KR20190038531A - Method and device for extracting nucleic acids using nano-filters - Google Patents

Abstract

Provided is a nucleic acid extraction container which comprises: a first container including a nanofilter having a uniform nano-sized pore at the bottom surface, containing a first liquid, and having at least one or two first wells including a first electrode capable of being immersed in the first liquid; and a second container accommodating the first well, containing a second liquid, and having at least one or two second wells including a second electrode capable of being immersed in the second liquid. According to the present invention, it is possible to extract high purity nucleic acid per unit time by isolating nucleic acid in a quick and efficient way even with low electric power.

Description

[Method and device for extracting nucleic acids using nano-filters]

The present invention relates to a nucleic acid extraction apparatus and method, and more particularly, to a nucleic acid extraction apparatus and method using a nano filter.

Recently, a lot of research has been conducted on techniques for extracting nucleic acids from biological samples such as cells, bacteria or viruses. In general, there are three methods of separating nucleic acids from biological materials. A method of separating magnetically adsorbed nucleic acids using magnetic beads, and a method of eluting a solution using a column filled with silica gel. After filling a liquid containing DNA, applying negative pressure to remove the liquid, and then dropping the DNA from silica gel, and recently, silica or glass fibers that specifically bind to nucleic acids are prepared as a membrane, and the membrane is A method of separating DNA molecules by centrifugation after inserting a filter kit provided on the lower surface into a tube for electrophoresis is also used. Since the silica or glass fiber has a low binding ratio with proteins and cell metabolites, a relatively high concentration of nucleic acid can be obtained. However, most of the nucleic acid extraction equipment using magnetic particles is a simple automation of the conventional technology, and the volume of the equipment is large, and there is a problem that the solution on the pipette during the extraction process falls into another test container, and cross-contamination may occur. The centrifugal separation method has a limitation in the number of samples that can be processed, and the method using glass fibers has a disadvantage that it takes a lot of time because many processing steps must be performed. In addition, the current nucleic acid extraction method may cause problems such as a change in removal efficiency depending on the type of sample, a decrease in the amount of extracted nucleic acid, or an increase in experiment time. In this regard, Korean Patent Application Publication No. 2015-0127917 discloses a method for extracting and amplifying nucleic acids using magnetic particles.

However, in the case of the prior art, a lot of time is required and production efficiency is low due to a complicated nucleic acid extraction process.

The present invention is to solve various problems including the above problems, and an object of the present invention is to provide a nucleic acid extraction apparatus using a nano filter capable of very quickly and efficiently separating nucleic acids even at low power. However, these problems are exemplary, and the scope of the present invention is not limited thereby.

According to one aspect of the present invention, one or two including a first electrode that can be immersed in the first liquid is provided with a nano filter having uniform nano-sized pores on the lower surface of the first liquid. A first container having more than one first well; And a second container having one or more second wells including a second electrode capable of being immersed in the second liquid in which the first well may be accommodated and a second liquid may be supported. Courage is provided.

According to another aspect of the present invention, a first liquid containing a sample containing a nucleic acid molecule to be extracted and a nucleic acid molecule after extraction into two compartments separated by a nanofilter having uniform nano-sized pores, respectively. Positioning a second liquid for carrying; Supporting a negative electrode and a positive electrode in the first liquid and the second liquid, respectively; Applying a DC current by connecting a DC power source to the cathode and anode electrodes; And stopping the application of a current and recovering the second liquid.

According to an embodiment of the present invention made as described above, it is possible to achieve the effect of extracting high-purity nucleic acids per unit time by separating nucleic acids very quickly and efficiently at lower power than conventional methods using a nano filter. have. Of course, the scope of the present invention is not limited by these effects.

1 is a schematic diagram comparing a method of extracting a nucleic acid according to an embodiment of the present invention and a method of extracting a nucleic acid using a nanofilter of the present invention.
2 is a schematic diagram showing the structure of a nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention.
3 is a schematic diagram showing the structure of a nucleic acid extraction apparatus 200 using a plurality of nanofilters according to an embodiment of the present invention.
4 is a schematic diagram showing a manufacturing process of a nanofilter 150 having uniform nanopores embedded in a nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention.
5 is a photograph taken by varying magnification of the nanofilter 150 on which the manufacturing process was completed according to an embodiment of the present invention, respectively, 20k(a), 50k(b), and 100k. (c) It shows the magnification.
6 is a schematic diagram showing a process of extracting DNA from a cell lysate using the nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention.
7 is a photograph showing a process of extracting DNA from a cell lysate using the nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention.
8 is a picture of a gel observing amplification through electrophoresis using DNA extracted according to an embodiment of the present invention.
9 is a graph showing DNA extraction yield analysis over time using the nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention.
10 is a graph showing quantitative analysis of DNA extracted through the nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention by real-time PCR.

Definition of Terms:

"Nucleic acid" used in this document is a high molecular substance composed of purine base, pyrimidine base, sugar, and phosphoric acid. Discoverer F. Miescher called the new substance found in the cell nucleus nuclein, and later in the nucleus. Nucleic acid was given the name meaning that it is an acidic substance present in a large amount. Nucleotides composed of base, pentose and phosphoric acid are polymerized with a phosphate diester bond to form a long chain-shaped molecule. Nucleic acid molecules have an orientation (polarity) of 5'→ 3', and this is one of the important characteristics related to the structure and function of nucleic acids. The sugar is deoxyribonucleic acid (DNA), which is deoxyribose, and ribonucleic acid (RNA), which is ribose. It is roughly different.

The "semiconductor device" used in this document is a circuit device made of a semiconductor material, and the semiconductors used here are silicon, germanium, gallium arsenide, and the like. It has been replacing thermoelectric devices in most applications. These semiconductors are classified in terms of properties such as n-type, p-type, and intrinsic, and they are used as a single substance or by bonding several of them together. There are various characteristics such as detecting light or radiation, sensing temperature, sensitive to magnetic field or pressure, such as diodes, transistors, thyristors, etc., and are widely used in various fields.

The "wafer" used in this document is a thin disk that is used to make semiconductor integrated circuits. Ingots (single crystal pillars) obtained by growing silicon (Si) or gallium arsenide (GaAs) are thinly cut into appropriate diameters. Refers to the round plate.

Detailed description of the invention:

According to one aspect of the present invention, one or two including a first electrode that can be immersed in the first liquid is provided with a nano filter having uniform nano-sized pores on the lower surface of the first liquid. A first container having more than one first well; And a second container having one or more second wells including a second electrode capable of being immersed in the second liquid in which the first well may be accommodated and a second liquid may be supported. Courage is provided.

In the nucleic acid extraction container, the first liquid may be distilled water, a buffer solution, or a PCR reaction solution, the first electrode may be connected to an anode of a power supply device, and the second liquid may be a sample containing nucleic acid molecules. .

In the nucleic acid extraction container, the first liquid may be a sample containing a nucleic acid molecule, and the sample containing the nucleic acid molecule may be blood, serum, plasma, urine, sweat, tears, or tissue or cell lysate. The 2 liquid may be distilled water, a buffer solution, or a PCR reaction solution.

In the nucleic acid extraction container, the pore may have a diameter of 3 to 300 nm, may have a diameter of 10 to 280 nm, 25 to 270 nm, 50 to 250 nm or 100 to 220 nm, and the nano The thickness of the filter may be 10 nm to 5 μm, and the material of the nanofilter is semiconductor, metal, metal nitride, semiconductor nitride, metal sulfide, semiconductor sulfide, metal phosphide, semiconductor phosphide, metal arsenide, semiconductor arsenide, metal A material that can be an oxide or a semiconductor oxide, and can form a thin film on a substrate through a thin film formation method such as chemical vapor deposition (CVD) or physical vapor deposition (PVD). Anything can be used

In the nucleic acid extraction container, the metal may be titanium, copper, silver, gold, gallium, indium, molybdenum, tin, aluminum, zinc, rucenium, tantalum, or tungsten, and the semiconductor may be doped or undoped silicon, germanium , Gallium compound, silicon carbide (SiC), selenium, boron nitride (BN), boron phosphide (BP), boron arsenide (BAs), and the metal nitride is titanium nitride (TiN), aluminum nitride (AlN) , Tantalum nitride (TaN), indium nitride (InN), gallium nitride (GaN), the semiconductor nitride may be silicon nitride (SiN _x ), the metal phosphide is aluminum phosphide (AlP), May be indium phosphide (InP), gallium phosphide (GaP), the metal sulfide may be molybdenum disulfide (MoS), the metal arsenide may be gallium arsenide (GaAs), indium arsenide (InAs), aluminum arsenide (AlAs) or cadmium arsenide (Cd ₃ As ₂ ), and the metal phosphide is cobalt poaspide (CoP), iron phosphide (FeP), nickel phosphide (NiP), vanadium phosphide (VP), tungsten phosphide Pide (WP) may be aluminum phosphide (AlP), indium phosphide (InP), and the metal oxide is a first copper oxide (Cu ₂ O), a second copper oxide (CuO), bismuth oxide (Bi ₂ O _{3 ).} ), titanium oxide (TiO ₂ ), hafnium oxide (HfO ₂ ), and the semiconductor oxide may be silicon oxide (SiO ₂ ) or germanium oxide (GeO ₂ ).

In the nucleic acid extraction container, the first well and the second well may be composed of 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 192 wells or 384 wells.

According to another aspect of the present invention, a first liquid containing a sample containing a nucleic acid molecule to be extracted and a nucleic acid molecule after extraction into two compartments separated by a nanofilter having uniform nano-sized pores, respectively. Positioning a second liquid for carrying; Supporting a negative electrode and a positive electrode in the first liquid and the second liquid, respectively; Applying a DC current by connecting a DC power source to the cathode and anode electrodes; And stopping the application of a current and recovering the second liquid.

In the extraction method, the voltage of the DC current may be 0.1 to 200 V, the first liquid may be blood, serum, plasma, urine, sweat, tears, or tissue or cell disruption, and the second liquid may be distilled water. , A buffer solution, or a PCR reaction solution.

More preferably, the voltage of the DC current may be 0.2 to 150 V, 0.3 to 110 V, or 0.4 to 100 V. Alternatively, the voltage of the DC current may be 0.1 to 2.0 V, 0.1 to 1.8 V, 0.3 to 1.7 V, 0.4 to 1.6 V, 0.5 to 1.5 V. In particular, the method of the present invention is a very great advantage that it can operate at a low voltage, and it can be used to quickly extract nucleic acids from unidentified samples at outdoor sites through portable equipment powered by 1.5 V batteries. It can be used very efficiently to check environmental conditions. In addition, it can be very efficiently used for metagenomic research through rapid separation of the metagenome.

In the extraction method, the pore may have a diameter of 3 to 300 nm, the thickness of the nanofilter may be 10 nm to 5 μm, and the material of the nano filter may be a semiconductor, a metal, a metal nitride, a semiconductor nitride, It may be a metal sulfide, a semiconductor sulfide, a metal phosphide, a semiconductor phosphide, a metal arsenide, a semiconductor arsenide, a metal oxide, or a semiconductor oxide.

The present inventors prepared E. coli cells cultured using a lysis buffer, which is a method used to extract nucleic acids, in the form of cell lysate, and centrifuged the cell lysate. The method of separating the DNA from the silica membrane by adsorbing DNA on the silica membrane and treating the elusion buffer does not cleanly remove the lysis buffer or cell debris used in the cell lysis step. If a chaotropic reagent is used to immobilize DNA on the surface of a silica membrane, it may affect the next experiment and PCR amplification may not proceed smoothly, and exhibit a low extraction efficiency of less than 60%, and various reagents As a result of recognizing the occurrence of various problems, such as impossibility of on-site detection, which can be used immediately due to use and time consuming, and making diligent efforts to solve these problems, a nano filter having a uniform pore size was developed. A nucleic acid extracting apparatus 100 using a nano-filter that extracts high-purity nucleic acids quickly and efficiently with low power was developed (FIG. 1).

In the above method, the step of applying the DC current may be performed for 30 seconds to 20 minutes, 1 to 10 minutes, 1 to 3 minutes, or 1 to 5 minutes.

Hereinafter, various exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The embodiments of the present invention are provided to more completely describe the present invention to those of ordinary skill in the art, and the following examples may be modified in various other forms, and the scope of the present invention is as follows. It is not limited to the examples. Rather, these embodiments are provided to make the present disclosure more faithful and complete, and to completely convey the spirit of the present invention to those skilled in the art. In addition, in the drawings, the thickness or size of each layer is exaggerated for convenience and clarity of description.

Throughout the specification, when referring to one component such as a film, region or substrate positioned "on", "connected", "stacked" or "coupled" another component, the one component It can be interpreted that an element may be directly in contact with another element “on”, “connected”, “stacked” or “coupled”, or that there may be other elements interposed therebetween. On the other hand, when it is mentioned that one component is positioned "directly on", "directly connected", or "directly coupled" of another component, it is interpreted that there are no other components intervening therebetween. do. Uniform symbols refer to uniform elements. As used herein, the term “and/or” includes any and all combinations of one or more of the corresponding listed items.

In this specification, terms such as first and second are used to describe various members, parts, regions, layers and/or parts, but these members, parts, regions, layers and/or parts are limited by these terms. It is self-evident. These terms are only used to distinguish one member, part, region, layer or portion from another region, layer or portion. Accordingly, a first member, part, region, layer or part to be described below may refer to a second member, part, region, layer or part without departing from the teachings of the present invention.

Also, relative terms such as “top” or “above” and “bottom” or “below” may be used herein to describe the relationship of certain elements to other elements as illustrated in the figures. It can be understood that relative terms are intended to include other orientations of the device in addition to the orientation depicted in the figures. For example, if an element is turned over in the figures, elements depicted as being on the top side of other elements will have orientation on the bottom side of the other elements. Therefore, the term “top” as an example may include both “bottom” and “top” directions depending on the particular orientation of the drawing. If the device is oriented in a different direction (rotated by 90 degrees with respect to the other direction), the relative descriptions used herein can be interpreted accordingly.

The terms used in this specification are used to describe specific embodiments, and are not intended to limit the present invention. As used herein, the singular form may include the plural form unless the context clearly indicates another case. Also, as used herein, “comprise” and/or “comprising” specify the presence of the mentioned shapes, numbers, steps, actions, members, elements and/or groups thereof. And does not exclude the presence or addition of one or more other shapes, numbers, actions, members, elements and/or groups.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing ideal embodiments of the present invention. In the drawings, for example, depending on manufacturing techniques and/or tolerances, variations of the illustrated shape can be expected. Therefore, the embodiments of the inventive concept should not be construed as being limited to the specific shape of the region shown in the present specification, but should include, for example, a change in shape caused by manufacturing.

2 schematically shows the principle and structure of a nucleic acid extraction apparatus 100 using a nanofilter according to an embodiment of the present invention. The nucleic acid extraction apparatus 100 using the nanofilter of the present invention uses an electric field (electric field) without a separate extraction or purification process of nucleic acid from the second liquid 135 by using a filter having a built-in uniform nanopores. ) Is a new method of extracting or purifying nucleic acids that selectively moves only nano-sized nucleic acids through the nano-filter. Nucleic acid extraction is an essential step in the field of biological research and requires a large amount of purified nucleic acid for polymerase chain reaction (PCR) for DNA amplification. As it takes a lot of time due to a complicated process, there are several limitations, such as high cost and low production yield. In order to overcome this limitation, many studies have been conducted on the manufacture of an automated device for extracting a desired biological material or nucleic acid from various biological samples. In this respect, the nucleic acid extraction device 100 using the nanofilter of the present invention is conventionally used. It can be said to be a nucleic acid extraction device that overcomes several limitations such as the complex process and low production yield.

The structure of the device is a first well 125 in which a space capable of accommodating a solution and a nanofilter 150 having uniform nanopores at the bottom of the space are embedded, as shown in FIG. 2. ) Is configured at the top and there is a cover 140 that can be selectively opened and closed to inject the first liquid 127 for storing the extracted nucleic acid molecules into the first well 125, and the first well 125 Inside, an anode 115 connected to an external power supply 155 is installed. In addition, the first well 125 may be accommodated with a space capable of holding the second liquid 135, which is a sample containing a nucleic acid molecule such as a cell disruption solution, and a cathode connected from the power supply device 155 ( It is composed of the second well 130 in which the cathode 120 is installed. In order to extract a nucleic acid using the above device, a first liquid 127 containing a nucleic acid molecule to be extracted, such as a cell lysate, is injected into the second well 130, and the extracted nucleic acid molecule is injected into the first well 125. After the second liquid 127, such as distilled water, buffer or PCR reaction solution, is injected and combined, DC current is supplied to both electrodes. Nucleic acid molecules carrying negative charges move to the first well 125 toward the anode 115 by the applied direct current. At this time, the second well through a nanofilter 150 installed on the lower surface of the first well 125 Among the various biological substances contained in the liquid 135, only nucleic acids having a nano size pass through the nano filter 150. Accordingly, high-purity nucleic acids can be quickly and efficiently extracted without a separate extraction device or extraction process, and the extracted nucleic acids can be directly used for research. Optionally, the positions of the first liquid 127 and the second liquid 135 may be interchanged. In this case, the anode 115 is in the second well 130 and the cathode 120 is in the first well 125 You just need to be located at. At this time, the sample containing the nucleic acid molecule may be blood, serum, plasma, urine, sweat, tears, tissue or cell disruption fluid, and the voltage of the applied current may be 0.1 to 200 V, and the current application time may be from 30 seconds to With a time of 5 minutes, it can be appropriately adjusted according to the voltage of the applied current and the amount or extraction amount of the sample. In addition, for power supply, a plug (not shown) that can be connected to the port of the power supply device 155 is configured at the other end of the wire to which the electrodes 120 and 115 are connected, so it can be easily connected and used. 155) can be used for both general and exclusive use. The manufacturing process of the nanofilter having the nanopores will be described in detail with reference to FIG. 5.

2 is a diagram showing the configuration and principle of the nucleic acid extraction apparatus 100 using the nanofilter of the present invention in a single configuration for easy understanding, which is a nucleic acid extraction apparatus using a plurality of nanofilters according to the amount of sample or the purpose of the researcher ( 100) can be connected to improve the efficiency of nucleic acid extraction, which can be easily understood by referring to FIG. 3. As shown in FIG. 3, a nucleic acid extracting apparatus 200 using a nanofilter composed of a plurality of combined forms rather than a single configuration is shown. In the method of extracting nucleic acids, as in the single configuration of FIG. 2, the second liquid 135 is injected into the second well 130, the first liquid 127 is injected into the first well 125, and the cover 140 is opened. It is coupled to the upper part of the first well 125. In the cover 140, the anode 115 is configured to supply power to each well to supply positive charge to the first well 125, and the cathode 120 to supply negative charge to the bottom of the second well 130 Each of these are made up. A plurality of extraction devices are suitable for the purpose of extracting a large amount of nucleic acids from a single sample at one time or extracting multiple types of samples at the same time, but careful attention is required in the injection of samples, supply of power, and recovery of the extract. 3 shows a predetermined number of binding forms in order to easily understand the configuration of the nucleic acid extraction apparatus 200 using a plurality of nanofilters, but this can be adjusted according to the amount of the sample or the purpose of the researcher. For example, 6 wells, 12 Well, 24 well, 48 well, 96 well, 192 well, or 384 well, such as parallel and series can be produced by adding a combination. In this case, the material of the well may be glass, iron, or resin, and the iron may be carbon steel, stainless steel, or alloy steel. In addition, the resin may be a thermoplastic resin or a thermosetting resin, and the thermoplastic resin may be polyvinyl chloride, polyethylene, polystyrene, It may be polypropylene, acrylic, nylon, or PET (polyethylene terephthalate), and the thermosetting resin may be phenol, melamine, or epoxy.

4 schematically shows a manufacturing process of a nanofilter 150 having uniform nanopores embedded in the nucleic acid extraction apparatus 100 using a nanofilter. The manufacturing process is the same as a general semiconductor device manufacturing process. First, a thin and uniform semiconductor by spraying oxygen or water vapor at a high temperature (800 ~ 1200°C) on the upper and lower membranes of the wafer used as the semiconductor material. An oxidation process of depositing silicon nitride (SiN) as a material in nanoscale is performed. Thereafter, the membrane is coated with a photoresist, and when light is received, a chemical reaction occurs and properties are changed. Using a mask of a desired pattern, the light is selectively irradiated and the A photolithograpy process of forming the same pattern as the mask pattern is performed. A 200 nanometer pore is created by the above process, and an etching process is performed in which only the exposed part without a photoresist is corroded by using a chemical material. Subsequently, a photoresist is evenly applied to the wafer surface and SiN on the rear surface is etched to complete the manufacturing process of the nanofilter 150.

FIG. 5 is a photograph of the rear surface of the nanofilter 150 on which the manufacturing process has been completed after backside etch at different magnifications with a scanning electron microscope (SEM). The material of the manufactured nanofilter 150 may be a semiconductor, a metal, a metal nitride, a semiconductor nitride, a metal sulfide, a semiconductor sulfide, a metal phosphide, a semiconductor phosphide, a metal arsenide, a semiconductor arsenide, a metal oxide, or a semiconductor oxide. , The semiconductor may be doped or undoped silicon, germanium, gallium compound, silicon carbide (SiC), selenium, boron nitride (BN), boron phosphide (BP), boron arsenide (BAs), and the metal nitride is titanium It may be nitride (TiN), aluminum nitride (AlN), tantalum nitride (TaN), indium nitride (InN), gallium nitride (GaN), and the semiconductor nitride may be silicon nitride (SiN _x ). The metal phosphide may be aluminum phosphide (AlP), indium phosphide (InP), gallium phosphide (GaP), and the metal sulfide may be molybdenum disulfide (MoS), and the metal arsenide may be gallium arsenide (GaAs ), indium arsenide (InAs), aluminum arsenide (AlAs), or cadmium arsenide (Cd ₃ As ₂ ), and the metal phosphide is cobalt poaspide (CoP), iron phosphide (FeP), nickel phosphide ( NiP), vanadium phosphide (VP), tungsten phosphide (WP) aluminum phosphide (AlP), indium phosphide (InP), and the metal oxide is a first copper oxide (Cu ₂ O), a second oxidation It may be copper (CuO), bismuth oxide (Bi ₂ O ₃ ), titanium oxide (TiO ₂ ), hafnium oxide (HfO ₂ ), and the semiconductor oxide may be silicon oxide (SiO ₂ ) or germanium oxide (GeO ₂ ). have. remind The thickness of the nanofilter 150 may be 10 nm to 5 μm, but it may be properly adjusted and manufactured, and the pore size may be appropriately adjusted according to the type of sample and the purpose of the researcher with a diameter of 3 to 300 nm.

Hereinafter, the present invention will be described in more detail through examples. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various different forms, and the following embodiments are intended to complete the disclosure of the present invention, and the scope of the invention to those of ordinary skill in the art. It is provided to fully inform you.

Example 1: Preparation of nanofilter

_{A silicon nitride (SiN x} ) layer having a thickness of about 500 nm was deposited on a silicon wafer (diameter 50 mm) by low pressure chemical vapor deposition (LPCVD). Then, a photosensitive material was applied to the deposited silicon nitride layer, and then imprinted with a circular mold having a diameter of 200 nm to form a pattern on the photosensitive material layer. Then, exposure and development were performed on the patterned photosensitive material layer (left of FIG. 4). Subsequently, partial dry etching was performed on the silicon nitride layer.

Then, after applying a photosensitive material on the backside, exposing and developing to expose the silicon nitride layer, dry etching is performed to remove the silicon nitride layer on the backside, and wet etching using potassium hydroxide (KOH). The silicon substrate layer was removed using a process. Subsequently, the photosensitive material was peeled off, and dry etching was performed on the back side of the silicon nitride layer to prepare a completely perforated silicon nitride nanofilter having a thickness of about 300 nm.

As a result of observing the prepared nanofilter with a scanning electron microscope, it was confirmed that nanopores having a uniform diameter of 200 nm were normally formed (FIG. 5).

Example 2: Fabrication of a nucleic acid extraction container using a nanofilter

The present inventors produced a well-formed PDMS block with a completely non-perforated lower part by punching a PDMS block (polydemthylsilosane), and then placed a silicon nitride nanofilter (700 X 700 μm) prepared in Example 1 on the well-formed PDMS block. After loading, the upper well was formed by stacking a partition wall made of PDMS material on the nanofilter. Then, fix it using a stand so that the platinum electrode is located in the upper well, and the platinum electrode is placed in the lower well by plugging the platinum electrode in the well-forming PDMS block, and then the platinum electrode connected to the upper well and the lower well is supplied with a DC power supply. It was connected to the positive and negative poles of the power supply.

Experimental Example 1: DNA extraction and amplification

DNA was extracted from the cell lysate using the nucleic acid extraction container 100 prepared in Example 2, and amplification was verified using the extracted DNA. In the second well 130, which is the lower well, 300 µl of a cell lysate obtained by crushing E. coli cells using a lysis buffer was injected (FIG. 6). Then, the nanofilter prepared in Example 1 was placed on the PDMS block, and the first well 125, which is an upper well, was formed by placing the partition wall of the PDMS material on the PDMS block, and then 300 µl of the PCR reaction solution was injected. Then, a DC current was applied for 1 minute by dividing the voltage into 50V and 100V through the DC power supply 155 (FIG. 7). After the extraction was completed, the solution in the upper well was transferred to a PCR reaction tube, and the following primer sets specific for E. coli were added, followed by PCR reaction (see Table 1). As a result, as shown in FIG. 8, it was confirmed that a PCR reaction using the extracted DNA as a template by applying a DC current of 50V and 100V for 1 minute was successfully performed. In the case of lane 1 of FIG. 8, the result of the negative control group using the solution contained in the upper well to which no current was applied.

primer Base sequence (5'->3') Sequence number Forward GGG CAG TTA TTT TGC TGT GGA One Reverse TGT TGC CGT ATT AAC GAA CCC 2 Taqman FAM-CTA TCA GGC GCG TTT TGA CCA TCT TCG-TAMRA 3

Experimental Example 2: Low-power nucleic acid extraction and real-time PCR reaction

From the above results, the present inventors performed the same experiment as in Experimental Example 1 by lowering the used voltage to 1.0 V in order to confirm whether the nucleic acid extraction method according to an embodiment of the present invention can be performed with low power such as a battery.

Specifically, instead of the cell lysate, 150 µl of an aqueous DNA solution (65.8 ng/µl) of known concentration was injected into the second well 130 of the well-forming PDMS block, and then the nanofilter prepared in Example 1 was placed on the block. After loading, 20 µl of the PCR reaction solution was dropped onto the nanofilter. Then, after connecting the platinum electrode as in Experimental Example 2, a direct current of 1.0V was applied for 1 minute, 2 minutes, 3 minutes, 5 minutes, 7 minutes and 10 minutes, respectively. Thereafter, the upper PCR reaction solution was recovered _{to obtain OD 260} and OD ₂₈₀ to measure the amount of DNA purity.

As a result, it was observed that the concentration of DNA increased with time (FIG. 9A), and the A260/280 value, an index indicating the purity of nucleic acid, was close to 2.0, indicating that the purity was very high (FIG. 9B). In addition, the present inventors applied a direct current of 1.0V for 9 minutes to perform a real-time PCR reaction using the extracted DNA as a template, and as a result, the same result as the real-time PCR result using the DNA before extraction as a template was calculated (Fig. 10). . This allows the nucleic acid extraction method and apparatus according to an embodiment of the present invention to rapidly extract nucleic acid molecules such as DNA or RNA from a sample in which a nucleic acid molecule to be amplified, such as a cell lysate, is present, as well as extract the extracted nucleic acid. It means that the purity is high enough to perform precise analysis such as real-time PCR.

In conclusion, high purity nucleic acids can be quickly and efficiently extracted from various biological samples at low power by using the nucleic acid extraction device 100 using a nanofilter having a uniform pore size manufactured according to an embodiment of the present invention. Therefore, we manufacture portable equipment that uses 1.5 V batteries as a power source to quickly extract nucleic acids from the field and then use them for experiments after moving to a laboratory, or so-called on-site detection, which is used for detection directly in the field. It can be used for the purpose of.

The present invention has been described with reference to the above-described embodiments, but these are merely exemplary, and those of ordinary skill in the art will appreciate that various modifications and equivalent other embodiments are possible therefrom. Therefore, the true technical protection scope of the present invention should be determined by the technical spirit of the appended claims.

<110> SNU R&DB FOUNDATION <120> Method and device for extracting nucleic acids using nano-filters <130> PD16-5433 <160> 3 <170> KopatentIn 2.0 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Forward <400> 1 gggcagttat tttgctgtgg a 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Reverse <400> 2 tgttgccgta ttaacgaacc c 21 <210> 3 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Taqman <400> 3 ctatcaggcg cgttttgacc atcttcg 27

Claims (19)

A first electrode having one or more first wells including a first electrode capable of being immersed in the first liquid and having a nanofilter having a uniform nano-sized pore on a lower surface thereof and capable of supporting a first liquid, Vessel; And
A second container having one or more second wells comprising a second electrode capable of being immersed in the second liquid, wherein the first well can be received and the second liquid can be carried, . The method according to claim 1,
Wherein the first liquid is distilled water, a buffer solution, or a PCR reaction liquid. 3. The method of claim 2,
Wherein the first electrode is connected to the anode of the power supply. 3. The method of claim 2,
Wherein the second liquid is a sample containing nucleic acid molecules. The method according to claim 1,
Wherein the first liquid is a sample containing nucleic acid molecules. The method according to claim 4 or 5,
Wherein the sample containing the nucleic acid molecule is blood, serum, plasma, urine, sweat, tears or tissue or cell lysate. 5. The method of claim 4,
Wherein the second liquid is distilled water, a buffer solution, or a PCR reaction liquid. The method according to claim 1,
And the second electrode is connected to the cathode of the power supply. The method according to claim 1,
Wherein the pore has a diameter of 3 to 300 nm. The method according to claim 1,
Wherein the thickness of the nanofilter is 10 nm to 5 占 퐉. The method according to claim 1,
Wherein the material of the nanofilter is a semiconductor, a metal, a metal nitride, a semiconductor nitride, a metal sulfide, a semiconductor sulfide, a metal phosphide, a semiconductor phosphide, a metal arsenide, a semiconductor non-oxide, a metal oxide, or a semiconductor oxide. The method according to claim 1,
Wherein the first well and the second well are comprised of 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, 192 wells or 384 wells. A first liquid containing a sample containing nucleic acid molecules to be extracted and a second liquid for carrying nucleic acid molecules after extraction are placed in two sections bounded by a nanofilter having a uniform nano-sized pore step;
Supporting a cathode and an anode electrode on the first liquid and the second liquid, respectively;
Connecting a DC power source to the cathode and the anode electrode to apply a DC current; And
And stopping current application and recovering said second liquid. 14. The method of claim 13,
Wherein the voltage of the direct current is 0.1 to 200 V. 14. The method of claim 13,
Wherein the first liquid is blood, serum, plasma, urine, sweat, tears or tissue or cell lysate. 14. The method of claim 13,
Wherein the second liquid is distilled water, a buffer solution, or a PCR reaction liquid. 14. The method of claim 13,
Wherein the pores have a diameter of from 3 to 300 nm. 14. The method of claim 13,
Wherein the thickness of the nanofilter is between 10 nm and 5 mu m. 14. The method of claim 13,
Wherein the material of the nanofilter is a semiconductor, a metal, a metal nitride, a semiconductor nitride, a metal sulfide, a semiconductor sulfide, a metal phosphide, a semiconductor phosphide, a metal arsenide, a semiconductor non-oxide, a metal oxide, or a semiconductor oxide.

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