

  As a result, it was found that when the ink composition of the present invention was used, the DNA extraction time was sufficient even for several minutes, and the time required for extraction was sufficient for several minutes. This result also suggests that the printed DNA easily leaks out. Therefore, the following evaluation was performed.

(Multiple collection of DNA)
Spot 0.2 μl of a preparation with a final DNA concentration of 100 ng / μl from Examples 2 and 4 onto 4 mm ² , dry at 150 ° C. for 10 minutes, collect DNA once from the spot with 1 μl of water, and place the spot again at 150 ° C. It was dried in 10 minutes, and DNA was collected with another 1 μl of water. This operation was repeated 5 times, and how many times the DNA in the collected solution could be amplified by PCR was examined. As a result, in the water-resistant transparent ink of Example 2, the same amount of amplification was observed from the collected liquid at the fifth time. On the other hand, in the case of the water-resistant black ink in Example 4, the amount of amplification was drastically decreased from the PCR using the fourth extract, and the band was not confirmed from the fifth extract. In water-resistant transparent ink, amplification is observed from the sixth extract, but in water-resistant black ink, it is often observed even at the sixth time, but when it is not observed, it is divided into cases where the amount is observed but small There was found. As a result, water-resistant transparent ink can be analyzed many times from the same printing surface, and water-resistant black ink can be analyzed three or four times. In particular, in a water-resistant transparent ink, a small amount of DNA leaks when treated with a small amount of water, but it does not flow out to the extent that it is impossible to analyze. In particular, in the case of water-resistant transparent ink (Example 2), according to water resistance verification, the spot was treated with running water (tap water) for 48 hours, rubbed with a polyethylene cloth, dried once again, and 20 μl of water from the spot. When DNA was collected and confirmed by PCR, it was observed in all three of the three verifications.

(Implementation of direct PCR from printing)
Next, apply ² μl each of the compositions up to Examples 1 and 4 in 1 cm ² so as to be as uniform as possible while using a pipetman, dry overnight at room temperature in the dark, and then use a razor. Then, 4 mm ² each was cut out and PCR was performed using this piece of paper. In each of the PCR tubes, it is assumed that 0.4 ng, 4 ng, 40 ng, and 400 ng of DNA were added in Examples 1 and 3 as 0.8 ng and 8 ng templates in Examples 2 and 4, respectively. When PCR products were examined by agarose electrophoresis, amplification was confirmed in all cases in Examples 1, 2, and 3, but amplification was not observed in all cases in Example 4. As a result, it turned out that DNA amplification by PCR was possible using a 4 mm ² paper piece itself coated with water-resistant transparent DNA ink as a template, and with water-resistant black DNA ink, if the concentration of DNA is extremely low, It turned out to be impossible. Similarly, in Comparative Example 4, amplification was not observed in all cases under the condition that 0.4 ng, 4 ng, 40 ng, and 400 ng of DNA were added to the PCR tube as a template.

(Environmental resistance evaluation)
Furthermore, in order to investigate whether the environmental resistance can be expected to be improved by actually increasing the ink content, the resistance to ultraviolet rays, which is considered to have the most influence on DNA, was examined. After the ink compositions of Examples 1 to 3 were dried at 60 ° C. for 16 hours, ultraviolet irradiation was started, 4 mm ² portions were cut out every 4 hours with a disposable razor, and this piece of paper was added to the PCR tube. In Example 4, since PCR with a piece of paper is impossible, 0.2 μl is spotted on 4 mm ² , UV irradiation is started, 1 μl of water is placed on the spot every few hours for 10 seconds, and lightly on 4 mm ² After pipetting, the solution was collected and added to the PCR tube, and whether or not the target DNA could be amplified by PCR was used as a criterion for judgment. As a result, in Examples 1 and 3, amplification may not occur in 24 hours, whereas in the case of the water-resistant transparent ink, 8 ng per 4 mm ^2, and 1/10 amount of 0.8 ng (Example 2). ), Amplification by PCR was confirmed with UV irradiation for 24 hours, and although no band was confirmed after 72 hours of irradiation, it could be confirmed even after 48 hours. Furthermore, when the black water-resistant DNA ink was examined for 20 ng and 2 ng per 4 mm ² (Example 4), amplification by PCR could be confirmed reliably even after 48 hours of UV irradiation, and further amplification after 72 hours. It was confirmed with certainty.

On the other hand, as a comparison, the DNA solution is diluted as it is with water to make final DNA amounts of 100 ng / μl and 10 ng / μl, 0.2 μl is spotted on 4 mm ² , UV irradiation is started, and spotted every few hours. After adding 1 μl of water for 10 seconds and pipetting lightly on 4 mm ^{2 of} the solution, collect the solution, add it to the PCR tube, and examine whether the target DNA can be amplified by PCR. It was found that the band could not be confirmed.

Claims (9)

  A water-resistant ink composition containing DNA.   2. The water-resistant ink composition according to claim 1, wherein the water-resistant ink composition is a mixture of water-resistant ink and DNA.   The water-resistant ink composition according to claim 1, comprising water, a colorant, a resin, and DNA.   4. The water-resistant ink composition according to claim 3, wherein the resin is at least one selected from the group consisting of a urethane resin, an acrylic resin, and an epoxy resin. 5. The ink composition according to claim 1, wherein the DNA content is 3 × ^{10 −10} μg or more and 20 μg or less per 1 μl of the ink composition.   A method for extracting DNA from a print on a medium, the method comprising a step of contacting a print produced using the ink composition according to any one of claims 1 to 5 with water or an aqueous solution.   The method of claim 6, wherein the medium is paper, plastic resin, or fabric. A method for appraising the authenticity of printing on a medium,
A step of contacting a print made using the ink composition according to claim 1 with water or an aqueous solution;
A step of preparing a DNA-containing aqueous solution obtained by contacting the print with water or an aqueous solution; and a step of analyzing the DNA in the prepared DNA-containing aqueous solution,
Including a method.   The method of claim 8, wherein the medium is paper, plastic resin, or fabric.