[Technical Field] The present invention relates to gene disk prepared using filter paper and a method for storing DNA semi-permanently using the same.

[Background Art] It is important to conserve efficiently many genetic resources to utilize effectively a variety of genetic information in living things. Therefore, the role of a gene bank that keeps DNA for a future use has been gradually generalized. For example, the DNA kept in the gene bank can be used as a crucial data for making identification of corpses on the event of unexpected accidents as well as paternity testing. Further, it can be applied to diagnosis of genetic diseases or analysis of a family tree about genetic diseases. Also, the stored DNA will be used in more variable manners according to advance in biotechnology. DNA is now stored generally by freezing. That is, skin or blood from human body is stored at a low temperature of -20 ~ -70 °C . However, such storage requires special freezing equipment and is a burden economically due to requirement of continuous cost for the equipment maintenance. Also, methods for storing DNA by purifying and drying it from biological sample and by putting it in ethanol at room temperature are well known. However, these methods are unsuitable to store DNA for a long period at a facility which do not provide specific conditions. Because the DNA stored using these methods is not observed visually. hi connection with methods for storing DNA, the Korean Patent Application No. 1999-0019574 disclosed a method for storing DNA size marker by lyophilizing it mixed with buffer, dyeing, stabilizer, specific gravity synergist and water. The Utility Model Registration No. 0271825 disclosed a human DNA storage container, in which has a storage room in interior part, wherein the room possesses a storage compartment to store DNA, and entrance of the compartment is plugged with a stopper and on the inside of the compartment is filled with inactive gas through a gas injection port and a vent, and then a joint part of the vent and the stopper is sealed. And, the Korean Patent Application No. 2002- 0065801 disclosed a method for storing DNA in vessel, wherein the DNA is kept as a form combined with diatomite or apatite hydroxide. But, method for storing DNA by mixing or combining with specific materials has problems in denaturalization of DNA itself after long period and recovery of DNA.

[Detailed Description of the invention] [Technical Problems] Therefore, as the present invention has been made in view of the above- mentioned problems and limitations, the present invention is to provide a method for storing DNA effectively. It is an object of the present invention to provide gene disk to store DNA effectively and a method for preparing thereof. Also, it is another object of the present invention to provide a method for storing DNA using the gene disk in a simple and convenient, economic and safe for a long period.

[Technical Solutions] The present invention relates to gene disk prepared using filter paper and a method for storing DNA semi-permanently using the same. The gene disk of the present invention is prepared by dropping DNA solution on filter paper and drying it; covering the dried paper with oilpaper back and forth; coating the filter paper covered with oilpaper by putting it in coating film; and obtaining the gene disk. The gene disk of the present invention can be prepared in a simple and economic manner as described in the above, makes it possible to store DNA safely for a long period and use DNA by recovering it conveniently and effectively, if necessary. According to the present invention, the quality of the filter paper used for preparing the gene disk does not care if dryable. For example, the filter paper comprises Whatman paper, Avantek paper, etc. Methods for storing DNA using the gene disk of the present invention comprise a form including gene disk on one side of crystal product or a form including by setting up storage space such as groove, and sealing devices on necklace or various accessories as well as card form. DNA storage card including the gene disk of the present invention comprises IC memory chip or 2D code and/or metal material to identify storage information of the gene disk. The metal material is an aluminum alloy corroded on iron plate, and it is described a serial number given to genetic resource and storage information of the gene disk on the metal material, and is thermostable at 1000°C . The DNA storage card can include both or any one of the chip or the code and the metal material. Specifically, it makes possible to primarily make identification of genetic resource by inserting the corroded metal material in the card on DNA damage by fire or unexpected accidents. Also, the 2D code has a smaller size than common bar code, however as it is connected with a computer which inputs information about identification of genetic resource due to be stored as a numerical formula, identification of genetic resource can be distinguished. Reference will now be made in detail to the preferred embodiments of the present invention. It is to be understood that the following examples are illustrative only and the scope of the present invention is not limited thereto.

[Advantages of the invention] The gene disk of the present invention can store DNA of the individual genetic information in a simple, safe and semi-permanent manner. And, DNA recovered conveniently and efficiently from the opened gene disk can be applied to gene test for a study and other objects, if necessary. [Brief description of the Drawings] Fig. 1 shows a gel photograph as a result of electrophoresis on DNA extracting from human skin epithelial cell. Fig.2 shows a gel photograph as a result of electrophoresis on PCR products of DNA extracting from human skin epithelial cell. Fig. 3 shows a gel photograph as a result of electrophoresis on DNA extracting from the gene disk of the present invention. Fig. 4 shows a gel photograph as a result of electrophoresis on PCR products of DNA extracting from the gene disk of the present invention. Fig. 5 is a photograph of DNA storage card including IC memory chip with the gene disk of the present invention. Fig. 6 is a photograph of DNA storage card including 2D code with the gene disk of the present invention.

[Preferred Embodiment for Carrying Out the invention] Example 1: DNA extraction To prepare gene disk including DNA, DNA was extracted and purified from cells as follows: First all, human skin epithelial cells were collected by attaching sticker on the inside skin of subject's elbow for 20 seconds. After mixing 2mL of TE buffer(10mM Tris-HCl/lmM EDTA), 120/z£ of 10% SDS and 2μ& of 20μg/ μJt proteinase K in 15mL-conical tube, the sticker was rolled and the rolled sticker was put into the tube, and then agitated strongly by inverting. The mixture was incubated in dry bath at 56°C on overnight, and 5Q0μJL of the supernatant was put into 0.5mL-conical tube followed by adding protein precipitation solution. The above mixture was incubated at 4°C for 20mins and after centrifuging(13,000rpm, lOmin) the mixture, approximately 500μi of the supernatant was put into new tube and was added by 50/^£ of 3M sodium acetate and ImL of 99.5% cold ethanol, and then was mixed with inverting mildly. The mixture was kept at -20 °C for lhr and centrifuged at 13,000rpm for lOmins at room temperature, and then the supernatant was discarded. ImL of 70% cold ethanol was added in and after inverting it, centrifuged at 13,000rpm for lOmins at room temperature, and then the supernatant was discarded. After upsetting and air-drying the tube, the tube was dried in dry bath incubator at 60 °C for over lOmins. The pellet was dissolved in 40 μi of TE buffer for 1 hour with tapping occasionally. We extracted DNA from sticker by the above process. Fig. 1 shows a gel photograph as a result of electrophoresis on DNA extracting from human skin epithelial cell using the sticker, Fig. 2 shows a gel photograph as a result of electrophoresis on Apo-E PCR product from the DNA extracted. As a result of the test on Apo-E DNA without enzyme cutting, we confirmed Apo-E DNA and, however total amounts of DNA was small.

[Embodiments for Carrying Out the invention] Example 2: Preparation of gene disk Gene disk including the DNA extracted from Example 1 was prepared by the following process. Oil paper, coating film and filter paper(Whatman paper) used in the process were available in commercial market. Filter paper(Whatman paper) was cut into a circular form of 6mm in diameter. \Qμi of total 50μl of purified DNA solution(10ng//^, concentration) obtained from Example 1 was dropped on the Whatman paper. The filter paper soaked with the DNA solution was dried completely by blocking airflow in clean bench. After drying DNA completely, the DNA was covered with oilpaper back and forth. It was put into coating film and coated by coating machine at 60°C~70°C. Gene disk obtained from the above was cold quickly and stored in the darkroom at room temperature.

Example 3: Recovery and Confirmation DNA from the gene disk To recover DNA stored on the gene disk prepared from Example 2 was carried out by the following process. DNA was recovered by four kinds of methods. The gene disk prepared from Example 2 included lOμβ of DNA solution(approximately 10ng/μβ concentration).

(1) TE buffer dissolution method The gene disk coated paper was cut with scissors and put into E- tube(1.5mL). 50μβ of TE buffer(10mM Tris-HCl/lmM EDTA) was added into the E-tube and vortexed mildly. After spinning it down, it was dissolved with tapping and spinning down irregularly at 40 °C for lhr. The disk was removed with forceps and spun it down over again.

(2) D.W drying method The gene disk coated paper was cut with scissors and put into E- tube(1.5mL). 50 βl of D.W. was added into the E-tube and tapped. It was kept at 40 °C for lhr in order to come out sufficiently DNA from the disk. After removing disk, it was dried completely in dry bath at 60 "C with tapping. And then, 20 μl of D.W. was added into the E-tube with tapping and dissolved at 40 °C for lhr.

(3) Isopropyl alcohol collection method The gene disk coated paper was cut with scissors and put into E-tube. 200//£ of D.W. was added into the E-tube and tapped. It was kept at 40 °C for lhr in order to come out sufficiently DNA from the disk. After removing disk, 200 μi of isopropyl alcohol was added in the tube and inverted, and then incubated at - 20 °C. It was centrifuged at 13,000 rpm for lOmins and the supernatant was discarded. The tube was upset and dried at room temperature for a few minutes, and then kept in dry bath at 60 °C . 20 μJL of TE buffer was added into the E-tube with tapping and dissolved at 60 "C for a short time or at 40 °C for a long time. (4) Phenol/Chloroform treatment method The gene disk coated paper was cut with scissors and put into E-tube. 200/z£ of D.W. was added into the E-tube and tapped. It was kept at 40 °C for lhr in order to come out sufficiently DNA from the disk. After removing disk, 200μ€ of phenol/chloroform^ :1) was added into the tube and mixed by inverting for lOmins, and then centrifuged at 13,000 rpm for lOmin. 200μβ of the supernatant was poured into new tube. 20 μi of 3M sodium acetate and 400 βi of 100% cold ethanol was added into the tube. The mixture was kept at -20 °C for lhr and centrifuged at 13,000rpm for lOmins, and then the supernatant was discarded. The tube was upset and dried at room temperature for a few minutes, and then kept in dry bath at 60 °C for over lOmins. 60 βi of TE buffer was added into the E-tube with tapping and dissolved at 40 "C for a long time after kept at 60 °C for lhr. Electrophoresis on the solutions collected by four methods described in the above was carried out and DNA band obtained from was observed. As a result of the electrophoresis, DNA band was observed in all of four methods. In detail, DNA band obtained from relatively simple methods, D.W. dry method and TE buffer dissolving method showed more darker color than that of obtained from more complex methods, isoprophyl alchol collection method and phenol/chloroform treatment method. Therefore, to recover DNA from gene disk can available any one of four methods, preferably, D.W. dry method and TE buffer dissolving method can be used to minimize DNA loss. Fig. 3 shows a gel photograph as a result of electrophoresis on DNA extracting from the gene disk after a lapse of one year of storage using TE buffer dissolving method. Fig. 4 shows a gel photograph as a result of electrophoresis on Apo-E PCR products of DNA extracting from the gene disk after a lapse of one year of storage using TE buffer dissolving method. According to the experimental result on the gene disk after a lapse of one year of storage, even though DNA storage for a long time, DNA stored on the gene disk was not denaturized and recovered effectively. Example 4: Preparation of DNA storage card According to Example 2 and 3, DNA stored on the gene disk of the present invention was collected without denaturation. The gene disk of the present invention can be stored as a form including gene disk on one side of crystal product or a form including by setting up storage space such as groove, and sealing devices on necklace or various accessories as well as card form. DNA storage card of a card form was prepared in the Example. Fig. 5 shows DNA storage card including IC memory chip and Fig. 6 shows DNA storage card including 2D code with the gene disk. Fig. 5 shows a storage card for genetic resource of the gene disk(a) and a portable card(b). The storage card of fig. 5(a) has an IC memory chip on upper part and a gene disk on lower part of its left side. And on its right side, name and issue date of card is described on upper part and metal material of aluminum alloy corroded on iron plate is included on lower part. It is described a serial number given to genetic resource and storage information of the gene disk on the metal material. The card can include both or any one of the chip and the metal material to make identification of genetic resource. Specifically, it makes possible to primarily make identification of genetic resource by inserting the corroded metal material in the gene disk on DNA damage by fire or unexpected accidents. Fig. 6 shows DNA storage card including 2D code and/or metal material with the gene disk and also, does a storage card(a) and a portable card(b). The above 2D code was stored as a numerical formula, and has a smaller size than common bar code, and as it is connected with a computer which inputs information about identification of genetic resource, identification of genetic resource can be distinguished.

[Industrial applicability] The gene disk of the present invention can store DNA of the individual genetic information in a simple, safe and semi-permanent manner. And, DNA recovered conveniently and efficiently from the opened gene disk can be applied to gene test for a study and other objects, if necessary. Accordingly, the present invention is very useful invention in biotechnology and pharmaceutical industry.