KR20050120549A - The method of preparing an article containing dna by using sol-gel technique - Google Patents

Abstract

The present invention comprises the steps of extracting DNA; Amplifying the extracted DNA; Preparing a starting solution by adding a DNA solution obtained by dissolving the amplified DNA in water to a mixed solution of metal alkoxide and alcohol and stirring at 35 to 45 ° C; Injecting the starting solution into a target product; And it relates to a method for producing a DNA-containing product comprising the step of drying and maturing the starting solution in the target product into a dried gel body in the target product. According to the present invention, by applying the sol-gel method used for low temperature synthesis of ceramics to form DNA as a dry gel body, it is possible to minimize the influence of external conditions or stimulation, and to maintain the original DNA of DNA without destroying the arrangement of the DNA. As a result, processing to the final product is easier and the aesthetics of the final product can be improved.

Description

The method of preparing an article containing DNA by using sol-gel technique}

The present invention relates to a method for producing a DNA-containing product using the sol-gel method, and more particularly, to a method for producing a DNA-containing product using the sol-gel method to enable long-term storage without damaging the DNA.

DNA is a basic substance of nuclear chromosomes and a chemical substance that contains various genetic information and is considered as a key research subject in medicine, biotechnology related to medicines and genetic manipulation. However, since DNA cannot be produced artificially and the amount extracted from biological tissues is limited, there is a problem in that it cannot meet the demand for various research and development that is active in the medical and biotechnology fields. Therefore, in order to overcome such a problem, a technique for amplifying and culturing DNA extracted from biological tissues has been developed in the past, and a typical PCR (Polymerase Chain Reaction) method has been proposed and used (US Pat. No. 4,683,202). PCR is a way to selectively amplify specific DNA in vitro, not in living organisms, by accurately understanding the DNA replication process in cells, by separating the enzymes involved in the replication, and by understanding their role. PCR is the most widely used method for amplifying DNA, and is widely used for paternity identification and criminal arrest through gene inspection, gene analysis, pathogen detection, and biological evolution analysis.

As the amplification culture technology of DNA has been developed, various research and development using DNA has been active, and on the other hand, attempts have been made to develop various products using DNA. In other words, the idea that DNA can be a unique symbol of living things including humans is one of the attempts to contain and commercialize DNA of living things including general people, celebrities, humans and pets. A method of manufacturing a product containing DNA has already been disclosed in Korean Unexamined Patent Publication No. 2000-63098 and Japanese Patent No. 334577. However, the above literature suggests only a method of manufacturing a product in which a DNA-like electrophoresis is used to print a barcode-like image or inserts a DNA solution into a tangible or intangible capsule, and the DNA is solidified and inserted into the product. Or the technique to be contained is not specifically mentioned.

If the DNA is solidified into a solid or gel body, it can be used in a variety of products. In other words, when the DNA is processed into a solid or gel body, it is very useful because it minimizes the effects of external conditions or stimuli and maintains the original shape without destroying the DNA sequence. Furthermore, the use of DNA in solid or gel form not only facilitates processing into the final product, but also has the side effect of improving the aesthetics of the final product. However, since DNA is very vulnerable to heat or chemical stimulation, it is difficult to be easily processed into a solid or gel body. For example, when the general glass molding technique is used to process DNA into a solid or gel body, the molding temperature should be set at approximately 600 to 1000 ° C. This temperature condition is a high temperature exceeding a threshold temperature at which DNA array is destroyed. Corresponds to DNA has a limit to break down and fail to maintain its alignment at high temperatures above about 150 ° C.

The inventors have noted that the sol-gel method, a technique used for low temperature synthesis of ceramics, can be used to overcome this limitation. The sol-gel method is a method for synthesizing inorganic materials such as amorphous ceramics (glass), crystallized glass, and ceramics (sintered polycrystal, thin film, ultra fine powder) from a starting material of a metal alkoxide or metal salt at low temperature. The process of converting a liquid into a continuous solid network. That is, the organic or inorganic compound of the metal is made into a solution together with a solvent of a suitable liquid, and the hydrolysis-polycondensation reaction of the compound is carried out in the solution to form a sol in which the fine particles are suspended in the liquid. This structure is a method of synthesizing glass or ceramics by appropriately heat-treating what has become a gel state containing a liquid or gas between the poles. The sol-gel method is divided into an alkoxide sol-gel (polymeric gel) and a colloidal sol-gel (particulate gel) according to the kind of molecular precursors used therein.

In general, the sol-gel method is carried out by preparing a starting solution, stirring it, and then drying at a low temperature (about 80 ° C.), followed by drying at a high temperature (600 ° C.). In the present invention, the high temperature drying process, which is the final step of the process step, is omitted, and only the low temperature drying process is performed. This is because when the high temperature drying process prevents the DNA from being broken, and only the low temperature drying process is performed, DNA of a solid or gel body that can be used for processing into a final product can be obtained.

One technical problem to be achieved by the present invention is to provide a method for effectively producing a DNA-containing product by processing the DNA into a solid or gel body using the sol-gel method.

One embodiment of the present invention for achieving the above technical problem, the step of extracting DNA; Amplifying the extracted DNA; Preparing a starting solution by adding a DNA solution obtained by dissolving the amplified DNA in water to a mixed solution of metal alkoxide and alcohol and stirring at 35 to 45 ° C; Injecting the starting solution into a target product; And it provides a method for producing a DNA-containing product comprising the step of drying the starting solution added to the target product at 50 ~ 60 ℃ in the target product and aged at 75 ~ 85 ℃ to form a dried gel body.

Another embodiment of the present invention for achieving the above technical problem, extracting DNA; Amplifying the extracted DNA; Preparing a starting solution by adding a DNA solution obtained by dissolving the amplified DNA in water to a mixed solution of metal alkoxide and alcohol and stirring at 35 to 45 ° C; Drying the starting solution at 50 to 60 ° C. and aging at 75 to 85 ° C. in a separate container to form a dried gel body; And it provides a method for producing a DNA-containing product comprising the step of including the formed dry gel body in the target product.

Among the two preferred embodiments of the present invention mentioned above, the former is a method of aging and drying by adding a starting solution to a target product in forming a DNA into a dry gel body using a sol-gel method, while the latter separately separates the starting solution. There is a difference in adopting a method of introducing into a target product after aging and drying.

Extraction of the DNA in the present invention can be carried out according to a known method. In the case where the DNA extraction target is a human, blood, hair, nails, and other body parts capable of extracting DNA may be collected and used as a sample without limitation. Methods of extracting DNA from the sample taken vary depending on the type of sample and the details thereof are also known in the art. According to a preferred embodiment of the present invention, the DNA may be extracted by using a human scalp (including the hair root) through a cell lysis step, an impurity removal step, and an aggregation and drying step of the DNA.

According to the present invention, the DNA extracted from the above can be used in a natural state or amplified. Various known methods can be used to amplify DNA, but according to a preferred embodiment of the present invention is carried out by a PCR process. In the present invention, the method for carrying out the PCR process in detail and the apparatus used therein may be appropriately selected from those known in the art.

The present invention is characterized in that it comprises the step of molding the amplified DNA into a dry gel body using a sol-gel method used in the field of low temperature synthesis of ceramics. According to a preferred embodiment of the present invention, an alkoxide sol-gel method using a metal alkoxide as a precursor is applied. As for the general sol-gel method including the alkoxide sol-gel method, the reaction mechanism is known in the art. It is also well known how to specifically perform the sol-gel method. In the present invention, the sol-gel method is applied in order to form DNA into a solid or gel body, through which DNA can be effectively introduced into a target product and the added DNA can be preserved without circular destruction. In particular, in the present invention, by omitting the high temperature drying process, which is the final step in the known sol-gel process step, the defect of the arrangement of DNA at high temperature is destroyed. The present invention has been made based on the technical idea of applying the sol-gel method used for low temperature synthesis of ceramics to form DNA into a solid or gel body, and this breakthrough application is original, which has not been tried to date.

The present invention includes the step of preparing a starting solution using a metal alkoxide as a precursor. In the present invention, it is preferable to use Si-alkoxide such as Si (OCH ₃ ) ₄ as the metal alkoxide, but any other known metal alkoxide may be used without limitation as long as it is within the scope of achieving the effects of the present invention. Metal alkoxides are preferred as precursors because they have advantages such as easy reaction with water, easy transition to inorganic materials, ease of synthesis, and generally no toxicity.

In the method for producing a DNA-containing product according to the present invention, application of the sol-gel method is started from the step of preparing a starting solution. The starting solution according to the present invention is prepared by dissolving amplified DNA in water to prepare a DNA aqueous solution by adding a metal alkoxide and alcohol to the mixture, and agitating at 35 to 45 ° C. The DNA contained in the starting solution is converted into a sol state through hydrolysis, condensation or chemical substitution reaction, and then formed into a dried gel body through aging at 50 to 60 ° C. and drying at 75 to 85 ° C. The formed dried gel body is not high in itself, but can be effectively used for commercialization by properly selecting the target product. For example, when inserted into a product such as jewelry or cubic, it can play an excellent role in fixing and preserving DNA. On the other hand, the dried gel body may be used as a cultured pearl nucleus may be prepared from cultured pearls with DNA inserted.

According to the present invention, it comprises the step of introducing the prepared starting solution to the target product. The subject product referred to in the present invention refers to a product that can be commercialized with the DNA inserted or contained therein. Specific target products to which the present invention can be applied include various accessories, personal collections, stationery, precious metals, stationery, books, etc. that can be used as souvenirs or gifts, but in addition, if it can be commercialized by including dry gel chain DNA, It can be considered without limitation. According to a preferred embodiment of the present invention, DNA may be inserted or contained in cubic or cultured pearls as a target product.

According to one embodiment of the present invention, the starting solution added to the target product comprises the step of maturing and drying in the target product to form a dry gel body. Meanwhile, according to another embodiment of the present invention, the starting solution may be aged and dried in a separate container to form a dried gel body, and then inserted into the target product.

In the present invention, the gelling is a starting solution is subjected to the polymerization, coarsening and phase transition while the aging step, according to a preferred embodiment of the present invention to be aged at 50 ~ 60 ℃, 48 to 52 hours desirable.

On the other hand, the drying step refers to an operation of burning off the organic solvent or the remaining organic material used in the sol-gel method, and finally forming an amorphous inorganic thin film. To this end, it is preferable to slowly evaporate the volatile component over about 6 hours while keeping the sealed container slightly open at 50 to 60 ° C., and then heating under vacuum to dry at 75 to 85 ° C. for 20 to 30 hours.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings and specific examples. However, the following examples are only illustrated to describe the present invention in detail, and the scope of the present invention is not limited by the description of the embodiments.

Figure 1 illustrates a form in which the DNA dry gel body is inserted into the cubic in accordance with an embodiment of the present invention, reference numeral 1 shows a perforation provided to insert the DNA is formed in the cubic. Reference numeral 2 denotes DNA in a dry gel state inserted in the perforation, and d denotes 0.2 to 0.5 mm as a preferable perforation width.

Figure 2 shows the form in which the DNA dry gel body is used as a nucleus of cultured pearls according to another embodiment of the present invention. In Fig. 2, reference numeral 1 denotes a cultured pearl and reference numeral 2 denotes a DNA dried gel body inserted as a nucleus of the cultured pearl.

Example 1

DNA  Gemstones ( Cubic  Form)

Extraction of DNA

As a sample, human hair (5 hairs including hair roots) was taken, and the collected 5 hairs were cut into 1 cm size from the hair root where the cells are most attached and placed in sterile 1.5 ml E-tube. . Cell lysate was added to 1.5 ml E-tube containing the cut hair, and the cells were stagnated at 56 ° C. for at least 2 hours to destroy cells. Through this process, substances such as DNA, RNA, protein, and polysaccharide are mixed in the solution. Subsequently, impurities such as cell masses, RNA, proteins, and polysaccharides were removed using phenol or chloroform to obtain a solution in which only DNA was left. After mixing 1 / 10-fold salt (Salt: 3M Sodium acetate, 7.5M Ammonium acetate) with 2.5 times 70% ethanol in a solution containing only DNA, it is left in a freezer at -20 ° C for at least 2 hours and then 12,000 at 4 ° C. DNA was aggregated by centrifuge for 15 minutes at rpm. Then ethanol was removed and dried to extract DNA.

PCR amplification of DNA

The DNA collected above was prepared in a vessel (PCR) with a reagent for PCR (1.0 μl of taq-polymerase 1.5 unit, 1 μM 0.25 μl × 2 of primer, 0.2 μm of dNTPs 0.2 μm, 4.0 μl of PCR buffer) and 40 μl of tertiary distilled water. -tube). In order to separate the double strand structure of the DNA into a single strand structure was maintained for 3 minutes at 94 ℃. Samples were rapidly cooled to 60 ° C. for single-strand binding and maintained for 35 seconds. The DNA-polymerase activity was maintained at 72 ° C. for 1 minute. The process was repeated 30 times. Subsequently, the extract solution was centrifuged at 13000 rpm, the supernatant was discarded, and the precipitate (DNA) was dried and stored at -20 ° C.

<Preparation of starting solution>

An alkoxide mixture was prepared by adding 4.71 ml of CH₃OH to 13.60 g of Si (OCH₃) ₄. The amplified DNA was mixed with 1.00 ml of H 2 O, and then added to the alkoxide mixture with 2.60 ml of HCl, followed by stirring while heating to 40 ° C. to prepare a starting solution.

<Processing of cubic>

A 0.2 mm perforation was drilled into the cubic at a slow speed so as not to damage the cubic. The starting solution prepared above was added to the perforations formed, and the inlet was blocked. The starting solution inserted into the cubic was aged at 55 ° C. for 48 hours. The inlet sealed at 55 ° C. was slightly opened and dried slowly for 6 hours until the volatiles of the gel were almost volatilized. It was heated in vacuo again to 80 ° C. and dried for 24 hours.

Example 2

DNA  Preparation of Cultured Pearls Containing

Extraction of DNA

As a sample, human hair (5 hairs including hair roots) was taken, and the collected 5 hairs were cut into 1 cm size from the hair root where the cells are most attached and placed in sterile 1.5 ml E-tube. . Cell lysate was added to 1.5 ml E-tube containing the cut hair, and the cells were stagnated at 56 ° C. for at least 2 hours to destroy cells. Through this process, substances such as DNA, RNA, protein, and polysaccharide are mixed in the solution. Subsequently, impurities such as cell masses, RNA, proteins, and polysaccharides were removed using phenol or chloroform to obtain a solution in which only DNA was left. After mixing 1 / 10-fold salt (Salt: 3M Sodium acetate, 7.5M Ammonium acetate) with 2.5 times 70% ethanol in a solution containing only DNA, it is left in a freezer at -20 ° C for at least 2 hours and then 12,000 at 4 ° C. DNA was aggregated by centrifuge for 15 minutes at rpm. Then ethanol was removed and dried to extract DNA.

PCR amplification of DNA

The DNA collected above was prepared in a vessel (PCR) with a reagent for PCR (1.0 μl of taq-polymerase 1.5 unit, 1 μM 0.25 μl × 2 of primer, 0.2 μm of dNTPs 0.2 μm, 4.0 μl of PCR buffer) and 40 μl of tertiary distilled water. -tube). In order to separate the double strand structure of the DNA into a single strand structure was maintained for 3 minutes at 94 ℃. Samples were rapidly cooled to 60 ° C. for single-strand binding and maintained for 35 seconds. The DNA-polymerase activity was maintained at 72 ° C. for 1 minute. The process was repeated 30 times. Subsequently, the extract solution was centrifuged at 13000 rpm, the supernatant was discarded, and the precipitate (DNA) was dried and stored at -20 ° C.

<Preparation of starting solution>

An alkoxide mixture was prepared by adding 4.71 ml of CH₃OH to 13.60 g of Si (OCH₃) ₄. The amplified DNA was mixed with 1.00 ml of H 2 O, and then added to the alkoxide mixture with 2.60 ml of HCl, followed by stirring while heating to 40 ° C. to prepare a starting solution.

<Production of cultured pearl core>

The starting solution prepared above was put into a perforation in a separate container, and the inlet was closed to seal it. The starting solution inserted in the perforation was aged at 55 ° C. for 48 hours. The inlet, which was sealed at 55 ° C., was slightly opened and dried slowly until the volatiles of the gel were almost volatilized. This was heated to 80 ℃ again in a vacuum state and dried for 24 hours to form a dry gel body of DNA. The formed dried gel body was polished to a sphere having a diameter of 5.5 mm, and then attached to Epodelium cell pieces to form a pearl nucleus. After inserting the pearl core into the pearl pearl, the secretion layer was to be coated on the pearl core. Cultured pearls were prepared by cultivating cultured pearl shells with nuclear cores for 6 months.

According to the method of the present invention, by applying the sol-gel method used for low-temperature synthesis of ceramics to form DNA as a dry gel body, to minimize the effects of external conditions or stimulation, and the DNA sequence without breaking the DNA sequence In addition, the processing to the final product is easier and the aesthetics of the final product can be improved.

Figure 1 shows a form in which the DNA dry gel body is inserted into the cubic in accordance with an embodiment of the present invention.

Figure 2 shows the form in which the DNA dry gel body is used as a nucleus of cultured pearls according to another embodiment of the present invention.

Claims (6)

Extracting DNA; Amplifying the extracted DNA; Preparing a starting solution by adding a DNA solution obtained by dissolving the amplified DNA in water to a mixed solution of metal alkoxide and alcohol and stirring at 35 to 45 ° C; Injecting the starting solution into a target product; And aging the starting solution added to the target product at 50 to 60 ° C. and drying at 75 to 85 ° C. to form a dried gel body in the target product. The method of claim 1, wherein the subject product is cubic. Extracting DNA; Amplifying the extracted DNA; Preparing a starting solution by adding a DNA solution obtained by dissolving the amplified DNA in water to a mixed solution of metal alkoxide and alcohol and stirring at 35 to 45 ° C; Aging the starting solution in a separate vessel at 50 ~ 60 ℃ and dried at 75 ~ 85 ℃ to form a dry gel body; And Method of producing a DNA-containing product comprising the step of including the formed dried gel body in the target product. The method of claim 3, wherein the subject product is cultured pearls. The method according to claim 1 or 3, wherein the amplification of the DNA is performed by a PCR method. The method of claim 1, wherein the metal alkoxide is Si (OCH ₃ ) ₄ .