KR20000063098A - Method of preparing objects containing dna - Google Patents

Abstract

PURPOSE: A preparation method of DNA-containing product is provided. The resultant DNA-containing product is a unique product for a donor only and is excellent in storage. CONSTITUTION: The preparation method of DNA-containing product comprises steps of : obtaining a sample containing DNA from the donor; carrying out the DNA preparation from the sample; amplifying the prepared DNA using PCR; optionally, drying the amplified DNA-containing solution; and infusing and sealing hermetically the dried DNA in the finite product or mixing the DNA solution into the non-finite product. The DNA amplification is characterized in comprising a first amplification using PCR; and a second amplification consisting of inserting the amplified DNA into a vector to obtain a recombinant plasmid, transforming a host organism with the plasmid, culturing the host organism and isolating the produced recombinant plasmid.

Description

METHOD OF PREPARING OBJECTS CONTAINING DNA}

The present invention relates to a method for producing a DNA-containing product, and more particularly, obtaining a sample containing DNA from a donor; Extracting DNA from the sample; Amplifying the extracted DNA; A method of producing a DNA-containing product comprising the step of selectively drying the amplified DNA-containing solution, and then injecting into a space formed in a product having a certain shape and sealing or mixing with a product having no shape. .

DNA is a basic substance of cell nucleus chromosome and has various genetic information. However, since only a small amount of DNA is separated from living organisms, it is necessary to amplify a large amount of a specific sequence of DNA in order to study it, and various methods and apparatuses that can be used are known (Mulis, K. et al, Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction.Cold spring Harbor Symp.Quant. Biol. 51, 263-273, 1986; United States Patent Nos. 4,683,195, 4,683202, 4,889,818, and Korean Patent No. 126,231 Arc, etc.)

The present inventors consider that DNA is a material that can uniquely express a donor's differentiated character, and injects or mixes DNA into various products in demand to carry the donor's character, thereby providing excellent preservation and aesthetic beauty. The present invention has been completed by finding out that a containing product can be obtained.

Accordingly, an object of the present invention is to prepare a product containing DNA by injecting or mixing the DNA showing the characteristics of the donor into various products such as accessories, records, stationery, perfume, etc. using techniques such as DNA extraction and amplification. To provide a way.

1 shows a key ring product decorated to contain a DNA solution and a HLA DNA band strip in accordance with one embodiment of the present invention.

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

3 shows a necklace product decorated according to another embodiment of the present invention.

※ Explanation of code for main part of drawing

1: key chain 2: HLA DNA band strip

3: photo of DNA donor 4: silk-printed autograph

5: DNA solution 6: water

10, 20: necklace

To this end, according to one embodiment of the invention, obtaining a sample containing DNA from the donor; Extracting DNA from the sample; Amplifying the extracted DNA; A method for producing a DNA-containing product is provided, which comprises the step of selectively drying the amplified DNA-containing solution, and then injecting and sealing the space formed in the product having a certain shape.

According to another embodiment of the invention, obtaining a sample containing DNA from the donor; Extracting DNA from the sample; Amplifying the extracted DNA; A method for producing a DNA-containing product is provided, which comprises mixing the amplified DNA solution with a product that has been selectively dried and then has no shape.

Donors of DNA to be used in the methods of the invention can be any organism having DNA, including humans. Given the demand for DNA-containing products, it is desirable that celebrities, such as lovers, sports stars, religious leaders, political leaders, writers, be DNA donors, and family members, members of certain groups, pets, etc. desirable.

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

Although there are some differences in the method depending on the type of sample, all can be extracted and recovered by known methods (Buffone et al., Isolation of DNA from biological specimens without extraction with phenol.Cln.Chem. 31: 164). 165, 1985) For example, the mixture of EDTA blood and RBC lysis buffer may be centrifuged, and the obtained residue may be mixed with Bolex resin, boiled and extracted.

When producing a DNA-containing product according to the present invention may be used in the natural state of the whole DNA, it is preferable to amplify and use because the amount is a small amount.

DNA amplification may be performed simply by a polymerase chain reaction (PCR) process, and may include primary DNA amplification by polymerase chain reaction (PCR); The first amplified DNA is inserted into a vector to obtain a recombinant plasmid, introduced into the host organism, transformed, and then the transformed host organism is cultured, followed by a second DNA amplification process for mass separation of the resulting recombinant plasmid. You can also do it.

A PCR process can be performed using a normal PCR apparatus, for example using the method of Mulis. More simply, the gene kit (HLA Genotyping kit available from Inno-lipa) can be amplified.

In the second amplification using the plasmid, the amplified DNA is first cloned into a vector using a TA cloning kit (Invitrogen). If you do not use a cloning kit, you can vector clone as follows: T4 DNA polymerase was added to the amplified DNA for 15 minutes without dNTP, and dNTP was reacted for 15 minutes to remove the blunt ends from which T and A (one on each side) protruding DNA were removed. blunt end). The vector is also cleaved with a blunt end restriction enzyme such as SmaI, and the blunt-ended DNA is ligated to the vector using T4 DNA ligase to obtain a production plasmid.

The cloned plasmid can be introduced into a suitable host cell and separated in large quantities. As host cells, E. coli HB 101, E. coli CSH 41, and the like can be used. The host cells transformed by introducing the plasmid according to the present invention are cultured in LB (Luria broth) medium and the like, and Birnboin & Doly (1979). The plasmid can be extracted and separated according to methods such as).

The amplified DNA can be injected into a space formed in a product having a uniform shape in a solution state and sealed to obtain a DNA-containing product. Alternatively, the DNA solution can be dried and gelled or injected into a solid state. DNA solution or dried DNA can be stained and inserted into the product to enhance the visual effect.

For example, the DNA solution may be dyed by adding dyeing reagents such as bromophenol blue and phenol red, and further mixed with glycerol or the like and injected into the product.

In the present invention, the product having a certain form means an article having a fixed shape and shape. For example, toys such as dolls, ornaments, accessories such as key chains, necklaces, and earrings, records such as CDs, packaging boxes, books, writing instruments, pencils, books and stationery, sporting goods, bags, clothing, Footwear and the like include, but are not limited thereto.

The space in which the DNA is to be injected may be formed according to a conventional method, and in order to increase the decorative effect when the DNA is stained, it is preferable to use a transparent material for the product portion into which the DNA is inserted. The shape formed by the transparent material can be variously selected. Furthermore, a double helix picture of a general DNA, a signature and a picture of a DNA donor, etc., which are characteristically showing that the DNA-containing product contains DNA, may be added.

According to another embodiment of the present invention, it is also possible to prepare a DNA-containing product by selectively amplifying the amplified DNA solution and then mixing it with a product having no form. In the present invention, a product that does not have a predetermined form means a liquid, powder, or the like, having no fixed shape and shape. For example, cosmetics such as perfumes are included, but are not limited thereto.

According to another embodiment of the present invention, the amplified DNA is electrophoresed on a polyacrylamide gel, then stained and visualized, the gel is dried to form a film, and then the DNA is applied to a product having a certain form. A product containing can be obtained.

In the case of using a blood sample as a DNA extraction sample, a product containing DNA may be obtained by making a HLA (Human Leukocyte Antigen) DNA band strip and applying it to a product having a predetermined shape. HLA DNA band strips have the effect of visualizing features unique to DNA donors.

Since the DNA-containing product produced according to the method of the present invention contains DNA, which is a unique feature of the donor, it is meaningful as a souvenir to remember the donor's celebrities, family members, lovers, pets and the like. Therefore, it can be used as a funeral article, a coma, a religious offering, and the like.

On the other hand, due to the nature of the DNA, the DNA contained in the product prepared according to the present invention can be preserved without a long-term change. Therefore, after a certain period of time, by comparing the DNA contained in the product according to the present invention and the DNA of the subject to be identified, whether the subject is the same person or a certain family relationship with the donor who provided the DNA when manufacturing the product according to the present invention. It can also be used to check whether it is present. For this purpose, for example, various cards containing DNA according to the method of the present invention can be produced.

Hereinafter, the present invention will be described by way of examples. The following examples are provided only to facilitate understanding of the present invention, but the present invention is not limited to these examples.

Example 1

<DNA extraction and acquisition>

3 ml of whole blood was collected in an EDTA coated tube from a donor of DNA and refrigerated at 2-8 ° C.

50 µl of EDTA blood and RBC lysis buffer were added to a sterile 1.5 ml microcentrifuge tube and mixed well. Centrifugation was performed for 1 minute at 13,000 rpm to obtain acupuncture. The needles were washed with 500 μl sterile distilled water. If RBC remained, the centrifugation and washing were repeated. 200 μl of Chelex resin was added to the washed salivary solution, and the solution was well released. After boiling for 10 minutes, the mixture was centrifuged and 10 µl of the supernatant was used for DNA amplification. After boiling for 10 minutes, cool in a chilled condition and then boil again for 10 minutes.

<DNA amplification>

An amplification mixture was prepared with the following composition, and DNA was amplified using a PCR instrument (Inno-lipa, HLA Genotyping kit) under the following conditions.

I. Amplification mixture (50 μl total)

Amplification Buffer 10 μl (with dATP mix) Primer mixture 10 μl MgCl ₂ 10 μl Taq Enzyme (5unit / ml) 0.6 μl Extracted DNA 5.0 μl ddH ₂ O 14.4 μl

II. PCR conditions

95 ℃ 5 minutes 1 cycle 95 ℃ 20 seconds 30 cycles 55 ℃ 20 seconds 72 ℃ 30 seconds 72 ℃ 10 minutes 1 cycle

<Pretreatment>

1. HLA DNA Band Strip Manufacturing

HLA DNA band strips were prepared from the amplified DNA using an HLA kit (manufactured by Inno-lipa).

2. DNA Insert Preparation

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. E. coli HB101 was used as a strain during mass separation, and the strain culture conditions were shaken incubated for 18 hours at 37 ° C. In general, LB (Luria broth) medium was used to culture primary HB101 bacteria overnight, and then cultured by inoculating 1/100 diluted cultured bacteria in 500ml LB. Plasmid separation was performed as follows (Birnboin & Doly, 1979). Escherichia coli (E. coli HB101) having plasmid DNA was incubated at 37 DEG C for 18 hours, and plasmid separation reagent SolI [50 mM glucose, 25 mM Tris-Cl (pH 8.0)], 10 mM EDTA (pH 8.0)], Sol II [0.2N NaOH, 1% SDS], SolIII [60M potassium acetate 60ml, glacial acetic acid 11.5ml, H ₂ O 28.5ml] were added sequentially, mixed gently, centrifuged at 14000 rpm for 10 minutes, and the supernatant was collected and phenol. : Chloroform (1: 1) solution was mixed in the same amount and mixed vigorously. This was again centrifuged and the supernatant was collected, and the procedure was repeated 2-3 times to take only the clean supernatant, and then ethanol (100%) was added twice (volume) and left in the freezer for 10 minutes. After centrifugation, the supernatant was removed and dried, washed once with 1 ml of 70% ethanol, and dried to dissolve DNA in TE buffer and used after storage in a freezer.

Glycerol and bromophenol blue were added to the isolated DNA.

<After treatment>

Two circular acrylic plates 5 cm in diameter and 0.5 cm thick (one having a liquid inlet and a ring connection) with concave inner portions were prepared in a conventional manner, and the HLA DNA band strips (sides) as shown in FIG. Attached), photographs of DNA donors and silk-printed signs, and then spray strongly adhered to both sides of the acrylic. Through the liquid inlet, the DNA solution prepared during the pretreatment was filled up and blocked. Both sides were made to have the same shape, and the overall thickness was 1 cm. A key ring is attached to the hook to complete the accessory.

Example 2

<DNA extraction and acquisition>

The hair root portion of the hair of the donor of DNA was cut to a size of 5 mm. The cut hair roots were placed in a 1.5 ml tube, 200 μl of resin was added to remove other cell debris except DNA, and then boiled for 10 minutes. Subsequently, the supernatant separated by centrifugation at 12,000 rpm was used for DNA amplification.

<DNA amplification>

An amplification mixture was prepared with the following composition, and DNA was amplified by PCR reaction under the following conditions.

I. Amplification mixture (50 μl total)

Amplification Buffer [Tris-Cl (pH 9.0), AMS, BSA, MgCl ₂ ] 5 μl Primer Mixture [D17S695, D21S11, NerR, hTPO] 4 μl dNTP 1 μl Taq Enzyme (5unit / ml) 0.6 μl Extracted DNA 5.0 μl ddH ₂ O 34.4 μl

II. PCR conditions

95 ℃ 1 minute 30 cycles 62 ℃ 1 minute 72 ℃ 1 minute

<Pretreatment>

1. Preparation of DNA-containing polyacrylamide gel film

A 10% polyacrylamide gel was prepared to load amplified DNA. A 200 V current was applied to electrophores the gel DNA. After the electrophoresis was completed, the gel was immersed in 0.4% silver nitrate aqueous solution for 30 minutes to stain the DNA. After dyeing, cellophane paper was placed on the upper and lower surfaces of the gel and dried to obtain a thin film-like DNA-containing gel (thickness 1 mm, length 3 cm).

2. DNA Insert Preparation

DNA inserts were prepared by adding glycerol and phenol red to the DNA solution obtained in the amplification process.

<After treatment>

A key ring ornament containing DNA was prepared in the same manner as in Example 1 except that the prepared polyacrylamide gel film was used instead of the HLA DNA band strip.

According to the method of the present invention, it is possible to uniquely express a donor's differentiated character, and to obtain a DNA-containing product having excellent preservability that can be retained for a long time.

Claims (7)

Obtaining a sample containing DNA from the donor; Extracting DNA from the sample; Amplifying the extracted DNA; Injecting and amplifying the amplified DNA-containing solution into a space formed in a product having a certain shape, the product being a toy, accessories, ornaments, records, books, packaging, sports goods, shoes, bags , Clothing, cards, photos, souvenirs, works of art, funerals, coma or religious products, characterized in that the production of DNA-containing products. The method of claim 1, The method of producing a DNA-containing product, characterized in that the DNA amplification step is carried out by a polymerase chain reaction (PCR) process. The method of claim 1, The DNA amplification step is a primary DNA amplification step by a polymerase chain reaction (PCR); The first amplified DNA was inserted into a vector to obtain a recombinant plasmid, introduced into the host organism, transformed, followed by culturing the transformed host organism, and through a second DNA amplification step of mass separation of the resulting recombinant plasmid. Method for producing a DNA-containing product, characterized in that carried out. The method according to any one of claims 1 to 3, The method of producing a DNA-containing product further comprises the step of drying the amplified DNA-containing solution before injecting the product. The method according to any one of claims 1 to 3, The method of producing a DNA-containing product further comprises the step of visualizing by injecting the amplified DNA-containing solution into the product. Obtaining a sample containing DNA from the donor; Extracting DNA from the sample; Amplifying the extracted DNA; Method for producing a DNA-containing cosmetic comprising the step of mixing the amplified DNA solution with a cosmetic. The method of claim 6, Method for producing a DNA-containing cosmetic, characterized in that further comprising the step of drying the amplified DNA-containing solution before mixing with the cosmetic.