KR20200130087A - Nucleic Acid Purification Device and Nucleic Acid Purification Method - Google Patents

Abstract

A nucleic acid purification apparatus according to the present invention comprises an activated carbon fiber filter, and the activated carbon fiber filter comprises an activated carbon fiber and a potassium compound coated on the surface of the activated carbon fiber. By using the nucleic acid purification apparatus and a nucleic acid purification method according to the present invention, it is possible to quickly and effectively purify nucleic acids through a simple filtration process.

Description

Nucleic Acid Purification Device and Nucleic Acid Purification Method}

The present invention relates to a nucleic acid purification apparatus and a nucleic acid purification method.

As genetic engineering advances, molecular diagnostics using PCR (polymerase chain reaction), microarray, and NGS (Next generation sequencing, next generation sequencing) have been developed, enabling diagnosis and prediction of various diseases based on genes. In addition, there is a need for a method for efficiently extracting DNA from various samples. With the development of molecular diagnosis technology, PCR has developed to the extent that it is possible to proceed in a short time of about 10 spares, but the extraction and purification process of nucleic acid, which is a pretreatment required for molecular diagnosis, generally takes about 1 hour. It is a limited situation to use molecular diagnosis for on-site diagnosis, etc. that require rapidity due to this requirement.

A problem to be solved by the present invention is to provide a nucleic acid purification apparatus and a nucleic acid purification method capable of simplifying a nucleic acid purification process and obtaining a nucleic acid with a high yield.

The nucleic acid purification apparatus according to embodiments of the present invention includes an activated carbon fiber filter, and the activated carbon fiber filter is activated carbon fiber and coated on the surface of the activated carbon fiber. Potassium compounds may be included.

According to some embodiments, the activated carbon fiber may have a specific surface area of 500 m ² /g to 3000 m ² /g.

According to some embodiments, the activated carbon fiber may include pores having a diameter of 5 Å to 500 Å.

According to some embodiments, the potassium compound is potassium chloride (KCl), potassium iodide (KI), potassium hydroxide (KOH), potassium dihydrogen phosphate (KH ₂ PO ₃ ), potassium hydrogen carbonate (KHCO ₃ ), potassium oxide ( It may include at least any one of K ₂ O).

According to some embodiments, a column tube having an inlet disposed at one end and an outlet disposed at the other end, a retaining ring disposed in the column tube and fixing the activated carbon fiber filter, and contacting the activated carbon fiber filter Further comprising a filter paper, wherein the activated carbon fiber filter is disposed in the column tube, the fixing ring is disposed closer to the inlet than the activated carbon fiber filter, and the filter paper is disposed closer to the outlet than the activated carbon fiber filter Can be.

The nucleic acid purification method according to embodiments of the present invention may include disrupting cells and filtering the disrupted cells through an activated carbon fiber filter coated with a potassium compound.

According to some embodiments, disrupting the cells may include adding an anionic surfactant to the cells.

According to some embodiments, filtering the crushed cells may include a centrifugation process.

Using the nucleic acid purification apparatus and nucleic acid purification method according to the present invention, it is possible to quickly and effectively purify nucleic acids through a simple filtration process. The nucleic acid purification apparatus according to the present invention includes an activated carbon fiber filter, and the activated carbon fiber filter may include an activated carbon fiber and a potassium compound coated on the activated carbon fiber. Activated carbon fibers can adsorb the rest of the cell lysate except for nucleic acid, and the potassium compound reacts with the anionic cell lysis solution, thereby obtaining a high purification rate of nucleic acid in the filtrate.

1 is a perspective view of a nucleic acid purification apparatus according to the technical idea of the present invention.
2 is an inner perspective view of FIG. 1.
3 is a schematic diagram of an activated carbon fiber filter.
4 is a flowchart illustrating a nucleic acid purification method according to the technical idea of the present invention.
5 is a photograph of a DNA sample extracted by the apparatus and method according to the present invention and a DNA sample extracted by a commercial kit for genomic DNA extraction by electrophoresis analysis.
6 is a graph showing the degree of amplification of nucleic acids in a filtrate.

In order to fully understand the configuration and effects of the present invention, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but may be implemented in various forms and various modifications may be made. However, it is provided to complete the disclosure of the present invention through the description of the present embodiments, and to fully inform the scope of the present invention to those of ordinary skill in the art. In the accompanying drawings, the size of the constituent elements is enlarged compared to the actual size for convenience of description, and the ratio of each constituent element may be exaggerated or reduced.

Terms used in the embodiments of the present invention may be interpreted as meanings commonly known to those of ordinary skill in the art, unless otherwise defined. Hereinafter, the present invention will be described in detail by describing exemplary embodiments of the present invention with reference to the accompanying drawings.

1 is an external perspective view of a nucleic acid purification apparatus according to the technical idea of the present invention. 2 is an inner perspective view of FIG. 1.

1 and 2, the nucleic acid purification apparatus 100 according to the present invention may include a column tube 110, a fixing ring 140, an activated carbon fiber filter 120, and a filter paper 130. .

The column tube 110 may include an injection port IN through which a solution before purification is injected and an outlet OUT through which a solution after purification is extracted. The outlet OUT of the column tube 110 may have a hopper type, for example, so that the solution can be easily drained.

A filter paper 130 and a fixing ring 140 may be disposed in the column tube 110 with the activated carbon fiber filter 120 therebetween. The inner diameter of the column tube 110 may be circular, for example. The activated carbon fiber filter 120 may be disposed closer to the outlet (OUT) than to the inlet (IN). The fixing ring 140 may be disposed closer to the inlet IN than the carbon fiber filter 120, and the filter paper 130 may be disposed closer to the outlet OUT than the carbon fiber filter 120.

3 is a schematic diagram of an activated carbon fiber filter.

Referring to FIG. 3, the activated carbon fiber filter 120 may include an activated carbon fiber 120a and a potassium compound 120b coated on the activated carbon fiber 120a.

The activated carbon fibers 120a may be in the form of fibers composed of only carbon. Specifically, activated carbon fiber (120a) pyrolyzes rayon, acrylic fiber (Polyacrylonitrile, PAN), pitch (pitch obtained from coal tar or heavy petroleum oil as a raw material) fiber into chemicals, water vapor, carbon dioxide, or oxidizing gas such as oxygenation. You can get it through.

For example, the activated carbon fiber 120a may have a specific surface area of 500 m ² /g to 3000 m ² /g. The activated carbon fiber 120a may include a plurality of pores (H).

이하를 미세공, 500

이하를 중세공으로 정의하고, 500

초과는 대세공으로 정의한다.In IUPAC, pores are classified into macropores, mesopores, and micropores according to their diameter.

Micropore, 500

The following is defined as medieval, and 500

Excess is defined as grand work.

내지 500

의 직경을 가질 수 있다. 즉, 활성 탄소 섬유(120a)는 중세공들(H1) 및 미세공들(H2)을 주로 포함할 수 있다. 중세공들(H1) 및 미세공들(H2) 내에 짧은 가닥의 핵산 단편, 단백질, 및 유기 용매의 흡착이 잘 이루어질 수 있다. The pores (H) of the activated carbon fiber (120a) are on average 5

To 500

Can have a diameter of. That is, the activated carbon fiber 120a may mainly include mesopores H1 and micropores H2. Adsorption of short-stranded nucleic acid fragments, proteins, and organic solvents within the mesopores (H1) and micropores (H2) can be performed well.

In addition, since the distribution of large pores is small, adsorption of relatively large nucleic acids such as plasmid DNA or genomic DNA (gDNA) can be minimized.

The potassium compound 120b may be formed in the form of particles on the surface of the activated carbon fiber 120a. Potassium compound (120b) is among potassium chloride (KCl), potassium iodide (KI), potassium hydroxide (KOH), potassium dihydrogen phosphite (KH ₂ PO ₃ ), potassium hydrogen carbonate (KHCO ₃ ), and potassium oxide (K ₂ O). It may include at least any one.

The activated carbon fiber filter 120 can be formed by immersing the activated carbon fiber 120a with a solution in which the potassium compound 120b is dissolved, and then completely drying the activated carbon fiber in a wet state in an oven at room temperature or high temperature. have. In addition, the activated carbon fiber filter 120 may remove a part of the solution deposited in the activated carbon fiber 120b by centrifugation before drying.

The fixing ring 140 may prevent the activated carbon fiber filter 120 from being separated. When the filter paper 130 is centrifuged during the filtration process, the activated carbon fiber filter 120 may be prevented from being deformed, and particulate foreign substances having a relatively large size included in the solution to be filtered may be filtered out.

4 is a flow chart showing a nucleic acid purification method according to the present invention.

4, the nucleic acid purification method according to the present invention may include disrupting the cells (S100), and filtering the disrupted cells through an activated carbon fiber filter coated with a potassium compound (S200). have.

Prior to disrupting the cells (S100), it may further include extracting the target cells. For example, when white blood cells are extracted from blood, other red blood cells, platelets, and plasma may be removed. By adding a nonionic surfactant in the blood, red blood cells, platelets, and plasma can be disrupted. Thereafter, centrifugation is performed to remove the upper liquid, and white blood cells collected on the bottom may be extracted.

Crushing the cells (S100) may use at least one of physical methods and chemical methods. Physical methods include thermal lysis, ultrasonic lysis, high pressure homogenizer, ball mill/bead mill, and freeze-thaw. Can include.

Chemical methods may include adding to the cells a cell lysis solution comprising at least one of an anionic surfactant, an enzyme, and a salt. The anionic surfactant may include sodium lauryl sulfate (SLS) or sodium dodecyl sulfate (SDS), for example. In some embodiments, a cell decomposition solution may be added to the white blood cells, followed by heating and stirring. In addition, it can be diluted by adding distilled water or alcohol to the crushed cells (solution state).

The disrupted cells can be filtered through an activated carbon fiber filter coated with a potassium compound. (S200) This filtration process may be performed in the nucleic acid purification apparatus 100 described with reference to FIGS. 1 to 3. The filtration process may include any one of a centrifugation process, a pressurized process, or a decompression process.

Anionic surfactants such as sodium dodecyl sulfate used for cell disruption may bind with K ⁺ ions coated on the surface of the activated carbon fiber and aggregate on the surface of the activated carbon fiber. Therefore, it may be possible to remove from the filtrate afterwards without a separate process. In addition, in the case of micropores and mesopores of activated carbon fibers, the adsorption rate of nucleic acid fragments and organic solvents is excellent, so that they may not be observed in the filtrate. The active carbon fiber has a small distribution of large pores, so it can be filtered with minimal adsorption of large nucleic acids such as plasmid DNA or genomic DNA. The observed nucleic acid may be any one selected from the group consisting of genomic DNA, RNA, plasmid DNA, mRNA, and rRNA.

<Example 1> Preparation of nucleic acid purification device

An activated carbon fiber with a thickness of 1 mm is cut into a disk with a diameter of 7 mm. The disk-shaped activated carbon fiber (120a) is supported in 600 μL of a 100 mM potassium chloride solution.

에서 5시간동안 열처리시켰다. 염화 칼륨 용액의 용매가 제거되고, 활성 탄소 섬유(120a) 상에 염화 칼륨 화합물(120b)이 코팅됨으로써, 활성 탄소 섬유 필터(120)가 형성된다. 이후, 활성 탄소 섬유 필터(120)를 7mm의 컬럼관(110) 내의 여과지(130) 상에 위치시켰다. 이후 컬럼관의 내경(inner diameter)과 일치하는 고정링을 활성 탄소 섬유 필터 상에 배치시켰다.After removing the wet activated carbon fiber 120a, the solution on the surface of the activated carbon fiber 120a was primarily removed through centrifugation at 12,000 ХG for 2 minutes. Activated carbon fiber treated with potassium chloride solution is 70

It was heat-treated for 5 hours at. The solvent of the potassium chloride solution is removed, and the potassium chloride compound 120b is coated on the activated carbon fiber 120a, thereby forming the activated carbon fiber filter 120. Then, the activated carbon fiber filter 120 was placed on the filter paper 130 in the 7mm column tube 110. Thereafter, a retaining ring matching the inner diameter of the column tube was placed on the activated carbon fiber filter.

<Example 2> Nucleic acid purification method

Nonionic surfactant was added to 200 µl of blood, red blood cells were crushed, centrifuged at 6,000 ХG for 1 minute to remove the supernatant, and then white blood cells collected on the bottom were extracted.

에서 10분간 가열과 교반하여 백혈구를 파쇄하였다. To the extracted white blood cells, 35 µl of a cell lysis solution containing sodium dodecyl sulfate was added, and 56

The white blood cells were crushed by heating and stirring for 10 minutes.

After 50 µl of distilled water was added, it was put into the nucleic acid separation apparatus of Example 1 and centrifuged at 12,000 ХG for 1 minute to purify DNA.

5 is a photograph comparing a DNA sample extracted by the apparatus and method according to the present invention and a DNA sample extracted by a commercial kit for genomic DNA extraction by electrophoresis analysis.

In Figure 5, Experimental Example A is an electrophoresis diagram of genomic DNA according to Example 2, Comparative Example A and Comparative Example B are electrophoretic diagrams of genomic DNA when a commercially available kit is used, and Comparative Example C is active. It corresponds to the electrophoresis diagram of genomic DNA when carbon fiber was used but the potassium compound was not coated.

In the case of Comparative Examples A and B, in order to extract genomic DNA, cells were subjected to lysis, DNA adsorption, washing, and elution processes including three or more centrifugations.

Referring to FIG. 5, in Experimental Example A, it was confirmed that genomic DNA was purified to a similar level compared to a commercially available product. Therefore, in the present invention, since it can be simply composed of cell disruption, filtering, and one centrifugation, DNA can be conveniently purified.

6 is a graph showing the degree of amplification of nucleic acids in the filtrate.

에서 10분간 가열하고 교반하였다. 증류수 50 ㎕를 첨가하여 상기의 방법으로 제작된 핵산정제용 활성 탄소섬유가 포함된 핵산 분리 장치(도 1 내지 도 3)에 넣고 12,000ХG에서 1분간 원심 분리하여 DNA를 정제하였고, real-time PCR을 통해 핵산 증폭을 진행하였다. Referring to FIG. 6, for Experimental Example B, 200 µl of blood was centrifuged to obtain 35 µl of a cell lysis solution containing sodium dodecyl sulfate to leukocytes.

Heated and stirred for 10 minutes. 50 µl of distilled water was added to the nucleic acid separation device (Figs. 1 to 3) containing activated carbon fibers for nucleic acid purification prepared by the above method, and centrifuged at 12,000 ХG for 1 minute to purify DNA, and real-time PCR Nucleic acid amplification was performed through

Likewise, the amplification amount of DNA in the solution filtered through activated carbon fiber not treated with potassium chloride was confirmed through real-time PCR.

In FIG. 6, the solution was filtered through an activated carbon fiber filter treated with potassium chloride, and when performing real-time PCR, sodium dodecyl sulfate and impurities were sufficiently removed, thereby confirming that nucleic acid amplification was good (Experimental Example B). . The solution filtered through activated carbon fiber not treated with potassium chloride has the effect of removing impurities to some extent, but nucleic acid amplification by real-time PCR is sufficiently achieved due to the effect of the residual sodium dodecyl sulfate and impurities when viewing the amplification curve. Did not lose (Comparative Example D). The solution that did not undergo the filtration process did not undergo any nucleic acid amplification by real-time PCR due to inhibition by sodium dodecyl sulfate and other impurities (Comparative Example E).

In the above, embodiments of the present invention have been described with reference to the accompanying drawings, but those of ordinary skill in the art to which the present invention pertains can be implemented in other specific forms without changing the technical spirit or essential features. You can understand that there is. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not limiting.

100: nucleic acid purification device 120b: potassium compound
130: filter paper 140: fixing ring
H: void 110: column tube
120: activated carbon fiber filter 120a: activated carbon fiber

Claims (8)

Including an activated carbon fiber filter,
The activated carbon fiber filter is:
Activated carbon fiber; And
A nucleic acid purification apparatus comprising a potassium compound coated on the surface of the activated carbon fiber.
The method of claim 1,
The activated carbon fiber has a specific surface area of 500 m ² /g to 3000 m ² /g nucleic acid purification device.
The method of claim 1,
The activated carbon fiber is a nucleic acid purification apparatus comprising pores having a diameter of 5 Å to 500 Å.
The method of claim 1,
The potassium compound is:
Potassium chloride (KCl), potassium iodide (KI), potassium hydroxide (KOH), potassium dihydrogen phosphite (KH ₂ PO ₃ ), potassium hydrogen carbonate (KHCO ₃ ), potassium oxide (K ₂ O) containing at least one of Nucleic acid purification device.
The method of claim 1,
A column tube having an inlet disposed at one end and an outlet disposed at the other end;
A fixing ring disposed in the column tube and fixing the activated carbon fiber filter; And
Further comprising a filter paper in contact with the activated carbon fiber filter,
The activated carbon fiber filter is disposed in the column tube,
The retaining ring is disposed closer to the inlet than the activated carbon fiber filter,
The nucleic acid purification device is disposed closer to the outlet than the filter paper is the activated carbon fiber filter.
Disrupting cells; And
A nucleic acid purification method comprising filtering the crushed cells through an activated carbon fiber filter coated with a potassium compound.
The method of claim 6,
Breaking the cells comprises adding an anionic surfactant to the cells.
The method of claim 6,
Filtering the disrupted cells comprises a centrifugation process.

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