JP2002518040A - Method for producing DNA-containing products - Google Patents

Abstract

(57) Abstract: The present invention relates to a method for producing a DNA-containing product, and more specifically, a step of obtaining a sample containing DNA from a donor, a step of extracting DNA from the sample, and a step of extracting the extracted DNA. Amplifying, and injecting the amplified DNA-containing solution into a space formed in a product having a certain form and sealing or mixing with a product having no certain form. It relates to a method of manufacturing a product.

Description

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL FIELD The present invention relates to a method for producing a DNA-containing product.
Obtaining a sample comprising NA; extracting DNA from said sample;
Amplifying the extracted DNA, and injecting the amplified DNA-containing solution into a space formed in the product having a certain shape and sealing or mixing the solution with the product having no certain shape. The present invention relates to a method for producing a DNA-containing product.

BACKGROUND ART DNA is a basic substance of a nuclear chromosome and has various kinds of genetic information.
It has been studied and analyzed as an important research target in the fields of medicine and biotechnology. However, since only a small amount of DNA is separated from living organisms, a process of amplifying a specific sequence in DNA in a large amount is required to study the DNA, and various methods and apparatuses that can be used therefor are required. Are known (Mulis, K. et al, Specific enzym
atic amplification of DNA in vitro: the polymerase chain reaction.
Spring Harbor Symp. Quant. Biol. 51, 263-273, 1986; U.S. Patent Nos. 4,683,195, 4,683,202, 4,889,818, and Korean Patent No. 126,231).

In view of the fact that DNA is a substance that can uniquely express a differentiated character of only the donor, the present inventors have developed the DNA into various products that are in demand for carrying the donor's character. It has been found that by injecting or mixing, a DNA-containing product having excellent preservability and excellent aesthetic appearance can be obtained, thereby completing the present invention.

DISCLOSURE OF THE INVENTION [0004] An object of the present invention is to use accessories, a sound board,
Provided is a method for producing a DNA-containing product by injecting or mixing DNA exhibiting the characteristics of a donor into a product having a certain form such as stationery and a product having no certain form such as perfume and ink. It is in.

To this end, according to one embodiment of the present invention, a step of obtaining a sample containing DNA from a donor, a step of extracting DNA from the sample, a step of amplifying the extracted DNA, Injecting the prepared DNA-containing solution into a space formed in a product having a predetermined shape and sealing the space, and a method for producing a DNA-containing product.

[0006] Alternatively, the amplified DNA-containing solution may be dried in a gel or solid state, and the dried DNA may be placed in a space formed within a product having a certain shape.

According to another embodiment of the present invention, obtaining a sample containing DNA from a donor, extracting DNA from the sample, amplifying the extracted DNA, Mixing the solution with a product that does not have a morphology.

[0008] Alternatively, the amplified DNA-containing solution may be dried in a gel or solid state, and the dried DNA may be placed in a space formed within a product having a certain shape.

According to yet another embodiment of the invention, obtaining a sample containing DNA from a donor, extracting DNA from said sample, amplifying the extracted DNA, Electrophoresing the DNA on a polyacrylamide gel, staining the electrophoresed DNA, drying the gel containing the stained DNA to obtain a gel film,
Applying the method to a product having a certain morphology.

According to still another embodiment of the present invention, a step of obtaining a sample containing DNA from a donor, a step of extracting DNA from the sample, a step of amplifying the extracted DNA, The present invention provides a method for producing a DNA-containing product, comprising the steps of: producing an HLA DNA band strip from the obtained DNA; and applying the produced HLA DNA band strip to a product having a certain form.

[0011] The present invention further provides a DNA-containing product produced by any of the above methods.

The donor of the DNA to be used in the method of the present invention can be any organism having the DNA, including humans. In view of the demand for DNA-containing products, it is desirable that celebrities such as movie actors, sports stars, religion leaders, political leaders, writers, etc. become DNA donors, and families, members of specific groups, pets, etc. are DNA donors. It is also desirable that

When the DNA donor is a human, the DNA sample may be a part of the body such as blood, hair, skin tissue, nails, or any DNA-containing sample obtained from the body.

Although there is a slight difference in the method depending on the type of the sample, all methods are known in the art.
(Buffone, etc., Isolation of DNA from biological
specimens without extraction with phenol.Cln.Chem. 31: 164-165, 1985)
. In the case of blood, for example, a mixture of EDTA blood and RBC lysis buffer solution is subjected to centrifugation or the like, and the obtained residue is mixed with Chelex resin and boiled for extraction.

When producing a DNA-containing product according to the present invention, whole DNA in a natural state can be used, but since the amount is small, it is preferable to use it after amplification.

The DNA amplification can be performed simply by a PCR (polymerase chain reaction) step.
Next, the amplified DNA is inserted into a vector to obtain a recombined plasmid, which is introduced into a host organism, transformed, cultured, and the transformed host organism is cultured, and a large amount of the produced recombined plasmid is isolated. It can also be performed through a secondary DNA amplification process.

The PCR step can be performed, for example, by using a method such as Mulis or the like and using ordinary PCR equipment. More easily, a gene kit (Inno-lipa, HLA Genotyping
kit).

At the time of secondary amplification using a plasmid, the previously amplified DNA is cloned into a vector using a TA cloning kit (manufactured by Invitrogen). When a cloning kit is not used, vector cloning can be performed as follows. T4 DNA polymerase was added to the amplified DNA, and reacted for 15 minutes without dNTP. DNTP was added and reacted for 15 minutes. The DNA was allowed to react with T and A protruding at both ends (one on one side). (Protruding each) to make blunt end. The vector was also digested with a blunt-end restriction enzyme such as SmaI, and the blunt-ended DNA was ligated to the vector using T4 DNA ligase (ligation).
Obtain a recombined plasmid.

The isolated recombined plasmid can be introduced into an appropriate host cell and isolated in large quantities. As the host cells, E. coli HB101, E. coli CSH41 and the like can be used. Plasmids can be extracted and separated by methods such as Doly (1979).

The obtained DNA, in the form of a solution, is immediately injected into a space formed in a product having a certain form and sealed, and a DNA-containing product is obtained. Alternatively, the DNA solution may be dried and prepared in a gel or solid state and then put. DNA solutions or dried DNA can be stained and inserted into the product to enhance the visual effect. For example, a DNA solution can be stained by adding a staining reagent such as bromophenol blue or phenol red, and further mixed with glycerol or the like and injected into a product.

In the present invention, a product having a certain shape means a product having a fixed shape and a fixed pattern. For example, toys such as dolls, accessories, accessories such as key chains, necklaces, earrings, sound boards such as CDs, packaging boxes, books, writing utensils,
Examples include, but are not limited to, books and stationery, such as pencil cases, sports equipment, bags, clothing, footwear, and the like.

The space into which the DNA is injected into the product may be formed by a conventional method, and when the DNA is stained, in order to increase a decorative effect, the product portion into which the DNA is inserted may include: It is desirable to use a transparent material. The pattern formed of the transparent material can be variously selected. Furthermore, a general double helix diagram of DNA, which indicates that DNA is contained in the DNA-containing product, and a signature and a photograph of the DNA donor can also be added.

The amplified DNA solution may be selectively dried and then mixed with a product having no specific form to produce a DNA-containing product. In the present invention, a product having no fixed form means a liquid, a powder, or the like having no fixed shape and pattern. Examples include, but are not limited to, perfume, ink, ink and dye.

According to yet another embodiment of the present invention, the amplified DNA is electrophoresed on a polyacrylamide gel, then stained and visualized, and the gel is dried to form a film, By applying to a product having the above, a DNA-containing product can be obtained.

When a blood sample was used as the DNA extraction sample, HLA (Human Leukocyte A)
ntigen) By preparing a DNA band strip and applying it to a product having a certain form, a product containing DNA can also be obtained. HLA DNA band strips have the effect of visualizing features that only DNA donors have.

The HLA DNA band strip can be manufactured as follows. DNA extracted from a blood sample is amplified through PCR and reacted with, for example, a kit strip provided by Inno-lipa. Probes that can confirm various sites of leukocytes are adsorbed at regular intervals on the strip, and probes having a sequence corresponding to the extracted DNA bind to the DNA in a base pair. If a reagent that develops a color by reacting with a base-paired double-helix DNA is added, the bonded site will show a color, but since each person has a specific DNA base sequence, Various forms of strip band can be seen.

Since the DNA-containing product produced by the method of the present invention contains DNA which is a characteristic feature of the donor, it can be used as a souvenir to memorize the donor's famous person, family, lover, pet, etc. it makes sense. The product manufactured by the method of the present invention is DN
It can be distinguished from products that do not contain A. The method of the present invention can be used to determine which products are authentic.

The DNA contained in the product produced according to the present invention can be stored for a long period without change. Thus, after a certain period of time, by comparing the DNA contained in the product according to the present invention with the DNA of the person to be queried, the person to be identifiable when manufacturing the product according to the invention, Is the same person who provided the
It is also used to confirm whether or not a certain family relationship exists. For this purpose, for example, various cards containing DNA according to the method of the present invention can be manufactured.

Hereinafter, the present invention will be described based on examples. The following examples are provided only to facilitate understanding of the present invention, and the present invention is not limited to these examples.

BEST MODE FOR CARRYING OUT THE INVENTION Example 1 Extraction of DNA From a DNA donor, 3 ml of whole blood was collected in an EDTA-coated tube and stored refrigerated at 2 to 8 ° C.

In a sterilized 1.5 ml microcentrifuge tube, 50 μl of EDTA blood,
RBC lysis buffer solution was added and mixed well. The mixture was centrifuged at 13,000 rpm for 1 minute to obtain a precipitate. The precipitate was washed with 500 μl of sterile distilled water. If RBCs remained, the centrifugation and washing steps were repeated. Chelex resin 200 on the washed sediment
μl was added and dissolved well. After boiling for 10 minutes, centrifuge, and then
Was used for DNA amplification. After boiling for 10 minutes, cool to a refrigerated state,
Boil for minutes can be repeated.

DNA amplification An amplification mixture having the following composition was prepared, and a PCR device (manufactured by Inno-lipa,
DNA was amplified using HLA Genotyping kit).

[Table 1]

[Table 2]

Preprocessing 1. Production of HLA DNA band strip From the amplified DNA, use an HLA kit (Inno-lipa) to prepare HLA DNA
A band strip was manufactured.

[0034] 2. Production of DNA Insert The DNA amplified in the amplification process was cloned into a vector using a TA cloning kit (manufactured by Invitrogen), and the cloned plasmid was isolated in large quantities. At the time of large-scale isolation, E. coli HB101 was mainly used, and shaking incubation was performed at 37 ° C. for 18 hours under the main culture conditions. A commonly used LB (Luria broth) medium was prepared, and the primary HB101 bacteria were cultured overnight.
Cultured bacteria diluted 1/100 with 1 LB were inoculated again and cultured. Plasmid isolation was performed as follows (Birnboin & Doly, 1979). Escherichia coli (Escherichia coli HB101) having the plasmid DNA was cultured at 37 ° C. for 18 hours, and the plasmid separation reagent Sol was used.
I [50 mM glucose, 25 mM TrisCl (ph 8.0), 10 mM EDTA (ph 8.0)], Sol II [0.2N
NaOH, 1% SDS], SolIII [60 ml of 5M potassium acetate, glacial acetic acid]
11.5 ml, 28.5 ml of H ₂ O] were added successively, and then mixed gently.
After centrifugation for 1 minute, the upper layer solution was collected, and the same amount of a phenol: chloroform (1: 1) solution was mixed and mixed vigorously. This was centrifuged again and the upper layer solution was collected. The above process was repeated two or three times to obtain only a clean upper layer solution, and then ethanol (10
(0%) was added twice (by volume) and placed in a freezer for 10 minutes. After centrifugation, the supernatant was removed, dried, washed once with 1 ml of 70% ethanol, dried, dissolved in TE buffer, stored in a freezer, and used.

Glycerol and bromophenol blue were added to the separated DNA.

Post-Treatment Two circular acrylic plates (5 cm diameter, 0.5 cm thick) were positioned such that a space was created at the contact surface of the two plates. One has an injection port and a ring connection for the DNA solution. As shown in FIG. 1, an HLA DNA band strip was attached to the side of the acrylic plate, and a photograph of the DNA donor and a silk-printed signature were applied to the outer surface of the acrylic plate. The acrylic plates were glued on each other. Through the liquid injection hole,
The DNA solution produced during the pre-treatment process was filled and closed. It was manufactured so that both surfaces had the same shape, and the total thickness was 1 cm. The key ring was inserted between the ring connections to complete the accessory.

Example 2 Extraction of DNA The root of the hair of the donor of DNA was cut into a size of 5 mm. The cut hair roots were placed in a 1.5 ml tube, and 200 μl of a resin (chelex resin) was added to remove other cell debris excluding DNA, followed by boiling for 10 minutes. Then 1
After centrifugation at 2,000 rpm, the separated upper layer solution was used for DNA amplification.

DNA Amplification An amplification mixture having the following composition was prepared and subjected to PCR under the following conditions to amplify DNA.

[Table 3]

[Table 4]

Preprocessing 1. Production of DNA-containing polyacrylamide gel film A 10% polyacrylamide gel was prepared, and the amplified DNA was loaded. The gel DNA was electrophoresed by applying 200V. Upon completion of the electrophoresis, the gel was leached in a 0.4% silver nitrate aqueous solution for 30 minutes to stain the DNA. After staining
Cellophane paper was placed on the upper and lower surfaces of the gel and dried to obtain a strong film-like DNA-containing gel (thickness 1 mm, length 3 cm).

[0040] 2. Production of DNA Insert Glycerol and phenol red were added to the DNA solution obtained in the above amplification process to produce a DNA insert.

The post-treatment was carried out in the same manner as in Example 1, except that the DNA-containing polyacrylamide gel film prepared above was used instead of the HLA DNA band strip.
A key ring ornament containing A was manufactured.

INDUSTRIAL APPLICABILITY According to the method of the present invention, a DNA-containing product that can uniquely express a differentiated character of only a donor, can be stored for a long time, and has excellent preservability Is obtained.

[Brief description of the drawings]

FIG. 1 illustrates a key ring decorated to include a DNA solution and an HLA DNA band strip, according to one embodiment of the present invention.

FIG. 2 illustrates a necklace product decorated according to another embodiment of the present invention.

FIG. 3 illustrates a necklace product decorated according to yet another embodiment of the present invention.

[Explanation of symbols]

 1 Key holder 2 HLA DNA band strip 3 Photograph of DNA donor 4 Silk-printed signature 5 DNA solution 6 Water 10,20 Necklace

Claims (11)

[Claims] 1. a step of obtaining a sample containing DNA from a donor; a step of extracting DNA from the sample; a step of amplifying the extracted DNA; and a step in which the amplified DNA-containing solution has a certain form. Injecting into a space formed in the product and sealing the product. 2. The method according to claim 2, wherein the step of amplifying the DNA comprises a PCR (polymerase chain reac
2. The method for producing a DNA-containing product according to claim 1, wherein the method is carried out in the step of). 3. The method according to claim 1, wherein the DNA amplifying step comprises PCR (polymerase chain reac
), the primary amplified DNA is inserted into a vector to obtain a recombined plasmid, which is introduced into a host organism, transformed, and then transformed. 2. A secondary DNA amplification step of culturing and separating large quantities of the reassorted plasmids produced.
A method for producing the DNA-containing product according to the above. 4. The method for producing a DNA-containing product according to claim 1, wherein the amplified DNA-containing solution is dried in a gel or solid state and then put into a product. Method. 5. The method for producing a DNA-containing product according to claim 1, wherein the amplified DNA-containing solution is dyed and then injected into the product. 6. A step of obtaining a sample containing DNA from a donor; a step of extracting DNA from the sample; a step of amplifying the extracted DNA; Mixing the product with a product not to be produced. 7. The method for producing a DNA-containing product according to claim 6, wherein the product having no specific form is selected from the group consisting of perfume, ink, ink, and dye. 8. The method for producing a DNA-containing product according to claim 6, wherein the amplified DNA-containing solution is dried in a gel or solid state, and then mixed with the product. 9. A step of obtaining a sample containing DNA from a donor; a step of extracting DNA from the sample; a step of amplifying the extracted DNA; and electrophoresis of the amplified DNA on a polyacrylamide gel. Causing the gel containing the stained DNA to be dried to obtain a gel film; and applying the gel film to a product having a certain form. A method for producing a DNA-containing product, comprising: 10. A step of obtaining a sample containing DNA from a donor; a step of extracting DNA from the sample; a step of amplifying the extracted DNA; and producing an HLA DNA band strip from the amplified DNA. A method for producing a DNA-containing product, comprising the steps of: applying the produced HLA DNA band strip to a product having a predetermined form. 11. A DNA-containing product produced by the method according to any one of claims 1 to 10.