JP2007003257A - Method and device for transferring nucleic acid and genuineness judgment method of product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a nucleic acid transfer method which enables an efficient elution and transfer of nucleic acid without destructing a nucleic acid-containing part and requiring much labor, time and technique, a nucleic acid transfer device and a genuineness judgment method of a product. <P>SOLUTION: A nucleic acid-containing printed matter 7 is mounted on a specimen mounting stand and wet filter paper 8 is mounted on the printed matter 7 in a close contact. When electrodes 1 and 1' are brought into contact with the wet filter paper 8, a battery is formed to allow a current to flow and nucleic acid is transferred to the wet filter paper 8 by an electrophoretic theory. The transferred nucleic acid is extracted using a buffer solution to perform PCR amplification and the obtained electrophoretic pattern is compared with the electrophoretic pattern obtained by the transfer from the preliminarily intended place of a true product to judge genuineness. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a nucleic acid transfer method, a transfer apparatus, and a product authenticity determination method.

  Conventionally, methods for extracting nucleic acid (DNA) include a method in which a nucleic acid-containing portion is cut out and broken and used for extraction, a method using capillary action by blotting paper, an electrophoresis method, and the like.

  In the method of cutting out and rupturing a part containing nucleic acid and using it for extraction, the original shape cannot be preserved due to rupture, it takes a lot of time for rupture, and nucleic acid cannot be sufficiently eluted. There was a problem.

  In a method using a capillary phenomenon by blotting paper or the like (see, for example, Patent Document 1), there is a problem that extraction is not possible unless a sufficient amount of nucleic acid is included for analysis.

  Electrophoresis requires a high-voltage power supply device and a special device dedicated to elution transfer, and can only be separated by small fragments of the extracted nucleic acid, which is insufficient for analysis of nucleic acids extracted by the liquid method. there were.

  Further, by tilting the horizontal submarine gel electrophoresis tank, a method and apparatus for separating nucleic acids by generating a distorted electric field containing a direction component perpendicular to the electrophoresis direction in the agarose gel (for example, Patent Document 2). The method using this apparatus has a problem that it cannot be extracted unless it contains a sufficient amount of nucleic acid for analysis, and it takes a long time for analysis.

  Furthermore, since it is generally difficult to efficiently extract nucleic acids, primary detection has been performed using secondary means such as IR luminescence. For this reason, there are problems such as the cost of secondary materials such as IR emission and the need for equipment for detection.

Japanese Patent No. 3360270 JP 2001-264292 A

  The present invention has been made in view of the above-described problems, and the object thereof is to efficiently destroy nucleic acids without the need to destroy the portion containing the nucleic acids, and without requiring much labor, time, and techniques. It is intended to provide a method and apparatus capable of elution transfer.

  The nucleic acid transfer method of the present invention is a method for transferring a nucleic acid from a nucleic acid-containing base material to a transfer material without destroying the base material, wherein the transfer target is wetted on the base material containing the nucleic acid. A material is brought into contact, an electrode is brought into contact with the material to be transferred, and nucleic acid is eluted from the material to be transferred by electrophoresis, and is clearly transferred in a short time.

  The electrode is brought into contact with the transfer material at a constant pressure.

  Examples of the transfer material include filter paper, cloth, agarose gel, and polyacrylamide gel.

  The nucleic acid transfer device of the present invention is a device for eluting nucleic acid from a nucleic acid-containing substrate and transferring it non-destructively to a transfer material, a pair of electrodes for causing electrophoresis, and a power source for energization It is characterized by comprising.

  The nucleic acid transfer apparatus of the present invention may further include a sample mounting table on which a nucleic acid-containing substrate is placed and a support member that supports a pair of electrodes.

  The nucleic acid transfer apparatus of the present invention may further be provided with an alignment unit capable of performing alignment at a previously intended location of the nucleic acid-containing substrate.

  In the nucleic acid authenticity determination method of the present invention, a target product for determining authenticity is placed on a sample mounting table of a nucleic acid transfer device composed of a pair of electrodes and a power source. Then, a wet transfer material is adhered and laminated at the same position as the nucleic acid contained in the intended site of the genuine product, the pair of electrodes are brought into contact with the transfer material, and electricity is supplied from a power source. The nucleic acid is eluted from the target product by electrophoresis, transferred to a transfer material, the transfer material to which the nucleic acid has been transferred is broken, the nucleic acid is extracted using a buffer solution, the nucleic acid extract is PCR amplified, The obtained electrophoretic pattern is compared with the electrophoretic pattern of nucleic acid obtained by transferring from an intended place of a genuine product.

  Secret information and / or individual information is added to the nucleic acid used for product authenticity determination.

  Products used for authenticity determination are banknotes, passports, securities, and valuable printed matter.

According to the nucleic acid transfer method of the present invention, nucleic acid can be extracted from a nucleic acid-containing substrate in a short time without destroying the substrate.
In addition, a versatile nucleic acid transfer device can be obtained by devising the shapes of the material to be transferred and the electrodes.
Since the nucleic acid transfer method according to the present invention cannot be detected with the naked eye, it can be easily discriminated by applying it to a product for determining authenticity.

  Although the present invention uses a conventionally used electrophoresis method, specifically, since a nucleic acid is negatively charged, it can be moved to a positive pole and separated by applying a voltage. It is based on becoming.

Embodiments of the present invention will be described below with reference to the drawings.
The substrate of the nucleic acid-containing substrate is not particularly limited as long as it is a substrate that can contain nucleic acid, but when the substrate is paper, the nucleic acid-containing paper, the nucleic acid-coated paper, and the fiber DNA A printed matter or a card substrate printed using a printing ink in which a nucleic acid is contained in a printing ink, or a DNA fiber or the like to which is attached. In this example, a printed matter printed using a printing ink containing a nucleic acid as a nucleic acid-containing substrate and a filter paper as a transfer material will be described, but the printed matter and the filter paper are not particularly particular. FIG. 1 is a block diagram showing an embodiment of a nucleic acid transfer apparatus. The nucleic acid transfer apparatus according to the present embodiment includes electrodes 1, 1 ′, a power source 2, a voltmeter 3, an ammeter 4, an electrode support 5, and a sample mounting table 6.

Furthermore, as shown in FIG. 1, light may be applied from the lower part of the sample mounting table 6 so that the specific position of the nucleic acid-containing substrate and the material to be transferred can be aligned.
At this time, the size of the hole drilled as the light introduction path in the sample mounting table 6 is not particularly limited, but the size of the hole is suitably 1 to 5 mm, and the light source is not particularly limited. Then, LED which does not have heat was used.

  A printed material 7 containing nucleic acid is placed on the sample mounting table 6, and a moistened filter paper 8 is placed in close contact with the printed material 7. Although the shape of the wet filter paper 8 is not particular, it is set to a size that can cover part or all of the portion of the printed matter 7 containing the nucleic acid. Further, the wet state of the filter paper is not particularly limited, but the current required for the transfer is set to such a level as to flow. In this example, 5 × 10 mm filter paper was used by moistening with 5 μl of buffer. If the wet state is insufficient, the resistance value becomes large and the transfer efficiency is deteriorated. If it is excessive, the printed matter 7 containing the nucleic acid may be damaged.

Next, when the electrodes 1 and 1 ′ are brought into contact with the wet filter paper 8, a battery is formed and a current flows. As a result, the nucleic acid is transferred to the wet filter paper 8 by the principle of electrophoresis. The elution transfer in this example is sufficiently possible in a short time (within 10 minutes). At this time, when a constant DC voltage is applied, the nucleic acid transfer efficiency is further improved.
The voltage at this time is 0.1 to 100V, preferably 1 to 50V. It is also possible to control with an electric current using the ammeter 4. In that case, a current of 0.0001 to 100 mA, preferably 0.001 to 10 mA, flows through the wet filter paper 8. The contact time may be about 10 seconds to 3 minutes, and preferably about 30 seconds to 1 minute.

  Furthermore, when the electrodes 1 and 1 ′ are brought into contact with each other, the transfer efficiency of the nucleic acid is further improved by controlling the weight of the electrode support 5 so that a constant pressure is applied to the wet filter paper 8 and the printed matter 7. To do. Although the applied pressure varies depending on the electrode used, in this embodiment, an electrode having a diameter of 2 mm is applied with a weight of about 100 g.

  After the transfer, the moist filter paper is broken, and extraction is performed using a buffer. The extract is subjected to PCR amplification, and the amplified PCR product is confirmed. In this example, the concentration / cleanup operation such as ethanol precipitation of nucleic acid, which is generally required in the nucleic acid extraction / amplification process, is unnecessary.

  In this embodiment, filter paper is used as the material to be transferred. However, the present invention is not limited to this, and cloth, agarose gel, polyacrylamide, or the like may be used.

  FIG. 2 shows the result of rupturing the filter paper after completion of transfer, extracting and amplifying the nucleic acid transferred to the filter paper, and detecting using gel electrophoresis.

  Lanes 1 and 6 are molecular weight markers, lane 2 is nucleic acid mixed with the printed material, lane 3 is a PCR product obtained by PCR amplification of nucleic acid extracted from the printed material containing nucleic acid, and lane 4 is wet on the printed material containing nucleic acid. After removing the filter paper and contacting the electrode for 30 seconds, only the filter paper is taken out, the transferred nucleic acid is extracted and PCR amplified, and Lane 5 is a wet filter paper placed on the print containing the nucleic acid. This is a PCR product obtained by contacting the electrodes and applying a voltage of 10 V for 30 seconds, then taking out only the filter paper, extracting nucleic acid from the filter paper, and performing PCR amplification.

  From FIG. 2, it can be seen that the nucleic acid contained in the printed material is transferred to the filter paper (lanes 4 and 5). Moreover, it turns out that the effect is performed more efficiently by applying a voltage (lane 5).

FIG. 3 is a diagram showing an example in which no transfer was performed as a comparative control. Extraction and amplification of nucleic acid from filter paper were performed under the same conditions.
Lanes 7 and 8 are molecular weight markers, lane 9 is a nucleic acid mixed with the printed material, lane 10 is a PCR product obtained by PCR amplification of nucleic acid extracted from the printed material, and lane 11 is a dry filter paper placed on the printed material containing nucleic acid. After contacting the electrode for 30 seconds, only the filter paper is taken out, the transferred nucleic acid is extracted, the product after PCR amplification, lane 12 places the wet filter paper on the printed matter containing the nucleic acid, and the electrode is contacted This is a PCR product obtained by leaving only for 30 seconds without removing the filter paper, extracting the nucleic acid from the filter paper, and performing PCR amplification.

  From FIG. 3, when the electrode is contacted with dry filter paper (lane 11), and when the wet filter paper is not contacted with the electrode (lane 12), the nucleic acid contained in the printed material is applied to the filter paper. It was not transcribed. This is because a battery is not formed.

  An embodiment of a method used for authenticating a product by applying the nucleic acid transcription method of the present invention will be described. Arbitrary designs, patterns, symbols, etc. are applied to produce a genuine printed material containing nucleic acid at the intended location in advance. By adding individual information and confidential information to the nucleic acid, it is possible to further facilitate authenticity determination. Since the site to which the nucleic acid is added cannot be detected with the naked eye, it is information that only the manufacturer can know. Various methods can be considered as a method for producing the genuine printed material. In this embodiment, the authentic printed material is a printed material disclosed in Japanese Patent Application Laid-Open No. 2004-157021 filed earlier by the present inventors. An ink containing at least one kind of DNA to which identification information is given to a part of a pattern applied on a substrate, and an ink containing DNA different from at least one kind of said DNA to which no identification information is given , And a printed matter produced by printing or applying an arbitrary pattern, pattern, symbol or the like.

  The target printed matter for determining authenticity was placed on a sample mounting table of a nucleic acid transfer device composed of a pair of electrodes and a power source, and the placed target printed matter was contained in a place intended in advance of the genuine printed matter. First, it is confirmed whether or not the nucleic acid is present at the same position as the nucleic acid. As a confirmation method, the transfer material in a wet state is laminated in close contact with the nucleic acid-containing position of the target print, that is, the same position as the position of the nucleic acid applied to the authentic print, and the pair of electrodes is placed on the transfer material. , The electricity is supplied from the power source, the nucleic acid is eluted from the target print by electrophoresis and transferred to the transfer material, the transfer material to which the nucleic acid is transferred is broken, the nucleic acid is extracted using a buffer, and the extraction is performed. The solution is subjected to PCR amplification, and the obtained electrophoresis pattern is compared with the electrophoresis pattern of the nucleic acid obtained by transferring from a previously intended portion of the authentic print, and whether or not the target print is authentic is determined. Make a false decision. When an electrophoresis pattern cannot be obtained, no nucleic acid is contained.

  If necessary, the DNA base sequence information may be decoded. In this example, the material to which the nucleic acid was transferred was broken, the nucleic acid was extracted using a buffer, the extract was subjected to PCR amplification, and the resulting electrophoretic pattern and the authentic print were intended in advance. Compared with the electrophoresis pattern of the nucleic acid obtained by transferring from the location, the authenticity of whether or not the target printed matter is authentic is determined, but as an example, isothermal gene amplification using the extract Other known techniques such as amplification of DNA using a method and detection of an amplified product using a probe and an antibody may be used.

It is a block diagram of the nucleic acid transcription | transfer apparatus of this Embodiment. It is a figure which shows the result of gel electrophoresis. It is a figure which shows the result of the gel electrophoresis of a comparative example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1, 1 'electrode 2 Power supply 3 Voltmeter 4 Ammeter 5 Electrode support stand 6 Sample mounting stand 7 The thing containing nucleic acid 8 Wet filter paper

Claims (10)

A method for transferring a nucleic acid from a nucleic acid-containing substrate to a transfer material without destroying the substrate,
The wet transfer material is brought into contact with the substrate containing the nucleic acid and brought into close contact, the electrode is brought into contact with the transfer material, and the nucleic acid is eluted from the transfer material by electrophoresis. A method for transcription of nucleic acid, which comprises transcription. The method according to claim 1, wherein the electrode is brought into contact with the transfer material at a constant pressure. The method according to claim 1 or 2, wherein the transfer material is filter paper, cloth, agarose gel, or polyacrylamide. An apparatus for eluting nucleic acid from a nucleic acid-containing substrate and transferring it non-destructively to a transfer material, comprising a pair of electrodes for causing electrophoresis and a power source for energizing Transfer device. The transfer apparatus according to claim 4, further comprising a sample mounting table on which the nucleic acid-containing substrate is placed and a support member that supports the pair of electrodes. The transfer device according to claim 5, wherein the weight of the support member is controllable. The transfer device according to claim 5 or 6, wherein an alignment portion is provided on the sample mounting table. A method for determining the authenticity of a product containing nucleic acid,
The target product for determining authenticity is placed on the sample stage of the nucleic acid transfer device according to claim 4,
For the target product placed, the wet material to be transferred is adhered and laminated at the same location as the nucleic acid contained in the intended location in advance of the authentic product,
The pair of electrodes are brought into contact with the material to be transferred, electricity is supplied from a power source, nucleic acid is eluted from the target product by electrophoresis and transferred to the material to be transferred,
Breaking the transfer material to which the nucleic acid has been transferred, extracting the nucleic acid using a buffer solution,
A product authenticity determination method comprising PCR amplification of the nucleic acid extract and comparing the obtained electrophoretic pattern with a nucleic acid electrophoretic pattern obtained by transferring from a previously intended portion of the authentic product. 9. The product authenticity determination method according to claim 8, wherein secret information and / or individual information is given to the nucleic acid. The product authenticity determination method according to claim 8 or 9, wherein the product is a banknote, a passport, securities, or a precious printed matter.

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