KR20180031089A - Apparatus for extracting DNA using mesh unit and Extracing-Method of DNA - Google Patents

Apparatus for extracting DNA using mesh unit and Extracing-Method of DNA Download PDF

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KR20180031089A
KR20180031089A KR1020160118982A KR20160118982A KR20180031089A KR 20180031089 A KR20180031089 A KR 20180031089A KR 1020160118982 A KR1020160118982 A KR 1020160118982A KR 20160118982 A KR20160118982 A KR 20160118982A KR 20180031089 A KR20180031089 A KR 20180031089A
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dna
extractor
mesh structure
blood
dna extraction
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KR101893784B1 (en
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박성식
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바이오뱅크 주식회사
박성식
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay

Abstract

The present invention relates to a DNA extraction system and a DNA extraction method using the same. In particular, the DNA extraction system is configured to extract DNAs from blood, and comprises an extractor including a mesh structure coated with a reagent capable of bonding to DNAs. In particular, the DNA extraction method comprises the steps of: preparing extracted blood; exposing chromosomes of the blood, and destroying proteins and RNAs thereof; introducing an extractor including a mesh structure into the blood in which proteins and RNAs have been destroyed, and aggregating DNAs; washing the extractor in which DNAs are aggregated; and isolating DNAs from the washed extractor in which the DNAs are aggregated. By using the DNA extraction system using a mesh structure according to the present invention, a large number of DNAs can be extracted in a convenient and economical manner.

Description

메시구조체를 이용한 ＤＮＡ 추출시스템 및 이를 이용한 ＤＮＡ 추출방법{Apparatus for extracting DNA using mesh unit and Extracing-Method of DNA}[0001] The present invention relates to a DNA extraction system using a mesh structure, and a DNA extraction method using the same,

본 발명은 DNA 추출시스템 및 DNA 추출방법에 관한 것으로, 보다 상세하게는 면역학 검사를 위한 DNA추출을 하기 위하여 메시구조체를 이용함으로써 간편하고 경제적으로 다량의 DNA를 추출할 수 있는 메시구조체를 이용한 DNA 추출시스템 및 이를 이용한 DNA 추출방법에 관한 것이다.The present invention relates to a DNA extraction system and a DNA extraction method, and more particularly, to a DNA extraction system and a DNA extraction method using a mesh structure for extracting DNA for immunological examination, System and DNA extraction method using the same.

핵산을 이용한 분자진단법은 높은 정확성과 재현성, 신속성 등의 장점이 있어 최근 식품위생 분야와 법의학 분야에서 많은 이슈가 되고 있는 방법이다. 그러나 이런 장점에도 불구하고 여러 부가적인 측정 설비를 갖추어야 하기에 최근에는 이를 Lab on a Disc 형태로 구현하고자 하는 연구들이 많이 진행되어 있다. Nucleic acid-based molecular diagnostics have many advantages in terms of food hygiene and forensic science due to their high accuracy, reproducibility and rapidity. However, in spite of these advantages, various additional measurement equipments have to be equipped. Recently, many researches have been carried out to implement them in the form of Lab on a Disc.

이러한 핵산을 이용한 분자진단법을 Lab on a Disc 상에서 구현하는 데 있어 가장 큰 장애물은 DNA를 추출하기 위한 세포 파괴(cell lysis) 공정의 집적화이다.One of the biggest obstacles to implementing molecular diagnostic methods using nucleic acid on Lab on a Disc is the integration of cell lysis processes to extract DNA.

최근 세포 파괴를 위한 여러 집적 기술이 개발되어 효율성 및 경제성이 개선되었으나 기존 Lab on a Disc 형태의 세포 파괴 방법은 물리적 세포 파괴를 이용한 방법으로, 외부 장치가 필요하여 화학적 세포 파괴 방법에 비해 핵산의 추출 효율성이 떨어지거나, 마이크로 단위의 세밀한 구조가 필요하여 제작이 매우 어렵다는 단점이 있다. Recently, various integration technologies for cell destruction have been developed and efficiency and economical efficiency have been improved. However, the conventional method of destruction of cells in the form of Lab on a Disc is a method using physical cell destruction and requires an external device to extract nucleic acid It is disadvantageous in that it is very difficult to manufacture due to a lack of efficiency or a detailed structure of a micro unit.

그러나 물리적인 세포 파괴 방법은 정밀한 온도 제어가 필요한 화학적 세포 파괴 방법에 비해 구현이 훨씬 수월하며, 화학적 세포 파괴 방법은 효소를 온도 제어에 의해 활성화 시킴으로써 세포파괴의 효율을 극대화할 수 있다.However, physical cell destruction methods are much easier to implement than chemical cell destruction methods that require precise temperature control, and chemical cell destruction methods can maximize cell destruction efficiency by activating enzymes by temperature control.

따라서 화학적 세포 파괴 방법을 Lab on a Disc에 적용하되, 간편하게 DNA만 추출해 낼 수 있는 장치기술 개발이 절실히 필요하다.Therefore, it is urgently necessary to develop a device technology that can easily extract DNA only while applying the chemical cell destruction method to Lab on a Disc.

통상적인 화학적 세포 파괴 방법을 사용하여 혈액으로부터 DNA를 추출해 내는 방법은 다음과 같다.A method of extracting DNA from blood using a conventional chemical cell destruction method is as follows.

혈액 샘플(세포 등)을 추출한 다음, Lysis buffer라는 시약을 이용하여 세포막과 핵막을 파괴하여 염색체가 노출되도록 한다. 그런 다음, 단백질 분해효소 및 RNA 분해효소를 이용하여 단백질과 RNA를 파괴한다. 그런 다음, 철입자(비드)에 DNA가 잘 접합할 수 있는 이소프로파놀(Isopropanol)과 같은 시약을 코팅처리하여 DNA를 응집시키고, 마그네틱 추출기를 이용하여 응집된 DNA만 추출한뒤 워싱, 즉 에탄올(Ethanol)로 Isopropanol을 닦아내어 순수한 DNA를 추출한다.After extracting blood samples (cells, etc.), use a reagent called lysis buffer to break down the cell membrane and nuclear membrane to expose the chromosomes. Then, proteolytic enzymes and RNA degrading enzymes are used to destroy proteins and RNA. Then, the DNA is coagulated by coating a reagent such as isopropanol which can bind the DNA to the iron particles (beads) well, and the coagulated DNA is extracted using a magnetic extractor, followed by washing, that is, ethanol Ethanol) to remove pure DNA.

이와 같은 종래 방법의 마그네틱 추출기를 이용하는 경우, 별도의 마그네틱 바 및 비드를 이용하여야 하는 등 공정이 복잡한 문제가 있고, 마그네틱바의 한정된 표면에만 DNA가 응집되므로, 소량의 DNA만 분리해 내는 문제가 있다.When such a conventional magnetic extractor is used, there is a problem in that a separate magnetic bar and bead must be used, and there is a problem that only a small amount of DNA is separated because the DNA coagulates only on the limited surface of the magnetic bar .

한국공개특허 제10-2016-0096770호Korean Patent Publication No. 10-2016-0096770

본 발명은 상술한 문제점을 해결하기 위해 안출된 것으로서, 본 발명의 목적은 메시구조체를 구비한 추출시스템을 이용하여 간편하고 경제적으로 다량의 DNA를 추출하는 시스템 및 방법을 제공하는 것이다.SUMMARY OF THE INVENTION It is an object of the present invention to provide a system and method for extracting a large amount of DNA easily and economically by using an extraction system having a mesh structure.

상술한 문제를 해결하기 위하여, 본 발명에 따른 DNA 추출시스템은,In order to solve the above-mentioned problems, the DNA extraction system according to the present invention,

혈액으로부터 DNA를 추출하는 시스템에 있어서,A system for extracting DNA from blood,

DNA가 접합할 수 있는 시약이 코팅처리된 메시구조체를 구비한 추출기를 포함하여 구성되는 것을 특징으로 한다.Characterized in that the reagent to which the DNA can bind is constituted by an extractor having a coated mesh structure.

또한, 본 발명에 따른 DNA 추출시스템을 이용하여 DNA를 추출하는 방법은,In addition, a method of extracting DNA using the DNA extraction system according to the present invention is not particularly limited,

추출된 혈액을 준비하는 단계;Preparing an extracted blood;

상기 혈액의 염색체를 노출시키고 단백질과 RNA를 파괴시키는 단계;Exposing the chromosomes of the blood and destroying proteins and RNA;

상기 단백질과 RNA가 파괴된 혈액내에 메시구조체를 구비한 추출기를 투입하고 DNA를 응집하는 단계;Introducing an extractor having a mesh structure into the blood in which the protein and the RNA are destroyed, and aggregating the DNA;

상기 DNA가 응집된 추출기를 워싱하는 단계; 및Washing the extractor in which the DNA is aggregated; And

상기 워싱된 DNA가 응집된 추출기로부터 DNA를 분리하는 단계;를 포함하여 구성되는 것을 특징으로 한다.And separating the DNA from the extractor in which the washed DNA is aggregated.

또한, 본 발명에 따른 DNA 추출시스템을 이용하여 DNA를 추출하는 방법에서,In addition, in the method of extracting DNA using the DNA extraction system according to the present invention,

상기 추출기의 메시구조체에는 DNA가 접합할 수 있는 시약이 코팅처리 되는 것을 특징으로 한다.And a reagent to which DNA can be attached is coated on the mesh structure of the extractor.

또한, 본 발명에 따른 DNA 추출시스템을 이용하여 DNA를 추출하는 방법에서,In addition, in the method of extracting DNA using the DNA extraction system according to the present invention,

상기 메시구조체에 코팅된 시약은 이소프로파놀(Isopropanol)인 것을 특징으로 한다.And the reagent coated on the mesh structure is isopropanol.

또한, 본 발명에 따른 DNA 추출시스템을 이용하여 DNA를 추출하는 방법에서,In addition, in the method of extracting DNA using the DNA extraction system according to the present invention,

상기 DNA가 응집된 추출기로부터 DNA를 분리하기 위하여 분리시약을 투입하는 것을 특징으로 한다.Separating reagent is added to separate the DNA from the extractor in which the DNA is aggregated.

또한, 본 발명에 따른 DNA 추출시스템을 이용하여 DNA를 추출하는 방법에서,In addition, in the method of extracting DNA using the DNA extraction system according to the present invention,

상기 추출기는 표면적 치수가 상대적으로 큰 메시구조체로 되어 있어, 응집시약이 코팅될 수 있는 표면적과 표면적에 응집되는 DNA의 함량이 늘어나는 것을 특징으로 한다.The extractor is a mesh structure having a relatively large surface area, and is characterized in that the surface area at which the coagulation reagent can be coated and the content of DNA aggregated at the surface area are increased.

본 발명에 따른 메시구조체를 이용한 DNA 추출시스템 및 이를 이용한 DNA 추출방법은 면역학 검사를 위한 DNA추출을 하기 위하여 메시구조체를 이용함으로써 간편하고 경제적으로 다량의 DNA를 추출할 수 있는 효과가 있다.The DNA extraction system using the mesh structure according to the present invention and the DNA extraction method using the same have the effect of extracting a large amount of DNA easily and economically by using a mesh structure for extracting DNA for immunoassay.

도 1은 본 발명의 일실시예에 따른 메시구조체를 이용한 DNA 추출시스템 및 이를 이용한 DNA 추출방법을 간략하게 보여주는 도면.BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic view showing a DNA extraction system using a mesh structure and a DNA extraction method using the same according to an embodiment of the present invention. FIG.

이하, 첨부된 도면을 참조하여 본 발명인 메시구조체를 이용한 DNA 추출시스템 및 이를 이용한 DNA 추출방법의 바람직한 실시예를 더욱 상세하게 설명한다.
Hereinafter, preferred embodiments of a DNA extraction system using a mesh structure and a DNA extraction method using the same according to the present invention will be described in detail with reference to the accompanying drawings.

도 1은 본 발명의 일실시예에 따른 메시구조체를 이용한 DNA 추출시스템 및 이를 이용한 DNA 추출방법을 간략하게 보여주는 도면이다.
FIG. 1 is a schematic view showing a DNA extraction system using a mesh structure and a DNA extraction method using the same according to an embodiment of the present invention. Referring to FIG.

도 1에 도시된 바와 같이, 본 발명에 따른 메시구조체를 이용한 DNA 추출시스템은, 통상의 혈액으로부터 DNA를 추출하는 시스템에 DNA가 접합할 수 있는 시약이 코팅처리된 메시구조체를 구비한 추출기(1)를 포함하여 구성된다.
1, a DNA extraction system using a mesh structure according to the present invention includes an extractor 1 having a mesh structure coated with a reagent capable of DNA binding to a system for extracting DNA from ordinary blood ).

이하, 본 발명에 따른 메시구조체를 이용한 DNA 추출시스템을 이용하여 DNA를 추출하는 방법은 아래와 같다.
Hereinafter, a method of extracting DNA using a DNA extraction system using the mesh structure according to the present invention will be described.

(1) 추출된 혈액을 준비하는 단계(1) preparing the extracted blood

샘플 혈액을 추출하여 제1 용기(10)내에 담지하여 준비한다.
The sample blood is extracted and loaded in the first container 10 to prepare it.

(2) 혈액의 염색체를 노출시키고 단백질과 RNA를 파괴시키는 단계(2) exposing the chromosomes of blood and destroying proteins and RNA

혈액샘플이 담지된 용기내에 라이시스버퍼(Lysis buffer)와 같은 시약을 이용하여 세포막과 핵막을 파괴하여 염색체가 노출되도록 한다. Using a reagent such as a lysis buffer, the cell membrane and the nuclear membrane are destroyed to expose the chromosome in the container carrying the blood sample.

그런 다음, 염색체가 노출된 혈액내에 단백질 분해효소 및 RNA 분해효소를 투입하여 단백질과 RNA를 파괴한다.
Then, proteolytic enzymes and RNA degrading enzymes are injected into the exposed chromosomes to destroy proteins and RNA.

(3) 추출기 투입 및 DNA응집단계(3) Extractor injection and DNA aggregation step

바닥면이 메시구조체로 된 추출기(1)를 준비학고, 추출기의 메시구조체에 DNA가 잘 접합할 수 있는 이소프로파놀(Isopropanol)과 같은 특수시약을 코팅처리한다.An extractor (1) having a mesh structure on the bottom is prepared, and a special reagent such as isopropanol is coated on the mesh structure of the extractor so that the DNA can be adhered well.

그런 다음, 추출기를 단백질과 RNA가 파괴된 혈액내에 투입한다.Then, the extractor is put into the blood of the protein and the RNA is destroyed.

메시구조체에 코팅된 시약과 DNA가 반응하여 DNA는 메시구조체에 응집된다.
The DNA reacts with the reagent coated on the mesh structure, causing the DNA to aggregate in the mesh structure.

(4) 워싱단계(4) Washing step

DNA가 응집된 추출기를 제2 용기(20)내에 투입한 후, 에탄올(Ethanol)등으로 세탁처리한다.
The extractor in which the DNA is aggregated is put into the second container 20, and then washed with ethanol or the like.

(4) DNA분리단계(4) DNA separation step

워싱된 DNA가 응집된 추출기를 제3 용기(30)내에 투입한 후, 구조체에 코팅된 이소프로파놀(Isopropanol)과 같은 특수시약과 여기에 응집된 DNA를 분리하는 분리시약을 투입하여 DNA를 분리해 낸다.
After the extracted DNA aggregate is injected into the third container 30, a special reagent such as isopropanol coated on the structure and a separating reagent for separating the coagulated DNA are added to separate the DNA I do it.

상술한 메시구조체를 구비한 추출기는 표면적 치수가 상대적으로 큰 메시구조체로 되어 있어 표면적이 크므로 DNA가 응집될 수 있는 면적이 커 응집되는 DNA의 함량은 다량으로 크게 늘어나게 되고, 이를 통해 DNA를 분리해내는 경우, 간편하면서도 경제적으로 다량의 DNA검사를 수행할 수 있게 된다.
Since the extractor having the above-mentioned mesh structure has a relatively large surface area and has a large surface area, there is a large area where the DNA can flocculate, and the amount of aggregated DNA is greatly increased, If you do this, you will be able to perform a large amount of DNA tests conveniently and economically.

본 발명은 도면을 참조하여 실시예를 참고로 설명하였으나, 이는 예시적인 것에 불과하며, 당해 분야에서 통상의 지식을 가진자라면 이로부터 다양한 변형 및 균등한 실시예가 가능하다는 점을 이해할 것이다. 따라서, 본 발명의 진정한 기술적 보호범위는 첨부된 특허청구범위의 기술적 사상에 의해 정해져야 할 것이다.While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the true scope of the present invention should be determined by the technical idea of the appended claims.

1: 메시구조체를 구비한 추출기 10,20,30: 용기1: extractor with mesh structure 10, 20, 30: container

Claims (6)

혈액으로부터 DNA를 추출하는 시스템에 있어서,
DNA가 접합할 수 있는 시약이 코팅처리된 메시구조체를 구비한 추출기를 포함하여 구성되는 것을 특징으로 하는 DNA 추출시스템.
A system for extracting DNA from blood,
Wherein the DNA extraction system comprises an extractor having a mesh structure coated with a reagent to which DNA can be attached.
추출된 혈액을 준비하는 단계;
상기 혈액의 염색체를 노출시키고 단백질과 RNA를 파괴시키는 단계;
상기 단백질과 RNA가 파괴된 혈액내에 메시구조체를 구비한 추출기를 투입하고 DNA를 응집하는 단계;
상기 DNA가 응집된 추출기를 워싱하는 단계; 및
상기 워싱된 DNA가 응집된 추출기로부터 DNA를 분리하는 단계;를 포함하여 구성되는 것을 특징으로 하는 DNA 추출시스템을 이용하여 DNA를 추출하는 방법.
Preparing an extracted blood;
Exposing the chromosomes of the blood and destroying proteins and RNA;
Introducing an extractor having a mesh structure into the blood in which the protein and the RNA are destroyed, and aggregating the DNA;
Washing the extractor in which the DNA is aggregated; And
And separating the DNA from the extractor in which the washed DNA is aggregated. The method for extracting DNA using the DNA extraction system according to claim 1,
제2 항에 있어서,
상기 추출기의 메시구조체에는 DNA가 접합할 수 있는 시약이 코팅처리 되는 것을 특징으로 하는 DNA 추출시스템을 이용하여 DNA를 추출하는 방법.
3. The method of claim 2,
Wherein the extracting machine has a mesh structure coated with a reagent to which DNA can be attached.
제3 항에 있어서,
상기 메시구조체에 코팅된 시약은 이소프로파놀(Isopropanol)인 것을 특징으로 하는 DNA 추출시스템을 이용하여 DNA를 추출하는 방법.
The method of claim 3,
Wherein the reagent coated on the mesh structure is isopropanol.
제2 항에 있어서,
상기 DNA가 응집된 추출기로부터 DNA를 분리하기 위하여 분리시약을 투입하는 것을 특징으로 하는 DNA 추출시스템을 이용하여 DNA를 추출하는 방법.
3. The method of claim 2,
And a separation reagent is added to separate the DNA from the extractant in which the DNA is aggregated.
제2 항 내지 제 5 항 중 어느 한 항에 있어서,
상기 추출기는 표면적 치수가 상대적으로 큰 메시구조체로 되어 있어, 응집시약이 코팅될 수 있는 표면적과 표면적에 응집되는 DNA의 함량이 늘어나는 것을 특징으로 하는 DNA 추출시스템을 이용하여 DNA를 추출하는 방법.6. The method according to any one of claims 2 to 5,
Wherein the extractor is a mesh structure having a relatively large surface area so that the amount of DNA aggregated on the surface area and surface area on which the aggregating reagent can be coated is increased.
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