WO2017051939A1 - Method for extracting and amplifying nucleic acid using magnetic particles - Google Patents

Method for extracting and amplifying nucleic acid using magnetic particles Download PDF

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Publication number
WO2017051939A1
WO2017051939A1 PCT/KR2015/010008 KR2015010008W WO2017051939A1 WO 2017051939 A1 WO2017051939 A1 WO 2017051939A1 KR 2015010008 W KR2015010008 W KR 2015010008W WO 2017051939 A1 WO2017051939 A1 WO 2017051939A1
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dna
nucleic acid
pcr
magnetic particles
protein
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PCT/KR2015/010008
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French (fr)
Korean (ko)
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김태선
송금수
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(주)바이오메트릭스 테크놀로지
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Priority to PCT/KR2015/010008 priority Critical patent/WO2017051939A1/en
Publication of WO2017051939A1 publication Critical patent/WO2017051939A1/en

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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
    • 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

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  • the present invention relates to a nucleic acid based measurement technique having high sensitivity and efficiency, specifically, the DNA / RNA denatured in the denatured DNA-magnetic particle attachment resulting from the denaturation step, as well as being used on its own, as well as the aforementioned
  • the present invention relates to a method for extracting and amplifying nucleic acids including washing a denatured DNA-magnetic particle attachment with a protein-containing washing solution and then performing a primer binding step and a DNA chain extension step, and a method for diagnosing a viral infection using the same.
  • Molecular diagnostic tests including genetic diagnostic tests and immunohistochemical tests, are tests that evaluate genes, metabolic functions, drug metabolism, and disease-causing relationships based on biomarkers such as nucleic acids (DNA, RNA) and proteins.
  • biomarkers such as nucleic acids (DNA, RNA) and proteins.
  • nucleic acid diagnostics that analyzes and detects nucleic acids, which are genetic information substances derived from trace amounts of infectious agents, to diagnose infection.
  • Nucleic acid diagnostics is usually based on polymerase chain reaction (PCR), i) virus quantification and genetic analysis, ii) sexual and infectious tests, iii) disease diagnosis, monitoring and prognosis through detection, amplification and analysis of nucleic acids, iv Genetic tests used in drug genetics and drug genomics are conducted.
  • PCR polymerase chain reaction
  • the core of the technology using DNA / RNA is a nucleic acid extraction process, which extracts and purifies nucleic acids from a sample or a sample. Efforts and studies for this have been continued since the early 1900s. Nucleic acid extracted from the sample was removed from a large amount of salts and proteins such as proteins to prevent further processing, and concentrated in a small amount of buffer to enable more accurate and effective assays. As the purified nucleic acid was introduced by PCR method, a gene amplification technology developed in 1983, nucleic acid-based analysis technology has emerged very resiliently because it can be analyzed quickly and effectively due to high sensitivity and specificity. .
  • Nucleic acid extraction methods such as phenol-chloroform precipitation method, boom technology (solid based nucleic acid purification), solid phase reversible immobilization (SPRI), charge switch technology (CST) have been developed.
  • Boom technology is used mainly to fix nucleic acid on the surface of silica using chaotropic salt, and is used as a major technology of DNA / RNA isolaiton kit sold by Qiagen, MN.
  • Chaotropic salt is a representative water molecules network broker, which induces direct adsorption of DNA on the surface of a solid substrate to increase the recovery of nucleic acid and at the same time, cell debris, an impurity cell debris.
  • Membrane and other proteins can be dissolved to obtain high purity nucleic acid.
  • chaotropic salts are very toxic and, when they remain in DNA / RNA extraction solutions, cause degradation of DNA polymerase and act as a potent PCR inhibitor. Therefore, if chaotropic salt is used, the washing process is required, so it is necessary to wash 2 or 3 times.
  • This method is mainly purified using a column or magnetic particles, and DNA / RNA from the column or magnetic particles is dissolved in an aqueous solution due to the purification of nucleic acid in an open plastic ware, a large surface area, and a low DNA recovery rate. Melting or elution processes are necessary.
  • the volume of the sample containing the eluted DNA / RNA is in the amount of 100 to 200 ⁇ l, of which the sensitivity is lowered because only a small amount of 2 to 10 ⁇ l is consumed for the PCR analysis. The more the PCR efficiency is reduced.
  • due to the decrease in the recovery rate of the total DNA / RNA due to the desorption efficiency of the adsorbed DNA / RNA in the elution process there is no choice but to lose some of the DNA / RNA.
  • an automated device has been proposed to prevent the handling error of the experimenter.
  • the automated devices developed to date are mainly limited to the nucleic acid extraction function, and even though the nucleic acid extraction process and the nucleic acid amplification process are integrated, the movement of a plurality of nucleic acid extraction solutions and complex mechanical movements are required, resulting in a high unit cost. Rather, the problem of cross-contamination and the migration of samples that require the transfer of eluted nucleic acids to PCR tubes remains a significant challenge.
  • PCR method for extracting and amplifying nucleic acids using a solid support such as magnetic particles, solves the problems of sensitivity and low efficiency due to using only a part of the DNA / RNA present in the sample and contamination due to sample migration
  • An improved PCR method is proposed to
  • a method for extracting and amplifying a nucleic acid using a magnetic particle as a solid support includes a PCR process including a DNA denaturation step, a primer binding step, and a DNA chain extension step.
  • PCR can be performed with high sensitivity and high efficiency even for a sample having a low DNA content.
  • the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of the solid support
  • the present inventors have found that contamination of DNA can be prevented because the DNA denaturation step and the PCR step can be performed in the same container, thereby completing the present invention.
  • the PCR of DNA can be performed with high sensitivity and efficiency even for samples with low DNA content, the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of a solid support, and Denaturation and PCR can be performed in one container to prevent contamination of DNA.
  • FIG. 1 is a diagram schematically illustrating the integration of a nucleic acid extraction and amplification process according to an embodiment of the present invention
  • FIG. 2 is a diagram confirming the results of PCR reactions containing magnetic particles using BMT 9G DNA KIT due to integration of nucleic acid extraction and amplification process according to an embodiment of the present invention
  • FIG. 3 is a view showing the PCR results according to the amount of magnetic particles using the BMT 9G DNA KIT according to an embodiment of the present invention
  • FIG. 4 is a diagram showing a PCR result obtained by changing the concentration of the added skim milk using BMT 9G DNA KIT, and a diagram showing an electrophoresis pattern;
  • FIG. 5 is a diagram confirming the PCR results obtained by fixing the concentration of the scheme milk to 0.05% and changing the number of magnetic particles inserted into the PCR reaction using BMT 9G DNA KIT;
  • Figure 6 shows the PCR results obtained by changing the amount of magnetic particles inserted into the PCR reaction to 1mg / 20 ⁇ l and 2.5mg / 20 ⁇ l when adding 0.05% and 0.1% of the skim milk using BMT 9G DNA KIT To confirm by drawing;
  • FIG. 7 is a diagram illustrating the use of BMT 9G DNA KIT to confirm the PCR results obtained for various samples by fixing the concentration of skim milk to 0.1% and the amount of magnetic particles to 2.5 mg / 20 ⁇ l.
  • each color represents a fluorescence intensity
  • the specific fluorescence intensity having the order and intensity of white> red> yellow
  • blue 65000> 50000> 40000> 30000 >> 10000.
  • bright green has 35000 intensity and dark green has about 20000 intensity, so the bright color shows higher intensity.
  • a first object of the present invention is a method for extracting and amplifying a nucleic acid comprising at least one PCR process including a DNA denaturation step, a primer binding step, and a DNA chain extension step, the method comprising the following steps: Methods of extracting and amplifying nucleic acids are provided:
  • step (3) performing the primer binding step and the DNA chain extension step using the modified DNA-magnetic particle attachments resulting from step (2) by themselves.
  • step (2) may be carried out in detail as follows:
  • steps (2-1) to (2-4) are repeated one or more times.
  • the above-mentioned solid support may have one or more functional groups selected from the group consisting of siloxane groups, amino groups and hydroxyl groups on the surface, or silica, carbo It can be coated with hydrates, amines, polystyrenes, polypropylene and the like.
  • the size (diameter) of the magnetic particles described above may be 0.1 to 30 ⁇ m, specifically 0.2 to 25 ⁇ m, preferably 0.4 to 20 ⁇ m.
  • the amount or content of the above-described magnetic particles may be selected in the range of 0.10 to 10 mg / 20 ⁇ l, specifically 0.25 to 5 mg / 20 ⁇ l, preferably 0.5 to 2.5 mg / 20 ⁇ l.
  • the amount or content of the magnetic particles described above is a value selected to efficiently perform the amplification process of DNA, including a PCR process.
  • the magnetic particles described above may be 2.5 to 30 mg / 20 ⁇ l, specifically, Use in high amounts of 5-20 mg / 20 ⁇ l is also within the scope of the present invention.
  • the protein in the protein-containing washing solution described above may be selected from the group consisting of BSA, skim milk and casein, the content (concentration) is particularly limited However, it may have a h concentration of 0.01 to 0.1% (w / v), specifically 0.02 to 0.1% (w / v), preferably 0.03 to 0.08% (w / v), for ease of processing. .
  • the DNA chain extension step may be performed using a DNA amplification solution selected from the group consisting of PCR, RT-PCR, TMA, NASAB, SDA and RCA solutions.
  • the product obtained by the nucleic acid extraction and amplification reaction is subjected to gel electrophoresis, gel-electrophoresis (ELGA), electrochemiluminescent (ECL) or BMT 9G DNA KIT and BMT
  • the method may further include detecting by a method selected from the group consisting of Genotyping 9G Membrane KIT.
  • nucleic acid may mean DNA, RNA, fragments or oligomers thereof, and the method of the present invention may be applied to all of them, thus, “DNA chain extension step", “DNA amplification solution”
  • DNA-magnetic particle attachment (conjugate) and the like may be used to replace DNA with RNA.
  • the step of transforming double helix DNA into single helix for example DNA denature process, may be omitted.
  • FIG. 1 is a diagram schematically illustrating the integration of a nucleic acid extraction and amplification process according to an embodiment of the present invention.
  • the magnetic particles adsorbing nucleic acids are not moved out of the chamber by adding or removing the sample, the nucleic acid extraction solution and the washing solution by the nozzle.
  • the elution process is omitted as the PCR solution is added to the magnetic particles adsorbing the nucleic acid through the nozzle, and a method of increasing the sensitivity and accuracy while using the entire amount of the nucleic acid obtained from the sample for PCR is provided.
  • the nucleic acid extraction process is performed after adding the nucleic acid-containing sample to the chamber to which the nucleic acid extraction solution and the magnetic particles are added.
  • Nucleic acid separated from bacteria, viruses, or animal cells by the nucleic acid extraction solution is adsorbed to the magnetic particles.
  • the magnetic particles adsorbed by the nucleic acid by the magnetic bar located outside the chamber are fixed inside the chamber, and all the solutions except the magnetic particles are removed by suction.
  • the washing solution is injected into the chamber through a nozzle, and the washing solution is characterized in that a predetermined protein is added.
  • the solution is removed in the same manner as the extraction solution removal method, and only the magnetic particles adsorbed by the purified nucleic acid remain in the chamber. Thereafter, the PCR solution is added to the chamber, and the PCR reaction is performed by a heater located at the bottom of the chamber.
  • the above-described invention proposes a process method that can reduce contamination, improve sensitivity and accuracy by integrating a series of processes of nucleic acid extraction and amplification.
  • Nucleic acid extraction kits currently used are mostly used magnetic particles as a column or a solid support for adsorption, separation and purification of nucleic acids.
  • the use of the column and the magnetic particles has the advantage of easy extraction and purification since the nucleic acid can be separated from a large amount of sample by adsorbing the nucleic acid on a solid substrate.
  • this solid-based nucleic acid extraction method is required to be converted back to the liquid phase because it needs to be converted back to the liquid phase in order to be analyzed and measured using a subsequent process, for example, PCR.
  • the elution process when the adsorbed nucleic acid is desorbed again, the purified nucleic acid is lost according to the desorption efficiency.
  • the minimum amount of solution required for elution is required to be about 100 ⁇ l or more, most nucleic acids extracted from the sample are wasted because subsequent steps such as PCR use only 2 ⁇ l to 10 ⁇ l.
  • the present invention is an integrated process that can be easily and quickly measured by adding a PCR solution directly to the magnetic particles after eliminating the elution step after the lysis, nucleic acid adsorption, washing process in the nucleic acid extraction process using magnetic particles Developed.
  • the entire amount of nucleic acid adsorbed on the magnetic particles obtained from the sample is immediately added with a PCR solution to perform a PCR reaction.
  • the nucleic acid is effectively desorbed into the aqueous solution phase, which is immediately used for the PCR reaction. It has high sensitivity and accuracy because the entire amount of nucleic acid in the sample is used, and since the magnetic particles adsorbed with the nucleic acid do not move in one chamber, they do not lead to false positive results due to contamination due to the movement of the sample.
  • the present invention provides a method for adding a predetermined protein in the last washing step of the nucleic acid extraction process to solve the problem of inhibiting the PCR reaction for a large amount of magnetic particles.
  • the protein added to the final washing process may be non-specifically adsorbed on the surface of the magnetic particles other than the portion where the nucleic acid is adsorbed, thereby blocking the element that inhibits the PCR reaction at the source, leading to a more efficient PCR reaction.
  • the above-mentioned protein may be selected from the group consisting of BSA (bovine serum protein), skim milk and casein, and 0.005 to 0.2% (w / v), specifically 0.01% to 0.1% (w / v), preferably in an amount of 0.02 to 0.08% (w / v).
  • BSA bovine serum protein
  • skim milk and casein 0.005 to 0.2%
  • w / v 0.01% to 0.1%
  • w / v preferably in an amount of 0.02 to 0.08%
  • gelatin or commercially available non-animal protein blocking agents as the aforementioned proteins may also be included within the scope of the present invention.
  • Proteins such as BSA, skim milk and casein described above are proteins used as blocking buffers or blocking agents in Western blotting to analyze the presence or absence of specific proteins. It is used to block the non-specific binding of 1st Ab or 2nd Ab by coating the protein on the membrane surface that is not blotting.
  • blocking agents are not used in Southern Blotting to detect DNA, and thus the use of these blocking agents and their effects in PCR using a solid support are not known until now.
  • the surface of the chip solid may be coated with BSA or BSA may be added to the PCR solution, but it is used as a blocking agent as in the present invention. No difference.
  • the solid support may be separated from the washing solution by centrifugation, and the washing solution may be removed by decantation or pipetting.
  • step (2) when the solid support can be separated from the washing solution by centrifugation, the method of the present invention can be carried out without magnetic particles as described above.
  • step (2) may be performed in detail as follows:
  • PCR methods using magnetic particles can be found in many documents, and matters not specifically described in the present invention may use those described in the above documents.
  • the denatured DNA attached solid support is not only washed with a solution containing a specific protein, but also the denatured DNA is used on its own without eluting from the solid support, thereby providing a primer binding step and a DNA chain extension step.
  • the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of the solid support.
  • the attachment of the denatured DNA / RNA and the solid support described above can remain in the reaction vessel even after the reaction and washing steps and can proceed with subsequent PCR reactions without moving or changing the reaction vessel, resulting in DNA in the same vessel.
  • denaturation and PCR reaction contamination of DNA can be prevented.
  • HPV Human Papilloma Virus
  • Nucleic Acid Extraction Magnetic bead
  • the PCR process consists of (i) DNA denaturing, (ii) elution of denatured DNA, (iii) primer binding, and (iv) chain extension.
  • the above-mentioned elution includes washing of unreacted DNA denaturation step, heating process for mixing and separating the separation material.
  • the denatured DNA is separated from the magnetic particles in the elution step, and then amplified through the primer binding step and the DNA extension step.
  • the PCR process was carried out four times in conformity with the nucleic acid extraction procedure specified in the above-mentioned kit manual.
  • the amplified nucleic acid is used by mixing a hybridization solution with a PCR solution, it is not necessary to separate and purify the nucleic acid separately, and BMT HPV 9G DNA KIT [Biometrics Technology; Korea] to compare and analyze the fluorescence intensity.
  • the fluorescence intensity is expressed in the order of white-red-yellow-green-blue in the order of the strongest.
  • Photographs 1-4 of the left (without bead) of FIG. 2 show fluorescence photographs measured for the DNA thus amplified.
  • the cleaning process is performed in the elution step, and then the mixing and heating steps are omitted, except that the denatured DNA is separated from the magnetic particles and the amplification step is performed as it is.
  • the PCR procedure was performed four times using the detailed extraction method as it is.
  • the extracted DNA was denatured in the presence of magnetic particles, the resulting denatured DNA-magnetic particle attachment was washed and suspended by addition of a small amount of Tris-HCl (pH 8.0) buffer.
  • the suspended magnetic particles were quantified and PCR premix tube [Bionia; Korea] to perform PCR (once) to amplify the nucleic acid.
  • This amplified nucleic acid BMT HPV 9G DNA KIT [Biometrics Technology; Korea] to compare and analyze the fluorescence intensity.
  • the fluorescence intensity is expressed in the order of white-red-yellow-green-blue in the order of the strongest.
  • Photographs 1-4 of the right side (with bead) of FIG. 2 show fluorescence images measured on the DNA thus amplified.
  • Fig. 2 shows fluorescence photographs (with bead) obtained from PCR results in the state of denatured DNA-magnetic particle attachment (with experimental group) and fluorescence photographs (without bead) obtained from PCR results in the state of denatured DNA only without magnetic particle (control group). ) Is a comparison.
  • the blue spot of the control group was not expressed in the experimental group in sample 1, and the fluorescence difference was decreased the most from the white of the control group to the red of the experimental group in sample 3, but in samples 2 and 4, Fluorescence decreased rapidly from white in the control group to green in the experimental group.
  • FIG. 3 is a diagram showing PCR results of Reference Example 2a (Sample 1) and Reference Example 2b (Sample 2), when the amount of the magnetic particles used is 0.25mg / 20 ⁇ L slightly lower than the control group, but similar results (sensitivity) However, when the amount of use is more than 1 mg / 20 ⁇ l shows that the PCR efficiency is drastically reduced compared to the control, and when the amount of use is 2.5mg / 20 ⁇ l shows that the results are not obtained properly. Therefore, when the magnetic particles are used below 0.25 mg / 20 ⁇ l or less, it can be seen that the presence of the magnetic particles does not act as an inhibitor of PCR efficiency.
  • washing solution containing magnetic particles in an amount of 0.5 mg and containing a specific protein (eg skim milk) at a concentration of 0%, 0.01%, 0.03%, 0.05% or 0.1% (w / v) in the elution step.
  • a PCR procedure was performed in the same manner as in Reference Example 1 except that the reaction was performed.
  • As a control PCR was performed in the absence of magnetic particles, and the result obtained using the washing solution containing no skim milk (the same as the control of Reference Example 2a) was used.
  • FIG. 4a shows that the control group has a fluorescence intensity of white but has a very low sensitivity to green when no protein is used, but using a wash solution containing a skim milk protein in an amount of 0.01%, 0.03%, 0.05% and 0.1%. Shows that similar or the same sensitivity as the control can be obtained.
  • Figure 4b shows the results of the PCR of Figure 4a through electrophoresis, when using the skim milk protein of 0.05%, shows that the most distinct PCR band is confirmed.
  • Magnetic particles are used in amounts of 0.5 mg / 20 ⁇ l, 1 mg / 20 ⁇ l, 1.5 mg / 20 ⁇ l, 2.5 mg / 20 ⁇ l and 5 mg / 20 ⁇ l respectively, and specific proteins (eg skim milk)
  • the PCR procedure was performed in the same manner as in Example 1 except that the washing solution containing the concentration of 0.05% (w / v) was used.
  • PCR was performed in the absence of magnetic particles and the results obtained using a washing solution containing no skim milk were used.
  • Figure 5 shows that when using a washing solution containing 0.05% of the skim milk, using magnetic particles in an amount of 1 mg / 20ul obtained the same or similar results as the control.
  • the present invention it can be seen that by adding a protein such as BSA, skim milk, casein, etc. to the washing process, the protein is non-specifically bound to the magnetic particles, thereby blocking the inhibitory factor in the subsequent PCR efficiency.
  • a protein such as BSA, skim milk, casein, etc.
  • washing solution containing magnetic particles in amounts of 1 mg / 20 ⁇ l and 2.5 mg / 20 ⁇ l respectively and containing specific proteins (eg skim milk) at concentrations of 0.05% and 1% (w / v), respectively, in the elution step.
  • PCR procedure was performed in the same manner as in Example 1 except for using the following.
  • As a control PCR was performed in the absence of magnetic particles and the result obtained by using a washing solution containing no skim milk (same as the control of Example 2) was used.
  • FIG. 6 shows better results using a washing solution containing skim milk at a low concentration (0.05%) when the magnetic particles are used in a small amount (1 mg / 20 ⁇ l).
  • a large amount 2.5 mg / 20 ⁇ l shows that using a wash solution containing a high concentration (0.1%) of skim milk was obtained better results.
  • a washing solution containing more protein should be used.
  • a small amount of magnetic particles there is a high possibility that non-specifically adsorbed and remaining proteins remain on the surface of the magnetic particles, and these unbound residual proteins may be released into the PCR solution to act as an inhibitor in performing the PCR.
  • a large amount of magnetic particles since there is very little non-specifically adsorbed protein remaining on the surface of the magnetic particles, it is unlikely that they are released into the PCR solution. Therefore, a higher concentration of protein is required and can be used to perform the integration process of nucleic acid extraction and amplification using a large amount of magnetic particles.
  • FIG. 7 shows that Sample 1 increased from blue to green, Sample 2 increased from green to white, and Sample 3 and Sample 4 did not change to blue and white, respectively.
  • results of the present invention show that the PCR is carried out in the presence of magnetic particles and using protein-containing wash solution according to the present invention, rather than performing the conventional general nucleic acid extraction and amplification process.
  • nucleic acid extraction / amplification can be achieved by reducing the number of processes, increasing convenience and speed, and eliminating sample migration, resulting in false positive results due to contamination, and improving sensitivity and accuracy by using the entire amount of nucleic acid obtained from the sample. It is intended to clarify the development of an integrated process.
  • the present invention can be used industrially in the field of nucleic acid-based measurement techniques using PCR methods for extracting and amplifying nucleic acids and in the field of biotechnology using DNA / RNA.

Abstract

The present invention, with respect to a method for extracting and amplifying nucleic acid which repeats, one or more times, a DNA PCR process comprising the steps of denaturing DNA, attaching a primer, and extending the DNA chain, relates to a method for extracting and amplifying nucleic acid characterized by using the resulting modified DNA-magnetic particle precipitate as is without eluting the modified DNA/RNA from the magnetic particles, and executing the primer attachment step and DNA chain extension step after washing the modified DNA-magnetic particle precipitate by means of a protein-containing washing solution. According to the present invention, first, highly sensitive and effective PCR can be carried out even with a sample having a low DNA content as the DNA damage in the sample can be prevented, second, highly sensitive and effective PCR of DNA can be carried out even under the presence of a solid support as the surface of same is nonspecifically blocked with a protein, and third, DNA contamination can be prevented as the DNA denaturation step and PCR step can be carried out in the same container.

Description

자성입자를 이용한 핵산의 추출 및 증폭 방법Nucleic Acid Extraction and Amplification Method Using Magnetic Particles
본 발명은 높은 민감도 및 효율성을 갖는 핵산 기반 측정 기술에 관한 것으로, 구체적으로는, 변성단계에서 결과된 변성DNA-자성입자 부착물에서 변성된 DNA/RNA를 용리시키지 않고 그 자체로 사용할 뿐만 아니라 전술한 변성DNA-자성입자 부착물을 단백질-함유 세척용액으로 세척한 후에 프라이머 결합 단계 및 DNA 사슬 연장 단계를 수행하는 것을 포함하는 핵산의 추출 및 증폭 방법, 및 이를 이용한 바이러스 감염의 진단방법에 관한 것이다.The present invention relates to a nucleic acid based measurement technique having high sensitivity and efficiency, specifically, the DNA / RNA denatured in the denatured DNA-magnetic particle attachment resulting from the denaturation step, as well as being used on its own, as well as the aforementioned The present invention relates to a method for extracting and amplifying nucleic acids including washing a denatured DNA-magnetic particle attachment with a protein-containing washing solution and then performing a primer binding step and a DNA chain extension step, and a method for diagnosing a viral infection using the same.
DNA/RNA를 이용한 생명공학 분야의 연구는 DNA/RNA의 염기서열 분석 뿐만 아니라, 검체의 측정, 질병의 유무, 성질 분석 등을 위하여 1900년대 초반부터 꾸준히 개발되어 왔으며, 최근들어 바이러스 및 박테리아의 측정 및 검출하는 방법까지 개발되었다. Researches in the field of biotechnology using DNA / RNA have been developed steadily since the early 1900s for the analysis of DNA / RNA sequences as well as for the measurement of specimens, the presence of diseases, and the analysis of properties. And a method of detection has been developed.
유전자 진단 검사 및 면역 조직 진단 검사를 포함하는 분자 진단 검사는 핵산 (DNA, RNA) 및 단백질 등 생체지표물질(biomarker)을 기반으로 유전자와 대사 기능, 의약품 대사, 질병 유발 관계를 평가하는 검사로서, 현재는 극미량의 감염체에서 유래하는 유전 정보 물질인 핵산을 분석하고 검출하여 감염여부를 진단할 수 있는 핵산 진단학 (nucleic acid diagnostics)과 같은 의미로 사용되고 있다. Molecular diagnostic tests, including genetic diagnostic tests and immunohistochemical tests, are tests that evaluate genes, metabolic functions, drug metabolism, and disease-causing relationships based on biomarkers such as nucleic acids (DNA, RNA) and proteins. Currently, it is used in the same sense as nucleic acid diagnostics that analyzes and detects nucleic acids, which are genetic information substances derived from trace amounts of infectious agents, to diagnose infection.
핵산 진단학은 보통 중합효소연쇄반응(PCR)을 기반으로 하여, 핵산의 검출, 증폭, 분석을 통하여 i)바이러스 정량 및 유전자 분석, ii)성병 및 감염 검사, iii)질병 진단, 모니터링 및 예후, iv)약물유전학 및 약물 유전체학에 이용되는 유전자 검사 등을 실시하고 있다. Nucleic acid diagnostics is usually based on polymerase chain reaction (PCR), i) virus quantification and genetic analysis, ii) sexual and infectious tests, iii) disease diagnosis, monitoring and prognosis through detection, amplification and analysis of nucleic acids, iv Genetic tests used in drug genetics and drug genomics are conducted.
DNA/RNA를 이용하는 기술의 핵심은 핵산 추출 공정으로서, 검체 또는 시료로부터 핵산을 추출, 정제하는 것으로, 이를 위한 노력과 연구가 1900년대 초반부터 계속되어 왔다. 검체로부터 추출된 핵산은 후속 공정의 진행을 방해하는 다량의 염(salt)들과 단백질 등의 불순물이 제거되고, 소량의 버퍼에 농축되어 보다 정확하고 효과적인 분석 실험이 가능해졌다. 이렇게 정제된 핵산은 1983년대에 개발된 유전자 증폭 기술인 PCR 법이 소개되면서, 높은 민감도(sensitivity)와 특이성(specificity)로 인해 신속하고 효과적으로 분석이 가능해짐으로써, 핵산 기반 분석기술이 매우 탄력적으로 급부상하였다. The core of the technology using DNA / RNA is a nucleic acid extraction process, which extracts and purifies nucleic acids from a sample or a sample. Efforts and studies for this have been continued since the early 1900s. Nucleic acid extracted from the sample was removed from a large amount of salts and proteins such as proteins to prevent further processing, and concentrated in a small amount of buffer to enable more accurate and effective assays. As the purified nucleic acid was introduced by PCR method, a gene amplification technology developed in 1983, nucleic acid-based analysis technology has emerged very resiliently because it can be analyzed quickly and effectively due to high sensitivity and specificity. .
핵산 추출 방법으로는 페놀-클로로포름 침전법, Boom technology (solid based nucleic acid purification), SPRI (Solid Phase Reversible Immobilization), CST (Charge Switch Technology) 등 많은 기술들이 개발되었으며, 그 중 대표적인 핵산 추출 방법으로는 카오트로픽염(chaotropic salt) 를 이용하여 실리카 표면에 핵산을 고정하는 Boom technology가 주로 사용되고 있으며, 대표적으로 Qiagen, MN 사에서 판매되는 DNA/RNA isolaiton kit의 주요 기술로 사용되고 있다. 카오트로픽염은 대표적인 물분자 네트워크 파괴제 (water molec㎕es network broker)로서 고체기판 표면과 DNA의 직접적인 흡착을 유도하여 핵산의 회수율을 높이는 동시에 불순물인 셀데브리스(cell debris) 인 셀멤브레인(cell membrane)과 기타 단백질을 녹여내어 순도 높은 핵산을 얻을 수 있다.Nucleic acid extraction methods such as phenol-chloroform precipitation method, boom technology (solid based nucleic acid purification), solid phase reversible immobilization (SPRI), charge switch technology (CST) have been developed. Boom technology is used mainly to fix nucleic acid on the surface of silica using chaotropic salt, and is used as a major technology of DNA / RNA isolaiton kit sold by Qiagen, MN. Chaotropic salt is a representative water molecules network broker, which induces direct adsorption of DNA on the surface of a solid substrate to increase the recovery of nucleic acid and at the same time, cell debris, an impurity cell debris. Membrane) and other proteins can be dissolved to obtain high purity nucleic acid.
그러나 카오트로픽염은 독성이 매우 강해 DNA/RNA 추출 용액에 잔류해 있을 경우 DNA polymerase의 분해(degradation)을 유발하여 강력한 PCR 저해제로 작용한다. 따라서 카오트로픽염을 사용할 경우 세척공정이 반드시 요구되기 때문에 2번 또는 3번의 세척공정을 필수로 한다. 이러한 방식은 주로 컬럼이나 자성입자를 사용하여 정제되며, 오픈된 플라스틱제 기구(plastic ware)에서의 핵산의 정제나 많은 표면적, DNA 회수율 저하 등의 이유로 컬럼이나 자성입자로부터 DNA/RNA 를 수용액 상에 녹여내는 용출 또는 용리 (elution) 과정이 반드시 필요로 하게 된다. However, chaotropic salts are very toxic and, when they remain in DNA / RNA extraction solutions, cause degradation of DNA polymerase and act as a potent PCR inhibitor. Therefore, if chaotropic salt is used, the washing process is required, so it is necessary to wash 2 or 3 times. This method is mainly purified using a column or magnetic particles, and DNA / RNA from the column or magnetic particles is dissolved in an aqueous solution due to the purification of nucleic acid in an open plastic ware, a large surface area, and a low DNA recovery rate. Melting or elution processes are necessary.
용리된 DNA/RNA를 포함하는 샘플의 부피는 100~200 ㎕의 양이며, 그 중 PCR 분석을 위하여 2~10 ㎕ 정도의 적은 양만 소비되고 있기 때문에 민감도가 저하되는데, DNA/RNA의 양이 미량일수록 PCR의 효율이 감소하게 된다. 또한 용리(elution) 과정에서 흡착된 DNA/RNA의 탈착 효율에 기인한 전체 DNA/RNA의 회수율 저하 때문에 DNA/RNA의 일부 손실을 감수할 수 밖에 없다. The volume of the sample containing the eluted DNA / RNA is in the amount of 100 to 200 μl, of which the sensitivity is lowered because only a small amount of 2 to 10 μl is consumed for the PCR analysis. The more the PCR efficiency is reduced. In addition, due to the decrease in the recovery rate of the total DNA / RNA due to the desorption efficiency of the adsorbed DNA / RNA in the elution process, there is no choice but to lose some of the DNA / RNA.
또한, 추출된 핵산을 이용하여 PCR과 같은 핵산 증폭 공정을 수행할 경우, 특히 복수의 샘플을 사용할 때, 샘플의 잦은 이동으로 인해 교차오염 (cross-contamination)의 의한 위양성 결과를 빈번히 초래한다. In addition, when performing a nucleic acid amplification process such as PCR using the extracted nucleic acid, especially when using a plurality of samples, frequent movement of the sample often results in false positive results due to cross-contamination.
이러한 문제점들을 극복하기 위해서 실험자의 핸들링 에러를 방지하기 위한 자동화 기기가 제안되었다. 하지만, 현재까지 개발된 자동화기기는 주로 핵산추출 기능에 한정적이며, 핵산 추출 공정과 핵산 증폭 공정을 통합하였다 할지라도, 복수의 핵산 추출 용액의 이동과 복잡한 기계적 움직임이 요구되어 기기의 단가가 높아질 뿐만 아니라 용출된 핵산을 PCR 튜브로 옮겨야 하는 샘플의 이동 및 이로 인한 교차오염문제는 아직 극복하지 못하고 중대한 과제로 남아있다. In order to overcome these problems, an automated device has been proposed to prevent the handling error of the experimenter. However, the automated devices developed to date are mainly limited to the nucleic acid extraction function, and even though the nucleic acid extraction process and the nucleic acid amplification process are integrated, the movement of a plurality of nucleic acid extraction solutions and complex mechanical movements are required, resulting in a high unit cost. Rather, the problem of cross-contamination and the migration of samples that require the transfer of eluted nucleic acids to PCR tubes remains a significant challenge.
기존의 PCR 방법에서는, 샘플에 존재하는 DNA/RNA 중의 일부만을 이용하는 것에 기인한 저민감도 및 저효율의 문제점 및 샘플 이동으로 인한 오염의 문제점을 해결하기 위한 개선된 PCR 방법에 대한 필요성이 계속 있어 왔다.In conventional PCR methods, there has been a continuing need for improved PCR methods to address the problems of low sensitivity and low efficiency due to the use of only a portion of the DNA / RNA present in the sample and the contamination caused by sample migration.
자성입자와 같은 고체 지지체를 사용하여 핵산을 추출 및 증폭하는 PCR 방법에 있어서, 샘플에 존재하는 DNA/RNA 중의 일부만을 이용하는 것에 기인하는 민감도 및 저효율성의 문제점 및 샘플 이동으로 인한 오염의 문제점을 해결하기 위한 개선된 PCR 방법을 개발하고자 한다.PCR method for extracting and amplifying nucleic acids using a solid support such as magnetic particles, solves the problems of sensitivity and low efficiency due to using only a part of the DNA / RNA present in the sample and contamination due to sample migration An improved PCR method is proposed to
본 발명자들은 고체 지지체로서 자성입자를 사용하여 DNA 변성 단계, 프라이머 결합 단계 및 DNA 사슬 연장 단계를 포함하는 PCR 과정을 포함하는 핵산의 추출 및 증폭 방법에 있어서, In the inventors of the present invention, a method for extracting and amplifying a nucleic acid using a magnetic particle as a solid support includes a PCR process including a DNA denaturation step, a primer binding step, and a DNA chain extension step.
(1) 자성입자의 존재 하에 DNA를 변성시킴으로써, 자성입자에 변성된 DNA/RNA를 부착시키고, (1) attaching the modified DNA / RNA to the magnetic particles by denaturing the DNA in the presence of the magnetic particles,
(2) 상기 변성된 DNA/RNA가 부착된 자성입자 (이후, "변성 DNA/RNA 및 자성입자의 부착물" 또는 단순히 "변성DNA-자성입자 부착물"로도 칭함)을 세척할 때, 통상의 세척용액이 아니라 단백질을 함유하는 세척용액으로 세척하고, (2) a common washing solution when washing the denatured magnetic particles to which the denatured DNA / RNA is attached (hereinafter also referred to as “denatured DNA / RNA and magnetic particle attachments” or simply “denatured DNA-magnetic particle attachments”) Rinse with a washing solution containing protein,
(3) 상기 세척 후에, 변성된 DNA/RNA를 자성입자로부터 용리시키지 않고, 결과된 변성DNA-자성입자 부착물을 그 자체로 사용하여 프라이머 결합 단계 및 DNA 사슬 연장 단계를 수행함으로써, (3) after the washing, without eluting the denatured DNA / RNA from the magnetic particles, by performing the primer binding step and the DNA chain extension step using the resulting modified DNA-magnetic particle attachments per se,
첫째, 시료 내의 DNA의 손실을 방지할 수 있기 때문에, DNA 함량이 낮은 시료에 대해서도 고민감도 및 고효율성으로 PCR을 수행할 수 있으며, First, since the loss of DNA in the sample can be prevented, PCR can be performed with high sensitivity and high efficiency even for a sample having a low DNA content.
둘째, 고체 지지체의 표면을 단백질로써 비특이적으로 블로킹함으로써, 고체 지지체의 존재 하에서도 높은 민감도 및 효율로써 DNA의 PCR 과정을 수행할 수 있으며, Second, by non-specifically blocking the surface of the solid support with a protein, the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of the solid support,
셋째, 동일 용기 내에서 DNA의 변성단계 및 PCR 단계를 수행할 수 있기 때문에 DNA의 오염이 방지될 수 있음을 발견하고 본 발명을 완성하였다.Third, the present inventors have found that contamination of DNA can be prevented because the DNA denaturation step and the PCR step can be performed in the same container, thereby completing the present invention.
본 발명에 따르면, DNA 함량이 낮은 시료에 대해서도 높은 민감도 및 효율성으로 DNA의 PCR을 수행할 수 있고, 고체 지지체의 존재 하에서도 높은 민감도 및 효율로써 DNA의 PCR 과정을 수행할 수 있으며, 그리고 DNA의 변성 및 PCR 과정을 하나의 용기에서 수행할 수 있기 때문에 DNA의 오염을 방지할 수 있다.According to the present invention, the PCR of DNA can be performed with high sensitivity and efficiency even for samples with low DNA content, the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of a solid support, and Denaturation and PCR can be performed in one container to prevent contamination of DNA.
도 1은 본 발명의 구체예에 따른 핵산 추출과 증폭 공정의 통합을 개략적으로 도시하는 도면이고;1 is a diagram schematically illustrating the integration of a nucleic acid extraction and amplification process according to an embodiment of the present invention;
도 2 본 발명의 실시예에 따른 핵산 추출과 증폭 공정의 통합으로 인하여 자성입자를 포하는 PCR 반응 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면이고;2 is a diagram confirming the results of PCR reactions containing magnetic particles using BMT 9G DNA KIT due to integration of nucleic acid extraction and amplification process according to an embodiment of the present invention;
도 3은 본 발명의 실시예에 따라 자성입자 사용량에 따른 PCR 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면이고; 3 is a view showing the PCR results according to the amount of magnetic particles using the BMT 9G DNA KIT according to an embodiment of the present invention;
도 4는 첨가되는 스킴밀크의 농도를 변화시키면서 얻어진 PCR 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면과, 전기 영동 패턴을 나타내는 도면이고;4 is a diagram showing a PCR result obtained by changing the concentration of the added skim milk using BMT 9G DNA KIT, and a diagram showing an electrophoresis pattern;
도 5는 스킴밀크의 농도를 0.05%로 고정하고 PCR 반응에 삽입되는 자성입자의 개수를 변화시키면서 얻어진 PCR 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면이고;5 is a diagram confirming the PCR results obtained by fixing the concentration of the scheme milk to 0.05% and changing the number of magnetic particles inserted into the PCR reaction using BMT 9G DNA KIT;
도 6는 0.05%와 0.1%의 스킴밀크를 첨가할 경우에, PCR 반응에 삽입되는 자성입자의 사용량을 1mg/20㎕ 및 2.5mg/20㎕으로 변화시키면서 얻어진 PCR 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면이고;Figure 6 shows the PCR results obtained by changing the amount of magnetic particles inserted into the PCR reaction to 1mg / 20μl and 2.5mg / 20μl when adding 0.05% and 0.1% of the skim milk using BMT 9G DNA KIT To confirm by drawing;
도 7는 스킴밀크의 농도를 0.1%와 자성입자의 사용량을 2.5mg/20㎕으로 고정하고 여러 샘플에 대하여 얻어진 PCR 결과를 BMT 9G DNA KIT를 이용하여 확인하는 도면이다.FIG. 7 is a diagram illustrating the use of BMT 9G DNA KIT to confirm the PCR results obtained for various samples by fixing the concentration of skim milk to 0.1% and the amount of magnetic particles to 2.5 mg / 20 μl.
각 도면에서, 즉 각각의 색상은 형광 강도(intensity)를 나타내는데, 구체적인 형광강도는 백색 > 적색 > 황색 >> 청색 = 65000 > 50000 > 40000 > 30000 >> 10000의 순서 및 세기를 갖는다. 동일한 녹색(green)일 경우에도 밝은 녹색은 35000, 어두운 녹색은 20000 정도의 세기를 가지므로, 밝은 색이 더 높은 강도를 나타낸다.In each figure, ie each color represents a fluorescence intensity, the specific fluorescence intensity having the order and intensity of white> red> yellow >> blue = 65000> 50000> 40000> 30000 >> 10000. Even in the same green color, bright green has 35000 intensity and dark green has about 20000 intensity, so the bright color shows higher intensity.
본 발명의 첫 번째 목적은, DNA 변성 단계, 프라이머 결합 단계 및 DNA 사슬 연장 단계를 포함하는 DNA의 PCR 과정을 1회 이상 포함하는 핵산의 추출 및 증폭 방법에 있어서, 하기 단계를 포함하는 것을 특징으로 하는 핵산의 추출 및 증폭 방법이 제공된다:A first object of the present invention is a method for extracting and amplifying a nucleic acid comprising at least one PCR process including a DNA denaturation step, a primer binding step, and a DNA chain extension step, the method comprising the following steps: Methods of extracting and amplifying nucleic acids are provided:
(1) 자성입자와 같은 고체 지지체의 존재 하에 DNA를 변성시켜, 자성입자의 표면에 변성된 DNA/RNA를 부착시키고, (1) denaturing DNA in the presence of a solid support such as magnetic particles to attach the modified DNA / RNA to the surface of the magnetic particles,
(2) 상기 단계 (1)에서 결과된 변성DNA-자성입자 부착물을 단백질-함유 세척용액으로 세척하고, (2) washing the denatured DNA-magnetic particle attachment resulting from step (1) with a protein-containing washing solution,
(3) 상기 단계 (2)에서 결과된 변성DNA-자성입자 부착물을 그 자체로 사용하여 프라이머 결합 단계 및 DNA 사슬 연장 단계를 수행함. (3) performing the primer binding step and the DNA chain extension step using the modified DNA-magnetic particle attachments resulting from step (2) by themselves.
본 발명의 하나의 구현예에 따르면, 상기 단계 (2)는 세부적으로 다음과 같이 수행될 수 있다: According to one embodiment of the invention, step (2) may be carried out in detail as follows:
(2-1) 변성DNA-자성입자 부착물을 함유하는 반응용기에 단백질-함유 세척용액을 첨가하고, (2-1) A protein-containing washing solution is added to the reaction vessel containing the denatured DNA-magnetic particle deposit,
(2-2) 전술한 반응용기를 교반 또는 진탕하여 전술한 변성DNA-자성입자 부착물을 전술한 단백질-함유 세척용액으로 세척하고, (2-2) the above-mentioned modified DNA-magnetic particle attachment is washed with the above-described protein-containing washing solution by stirring or shaking the above-mentioned reaction vessel,
(2-3) 상기 (2-2)에서 결과된 혼합물 중의 자성입자를 자석으로 고정하고, (2-3) The magnetic particles in the mixture obtained in the above (2-2) are fixed with a magnet,
(2-4) 상기 (2-3)에서 결과된 혼합물에서 세척 용액을 분리 및 제거하고, (2-4) separating and removing the washing solution from the mixture resulting from (2-3) above,
(2-5) 경우에 따라서는, 상기 단계들, 예를들면 (2-1) ~ (2-4) 단계를 1회 또는 그 이상으로 반복함. (2-5) In some cases, the above steps, for example, steps (2-1) to (2-4) are repeated one or more times.
본 발명의 하나의 구현예에 따르면, 전술한 고체 지지체, 구체적으로 자성입자는, 표면에 실록산기, 아미노기 및 히드록실기로 구성된 군에서 선택될 수 있는 하나 이상의 관능기를 가질 수 있거나, 실리카, 카보히드레이트, 아민, 폴리스티렌, 폴리프로필렌 등으로 코팅될 수 있다. 전술한 자성입자의 크기(직경)는 0.1 ~ 30 ㎛, 구체적으로는 0.2 ~ 25 ㎛, 바람직하게는 0.4 ~ 20 ㎛일 수 있다. 전술한 자성입자의 사용량 또는 함량은 0.10 ~ 10 mg/20 ㎕, 구체적으로는 0.25 ~ 5 mg/20 ㎕, 바람직하게는 0.5 ~ 2.5 mg/20 ㎕의 범위에서 선택될 수 있다. 상술한 자성입자의 사용량 또는 함량은 PCR 과정을 포함하여 DNA의 증폭과정을 효율적으로 수행하기 위해 선택되는 수치이며, 경우에 따라서는, 전술한 자성입자를 2.5 ~ 30 mg/20㎕, 구체적으로는 5~20 mg/20㎕의 고함량으로 사용하는 것도 본 발명의 범주를 벗어나지 아니한다. According to one embodiment of the invention, the above-mentioned solid support, specifically the magnetic particles, may have one or more functional groups selected from the group consisting of siloxane groups, amino groups and hydroxyl groups on the surface, or silica, carbo It can be coated with hydrates, amines, polystyrenes, polypropylene and the like. The size (diameter) of the magnetic particles described above may be 0.1 to 30 μm, specifically 0.2 to 25 μm, preferably 0.4 to 20 μm. The amount or content of the above-described magnetic particles may be selected in the range of 0.10 to 10 mg / 20 μl, specifically 0.25 to 5 mg / 20 μl, preferably 0.5 to 2.5 mg / 20 μl. The amount or content of the magnetic particles described above is a value selected to efficiently perform the amplification process of DNA, including a PCR process. In some cases, the magnetic particles described above may be 2.5 to 30 mg / 20 μl, specifically, Use in high amounts of 5-20 mg / 20 μl is also within the scope of the present invention.
본 발명의 하나의 구현예에 따르면, 전술한 단백질-함유 세척용액에서 단백질은 BSA, 스킴밀크 (skim milk) 및 카제인(casein) 으로 구성된 군에서 선택될 수 있으며, 그 함량(농도)은 특별히 제한되지 않지만, 공정의 용이성을 위해 0.01~0.1%(w/v), 구체적으로는 0.02~0.1%(w/v), 바람직하게는 0.03~0.08%(w/v)의 h농도를 가질 수 있다. According to one embodiment of the invention, the protein in the protein-containing washing solution described above may be selected from the group consisting of BSA, skim milk and casein, the content (concentration) is particularly limited However, it may have a h concentration of 0.01 to 0.1% (w / v), specifically 0.02 to 0.1% (w / v), preferably 0.03 to 0.08% (w / v), for ease of processing. .
본 발명의 하나의 구현예에 따르면, DNA 사슬 연장 단계는 PCR, RT-PCR, TMA, NASAB, SDA 및 RCA 용액으로 구성된 군에서 선택되는 DNA 증폭용액을 사용하여 수행될 수 있다. According to one embodiment of the present invention, the DNA chain extension step may be performed using a DNA amplification solution selected from the group consisting of PCR, RT-PCR, TMA, NASAB, SDA and RCA solutions.
본 발명의 하나의 구현예에 따르면, 상기 핵산 추출 및 증폭 반응에 의해 수득된 결과물을 겔 전기영동 (gel electrophoresis), ELGA (enzyme-linked gel assay), ECL (electrochemiluminescent) 또는 BMT 9G DNA KIT 및 BMT Genotyping 9G Membrane KIT로 구성된 군에서 선택되는 방법에 의해 검출하는 단계를 더 포함할 수 있다. According to one embodiment of the present invention, the product obtained by the nucleic acid extraction and amplification reaction is subjected to gel electrophoresis, gel-electrophoresis (ELGA), electrochemiluminescent (ECL) or BMT 9G DNA KIT and BMT The method may further include detecting by a method selected from the group consisting of Genotyping 9G Membrane KIT.
이하에, 본 발명은 첨부된 도면을 참고로 하여 더욱 상세히 설명된다. 하기의 설명은 본 발명의 바람직한 구체예를 기술하는 것으로 이해되어야 하며, 본 발명이 반드시 이에 한정되는 것은 아님을 명시한다. Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. The following description is to be understood as describing preferred embodiments of the invention, noting that the invention is not necessarily limited thereto.
본 발명의 명세서에 있어서, 핵산은 DNA, RNA, 이들의 단편 또는 올리고머를 의미할 수 있으며, 본 발명의 방법은 이들 모두에 대해 적용될 수 있으며, 따라서"DNA 사슬연장 단계", "DNA 증폭용액", "DNA-자성입자 부착물(결합체)" 등의 용어들은 DNA를 RNA로 대체하여 사용될 수 있다. 더나가서, 본 발명의 방법을 RNA에 적용하는 경우에는, 이중나선인 DNA을 단일나선으로 변형하는 단계, 예를들면 DNA변성(denature) 과정을 생략할 수도 있다. In the context of the present invention, nucleic acid may mean DNA, RNA, fragments or oligomers thereof, and the method of the present invention may be applied to all of them, thus, "DNA chain extension step", "DNA amplification solution" The terms "DNA-magnetic particle attachment (conjugate)" and the like may be used to replace DNA with RNA. Furthermore, when the method of the present invention is applied to RNA, the step of transforming double helix DNA into single helix, for example DNA denature process, may be omitted.
도 1은 본 발명의 구체예에 따른 핵산 추출과 증폭 공정의 통합을 개략적으로 도시하는 도면이다. 1 is a diagram schematically illustrating the integration of a nucleic acid extraction and amplification process according to an embodiment of the present invention.
상기 도면에 따르면, 자성입자가 포함된 하나의 챔버에서 노즐에 의해 샘플과 핵산 추출 용액 및 세척 용액이 첨가되거나 용액이 제거됨으로써 핵산을 흡착한 자성입자는 챔버 외부로 이동하지 않는 형태를 취하고 있다. 또한 상기 노즐을 통하여 PCR 용액이 핵산을 흡착한 자성입자로 첨가되면서 용출(elution) 과정이 생략되고, 샘플로부터 얻어진 전량의 핵산을 PCR에 이용되면서 민감도와 정확도를 증가시키는 방법을 제시한다.According to the drawing, in one chamber containing magnetic particles, the magnetic particles adsorbing nucleic acids are not moved out of the chamber by adding or removing the sample, the nucleic acid extraction solution and the washing solution by the nozzle. In addition, the elution process is omitted as the PCR solution is added to the magnetic particles adsorbing the nucleic acid through the nozzle, and a method of increasing the sensitivity and accuracy while using the entire amount of the nucleic acid obtained from the sample for PCR is provided.
핵산 추출 용액과 자성입자가 첨가된 챔버에 핵산 함유 샘플을 첨가한 후 핵산 추출과정을 수행한다. 핵산 추출 용액에 의해 박테리아나, 바이러스, 동물세포로부터 분리된 핵산은 자성입자에 흡착된다. 챔버 외부에 위치한 마그네틱 바에 의해 핵산이 흡착된 자성입자는 챔버 내부에 고정되고, 흡인(suction)에 의해 자성입자를 제외한 용액이 모두 제거된다. 자성입자에 흡착된 핵산을 정제하기 위해 세척 용액이 노즐을 통해 챔버에 주입되고, 세척 용액에는 소정의 단백질이 첨가된 것을 특징으로 한다. 세척 공정 후 상기 추출용액 제거 방식과 마찬가지로 용액이 제거되고, 챔버에는 정제된 핵산이 흡착한 자성입자만이 남아있다. 이 후, PCR 용액이 챔버에 첨가되고, 챔버 하단에 위치한 가열기에 의해 PCR 반응이 수행된다.The nucleic acid extraction process is performed after adding the nucleic acid-containing sample to the chamber to which the nucleic acid extraction solution and the magnetic particles are added. Nucleic acid separated from bacteria, viruses, or animal cells by the nucleic acid extraction solution is adsorbed to the magnetic particles. The magnetic particles adsorbed by the nucleic acid by the magnetic bar located outside the chamber are fixed inside the chamber, and all the solutions except the magnetic particles are removed by suction. In order to purify the nucleic acid adsorbed on the magnetic particles, the washing solution is injected into the chamber through a nozzle, and the washing solution is characterized in that a predetermined protein is added. After the washing process, the solution is removed in the same manner as the extraction solution removal method, and only the magnetic particles adsorbed by the purified nucleic acid remain in the chamber. Thereafter, the PCR solution is added to the chamber, and the PCR reaction is performed by a heater located at the bottom of the chamber.
상기 명시된 발명은 핵산 추출과 증폭의 일련의 과정을 통합함으로써, 오염을 줄이고, 민감도와 정확도를 향상시킬 수 있는 공정 방법을 제시한다.The above-described invention proposes a process method that can reduce contamination, improve sensitivity and accuracy by integrating a series of processes of nucleic acid extraction and amplification.
1. 핵산 추출과 증폭의 통합 공정1. Integrated process of nucleic acid extraction and amplification
본 발명에 있어서, 핵산 추출과 증폭의 통합 공정에 의한 원리는 다음과 같이 설명될 수 있다: In the present invention, the principle by the integrated process of nucleic acid extraction and amplification can be explained as follows:
현재 사용되고 있는 핵산 추출 키트는 핵산의 흡착과 분리, 정제를 위해 대부분 컬럼이나, 고체 지지체로서 자성입자를 사용하고 있다. 컬럼과 자성입자의 사용은 고체상의 기질에 핵산을 흡착하여 다량의 샘플로부터의 핵산 분리가 가능하기 때문에 추출과 정제가 용이하다는 장점이 있다. 그러나, 이러한 고체 기반 핵산 추출 방식은 후속공정, 예를 들면 PCR 을 이용해 분석, 측정하기 위해서는 다시 액체상으로 전환되어야 할 필요성이 있기 때문에 다시 용액상으로 용출하는 과정이 반드시 요구된다.Nucleic acid extraction kits currently used are mostly used magnetic particles as a column or a solid support for adsorption, separation and purification of nucleic acids. The use of the column and the magnetic particles has the advantage of easy extraction and purification since the nucleic acid can be separated from a large amount of sample by adsorbing the nucleic acid on a solid substrate. However, this solid-based nucleic acid extraction method is required to be converted back to the liquid phase because it needs to be converted back to the liquid phase in order to be analyzed and measured using a subsequent process, for example, PCR.
용출(elution) 과정은 흡착된 핵산을 다시 탈착할 때, 탈착 효율에 따라서 정제된 핵산이 손실되고 있다. 또한 용출에 필요한 최소 용액량이 약 100㎕ 이상으로 반드시 요구되고 있음에도 불구하고 후속공정, 예를 들면 PCR의 경우 최소 2㎕에서 최대 10㎕ 만을 사용하기 때문에 샘플로부터 추출한 대부분의 핵산이 낭비되고 있다.In the elution process, when the adsorbed nucleic acid is desorbed again, the purified nucleic acid is lost according to the desorption efficiency. In addition, although the minimum amount of solution required for elution is required to be about 100 μl or more, most nucleic acids extracted from the sample are wasted because subsequent steps such as PCR use only 2 μl to 10 μl.
따라서 본 발명은 자성입자를 이용한 핵산 추출공정에서 라이시스(lysis), 핵산의 흡착, 세척공정을 마치고 용출 단계를 생략하고 곧바로 PCR 용액을 자성입자에 첨가함으로써 간단하고 신속하게 측정이 가능한 통합 공정을 개발하였다.Therefore, the present invention is an integrated process that can be easily and quickly measured by adding a PCR solution directly to the magnetic particles after eliminating the elution step after the lysis, nucleic acid adsorption, washing process in the nucleic acid extraction process using magnetic particles Developed.
샘플로부터 얻어진 자성입자에 흡착된 핵산 전량은 곧바로 PCR 용액이 첨가되어 PCR 반응이 이루어진다. 이때, 처음 단계인 변성(denature) 단계에서 94℃의 고온에서 진행되기 때문에 효과적으로 핵산이 수용액상으로 탈착이 되며, 이것은 곧바로 PCR 반응에 사용된다. 이것은 샘플 내의 전량의 핵산을 사용하기 때문에 높은 민감도와 정확성을 가지며, 핵산이 흡착된 자성입자는 하나의 챔버에서 이동하지 않기 때문에 샘플의 이동에 따른 오염에 의한 위양성 결과를 도출하지 않는다.The entire amount of nucleic acid adsorbed on the magnetic particles obtained from the sample is immediately added with a PCR solution to perform a PCR reaction. At this time, since it proceeds at a high temperature of 94 ° C. in the denature step, which is the first step, the nucleic acid is effectively desorbed into the aqueous solution phase, which is immediately used for the PCR reaction. It has high sensitivity and accuracy because the entire amount of nucleic acid in the sample is used, and since the magnetic particles adsorbed with the nucleic acid do not move in one chamber, they do not lead to false positive results due to contamination due to the movement of the sample.
2. 핵산의 증폭을 위한 최적화 공정2. Optimization Process for Amplification of Nucleic Acids
본 발명에서는 다량의 자성입자에 대한 PCR 반응 저해 문제를 해결하기 위해 핵산 추출 공정의 마지막 세척 공정에서 소정의 단백질을 첨가하는 방법을 제시한다. 마지막 세척 공정에 첨가되는 단백질은 핵산이 흡착된 부분 이외의 자성입자의 표면에 비특이적으로 흡착하여, PCR 반응에 저해되는 요소를 원천적으로 차단하여 보다 효율적인 PCR 반응을 도출할 수 있다. The present invention provides a method for adding a predetermined protein in the last washing step of the nucleic acid extraction process to solve the problem of inhibiting the PCR reaction for a large amount of magnetic particles. The protein added to the final washing process may be non-specifically adsorbed on the surface of the magnetic particles other than the portion where the nucleic acid is adsorbed, thereby blocking the element that inhibits the PCR reaction at the source, leading to a more efficient PCR reaction.
전술한 단백질로는 BSA (소의 세럼 단백질), 스킴밀크 (skim milk) 및 카제인(casein)으로 구성된 군에서 선택될 수 있으며, 0.005 내지 0.2%(w/v), 구체적으로는 0.01% 내지 0.1%(w/v), 바람직하게는 0.02 내지 0.08%(w/v) 의 양으로 함유될 수 있다. 또다르게는, 전술한 단백질로서 젤라틴 또는 시판되는 비동물성 단백질 블로킹제를 사용하는 것도 본 발명의 범주에 포함될 수 있다. The above-mentioned protein may be selected from the group consisting of BSA (bovine serum protein), skim milk and casein, and 0.005 to 0.2% (w / v), specifically 0.01% to 0.1% (w / v), preferably in an amount of 0.02 to 0.08% (w / v). Alternatively, the use of gelatin or commercially available non-animal protein blocking agents as the aforementioned proteins may also be included within the scope of the present invention.
전술한 BSA, 스킴밀크 및 카제인과 같은 단백질들은 특정 단백질의 유무 또는 양을 분석하는 웨스턴 블롯팅 (Western Blotting)에서 블로킹 버퍼 (blocking buffer) 또는 블로킹제 (blocking agent)로 사용되는 단백질로서, 블로팅(blotting)되지 않은 멤브레인 표면에 단백질을 코팅하여 1st Ab나 2nd Ab가 비특이적 결합을 하지 못하도록 블로킹하기 위해 사용되고 있다. 그러나 DNA를 검출하기 위한 사우던 블로팅 (Southern Blotting)에서는 이러한 블로킹제는 사용되지 않으며, 따라서 고체 지지체를 사용한 PCR에서 이러한 블로킹제의 사용 및 이의 효과에 대해서는 지금까지 알려져 있지 않았다. 다만 고형지지체 (예. micro fluidic chip)을 이용하여 PCR을 수행할 때, 칩고체 표면을 BSA로 코팅을 하거나 PCR 용액에 BSA를 첨가하는 경우가 있지만, 본 발명에서와 같은 블로킹제로서 사용되는 것은 아니라는 차이가 있다. Proteins such as BSA, skim milk and casein described above are proteins used as blocking buffers or blocking agents in Western blotting to analyze the presence or absence of specific proteins. It is used to block the non-specific binding of 1st Ab or 2nd Ab by coating the protein on the membrane surface that is not blotting. However, such blocking agents are not used in Southern Blotting to detect DNA, and thus the use of these blocking agents and their effects in PCR using a solid support are not known until now. However, when PCR is performed using a solid support (eg, micro fluidic chip), the surface of the chip solid may be coated with BSA or BSA may be added to the PCR solution, but it is used as a blocking agent as in the present invention. No difference.
본 발명자들은 고체 지지체의 존재 하에 프라이머 결합 단계 및 DNA 사슬 연장 단계와 같은 DNA 증폭 반응을 수행하는 경우, DNA 증폭 반응이 고체 지지체에 의한 교란 또는 저해되지만, 고체 지지체의 표면을 전술한 단백질로써 블로킹하면 고체 지지체에 의한 DNA 증폭 반응의 교란 또는 저해를 억제할 수 있음을 확인하였다.When the inventors perform DNA amplification reactions such as primer binding step and DNA chain extension step in the presence of a solid support, the DNA amplification reaction is disturbed or inhibited by the solid support, but blocking the surface of the solid support with the aforementioned protein It was confirmed that disturbance or inhibition of the DNA amplification reaction by the solid support can be suppressed.
한편, 본 발명의 하나의 변법에 따르면, 고체 지지체를 원심분리에 의해 세척용액 과 분리하고, 세척용액은 경사분리 또는 피펫팅에 의해 제거하는 방식으로 본 발명의 방법을 수행할 수 있다. Meanwhile, according to one variation of the present invention, the solid support may be separated from the washing solution by centrifugation, and the washing solution may be removed by decantation or pipetting.
본 발명의 더 이상의 변법에 따르면, 고체 지지체를 원심분리에 의해 세척용액과 분리할 수 있는 경우에는, 상술한 바처럼 자성입자가 아니어도 본 발명의 방법을 수행할 수 있다. 이럴 경우, 상기 단계 (2)는 세부적으로 다음과 같이 수행될 수 있다: According to a further variant of the present invention, when the solid support can be separated from the washing solution by centrifugation, the method of the present invention can be carried out without magnetic particles as described above. In this case, step (2) may be performed in detail as follows:
(2-1) 변성DNA-자성입자 부착물을 함유하는 반응용기에 단백질-함유 세척용액을 첨가하고, (2-1) A protein-containing washing solution is added to the reaction vessel containing the denatured DNA-magnetic particle deposit,
(2-2) 전술한 반응용기를 교반 또는 진탕하여 전술한 변성DNA-자성입자 부착물을 전술한 단백질-함유 세척용액으로 세척하고, (2-2) the above-mentioned modified DNA-magnetic particle attachment is washed with the above-described protein-containing washing solution by stirring or shaking the above-mentioned reaction vessel,
(2-3) 상기 (2-2)에서 결과된 혼합물을 원심분리하고, (2-3) centrifugation of the mixture obtained in (2-2) above;
(2-4) 상기 (2-3)에서 결과된 혼합물에서 세척 용액을 경사분리 및/또는 피펫팅에 의해 분리 및 제거하고, (2-4) separating and removing the washing solution from the mixture resulting from (2-3) by decantation and / or pipetting,
(2-5) 경우에 따라서는 상기 (2-1) ~ (2-4) 단계를 반복함. (2-5) If necessary, repeat steps (2-1) to (2-4).
자성입자를 사용한 PCR 방법은 많은 문헌에서 찾아볼 수 있으며, 본 발명에서 구체적으로 기술되지 않은 사항들은 전술한 문헌에서 기술된 것들을 사용할 수 있다. PCR methods using magnetic particles can be found in many documents, and matters not specifically described in the present invention may use those described in the above documents.
본 발명의 방법에 따르면, 변성DNA가 부착된 고체 지지체를 특정 단백질을 함유하는 용액으로 세척할 뿐 아니라 변성된 DNA를 고체 지지체에서 용출시키지 않고 그 자체로 사용하여 프라이머 결합 단계 및 DNA 사슬 연장 단계와 같은 후속 PCR 과정을 수행함으로써, 아래와 같은 이점을 얻을 수 있다:According to the method of the present invention, the denatured DNA attached solid support is not only washed with a solution containing a specific protein, but also the denatured DNA is used on its own without eluting from the solid support, thereby providing a primer binding step and a DNA chain extension step. By performing the same subsequent PCR process, the following benefits can be achieved:
첫째, 변성된 DNA/RNA를 고체 지지체와 분리하는 용출단계가 없어 DNA의 손실을 방지할 수 있기 때문에, DNA 함량이 낮은 시료에 대해서도 고민감도 및 고효율성으로 PCR을 수행할 수 있으며, First, since there is no elution step of separating the denatured DNA / RNA from the solid support, it is possible to prevent DNA loss, so that PCR can be performed with high sensitivity and high efficiency even for samples having a low DNA content.
둘째, 단백질-함유 세척용액을 사용하여 고체 지지체의 표면을 단백질과의 비특이적 결합에 의해 블로킹함으로써, 고체 지지체의 존재 하에서도 높은 민감도 및 효율로써 DNA의 PCR 과정을 수행할 수 있으며, Second, by blocking the surface of the solid support by non-specific binding with a protein using a protein-containing washing solution, the PCR process of DNA can be performed with high sensitivity and efficiency even in the presence of the solid support.
셋째, 전술한 변성된 DNA/RNA 및 고체 지지체의 부착물은 반응 및 세척 단계 이후에도 반응 용기 내에 잔류시킬 수 있고 반응 용기의 이동 또는 변경없이 후속 PCR 반응을 진행시킬 수 있는데, 결과적으로 DNA가 동일한 용기에서 변성되고 PCR 반응을 수행하게 되므로, DNA의 오염이 방지될 수 있다. Third, the attachment of the denatured DNA / RNA and the solid support described above can remain in the reaction vessel even after the reaction and washing steps and can proceed with subsequent PCR reactions without moving or changing the reaction vessel, resulting in DNA in the same vessel. By denaturation and PCR reaction, contamination of DNA can be prevented.
이하, 본 발명의 이해를 돕기 위해 바람직한 실시예를 제시하지만, 하기의 실시예는 본 발명을 보다 쉽게 이해하기 위하여 제공되는 것일 뿐 본 발명이 이에 한정되는 것은 아니다. Hereinafter, preferred examples are provided to aid in understanding the present invention, but the following examples are provided only for better understanding of the present invention, and the present invention is not limited thereto.
참고예 1Reference Example 1
핵산 함유 샘플로 HPV (인유두종 바이러스)를 사용하였으며, 핵산 추출 키트로서 Nucleic Acid Extraction (Magnetic bead) kit [He Nan Huier Nano Science And Technology Co., LTD; 중국]를 사용하였다. HPV (Human Papilloma Virus) was used as the nucleic acid-containing sample, and Nucleic Acid Extraction (Magnetic bead) kit [He Nan Huier Nano Science And Technology Co., LTD; China].
매뉴얼에 따르면, PCR 과정은 (i) DNA 변성(denaturing), (ii) 변성된 DNA의 용출 (elution), (iii) 프라이머 결합(annealing), 및 (iv) 사슬 연장(extending)로 구성된다.According to the manual, the PCR process consists of (i) DNA denaturing, (ii) elution of denatured DNA, (iii) primer binding, and (iv) chain extension.
전술한 용출(elution)은 DNA 변성단계 미반응물의 세척과정, 분리물질의 혼합 및 분리를 위한 가열 과정을 포함한다. 변성된 DNA는 용출단계에서 자성입자와 분리된 다음, 프라이머 결합단계 및 DNA 연장단계를 거쳐 증폭된다. The above-mentioned elution includes washing of unreacted DNA denaturation step, heating process for mixing and separating the separation material. The denatured DNA is separated from the magnetic particles in the elution step, and then amplified through the primer binding step and the DNA extension step.
(1) 대조군(1) control
대조군(control)으로서 전술한 키트(kit)의 사용 메뉴얼에 명시되어 있는 핵산 추출 절차를 그대로 준수하여 PCR 과정을 4회 수행하였다. As a control, the PCR process was carried out four times in conformity with the nucleic acid extraction procedure specified in the above-mentioned kit manual.
이렇게 증폭된 핵산은 PCR 용액에 혼성화용액(hybridization solution)을 혼합하여 사용하기 때문에 별도로 핵산의 분리, 정제가 필요하지 않으며, 그 자체로 BMT HPV 9G DNA KIT [바이오메트릭스 테크놀로지; 대한민국]를 이용하여 형광 세기를 비교 분석하였다. 형광세기는 강한 순서대로 흰색-빨강-노랑-초록-파랑의 순으로 표현된다. Since the amplified nucleic acid is used by mixing a hybridization solution with a PCR solution, it is not necessary to separate and purify the nucleic acid separately, and BMT HPV 9G DNA KIT [Biometrics Technology; Korea] to compare and analyze the fluorescence intensity. The fluorescence intensity is expressed in the order of white-red-yellow-green-blue in the order of the strongest.
도 2의 좌측 (without bead)의 사진 1~4 는 이렇게 증폭된 DNA에 대하여 측정된 형광 사진을 보여준다. Photographs 1-4 of the left (without bead) of FIG. 2 show fluorescence photographs measured for the DNA thus amplified.
(2) 실험군(2) experimental group
실험군으로서, 용출(elution) 단계에서 세척과정을 수행한 다음에 혼합과정 및 가열과정을 생략함으로써 변성된 DNA를 자성입자와 분리하지 않고 그대로 증폭단계를 수행하는 것을 제외하고는, 키트에 명시되어 있는 방법을 사용하여 세부 추출 방법을 그대로 사용하여, PCR 과정을 4회 수행하였다. As an experimental group, the cleaning process is performed in the elution step, and then the mixing and heating steps are omitted, except that the denatured DNA is separated from the magnetic particles and the amplification step is performed as it is. Using the method, the PCR procedure was performed four times using the detailed extraction method as it is.
추출된 DNA를 자성입자의 존재 하에 변성시키고, 결과로 수득된 변성된 DNA-자성입자 부착물을 세척하고, 여기에 소량의 Tris-HCl (pH 8.0) 버퍼를 첨가하여 부유시켰다. 이렇게 부유시킨 자성입자를 정량하여, PCR premix tube [바이오니아; 대한민국]에 삽입하여 PCR을 (1 회) 수행하여 핵산을 증폭하였다. The extracted DNA was denatured in the presence of magnetic particles, the resulting denatured DNA-magnetic particle attachment was washed and suspended by addition of a small amount of Tris-HCl (pH 8.0) buffer. The suspended magnetic particles were quantified and PCR premix tube [Bionia; Korea] to perform PCR (once) to amplify the nucleic acid.
이렇게 증폭된 핵산은 별도의 분리 또는 정제없이 BMT HPV 9G DNA KIT [바이오메트릭스 테크놀로지; 대한민국]를 이용하여 형광 세기를 비교 분석하였다. 형광세기는 강한 순서대로 흰색-빨강-노랑-초록-파랑의 순으로 표현된다. This amplified nucleic acid BMT HPV 9G DNA KIT [Biometrics Technology; Korea] to compare and analyze the fluorescence intensity. The fluorescence intensity is expressed in the order of white-red-yellow-green-blue in the order of the strongest.
도 2의 우측 (with bead)의 사진 1~4 는 이렇게 증폭된 DNA에 대하여 측정된 형광 사진을 보여준다. Photographs 1-4 of the right side (with bead) of FIG. 2 show fluorescence images measured on the DNA thus amplified.
결과 비교Compare results
도 2는 변성DNA-자성입자 부착물 상태에서의 PCR 결과에서 수득된 형광사진 (with bead) (실험군) 및 자성입자가 없이 변성DNA 만의 상태에서의 PCR 결과에서 수득된 형광사진 (without bead) (대조군)를 비교한 것이다. Fig. 2 shows fluorescence photographs (with bead) obtained from PCR results in the state of denatured DNA-magnetic particle attachment (with experimental group) and fluorescence photographs (without bead) obtained from PCR results in the state of denatured DNA only without magnetic particle (control group). ) Is a comparison.
각 4개의 샘플에 대하여, 1번 샘플에서는 대조군의 파랑 스팟이 실험군에서 발현되지 않았으며, 3번 샘플에서는 대조군의 흰색에서 실험군의 빨강으로 형광 차이가 가장 적게 감소하였지만, 2번과 4번 샘플에서는 대조군의 흰색에서 실험군의 초록색으로 형광이 급격히 감소했다. For each of the four samples, the blue spot of the control group was not expressed in the experimental group in sample 1, and the fluorescence difference was decreased the most from the white of the control group to the red of the experimental group in sample 3, but in samples 2 and 4, Fluorescence decreased rapidly from white in the control group to green in the experimental group.
결과적으로, 자성입자의 존재 하에 수행된 PCR (실험군)에서는 일반적으로 민감도가 감소하는 것으로 나타난다. As a result, in the PCR (experimental group) performed in the presence of magnetic particles, the sensitivity generally appears to decrease.
참고예 2a 및 2bReference Examples 2a and 2b
자성입자의 양을 0 mg (대조군), 0.25mg/20㎕, 0.5mg/20㎕, 1mg/20㎕, 1.5mg/20㎕ 및 2.5mg/20㎕으로 각각 변화시키는 것을 제외하고는, 참고예 1의 실험군에서와 동일하게 PCR 과정을 수행하였다. 대조군(사용량 0 mg)은 참고예 1의 대조군과 동일하게 PCR 과정이 수행된 것을 의미한다. Reference Examples except that the amount of magnetic particles was changed to 0 mg (control), 0.25 mg / 20 μl, 0.5 mg / 20 μl, 1 mg / 20 μl, 1.5 mg / 20 μl and 2.5 mg / 20 μl, respectively. PCR procedure was performed in the same manner as in the experimental group of 1. The control group (usage 0 mg) means that the PCR process was performed in the same manner as the control group of Reference Example 1.
도 3은 참고예 2a (샘플 1) 및 참고예 2b(샘플 2)의 PCR 결과를 보여주는 도면으로서, 자성입자의 사용량이 0.25mg/20㎕인 경우는 대조군에 비해 다소 낮지만 유사한 결과 (감도)를 보여주지만, 사용량이 1 mg/20㎕ 이상인 경우는 대조군에 비해 PCR 효율이 급격히 감소되는 것을 보여주며, 사용량이 2.5mg/20㎕인 경우에는 결과가 제대로 얻어지지 않는 것을 보여준다. 따라서, 자성입자는 0.25mg/20㎕ 또는 그 미만 사용시, 이하로 사용하는 경우에, 자성입자의 존재가 PCR의 효율의 저해요소로 작용하지 않는 것을 알 수 있다. 3 is a diagram showing PCR results of Reference Example 2a (Sample 1) and Reference Example 2b (Sample 2), when the amount of the magnetic particles used is 0.25mg / 20μL slightly lower than the control group, but similar results (sensitivity) However, when the amount of use is more than 1 mg / 20μl shows that the PCR efficiency is drastically reduced compared to the control, and when the amount of use is 2.5mg / 20μl shows that the results are not obtained properly. Therefore, when the magnetic particles are used below 0.25 mg / 20 μl or less, it can be seen that the presence of the magnetic particles does not act as an inhibitor of PCR efficiency.
실시예 1Example 1
자성입자를 0.5 mg의 양으로 사용하고 용출단계에서 특정 단백질 (예. skim milk)을 0%, 0.01%, 0.03%, 0.05% 또는 0.1% (w/v)의 농도로 포함하는 세척 용액을 사용하는 것을 제외하고는 참고예 1에서와 동일하게 PCR 과정을 수행하였다. 대조군으로서, 자성입자가 없는 상태에서 PCR을 수행하고 스킴밀크를 함유하지 않는 세척용액을 사용하여 수득된 결과물(참고예 2a의 대조군과 동일)을 사용하였다. Use a washing solution containing magnetic particles in an amount of 0.5 mg and containing a specific protein (eg skim milk) at a concentration of 0%, 0.01%, 0.03%, 0.05% or 0.1% (w / v) in the elution step. A PCR procedure was performed in the same manner as in Reference Example 1 except that the reaction was performed. As a control, PCR was performed in the absence of magnetic particles, and the result obtained using the washing solution containing no skim milk (the same as the control of Reference Example 2a) was used.
도 4a는 대조군의 형광세기가 흰색이지만 단백질을 사용하지 않은 경우에는 초록색으로 감도가 매우 감소되었지만, 스킴밀크 단백질을 0.01%, 0.03%, 0.05% 및 0.1%의 양으로 함유하는 세척 용액을 사용하면, 대조군과 유사하거나 동일한 감도를 수득할 수 있는 것을 보여준다. FIG. 4a shows that the control group has a fluorescence intensity of white but has a very low sensitivity to green when no protein is used, but using a wash solution containing a skim milk protein in an amount of 0.01%, 0.03%, 0.05% and 0.1%. Shows that similar or the same sensitivity as the control can be obtained.
도 4b는 도 4a의 PCR 결과를 전기영동을 통하여 확인한 결과, 0.05%의 skim milk 단백질을 사용할 때, 가장 뚜렷한 PCR 밴드가 확인되는 것을 보여준다. Figure 4b shows the results of the PCR of Figure 4a through electrophoresis, when using the skim milk protein of 0.05%, shows that the most distinct PCR band is confirmed.
실시예 2Example 2
자성입자를 0.5 mg/20㎕, 1 mg/20㎕, 1.5 mg/20㎕, 2.5 mg/20㎕ 및 5 mg/20㎕의 양으로 각각 사용하고 용출단계에서 특정 단백질 (예. skim milk)을 0.05% (w/v)의 농도로 포함하는 세척 용액을 사용하는 것을 제외하고는 실시예 1에서와 동일하게 PCR 과정을 수행하였다. 대조군으로서 자성입자가 없는 상태에서 PCR을 수행하고 스킴밀크를 함유하지 않는 세척용액을 사용하여 수득된 결과를 사용하였다. Magnetic particles are used in amounts of 0.5 mg / 20 μl, 1 mg / 20 μl, 1.5 mg / 20 μl, 2.5 mg / 20 μl and 5 mg / 20 μl respectively, and specific proteins (eg skim milk) The PCR procedure was performed in the same manner as in Example 1 except that the washing solution containing the concentration of 0.05% (w / v) was used. As a control, PCR was performed in the absence of magnetic particles and the results obtained using a washing solution containing no skim milk were used.
도 5는 0.05%의 스킴밀크를 함유하는 세척용액을 사용할 경우에, 자성입자를 1 mg/20㎕의 양으로 사용하면 대조군과 동일 또는 유사한 결과를 수득하였음을 보여준다. 이러한 결과는 스킴밀크를 함유하는 세척용액을 사용하면 자성입자를 0.25 mg/20㎕ 이하가 아닌 1 mg/20㎕ 까지의 양으로 사용할 수 있음을 나타내며, 이에 의해, 핵산 추출 효율이 증가될 뿐만 아니라 핵산 증폭 효율도 증가될 수 있어, 결과적으로 측정 민감도가 향상될 수 있다. Figure 5 shows that when using a washing solution containing 0.05% of the skim milk, using magnetic particles in an amount of 1 mg / 20ul obtained the same or similar results as the control. These results indicate that the use of the wash solution containing the skim milk can use the magnetic particles in an amount of up to 1 mg / 20 μl instead of 0.25 mg / 20 μl, thereby increasing the nucleic acid extraction efficiency as well. Nucleic acid amplification efficiency can also be increased, resulting in improved measurement sensitivity.
선행기술의 연구결과는, 자성입자의 존재하에 PCR을 수행하는 경우, 자성입자의 넓은 표면적으로 인해 PCR 효율이 저해되는 것으로 알려져 있다 (실시예 1 참조). PCR 과정에서 소량의 자성입자의 존재는 허용될 수 있지만 자성입자의 양이 적기 때문에 PCR의 감도가 낮으며, 다량의 자성입자의 존재는 PCR의 감도 뿐만 아니라 효율도 저하시킨다는 문제가 있다. 따라서, 변성된 DNA를 자성입자로부터 분리 및 용출을 수행한 다음, PCR을 수행하는 것이 필수적이었다. The results of the prior art have been known that when PCR is performed in the presence of magnetic particles, PCR efficiency is inhibited due to the large surface area of the magnetic particles (see Example 1). The presence of a small amount of magnetic particles may be acceptable in the PCR process, but the sensitivity of the PCR is low because the amount of the magnetic particles is small, and the presence of the large amount of magnetic particles lowers the sensitivity as well as the efficiency of the PCR. Therefore, it was essential to separate and elute the denatured DNA from the magnetic particles and then perform PCR.
본 발명에 따르면, BSA, 스킴밀크, 카제인 등의 단백질을 세척공정에 첨가하여 자성입자에 상기 단백질을 비특이적으로 결합시켜, 후속 공정인 PCR 효율에 저해요소를 차단할 수 있음을 보여준다. According to the present invention, it can be seen that by adding a protein such as BSA, skim milk, casein, etc. to the washing process, the protein is non-specifically bound to the magnetic particles, thereby blocking the inhibitory factor in the subsequent PCR efficiency.
실시예 3Example 3
자성입자를 1 mg/20㎕ 및 2.5 mg/20㎕의 양으로 각각 사용하고 용출단계에서 특정 단백질 (예. skim milk)을 0.05% 및 1% (w/v)의 농도로 각각 포함하는 세척 용액을 사용하는 것을 제외하고는 실시예 1에서와 동일하게 PCR 과정을 수행하였다. 대조군으로서 자성입자가 없는 상태에서 PCR을 수행하고 스킴밀크를 함유하지 않는 세척용액을 사용하여 수득된 결과 (실시예 2의 대조군과 동일)를 사용하였다. Washing solution containing magnetic particles in amounts of 1 mg / 20 μl and 2.5 mg / 20 μl respectively and containing specific proteins (eg skim milk) at concentrations of 0.05% and 1% (w / v), respectively, in the elution step. PCR procedure was performed in the same manner as in Example 1 except for using the following. As a control, PCR was performed in the absence of magnetic particles and the result obtained by using a washing solution containing no skim milk (same as the control of Example 2) was used.
도 6은 자성입자를 적은 양 (1 mg/20㎕)으로 사용하는 경우에는 낮은 농도 (0.05%)의 skim milk를 함유하는 세척용액을 사용하여 더욱 우수한 결과를 수득할 수 있지만, 반면, 자성입자를 많은 양 (2.5 mg/20㎕)으로 사용하는 경우에는 높은 농도 (0.1%)의 스킴밀크를 함유하는 세척용액을 사용하여 더욱 우수한 결과를 수득하였음을 보여준다. FIG. 6 shows better results using a washing solution containing skim milk at a low concentration (0.05%) when the magnetic particles are used in a small amount (1 mg / 20 µl). When using a large amount (2.5 mg / 20μl) shows that using a wash solution containing a high concentration (0.1%) of skim milk was obtained better results.
따라서, 높은 측정 효율을 얻기 위해 자성입자의 양을 증가시키기 위해서는 더많은 단백질을 함유하는 세척용액을 사용해야 함을 보여준다. 소량의 자성입자를 사용할 경우에는 자성입자의 표면에 비특이적으로 흡착되고 남은 단백질이 잔류해 있을 가능성이 높으며, 이러한 비결합 잔류 단백질이 PCR 용액으로 방출되어 PCR 수행에 저해제로 작용할 수 있다. 그러나, 다량의 자성입자를 사용할 경우에는 자성입자의 표면에 비특이적으로 흡착되고 남은 단백질이 매우 적기 때문에 이들이 PCR 용액으로 방출될 가능성이 낮다. 따라서 다량의 자성입자를 사용하여 핵산 추출과 증폭의 통합공정을 수행하기 위해서는 더 높은 농도의 단백질이 요구되며 또한 사용될 수 있다. Therefore, to increase the amount of magnetic particles in order to obtain high measurement efficiency, it is shown that a washing solution containing more protein should be used. When a small amount of magnetic particles are used, there is a high possibility that non-specifically adsorbed and remaining proteins remain on the surface of the magnetic particles, and these unbound residual proteins may be released into the PCR solution to act as an inhibitor in performing the PCR. However, when a large amount of magnetic particles are used, since there is very little non-specifically adsorbed protein remaining on the surface of the magnetic particles, it is unlikely that they are released into the PCR solution. Therefore, a higher concentration of protein is required and can be used to perform the integration process of nucleic acid extraction and amplification using a large amount of magnetic particles.
실시예 4Example 4
4개의 핵산 함유 샘플에 대하여, 자성입자를 2.5 mg/20㎕의 양으로 사용하고 용출단계에서 특정 단백질 (예. skim milk)을 1% (w/v)의 농도로 포함하는 세척 용액을 사용하는 것을 제외하고는 실시예 1에서와 동일하게 PCR 과정을 수행하였다. 대조군으로서, 각각의 핵산 함유 샘플에 대하여, 자성입자가 없는 상태에서 PCR을 수행하고 스킴밀크를 함유하지 않는 세척용액을 사용하여 수득된 결과들을 사용하였다. For four nucleic acid-containing samples, magnetic particles were used in an amount of 2.5 mg / 20 μl and a washing solution containing a specific protein (eg skim milk) at a concentration of 1% (w / v) was used in the elution step. A PCR procedure was performed in the same manner as in Example 1 except that. As a control, for each nucleic acid-containing sample, PCR was performed in the absence of magnetic particles and the results obtained using the wash solution containing no skim milk were used.
도 7은 샘플 1은 파랑에서 초록으로, 샘플 2는 초록에서 흰색으로 결과값이 증가하였고, 샘플 3 및 샘플 4는 각각 파랑과 흰색으로 결과값이 변하지 않은 것을 보여주고 있다. FIG. 7 shows that Sample 1 increased from blue to green, Sample 2 increased from green to white, and Sample 3 and Sample 4 did not change to blue and white, respectively.
본 발명의 결과는 기존의 일반적인 핵산 추출 및 증폭 공정을 수행하는 것보다 본 발명에 따라 자성입자 존재 하에 PCR을 수행하고 단백질-함유 세척용액을 사용할 때, 훨씬 향상된 민감도를 가지는 것을 보여준다. 또한 공정의 수를 감소시킴으로써 편의성과 신속성을 증가시키고, 샘플의 이동을 없앰으로써 오염에 의한 위양성 결과를 도출하지 않고, 샘플로부터 얻어진 핵산 전량을 사용함으로써 민감도와 정확도를 향상시킬 수 있는 핵산 추출/증폭의 통합 공정의 개발을 명시하는 바이다.The results of the present invention show that the PCR is carried out in the presence of magnetic particles and using protein-containing wash solution according to the present invention, rather than performing the conventional general nucleic acid extraction and amplification process. In addition, nucleic acid extraction / amplification can be achieved by reducing the number of processes, increasing convenience and speed, and eliminating sample migration, resulting in false positive results due to contamination, and improving sensitivity and accuracy by using the entire amount of nucleic acid obtained from the sample. It is intended to clarify the development of an integrated process.
본 발명의 단순한 변형 내지 변경은 모두 본 발명의 영역에 속하는 것으로, 본 발명의 구체적인 보호범위는 첨부된 특허청구범위에 의하여 명확해질 것이다.All simple modifications and variations of the present invention fall within the scope of the present invention, and the specific scope of the present invention will be apparent from the appended claims.
본 발명은 핵산을 추출 및 증폭하는 PCR방법을 사용하는 핵산기반측증기술분야 및 DNA/RNA를 이용한 생명공학 분야에서 산업적으로 이용될 수 있다.The present invention can be used industrially in the field of nucleic acid-based measurement techniques using PCR methods for extracting and amplifying nucleic acids and in the field of biotechnology using DNA / RNA.

Claims (12)

  1. DNA 변성 단계, 프라이머 결합 단계 및 DNA 사슬 연장 단계를 포함하는 DNA의 PCR 과정을 1회 이상 반복하는 핵산의 추출 및 증폭 방법에 있어서, 하기 단계를 포함하는 것을 특징으로 하는 핵산의 추출 및 증폭 방법:A nucleic acid extraction and amplification method comprising repeating a PCR process of DNA one or more times including DNA denaturation step, primer binding step, and DNA chain extension step, the method comprising the following steps:
    (1) 자성입자와 같은 고체 지지체의 존재 하에 DNA를 변성시켜, 자성입자의 표면에 변성된 DNA를 부착시키고, (1) denaturing DNA in the presence of a solid support such as magnetic particles to attach the modified DNA to the surface of the magnetic particles,
    (2) 상기 단계 (1)에서 결과된 변성 DNA-자성입자 부착물을 단백질-함유 세척용액으로 세척하고, (2) washing the denatured DNA-magnetic particle attachment obtained in step (1) with a protein-containing washing solution,
    (3) 상기 단계 (2)에서 결과된 변성 DNA-자성입자 부착물을 그 자체로 사용하여 프라이머 결합 단계 및 DNA 사슬 연장 단계를 수행함.(3) performing the primer binding step and the DNA chain extension step using the modified DNA-magnetic particle attachments resulting from step (2) by themselves.
  2. 제 1 항에 있어서, 전술한 고체 지지체는 표면에 실록산기, 아미노기, 히드록실기로 구성된 군에서 선택된 하나 이상의 관능기를 가지는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of extracting and amplifying a nucleic acid according to claim 1, wherein the solid support has at least one functional group selected from the group consisting of siloxane groups, amino groups, and hydroxyl groups on its surface.
  3. 제 1 항에 있어서, 전술한 고체 지지체는 실리카, 카보히드레이트, 아민, 폴리스티렌 및 폴리프로필렌으로 구성된 군에서 선택된 하나 이상의 물질로 코팅되어 있는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of claim 1, wherein the solid support is coated with one or more materials selected from the group consisting of silica, carbohydrates, amines, polystyrenes, and polypropylene.
  4. 제 1 항에 있어서, 사용되는 자성입자는 0.25 ~ 5 mg/20㎕의 양으로 사용되는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of extracting and amplifying a nucleic acid according to claim 1, wherein the magnetic particles used are used in an amount of 0.25-5 mg / 20 µl.
  5. 제 1 항에 있어서, 사용되는 자성입자는 5 ~ 20 mg/20㎕의 양으로 사용되는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of extracting and amplifying a nucleic acid according to claim 1, wherein the magnetic particles used are used in an amount of 5 to 20 mg / 20 µl.
  6. 제 1 항에 있어서, 전술한 단백질-함유 세척용액에서 단백질은 BSA, 스킴밀크 및 카제인으로 구성된 군에서 선택되는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of claim 1, wherein the protein in the protein-containing washing solution is selected from the group consisting of BSA, skim milk and casein.
  7. 제 5 항에 있어서, 전술한 단백질은 0.01 ~ 0.1%(w/v)의 양으로 함유되는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of extracting and amplifying a nucleic acid according to claim 5, wherein the aforementioned protein is contained in an amount of 0.01 to 0.1% (w / v).
  8. 제 1 항에 있어서, DNA 사슬 연장 단계는 PCR, RT-PCR, TMA, NASAB, SDA 및 RCA 용액으로 구성된 군에서 선택되는 DNA 증폭용액을 사용하여 수행되는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The method of claim 1, wherein the DNA chain extension step is performed using a DNA amplification solution selected from the group consisting of PCR, RT-PCR, TMA, NASAB, SDA, and RCA solutions.
  9. 제 1 항에 있어서, 상기 핵산 추출 및 증폭 반응에 의해 수득된 결과물을 겔 전기영동 (gel electrophoresis), ELGA (enzyme-linked gel assay), ECL (electrochemiluminescent) 또는 BMT 9G DNA KIT 및 BMT Genotyping 9G Membrane KIT로 구성된 군에서 선택되는 방법에 의해 검출하는 단계를 더 포함하는 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      According to claim 1, The product obtained by the nucleic acid extraction and amplification reaction is subjected to gel electrophoresis (gel electrophoresis), ELGA (enzyme-linked gel assay), ECL (electrochemiluminescent) or BMT 9G DNA KIT and BMT Genotyping 9G Membrane KIT Extraction and amplification method of nucleic acid, characterized in that it further comprises the step of detecting by a method selected from the group consisting of.
  10. 제 1 항에 있어서, 상기 단계 (2)는 하기 단계를 포함하는 것을 특징으로 하는 핵산의 추출 및 증폭 방법: The method of claim 1, wherein step (2) comprises the steps of:
    (2-1) 상기 단계 (1)에서 결과된 변성DNA-자성입자 부착물을 함유하는 반응용기에 단백질-함유 세척용액을 첨가하고, (2-1) adding the protein-containing washing solution to the reaction vessel containing the denatured DNA-magnetic particle deposit obtained in step (1) above,
    (2-2) 전술한 반응용기를 교반 또는 진탕하여 전술한 변성DNA-자성입자 부착물을 전술한 단백질-함유 세척용액으로 세척하고, (2-2) the above-mentioned modified DNA-magnetic particle attachment is washed with the above-described protein-containing washing solution by stirring or shaking the above-mentioned reaction vessel,
    (2-3) 상기 (2-2)에서 결과된 혼합물 중의 자성입자를 자석으로 고정하고, (2-3) The magnetic particles in the mixture obtained in the above (2-2) are fixed with a magnet,
    (2-4) 상기 (2-2)에서 결과된 혼합물에서 세척 용액을 분리 및 제거함.(2-4) Separating and removing the washing solution from the mixture resulting from (2-2) above.
  11. 제 1 항에 있어서, 전술한 핵산이 RNA인 것을 특징으로 하는 핵산의 추출 및 증폭 방법.      The nucleic acid extraction and amplification method according to claim 1, wherein the nucleic acid described above is RNA.
  12. 제 1 항의 핵산의 추출 및 증폭 방법을 사용하는, 바이러스 감염의 진단 방법.       A method for diagnosing a viral infection, using the method for extracting and amplifying the nucleic acid of claim 1.
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