WO1999067358A2

WO1999067358A2 - Method of preparing objects containing dna

Info

Publication number: WO1999067358A2
Application number: PCT/KR1999/000335
Authority: WO
Inventors: Yong-Bin Eym
Original assignee: Stargene Co., Ltd.
Priority date: 1998-06-24
Filing date: 1999-06-24
Publication date: 1999-12-29
Also published as: CN1306402A; KR19990064373A; WO1999067358A3; JP2002518040A; AU4655599A

Abstract

A method of preparing an object containing DNA, the method comprising the steps of: obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; introducing a solution of the amplified DNA into a cavity within the object through an opening of the cavity; and closing the opening of the cavity, wherein the object is with a defined shape, or mixing a solution of the amplified DNA with the object where the object is amorphous.

Description

METHOD OF PREPARING OBJECTS CONTAINING DNA

TECHNICAL FIELD

The invention relates to a method of preparing an object containing DNA.

BACKGROUND OF THE INVENTION

DNA is a basic generic material contained in chromosomes of cell organelles. Since DNA stores genetic information, it has been the subject of extensive study in both biological and medical fields. However, since only a small amount of DNA can be obtained from a living organism, the target sequences of DNA must be amplified before the DNA can be studied. Various methods and apparatuses for amplifying target sequences of DNA are known. (Mullis, K. et al, Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction., Cold Spring Harbor Symp. Quant.

Biol. 51, 263-273, 1986; US Patent Nos. 4,683,195, 4,683,202, and 4,889,818, Korean Patent No. 126,231)

The inventor has observed that DNA can represent differentiated characteristics of a donor. Exploiting this observation, the inventor has prepared objects containing DNA, specifically objects that can be preserved for a long time and have an attractive appearance, by introducing amplified DNA into the objects if they are solid, and by mixing amplified DNA with the objects if they are amorphous. The objects thus prepared represent the donor of the DNA by embodying the differentiated characteristics of the donor.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows a key ring that contains a DNA mixture prepared according to an embodiment of the present invention, and HLA DNA band strips.

Figure 2 shows a necklace prepared according to another embodiment of the present invention.

Figure 3 shows a necklace prepared according to a yet another embodiment of the present invention.

In the figures, each numeral has the following meaning :

1. a key ring 2. HLA DNA band strips

3. a picture of the donor of DNA

4. silk-screen signature

5. a solution containing DNA 6. water 10. 20. necklaces

BEST MODE FOR PRACTICING THE INVENTION

An object of the present invention is to provide a method of preparing solid objects, such as accessories, records and stationaries that contain DNA by injecting DNA of a donor into the objects and a method of preparing amorphous objects, such as perfume and ink, which contain DNA by mixing DNA of a donor with the objects. Both methods employ DNA extraction and amplification techniques. According to an embodiment of the invention, there is provided a method of preparing a solid object containing DNA, the method comprising the steps of: obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; introducing a solution of the amplified DNA into a cavity within the object through an opening of the cavity; and closing the opening of the cavity.

Alternatively, the solution of amplified DNA may be dried into a gel or solid state and the dried DNA introduced into the cavity of the solid object. According to another embodiment of the invention, there is provided a method of preparing an amorphous object containing DNA, the method comprising the steps of: obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; and mixing a solution of the amplified DNA with the object. Alternatively, the solution of amplified DNA may be dried into a gel or solid state and the dried DNA mixed with the amorphous object.

According to yet another embodiment of the invention, there is provided a method of preparing a solid object containing DNA, the method comprising the steps of: obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; subjecting the amplified DNA to a electrophoresis in a polyacrylamide gel; dyeing the electrophoresed DNA; drying the gel containing the dyed DNA to obtain a gel film; and applying the gel film to a surface of the object.

According to still another embodiment of the invention, there is provided a method of preparing a solid object containing DNA, the method comprising the steps of: obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; preparing HLA DNA band strips with the amplified

DNA; and applying the HLA DNA band strips to a surface of the object.

The present invention also provides objects containing DNA prepared by any one of the methods described above. The donor of DNA can be any living organism having DNA including a human being. In view of the demand for objects, celebrated figures associated with celebrities, such as political leaders, religious leaders, authors, movie stars, sports stars and singers, would be preferable donors of DNA. However, family members, members of a group, or pets could also be donors, since objects associated with these donors would be valuable to the other family members, group members, or pet owners, respectively.

The DNA sample may be any part or specimen that can be collected from the donor and contains the DNA of the donor. For a human DNA donor, blood, hair, skin tissue, fingernail, toenail, etc. may conveniently be used as the DNA sample.

DNA can be extracted and recovered by various known methods, though the methods could be modified depending on the DNA sample to be used. (Buffone et al, Isolation of DNA from biological specimens without extraction with phenol. Clin. Chem. 31 : 164-165, 1985) If, for example, the sample were blood, EDTA blood and RBC lysis buffer solution would be mixed with the sample and the mixture would be centrifuged. The residue thus obtained would be mixed with Chelex resin and boiled to extract DNA. Natural genomic DNA may be used for preparing objects containing DNA in accordance with the present invention. However, since only a small amount of natural DNA would be extracted from a sample, it is preferable to amplify the extracted natural DNA. DNA amplification may be carried out by polymerase chain reaction(s), or by two-stage amplifications, where the first amplification stage comprises a polymerase chain reaction, and the second amplification stage comprises the steps of inserting the DNA amplified in the first stage amplification into a vector to obtain a recombinant plasmid, introducing the recombinant plasmid into a host cell, culturing the host cell transformed by the recombinant plasmid, and separating the plasmids thus produced in large amounts.

The polymerase chain reaction (PCR) may be performed according to a method of Mullis et al., by means of a conventional polymerase chain reaction apparatus. Gene Kit (HLA Genotyping kit, Inno-lipa Co., Ltd. ) may be conveniently used for the amplification procedure.

In the second amplification stage using plasmids, the DNA amplified by the PCR may be cloned, for example, by using a TA cloning kit (Invitrogen). Otherwise, the amplified DNA may be reacted without dNTP in a medium containing T4 DNA polymerase for 15 minutes, and then reacted with dNTP for 15 minutes to obtain blunt ended DNA, where T and A bases protruding from each end of the DNA have been removed. Separately, an appropriate vector to be used in cloning process is cleaved by using a blunt end restriction enzyme such as Smal. To the vector thus obtained the blunt ended DNA is ligated by T4 DNA ligase.

The plasmid thus constructed may be introduced into an appropriate host cell for separating recombinant plasmids in large amounts. The host cell may be E. coli HB101, E. coli CSH 41, etc. The host cell transformed by the plasmid according to the present invention is cultured in Luria Broth (LB) medium, and then the plasmid produced in large amounts may be extracted and separated by a method of Birnboin & Doly (1979). The resulting DNA solution may be injected into a cavity within a solid object through an opening of the cavity, and the opening of the cavity may be closed to obtain an object containing DNA. Optionally, the DNA solution may be dried into a gel or solid state, and the dried DNA introduced into the cavity of the solid object. The DNA solution or dried DNA may be dyed to make it visible before it is introduced into the object. The DNA solution may be dyed with a dyeing reagent, such as bromophenol blue or phenol red and optionally mixed with glycerol, before being injected into the object. In the specification and claims, a "solid" object is any object having a defined shape, which may include, but is not limited to, jewelry such as rings, necklaces, ear rings, writing implements such as pens, fountain pens, pencils, pencil cases, and key rings, dolls, toys, shoes, bags, purses, wallets, records, CDs.

The cavity of the object into which DNA is to be injected may be formed by conventional processes. In order to enhance the decorative effect realized by dyed DNA, the cavity of the solid object preferably would be formed within a transformed pattern.

The transparent portion may have various shapes. The object containing DNA according to the present invention may be further embellished with a picture of the double stranded DNA helix, a signature and/or a picture of the donor of DNA to indicate that the object contains DNA of the donor.

The amplified DNA solution may optionally be dried, before being mixed with an amorphous object. An "amorphous object is any object not having a defined shape, which may include, but is not limited to perfume, ink, Chinese ink, and dyestuffs.

In accordance with an embodiment of the present invention, the amplified DNA may be subjected to electrophoresis in a polyacrylamide gel, dyed, and dried to obtain a gel film containing DNA. The resulting gel film may be applied to a surface of a solid object.

When DNA is extracted from a blood sample, HLA DNA band strips may be applied to a surface of the object to be decorated.

Specific characteristics of the DNA donor can thus be exhibited by the HLA DNA band strips.

The HLA DNA band strips may be prepared as follows. DNA is extracted from a blood sample and the extracted DNA is amplified. The amplified DNA may be reacted with, for example, a kit strip available from Inno-lipa Co., Ltd. Since probes for identifying various DNA sequences of leukocytes are present on the strip, these probes combine with the DNA by making base pairings. If a dyeing reagent which can react with the DNA were added to the strip, the dyeing reagent would react with DNA combined with the probe to form a color. The band strips would vary depending on the donor of DNA, since each donor has his or her own specific DNA base sequences.

Since objects prepared according to the methods of the present invention contain DNA, which represent specific characteristics of the DNA donor, one may remember the donor, such as celebrated figures, family members, lovers, pets, etc., by carrying the objects with one.

Further, objects prepared according to the methods of the present invention may be distinguished objects from which do not contain DNA. The method of the present invention may be used for determining whether a certain object is genuine or counterfeit. The DNA contained in the objects prepared according to the methods of the present invention may be preserved without substantial change for long periods of time. Therefore, the DNA may be used to confirm a person's identity: i. e. the DNA contained in an object prepared according to a method of the present invention may be compared with the DNA obtained from a person to be identified, thereby to determine, if required, whether the person is the donor or, is genetically related to the donor.

The following examples are provided to illustrate the method of preparing an object containing DNA according to the present invention.

EXAMPLE 1

Extraction of DNA 3m£ of whole blood collected from a donor were placed into a

EDTA coated tube, and at 2-8 °C in a refrigerator. 50/-£ of EDTA blood and RBC lysis buffer solution were placed into a sterilized 1.5mC micro-centrifuge tube and centrifuged at 13,000rpm for one minute to obtain a precipitated residue. The residue was washed with 500 £ of sterilized water. The centrifugation and the washing steps were repeated until all remaining RBCs were removed. 200μi of Chelex resin was added to the washed residue, boiled for 10 minutes and centrifuged. \0μϋ of the supernatant thus obtained was used for the further amplification procedure. The steps of boiling for ten minutes and subsequent refrigeration may be repeated.

DNA amplification

The materials shown in Table 1 below were mixed and the mixture was subjected to DNA amplification procedure under the conditions described in Table 2 below using a PCR apparatus (Inno-lipa Co., Ltd. HLA Genotyping kit).

Table 1. Amplification Mixture (50 μi in total)

Table 2. PCR Conditions

Pre-treatment

1. Preparation of HLA DNA band strips

HLA DNA band strips were prepared from the amplified DNA using HLA kit (Inno-lipa Co., Ltd)

2. Preparation of the DNA solution to be injected

The DNA obtained through the amplification procedure was cloned into a vector by means of a TA cloning kit (Invitrogen Co., Ltd.). The cloned plasmids were separated in large amounts. E. coli HB 101 was used, and the transformed E. coli HB101 was subjected to shaking incubation at 37 °C for 18 hours. The primarily HB 101 was cultured overnight in a conventional LB (Luria Broth) medium. The cultured bacteria were diluted to 1/100 with 500m6 of LB, and then innoculated in the medium and cultured. Plasmids were separated by the method of Birnboin & Doly (1979). E. coli HB101 having the desired plasmid DNA was cultured at 37 °C for 18 hours. The reagents for separating the plasmids, Sol I [50mM glucose, 25mM Tris • Cl(ρH 8.0), lOmM EDTA(pH 8)], Sol II [0.2N NaOH, 1 % SDS] and Sol III [5M potassium acetate 60m£, glacial acetic acid 1 1.5m£, H₂O 28.5 m£] were added sequentially, and the mixture thus obtained gently stirred, and centrifuged at 14,000 rpm for 10 minutes. To the supernatant thus obtained was added an equivalent amount of phenol : chloroform (1 : 1), mixed vigorously, and then centrifuged. The step was repeated twice or three times. 100% ethanol (two fold) was added to the clear supernatant thus collected, and the mixture was refrigerated for 10 minutes and then centrifuged. The supernatant was discarded, and the residue was dried, washed once with l m£ of 70% ethanol, dried, mixed with TE buffer solution to dissolve DNA, and refrigerated.

Glycerol and bromophenol blue were added to the DNA solution thus obtained.

Post-treatment

Two circular acrylic plates (each 5cm in diameter and 0.5cm in thickness) were placed next to one another such that a cavity was formed between the contiguous surfaces of the plates. One of the plate has a hole which communicates with the cavity and through which the DNA solution may be injected into the cavity. The plate also has a member to which a ring may be connected. As shown in Fig. 1, HLA band strips were attached to the sides of the plates, and a picture and silk-screen signatures of the donor of the DNA were applied to the outer surfaces of the plate. The acrylic plates touched to each other at their sides. The DNA solution prepared at the pre-treatment procedure was injected into the cavity through the hole in one of the plates, and the hole was then sealed. The acrylic object thus produced (1cm in thickness) is symmetrical about a plane. A key ring was completed by inserting a ring to the ring connection member of the acrylic object. EXAMPLE 2

Extraction of DNA

Hair obtained from a donor was cut to within 5cm from the roots. The hair was mixed with 2OO//0 of Chelex resin in a 5mC tube, and the mixture was boiled for 10 minutes to remove cell debris other than DNA. The mixture was then centrifuged at 12,000rpm. The supernatant thus obtained was used for the further amplification procedure.

DNA amplification

The amplification mixture was prepared as shown in Table 3 below and then subjected to a DNA amplification procedure under the PCR conditions shown in Table 4 below.

Table 3. Amplification Mixture (50 i£ in total)

Table 4. PCR Conditions

Pre-treatment

1. Preparation of polyacrylamide gel film containing DNA 10% polyacrylamide gel was prepared and the amplified DNA was loaded. The DNA was subjected to electrophoresis by applying 200V to the electrophoresis column. After electrophoresis was completed, the gel was precipitated in 0.4% silver nitrate solution for 30 minutes in order to dye the DNA in the gel. The gel was placed between two sheets of cellophane paper and dried to yield a stiff gel film containing DNA. (1mm in length, 3 cm in thickness)

2. Preparation of DNA solution to be injected

To obtain the DNA solution to be injected, phenol red and glycerol were added to the DNA obtained through the amplification procedure.

Post-treatment

A key ring was prepared according to the method of Example 1, except that the polyacrylamide gel containing DNA was used instead of HLA DNA band strips.

According to the present invention, one may prepare various objects containing DNA. These objects can represent differentiated characteristics of the donor of the DNA and be preserved for a long time.

Claims

1. A method of preparing a solid object containing DNA, the method comprising the steps of : obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; introducing a solution of the amplified DNA into a cavity within the object through an opening of the cavity; and closing the opening of the cavity.

2. The method of preparing an object containing DNA according to Claim 1, wherein the DNA amplification step is accomplished by means of polymerase chain reaction(s).

3. The method of preparing an object containing DNA according to Claim 1, wherein the DNA amplification step is accomplished by means of two-stage amplifications, the first amplification stage comprising a polymerase chain reaction, and the subsequent second amplification stage comprising the steps of inserting the DNA amplified in the first stage amplification into a vector to obtain a recombinant plasmid, introducing the recombinant plasmid into a host cell, culturing the host cell transformed by the recombinant plasmid, and separating the plasmids thus produced in large amounts.

4. The method of preparing an object containing DNA according to any one of Claims 1-3, wherein the solution of the amplified DNA is dried into a gel or solid state before being introduced into the cavity within the object.

5. The method of preparing an object containing DNA according to any one of Claims 1-3, wherein the solution of the amplified DNA is dyed before being introduced into the cavity within the object.

6. A method of preparing an amorphous object containing DNA, the method comprising the steps of : obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; and mixing a solution of the amplified DNA with the object.

7. The method of preparing an object containing DNA according to Claim 6, wherein the amorphous object is selected from the group consisting of perfume, ink, Chinese ink, and dyestuffs.

8. The method of preparing an object containing DNA according to Claim 6 or 7, wherein the solution of the amplified DNA is dried into a gel or solid state before being mixed with the object.

9. A method of preparing a solid object containing DNA, the method comprising the steps of : obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; subjecting the amplified DNA to electrophoresis in a polyacrylamide gel; dyeing the electrophoresed DNA; drying the gel containing the dyed DNA to obtain a gel film; and applying the gel film to a surface of the object.

10. A method of preparing a solid object containing DNA, the method comprising the steps of : obtaining a sample including DNA from a donor; extracting DNA from the sample; amplifying the extracted DNA; preparing HLA DNA band strips with the amplified DNA; and applying the HLA DNA band strips to a surface of the object.

11. An object containing DNA prepared by a method according to any one of Claims 1-10.