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WO2020096394A1 - Method, kit, and pcr device for detecting methylation of gene using reduction probe - Google Patents

Method, kit, and pcr device for detecting methylation of gene using reduction probe Download PDF

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WO2020096394A1
WO2020096394A1 PCT/KR2019/015112 KR2019015112W WO2020096394A1 WO 2020096394 A1 WO2020096394 A1 WO 2020096394A1 KR 2019015112 W KR2019015112 W KR 2019015112W WO 2020096394 A1 WO2020096394 A1 WO 2020096394A1
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gene
methylation
methylated
pcr
reduction probe
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Korean (ko)
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어칠 친바야바트
윤경주
전미향
이한우
박희경
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Seasunbio Materials Inc
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    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
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    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/107—Temperature of melting, i.e. Tm
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    • C12Q2531/00—Reactions of nucleic acids characterised by
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/164—Methylation detection other then bisulfite or methylation sensitive restriction endonucleases
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    • C12Q2537/00—Reactions characterised by the reaction format or use of a specific feature
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method, a kit and a PCR device for detecting methylation of a gene using PCR based on PNA (Peptide Nucleic Acid, hereinafter referred to as 'PNA'), and more specifically, specifically for a methylated (or non-methylated) gene.
  • PNA Peptide Nucleic Acid
  • Control PCR and PNA without using PNA for DNA samples for detecting unmethylated control DNA and methylation Tm value difference between the gene and the reduction probe and ⁇ Ct value of the PCR product using a binding reduction probe
  • Methylation analysis using a reduction probe Methylation analysis using a reduction probe
  • the amplification efficiency difference of PCR is measured to analyze and detect whether a gene is methylated, and a kit and a PCR device for use in the method.
  • DNA methylation plays a role in protecting against invasion of foreign genes in prokaryotes, and plays an important role in regulating gene expression during development in eukaryotes. Recently, it has been found that DNA methylation is also involved in genomic imprinting and stabilization or inactivation of the X chromosome, and also affects tissue-specific gene activity and increase or suppress expression of diseases-related genes.
  • the fifth base is present in the genomic DNA of mammalian cells, which is 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring.
  • 5-mC always comes only to C of the CG dinucleotide (5'-mCG-3 '), and this CG is often referred to as CpG.
  • Most of C of CpG is methylated with methyl group attached. This methylation phenomenon is known to be the cause of various diseases including cancer, and it is known that the expression of the corresponding gene is suppressed by methylation of CpG islands.
  • DNA methylation is a representative phenomenon that occurs at the earliest stages of cancer, and is recognized as an optimal tool for early diagnosis of cancer.
  • a diagnostic technology using a methylation phenomenon of a specific gene as a biomarker has been actively researched.
  • methods such as bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and pyrosequencing and methylation-specific polymerase chain reaction (PCR) have been actively attempted.
  • PNA was first reported in 1991 as a DNA similar to a nucleic acid base linked to a peptide bond rather than a phosphate bond (Nielsen et al., Science, 254: 1497-1500, 1991). PNA hybridizes with a natural nucleic acid of complementary base sequence to form a double strand. When the number of nucleic acid bases is the same, PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands. As the basic skeleton of PNA, it is most often used that N- (2-aminoethyl) glycine is repeatedly connected by an amide bond.
  • the basic skeleton of the peptide nucleic acid is different from the basic skeleton of a negatively charged natural nucleic acid. It is electrically neutral.
  • the four nucleobases present in the PNA occupy a space similar to the nucleic acid base of DNA, and the distance between the nucleic acid bases is almost the same as that of natural nucleic acids.
  • PNA is not only chemically stable than natural nucleic acids, but also biologically stable because it is not degraded by nuclease or protease. Since PNA is also electrically neutral, the stability of the PNA / DNA and PNA / RNA double strands is not affected by salt concentration. Because of these properties, PNA is able to recognize complementary nucleic acid sequences better than natural nucleic acids, so it is applied for diagnostic or other biological and medical purposes.
  • the most common method currently used by researchers to confirm DNA methylation is to perform bisulfite and methylation-specific enzymes, followed by MSP (methylation specific PCR).
  • MSP methylation specific PCR
  • the present inventors have confirmed that the melting temperature (Tm) between the target DNA and the PNA reduction probe is higher than the Tm between the non-methylated DNA and the PNA probe when the PNA reduction probe is bound to the methylated gene using the above-described advantages of the PNA. It was confirmed that the Ct value of the methylated DNA increased compared to the Ct value of the unmethylated DNA by selectively suppressing the amplification of the methylated gene using this.
  • the PNA reduction probe has a specific base sequence, it was confirmed that the Tm value difference between the PNA reduction probe and the methylated or unmethylated DNA is further increased, thereby completing the invention regarding a methylation detection method having high sensitivity and accuracy.
  • Another object of the present invention is to provide a kit for detecting whether a gene is methylated using a PNA-based reduction probe.
  • Another object of the present invention is to provide a PCR device that detects whether a gene is methylated using a PNA-based reduction probe.
  • the present invention comprises the steps of performing PCR on the gene in the presence of a reduction probe capable of specifically binding to a nucleotide sequence of a gene (target gene) where methylation can occur; Performing PCR on the unmethylated gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of the unmethylated gene (the same gene in the control sample); Measuring at least one of ⁇ Tm (melting temperature) and ⁇ Ct (cycle threshold) in the PCR; And determining whether a gene is methylated through a value of ⁇ Tm or determining a ⁇ Ct value to determine whether to methylate and the degree of methylation.
  • ⁇ Tm melting temperature
  • ⁇ Ct cycle threshold
  • the present invention provides a kit for use in a method for detecting methylation of a gene including a reduction probe capable of specifically binding to a nucleotide sequence of a gene where methylation can occur.
  • the present invention provides an apparatus for performing real-time PCR using a reduction probe, comprising: a Tm analysis unit acquiring the ⁇ Tm information; A Ct analysis unit obtaining the ⁇ Ct information; A control unit for determining whether a gene is methylated from the obtained ⁇ Tm and ⁇ Ct values based on preset ⁇ Tm and ⁇ Ct information; And an output unit that transmits information based on at least one of methylation, methylation degree (X), and ⁇ Tm and ⁇ Ct determined by the controller to the user. It provides a PCR device for detecting gene methylation comprising a.
  • 1 is a graph comparing Tm values between DNA probes targeting methylated or unmethylated DNA and PNA probes.
  • FIG. 2 is a graph comparing Tm values between sdPNA (self dimer PNA) targeting methylated or unmethylated DNA and a normal PNA probe.
  • 3 is a graph comparing Tm values between PNA probes targeting DNA containing 2 to 4 methylated or unmethylated cytosine.
  • 5 is a graph showing the results of the verification and sensitivity test of the methylation detection method using the sdPNA probe.
  • FIG. 6 is a graph showing the accuracy test results of the methylation detection method using a PNA probe.
  • the present invention comprises the steps of performing a PCR for the gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of a gene (target gene) in which methylation can occur; Performing PCR on the unmethylated gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of the unmethylated gene (the same gene in the control sample); Measuring at least one of ⁇ Tm (melting temperature) and ⁇ Ct (cycle threshold) in the PCR; And determining whether a gene is methylated through a value of ⁇ Tm or determining a ⁇ Ct value to determine whether to methylate and the degree of methylation.
  • ⁇ Tm melting temperature
  • ⁇ Ct cycle threshold
  • a reduction probe refers to a probe that inhibits amplification of a gene by binding to a methylated detection site.
  • the difference in amplification can be induced by binding more strongly to the methylated detection site than to the unmethylated detection site.
  • the methylated cytosine and the reduction probe make complementary binding. Achieve.
  • Complementary binding is the formation of strong intermolecular attraction forces by three hydrogen bonds between the methylated cytosine and the guanine base of the reduction probe.
  • Methylated cytosine refers to cytosine methylated on the fifth carbon of the pyrimidine ring. Since the methyl group corresponds to an electron donating group (EDG), methylated cytosine is a complementary base guanine and stronger hydrogen. It is combined. The increased intermolecular interaction of guanine with methylated cytosine is associated with an increase in the electron donating properties of the C5-substituent. In addition, only the pair containing 5-methyl cytosine binds stronger than the guanine and cytosine pair formed by standard cytosine. As a result, when performing PCR, the Tm between the methylated DNA and the reduction probe has a higher value than the Tm between the unmethylated DNA and the reduction probe.
  • EDG electron donating group
  • At least one of ⁇ Tm and ⁇ Ct values in the PCR performed in the above step is measured.
  • the amplification inhibitory effect is exhibited when performing PCR by making stronger binding with the reduction probe compared to the unmethylated DNA.
  • the Tm value or Ct value of the gene in which methylation occurred is measured high, and the Tm value of the gene in which methylation occurred is minus the Tm value of the gene in which methylation does not occur, or ⁇ Tm value or methylation in the Ct value of methylated DNA
  • a method for detecting methylation of a gene using a reduction probe that determines the degree of methylation (X) by specifying a ⁇ Ct value is provided.
  • DNA amplified in real time can be detected.
  • Control ⁇ Ct Ct (analysis PCR) -Ct (control PCR) ⁇ Ct (control DNA)
  • the result of high Ct value is ⁇ Ct> 0 of the methylated DNA, and has a value of ⁇ Ct ⁇ 0.
  • the degree of methylation (X) may represent a value between 0 and 1. The closer to 1, the lower the degree of methylation, and the closer to 0, the higher the degree of methylation. The analysis was based on the absolute condition that the X value of the unmethylated control DNA was 1.
  • the reduction probe according to an example of the present invention may be selected from the group consisting of oligonucleotides, LNA, PNA and mixtures thereof.
  • the base sequence of the target gene is a methylated base sequence, a hydroxy methylated base sequence, a formyl methylated base sequence, a carboxyl methylated base sequence or a combination of two or more of the methylated base sequence It may have a sequence.
  • ⁇ Tm according to an example of the present invention is characterized by satisfying the following equation (1).
  • k is a constant
  • mC is a difference in Tm when there is one methylated cytosine
  • n is the number of methylated cytosine.
  • the reduction probe according to an example of the present invention may include a self-dimer-formable sequence.
  • Self-dimer refers to the case of self-folding because it has a complementary base sequence inside.
  • FIG. 4 shows that in the case of non-methylated DNA, the PNA reduction probe self-folds to form a self dimer, and the inhibitory effect of PCR amplification is not exhibited, so that the Ct value is higher than that of the methylated DNA. have.
  • the base sequence of the PNA reduction probe (hereinafter referred to as sdPNA) capable of forming a self dimer is shown in Table 2 below, but is not limited thereto, and any base sequence complementary thereto may be used without limitation.
  • the Tm value between the sdPNA and the methylated target DNA is higher than the Tm that formed the PNA self dimer, and the Tm that formed the PNA self dimer appears to be higher than or equal to the Tm of the sdPNA and the unmethylated DNA.
  • ⁇ Tm is 2 ° C or more and 5 ° C or less when one methylated cytosine is used, and 3 ° C or more when sdPNA is used, and preferably 4 ° C or more.
  • the method for detecting methylation may be to analyze whether methylation and the degree of methylation by measuring ⁇ Tm of the non-methylated or methylated gene and the reduction probe without amplification of the gene.
  • the detection method according to an embodiment of the present invention is selected from standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), quantitative PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization) It can be one.
  • the detection method may include a method for detecting gene amplification efficiency or PCR products.
  • real-time PCR may be used, but is not limited thereto, and the PCR may be used without limitation as long as it is used in the art.
  • Real-time PCR is also called quantitative polymerase chain reaction (qPCR), and it is possible to amplify the target gene and perform quantitative analysis at the same time.
  • qPCR quantitative polymerase chain reaction
  • the present invention provides a kit for use in a method for detecting methylation of a gene including a reduction probe capable of specifically binding to a nucleotide sequence of a gene capable of methylation.
  • the present invention provides an apparatus for performing real-time PCR using a reduction probe, comprising: a Tm analysis unit for obtaining ⁇ Tm information; Ct analysis unit for obtaining ⁇ Ct information; A control unit for determining whether a gene is methylated or a degree of methylation (X) based on the obtained ⁇ Tm and ⁇ Ct values based on preset ⁇ Tm and ⁇ Ct information; And an output unit that transmits information based on at least one of methylation, methylation degree (X), and ⁇ Tm and ⁇ Ct determined by the controller to the user. It provides a PCR device for detecting gene methylation comprising a.
  • the output unit may be a visual transmission method to a user through a display or a voice transmission method through audio.
  • the PNA reduction probe (FAM-labeled, Dabcyl) used in the present invention was synthesized from Panagene (Korea), and DNA oligomer (Integrated DNA technologies, USA) was used.
  • PNA Reduction Probe SEQ ID NO: 16 or DNA Fluorescence Probe SEQ ID NO: 15 1 ⁇ M, unmethylated SEQ ID NO: 13 or methylated SEQ ID NO: 14 Target DNA oligomer 1 ⁇ M and mixed with PCR amplification solution (ENGNOMICS, Korea) and mixed with real-time gene amplifier (Real Time PCR machine, CFX96 TM Real Time PCR System, Bylarad, USA) reacts at 95 ° C for 5 minutes, lowers it to 30 ° C, and then increases fluorescence by increasing the temperature by 1 ° C from 30 ° C to 90 ° C. Dissolution curve analysis was performed. The sequences used in the experiment are shown in Table 3 below.
  • 1 is a graph showing the results of the above experiment, ⁇ Tm between a DNA fluorescent probe and a DNA target containing two unmethylated DNA or methyl cytosine is 1 ° C., PNA reduction probe and two unmethylated DNA or methyl cytosine ⁇ Tm between DNA targets was confirmed to be 4 ° C.
  • the PNA reduction probe contains a specific nucleotide sequence capable of having a self-dimer form.
  • the experimental method is the same as in Example 1-1, and the experimental results are shown in FIG. 2.
  • DNA primers SEQ ID NOs: 20 and 21 listed in Table 5 of 45 ng of bisulfite-treated gDNA were amplified by PCR using 0.1 uM and PCR amplification solution (Engineomyx, Korea), respectively. PCR conditions are as follows. After treating at 95 ° C for 5 minutes, a total of 25 times were performed at 95 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by reaction at 72 ° C for 5 minutes. The PCR product was subjected to pyrosequencing using SEQ ID NO: 22 listed in Table 5 (Genomic Tree, Daejeon).
  • SW480 gDNA which was determined to have 87% -91% methylation of cytosine at the target site of sdPNA, was 1% with jurkat gDNA, a control sample, which was determined to have 0% methylation of cytosine at the target site.
  • 5%, 20%, 40%, 75% mixed or unmixed gDNA was targeted and subjected to methylation analysis PCR (CFX-96 Real-time) using a PNA reduction probe.
  • PCR amplification conditions are as follows.
  • asymmetric PCR was used to generate single-stranded target nucleic acids.
  • the conditions of the asymmetric PCR are as follows. 10 ⁇ l PCR amplification solution (ENGNOMICS, Korea), 1 uM forward primer SEQ ID NO: 22, 0.1 uM reverse primer SEQ ID NO: 23, 0.1 uM sdPNA SEQ ID NO: 1, 45 ng gDNA is mixed, and distilled water is added so that the total volume is 20 ⁇ l Real-time PCR was performed.
  • Figure 5 is a graph showing the results of the experiment, the methylation detection performance through a combination of the ratio of DNA separated from standard cells (Jurkat) where Septin 9 gene is not methylated and SW480 cells where Septin 9 is methylated about 90% And sensitivity.
  • PCR CFX-96 Real-time amplification conditions are as follows. After 5 minutes of treatment at 95 ° C, a total of 45 times were performed at 95 ° C for 30 seconds, 58 ° C for 45 seconds, and 65 ° C for 30 seconds, and asymmetric PCR was used to generate single-stranded target nucleic acids.
  • the conditions of the asymmetric PCR are the same as in Experiment 2-2 above.

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Abstract

The present invention relates to a method, a kit, and a PCR device for detecting methylation of a gene using a reduction probe, which can detect methylation of a gene without treating bisulfite or a methylation-specific enzyme, and thus is simple and has high sensitivity and accuracy in detection, and thus is useful in fields for diagnosing various diseases including cancer.

Description

리덕션 프로브를 이용한 유전자의 메틸화 검출 방법, 검출 키트 및 PCR 장치Method for detecting methylation of gene using reduction probe, detection kit and PCR device

본 발명은 PNA(Peptide Nucleic Acid, 이하 'PNA'라 함) 기반의 PCR을 이용한 유전자의 메틸화 검출 방법, 키트 및 PCR 장치에 관한 것으로서, 보다 상세하게는 메틸화(또는 비메틸화) 유전자에 특이적으로 결합하는 리덕션 프로브를 이용하여 상기 유전자와 리덕션 프로브간의 Tm값 차이 및 상기 PCR 산물의 ΔΔCt값을 (비메틸화 된 컨트롤 DNA 및 메틸화를 검출 하고자 하는 DNA 시료를 대상으로 PNA를 사용하지 않은 컨트롤 PCR 및 PNA 리덕션 프로브를 이용한 메틸화 분석 PCR의 증폭 효율 차이) 측정하여 유전자의 메틸화 여부를 분석, 검출하는 방법, 상기 방법에 이용하기 위한 키트 및 PCR 장치에 관한 것이다.The present invention relates to a method, a kit and a PCR device for detecting methylation of a gene using PCR based on PNA (Peptide Nucleic Acid, hereinafter referred to as 'PNA'), and more specifically, specifically for a methylated (or non-methylated) gene. Control PCR and PNA without using PNA for DNA samples for detecting unmethylated control DNA and methylation (Tm value difference between the gene and the reduction probe and ΔΔCt value of the PCR product using a binding reduction probe) Methylation analysis using a reduction probe The amplification efficiency difference of PCR) is measured to analyze and detect whether a gene is methylated, and a kit and a PCR device for use in the method.

DNA 메틸화는 원핵생물에서는 외부 유전자의 침입으로부터 보호해 주는 역할을 하며, 진핵생물에서는 발달과정에서 유전자의 발현을 조절해주는 중요한 역할을 하는 것으로 알려져 있다. 최근의 연구에서는 DNA 메틸화가 그 외에도 유전체 각인 및 안정화나 X염색체의 불활성화 등에도 관여하며, 조직특이적 유전자 활성, 질병과 연관된 유전자들의 발현 증가 또는 억제에도 영향을 미친다는 것이 밝혀지고 있다. It is known that DNA methylation plays a role in protecting against invasion of foreign genes in prokaryotes, and plays an important role in regulating gene expression during development in eukaryotes. Recently, it has been found that DNA methylation is also involved in genomic imprinting and stabilization or inactivation of the X chromosome, and also affects tissue-specific gene activity and increase or suppress expression of diseases-related genes.

포유류 세포의 게놈 DNA에는 A, C, G, T 외에 5번째 염기가 존재하며, 이는 시토신 고리의 5번째 탄소에 메틸기가 붙은 5-메틸시토신(5-mC)이다. 5-mC는 항상 CG 다이뉴클레오타이드의 C에만 오며(5'-mCG-3'), 이러한 CG를 흔히 CpG라고 표시한다. CpG의 C는 대부분이 메틸기가 붙어서 메틸화되어 있다. 이러한 메틸화 현상은 암을 포함한 다양한 질병의 원인으로 알려져 있으며, CpG섬의 메틸화에 의해 해당하는 유전자의 발현이 억제된다고 알려져 있다. In addition to A, C, G, and T, the fifth base is present in the genomic DNA of mammalian cells, which is 5-methylcytosine (5-mC) with a methyl group attached to the fifth carbon of the cytosine ring. 5-mC always comes only to C of the CG dinucleotide (5'-mCG-3 '), and this CG is often referred to as CpG. Most of C of CpG is methylated with methyl group attached. This methylation phenomenon is known to be the cause of various diseases including cancer, and it is known that the expression of the corresponding gene is suppressed by methylation of CpG islands.

특히, 종양 억제 유전자의 CpG섬이 메틸화되어 발현이 차단됨으로써 암이 발생하는 것으로 알려져 있다. DNA 메틸화 현상은 암 발생의 가장 초기에 일어나는 대표적인 현상이기 때문에 암 조기진단을 위한 최적의 도구로 인식되고 있다. 최근에는, 암 조기진단 및 스크리닝을 위해서 특정 유전자의 메틸화 현상을 바이오마커로 이용한 진단기술의 개발이 활발히 연구되고 있다. 이러한 바이오마커 유전자와 연관된 CpG섬 메틸화를 검출하기 위하여 bisulfite sequencing, combined bisulfite restriction analysis(COBRA), 그리고 pyrosequencing과 메틸화특이 PCR(polymerase chain reaction) 등과 같은 방법들이 적극적으로 시도되고 있다. In particular, it is known that cancer occurs due to methylation of the CpG island of the tumor suppressor gene, thereby blocking expression. DNA methylation is a representative phenomenon that occurs at the earliest stages of cancer, and is recognized as an optimal tool for early diagnosis of cancer. Recently, for the early diagnosis and screening of cancer, the development of a diagnostic technology using a methylation phenomenon of a specific gene as a biomarker has been actively researched. In order to detect CpG island methylation associated with the biomarker gene, methods such as bisulfite sequencing, combined bisulfite restriction analysis (COBRA), and pyrosequencing and methylation-specific polymerase chain reaction (PCR) have been actively attempted.

PNA는 핵산염기가 인산 결합이 아니라 펩티드 결합으로 연결된 유사 DNA로 1991년에 처음 보고되었다(Nielsen et al., Science, 254:1497-1500, 1991). PNA는 상보적인 염기 서열의 천연 핵산과 혼성화(hybridization) 반응을 일으켜서 겹가닥을 형성한다. 핵산 염기의 수가 같은 경우 PNA/DNA 겹가닥은 DNA/DNA 겹가닥보다, PNA/RNA 겹가닥은 DNA/RNA 겹가닥보다 안정하다. PNA의 기본 골격으로는 N-(2-아미노에틸)글리신이 아미드 결합에 의해 반복적으로 연결된 것이 가장 흔히 쓰이고, 이 경우 펩티드 핵산의 기본 골격(backbone)은 음전하를 띠는 천연 핵산의 기본 골격과 달리 전기적으로 중성이다. PNA에 존재하는 4개의 핵산염기(nucleobase)는 DNA의 핵산 염기와 비슷한 공간을 차지하고 핵산 염기 사이의 거리도 천연 핵산의 경우와 거의 같다. PNA는 화학적으로 천연 핵산보다 안정할 뿐 아니라 핵산분해효소(nuclease)나 단백질분해효소(protease)에 의해 분해되지 않아 생물학적으로도 안정하다. PNA는 또한 전기적으로 중성이기 때문에 PNA/DNA, PNA/RNA 겹가닥의 안정성은 염 농도에 영향을 받지 않는다. 이러한 성질 때문에 PNA는 상보적인 핵산 염기 서열을 천연 핵산보다 더 잘 인식할 수 있어서 진단 또는 다른 생물학적, 의학적 목적으로 응용된다.PNA was first reported in 1991 as a DNA similar to a nucleic acid base linked to a peptide bond rather than a phosphate bond (Nielsen et al., Science, 254: 1497-1500, 1991). PNA hybridizes with a natural nucleic acid of complementary base sequence to form a double strand. When the number of nucleic acid bases is the same, PNA / DNA double strands are more stable than DNA / DNA double strands and PNA / RNA double strands are more stable than DNA / RNA double strands. As the basic skeleton of PNA, it is most often used that N- (2-aminoethyl) glycine is repeatedly connected by an amide bond. In this case, the basic skeleton of the peptide nucleic acid is different from the basic skeleton of a negatively charged natural nucleic acid. It is electrically neutral. The four nucleobases present in the PNA occupy a space similar to the nucleic acid base of DNA, and the distance between the nucleic acid bases is almost the same as that of natural nucleic acids. PNA is not only chemically stable than natural nucleic acids, but also biologically stable because it is not degraded by nuclease or protease. Since PNA is also electrically neutral, the stability of the PNA / DNA and PNA / RNA double strands is not affected by salt concentration. Because of these properties, PNA is able to recognize complementary nucleic acid sequences better than natural nucleic acids, so it is applied for diagnostic or other biological and medical purposes.

선행기술문헌Prior art literature

한국공개특허공보 10-2011-0130638 (2011.12.06.)Korean Patent Publication No. 10-2011-0130638 (2011.12.06.)

현재 연구자들이 DNA 메틸화 확인을 위해 가장 일반적으로 이용하고 있는 방법은 바이설파이트(bisulfite) 및 메틸화 특유한 효소를 처리한 후 MSP(메틸화 특이적 PCR)을 수행하는 것이다. 본 발명자들은 상기한 PNA의 장점을 이용하여 PNA 리덕션 프로브가 메틸화된 유전자에 결합되는 경우 표적 DNA와 PNA 리덕션 프로브 간의 용융온도(Tm)가 비메틸화 DNA와 PNA 프로브 간의 Tm 보다 높게 나타나는 것을 확인하였으며, 이를 이용하여 메틸화된 유전자의 증폭을 선택적으로 억제시킴에 따라 메틸화된 DNA의 Ct값이 비메틸화 DNA의 Ct값대비 증가하는 것을 확인하였다. 더불어 PNA 리덕션 프로브가 특정 염기서열을 가지게 되는 경우 PNA 리덕션 프로브와 메틸화 또는 비메틸화된 DNA간의 Tm 값 차이 더욱 높아지는 것을 확인하여 높은 민감도 및 정확도를 가지는 메틸화 검출 방법에 관한 발명을 완성하기에 이르렀다.The most common method currently used by researchers to confirm DNA methylation is to perform bisulfite and methylation-specific enzymes, followed by MSP (methylation specific PCR). The present inventors have confirmed that the melting temperature (Tm) between the target DNA and the PNA reduction probe is higher than the Tm between the non-methylated DNA and the PNA probe when the PNA reduction probe is bound to the methylated gene using the above-described advantages of the PNA. It was confirmed that the Ct value of the methylated DNA increased compared to the Ct value of the unmethylated DNA by selectively suppressing the amplification of the methylated gene using this. In addition, when the PNA reduction probe has a specific base sequence, it was confirmed that the Tm value difference between the PNA reduction probe and the methylated or unmethylated DNA is further increased, thereby completing the invention regarding a methylation detection method having high sensitivity and accuracy.

본 발명의 목적은 종래 기술보다 간편하게, 바이설파이트 또는 다른 효소 처리 없이 PNA 기반의 리덕션 프로브를 이용하여 유전자의 메틸화 여부를 PCR 증폭 후 추가 분석 단계 없이 검출하는 방법을 제공하기 위한 것이다.It is an object of the present invention to provide a method of detecting whether a gene is methylated using a PNA-based reduction probe without bisulfite or other enzymatic treatment, and then without a further analysis step, than the prior art.

본 발명의 다른 목적은 PNA 기반의 리덕션 프로브를 이용하여 유전자의 메틸화 여부를 검출하는 키트를 제공하기 위한 것이다.Another object of the present invention is to provide a kit for detecting whether a gene is methylated using a PNA-based reduction probe.

본 발명의 또 다른 목적은 PNA 기반의 리덕션 프로브를 이용하여 유전자의 메틸화 여부를 검출하는 PCR 장치를 제공하기 위한 것이다.Another object of the present invention is to provide a PCR device that detects whether a gene is methylated using a PNA-based reduction probe.

상기 목적을 달성하기 위하여 본 발명은 메틸화가 일어날 수 있는 유전자(표적 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재 하에, 상기 유전자에 대한 PCR을 수행하는 단계; 메틸화 되지 않은 유전자(컨트롤 시료의 동일 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재하에, 상기 메틸화 되지 않은 유전자에 대한 PCR을 수행하는 단계; 상기 PCR에서의 ΔTm(melting temperature)과 ΔCt(cycle threshold) 중 적어도 하나 이상을 측정하는 단계; 및 ΔTm의 수치를 통해 유전자의 메틸화 여부를 판단 또는 ΔΔCt값을 특정하여 메틸화의 여부 및 메틸화 정도를 판단하는 단계; 를 포함하는 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법을 제공한다[상기 ΔTm=(메틸화가 일어날 수 있는 유전자와 리덕션 프로브 간의 Tm)-(메틸화 되지 않은 동일 유전자와 리덕션 프로브 간의 Tm), ΔΔCt=(리덕션 프로브의 첨가 없는 유전자 시료의 ΔCt)-(리덕션 프로브를 첨가한 유전자 시료의 ΔCt), ΔCt=(메틸화되지 않은 유전자의 Ct)-(메틸화된 유전자의 Ct)이다].In order to achieve the above object, the present invention comprises the steps of performing PCR on the gene in the presence of a reduction probe capable of specifically binding to a nucleotide sequence of a gene (target gene) where methylation can occur; Performing PCR on the unmethylated gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of the unmethylated gene (the same gene in the control sample); Measuring at least one of ΔTm (melting temperature) and ΔCt (cycle threshold) in the PCR; And determining whether a gene is methylated through a value of ΔTm or determining a ΔΔCt value to determine whether to methylate and the degree of methylation. It provides a method for detecting the methylation of a gene using a reduction probe, characterized in that the ΔTm = (Tm between the gene and the reduction probe may occur methylation)-(Tm between the same unmethylated gene and the reduction probe), ΔΔCt = (ΔCt of the gene sample without the addition of a reduction probe)-(ΔCt of the gene sample to which the reduction probe is added), ΔCt = (Ct of the unmethylated gene)-(Ct of the methylated gene)].

본 발명은 메틸화가 일어날 수 있는 유전자의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브를 포함하는 유전자의 메틸화 검출 방법에 사용하기 위한 키트를 제공한다.The present invention provides a kit for use in a method for detecting methylation of a gene including a reduction probe capable of specifically binding to a nucleotide sequence of a gene where methylation can occur.

본 발명은 리덕션 프로브를 이용한 실시간 PCR을 수행하는 장치에 있어서, 상기 ΔTm 정보를 획득하는 Tm 분석부; 상기 ΔΔCt 정보를 획득하는 Ct 분석부; 사전에 설정된 ΔTm 및 ΔΔCt 정보를 바탕으로 상기 획득한 ΔTm 및 ΔΔCt값으로부터 유전자의 메틸화 여부를 판단하는 제어부; 및 상기 제어부에서 판단된 메틸화 여부, 메틸화 정도(X), 상기 ΔTm 및 ΔΔCt 중 적어도 하나에 기초한 정보를 사용자에게 전달하는 출력부; 를 포함하는 유전자 메틸화 검출용 PCR 장치를 제공한다.The present invention provides an apparatus for performing real-time PCR using a reduction probe, comprising: a Tm analysis unit acquiring the ΔTm information; A Ct analysis unit obtaining the ΔΔCt information; A control unit for determining whether a gene is methylated from the obtained ΔTm and ΔΔCt values based on preset ΔTm and ΔΔCt information; And an output unit that transmits information based on at least one of methylation, methylation degree (X), and ΔTm and ΔΔCt determined by the controller to the user. It provides a PCR device for detecting gene methylation comprising a.

본 발명에 따른 리덕션 프로브를 사용한 유전자 메틸화 검출 방법을 사용하는 경우, 유전자의 메틸화 여부를 우수한 민감도 및 정확도로 검출할 수 있으며, 최근 들어 많이 사용하고 있는 방법인 바이설파이트(bisulfite) 및 메틸화 특이적 제한효소 등을 처리하여 메틸화를 분석하는 방법과는 다르게 이러한 효소의 처리 없이 간편하게 일 단계 과정만으로 DNA 메틸화를 검출할 수 있다. In the case of using the gene methylation detection method using the reduction probe according to the present invention, it is possible to detect whether the gene is methylated with excellent sensitivity and accuracy, and bisulphite and methylation specific methods that are commonly used in recent years Unlike the method of analyzing methylation by treating restriction enzymes, etc., DNA methylation can be easily detected in one step without any processing of these enzymes.

도 1은 메틸화 또는 비메틸화 DNA를 표적으로 하는 DNA 프로브와 PNA 프로브 간의 Tm 값을 비교한 그래프이다. 1 is a graph comparing Tm values between DNA probes targeting methylated or unmethylated DNA and PNA probes.

도 2는 메틸화 또는 비메틸화 DNA를 표적으로 하는 sdPNA(self dimer PNA)와 일반 PNA프로브 간의 Tm 값을 비교한 그래프이다.FIG. 2 is a graph comparing Tm values between sdPNA (self dimer PNA) targeting methylated or unmethylated DNA and a normal PNA probe.

도 3은 2~4개의 메틸화 또는 비메틸화된 시토신을 포함한 DNA를 표적으로 하는 PNA 프로브 간의 Tm 값을 비교한 그래프이다.3 is a graph comparing Tm values between PNA probes targeting DNA containing 2 to 4 methylated or unmethylated cytosine.

도 4는 비메틸화된 DNA의 경우, PNA 리덕션 프로브의 self dimer 형성의 도식 및 그로 인한 메틸화된 DNA인 경우와의 Ct차이를 보여주는 그래프이다.4 is a graph showing the Ct difference between the case of unmethylated DNA, the schematic of self dimer formation of the PNA reduction probe, and the case of methylated DNA.

도 5는 sdPNA 프로브를 이용한 메틸화 검출법의 검증 및 민감도 테스트 결과를 나타낸 그래프이다.5 is a graph showing the results of the verification and sensitivity test of the methylation detection method using the sdPNA probe.

도 6은 PNA 프로브를 이용한 메틸화 검출법의 정확도 테스트 결과를 나타낸 그래프이다.6 is a graph showing the accuracy test results of the methylation detection method using a PNA probe.

이하에서 본 발명에 대하여 구체적으로 설명한다. 본 명세서에서 사용되는 용어는 따로 정의하지 않는 경우 해당 분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 내용으로 해석되어야 할 것이다. 본 명세서의 도면 및 실시예는 통상의 지식을 가진 자가 본 발명을 쉽게 이해하고 실시하기 위한 것으로 도면 및 실시예에서 발명의 요지를 흐릴 수 있는 내용은 생략될 수 있으며, 본 발명이 도면 및 실시예로 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail. Unless otherwise defined, terms used in this specification should be interpreted as contents generally understood by a person having ordinary skill in the relevant field. The drawings and examples in this specification are for those of ordinary skill in the art to easily understand and implement the present invention. In the drawings and examples, contents that may obscure the subject matter of the invention may be omitted, and the present invention may be used in the drawings and examples. It is not limited to.

본 발명은 메틸화가 일어날 수 있는 유전자(표적 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재 하에, 상기 유전자에 대한 PCR을 수행하는 단계; 메틸화 되지 않은 유전자(컨트롤 시료의 동일 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재하에, 상기 메틸화 되지 않은 유전자에 대한 PCR을 수행하는 단계; 상기 PCR에서의 ΔTm(melting temperature)과 ΔCt(cycle threshold) 중 적어도 하나 이상을 측정하는 단계; 및 ΔTm의 수치를 통해 유전자의 메틸화 여부를 판단 또는 ΔΔCt값을 특정하여 메틸화의 여부 및 메틸화 정도를 판단하는 단계; 를 포함하는 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법(상기 ΔTm=(상기 메틸화가 일어날 수 있는 유전자와 리덕션 프로브 간의 Tm)-(메틸화 되지 않은 동일 유전자와 리덕션 프로브 간의 Tm)이고, 상기 ΔΔCt=(리덕션 프로브의 첨가 없는 유전자 시료의 ΔCt)-(리덕션 프로브를 첨가한 유전자 시료의 ΔCt)이고, ΔCt=(메틸화되지 않은 유전자의 Ct)-(메틸화된 유전자의 Ct))에 관한 것이다.The present invention comprises the steps of performing a PCR for the gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of a gene (target gene) in which methylation can occur; Performing PCR on the unmethylated gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of the unmethylated gene (the same gene in the control sample); Measuring at least one of ΔTm (melting temperature) and ΔCt (cycle threshold) in the PCR; And determining whether a gene is methylated through a value of ΔTm or determining a ΔΔCt value to determine whether to methylate and the degree of methylation. It characterized in that it comprises a method for detecting methylation of a gene using a reduction probe (ΔTm = (Tm between the gene where the methylation can occur and the reduction probe))-(Tm between the same unmethylated gene and the reduction probe), the ΔΔCt = (ΔCt of the genetic sample without the addition of the reduction probe)-(ΔCt of the genetic sample to which the reduction probe is added), and ΔCt = (Ct of the unmethylated gene)-(Ct of the methylated gene).

본 발명에서 리덕션 프로브(저해 프로브; reduction probe)란 메틸화된 검출 부위에 결합하여 유전자의 증폭을 저해하는 프로브를 말한다. 비메틸화된 검출 부위보다 메틸화된 검출 부위에 더욱 강하게 결합하여 증폭의 차이를 유도할 수 있다.In the present invention, a reduction probe (reduction probe) refers to a probe that inhibits amplification of a gene by binding to a methylated detection site. The difference in amplification can be induced by binding more strongly to the methylated detection site than to the unmethylated detection site.

본 발명의 방법에서, 메틸화가 일어날 수 있는 유전자의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재 하에, 상기 유전자에 대한 PCR을 수행하는 단계에서는 메틸화된 시토신과 리덕션 프로브가 상보적 결합을 이룬다. 상보적 결합은 메틸화된 시토신과 리덕션 프로브의 구아닌 염기가 3개의 수소 결합을 함으로써 강한 분자 간의 인력을 형성하는 것이다. In the method of the present invention, in the presence of a reduction probe capable of specifically binding the nucleotide sequence of a gene where methylation can occur, in the step of performing PCR on the gene, the methylated cytosine and the reduction probe make complementary binding. Achieve. Complementary binding is the formation of strong intermolecular attraction forces by three hydrogen bonds between the methylated cytosine and the guanine base of the reduction probe.

메틸화된 시토신은 피리미딘 고리의 5번째 탄소에 메틸화가 되어 있는 시토신을 가리키는데 메틸기는 전자 주는 기(Electron Donation Group; EDG)에 해당하므로 메틸화된 시토신은 상보적 염기에 해당하는 구아닌과 더 강한 수소 결합을 하게 된다. 구아닌과 메틸화된 시토신의 분자간 상호 작용의 증가는 C5-치환체의 전자 공여 특성의 증가와 관련이 있다. 또한, 5-메틸 시토신을 포함하는 쌍만이 표준 시토신에 의해 형성된 구아닌과 시토신 쌍보다 더 강하게 결합한다. 이 결과 PCR 수행시 메틸화된 DNA와 리덕션 프로브 간의 Tm은 비메틸화된 DNA와 리덕션 프로브 간의 Tm 보다 높은 값을 가진다. Methylated cytosine refers to cytosine methylated on the fifth carbon of the pyrimidine ring. Since the methyl group corresponds to an electron donating group (EDG), methylated cytosine is a complementary base guanine and stronger hydrogen. It is combined. The increased intermolecular interaction of guanine with methylated cytosine is associated with an increase in the electron donating properties of the C5-substituent. In addition, only the pair containing 5-methyl cytosine binds stronger than the guanine and cytosine pair formed by standard cytosine. As a result, when performing PCR, the Tm between the methylated DNA and the reduction probe has a higher value than the Tm between the unmethylated DNA and the reduction probe.

상기 단계에서 수행된 PCR에서의 ΔTm과 ΔCt값 중 적어도 하나 이상을 측정한다. 메틸화된 시토신을 가진 표적 DNA의 경우 비메틸화된 DNA에 비하여 리덕션 프로브와 더 강한 결합을 함으로써 PCR 수행 시 증폭 억제 효과가 나타난다. 이를 이용하여 메틸화가 일어난 유전자의 Tm값 또는 Ct값이 높게 측정이 되고, 메틸화가 일어난 유전자의 Tm값에서 메틸화가 일어나지 않은 유전자의 Tm값을 뺀 ΔTm의 수치 또는 메틸화가 일어난 DNA의 Ct값에서 메틸화가 일어나지 않은 DNA의 Ct값을 뺀 ΔCt의 수치를 확인함으로써 표적 DNA의 메틸화 여부를 판단할 수 있다. At least one of ΔTm and ΔCt values in the PCR performed in the above step is measured. In the case of the target DNA having methylated cytosine, the amplification inhibitory effect is exhibited when performing PCR by making stronger binding with the reduction probe compared to the unmethylated DNA. Using this, the Tm value or Ct value of the gene in which methylation occurred is measured high, and the Tm value of the gene in which methylation occurred is minus the Tm value of the gene in which methylation does not occur, or ΔTm value or methylation in the Ct value of methylated DNA By determining the value of ΔCt minus the Ct value of the DNA that does not occur, it is possible to determine whether the target DNA is methylated.

ΔΔCt값을 특정하여 메틸화의 정도(X)를 판단하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법을 제공한다. 상기 ΔΔCt=(PNA 리덕션 프로브의 첨가 없는 유전자 시료의 ΔCt)-(PNA 리덕션 프로브를 첨가한 유전자 시료의 ΔCt)이고, ΔCt=(메틸화되지 않은 유전자의 Ct)-(메틸화된 유전자의 Ct)를 나타낸다.A method for detecting methylation of a gene using a reduction probe that determines the degree of methylation (X) by specifying a ΔΔCt value is provided. The ΔΔCt = (ΔCt of the gene sample without the addition of the PNA reduction probe)-(ΔCt of the gene sample with the addition of the PNA reduction probe), and ΔCt = (Ct of the unmethylated gene)-(Ct of the methylated gene) .

반응이 진행되는 동안 실시간으로 증폭된 DNA를 검출할 수 있다.During the reaction, DNA amplified in real time can be detected.

분석법Method 분석 공식Analytical formula Control ΔCtControl ΔCt Ct(분석PCR)-Ct(control PCR)=ΔCt(control DNA)Ct (analysis PCR) -Ct (control PCR) = ΔCt (control DNA)

Figure PCTKR2019015112-appb-img-000001
Figure PCTKR2019015112-appb-img-000002
Figure PCTKR2019015112-appb-img-000001
Figure PCTKR2019015112-appb-img-000002
Sample ΔCtSample ΔCt Ct(분석PCR)-Ct(control PCR)=ΔCt(분석 DNA)Ct (analysis PCR) -Ct (control PCR) = ΔCt (analysis DNA) 메틸화 정도(X)Methylation degree (X) ΔCt(control DNA)-ΔCt(분석 DNA)=메틸화 정도ΔCt (control DNA) -ΔCt (analysis DNA) = Methylation degree

증폭이 저해되면 Ct값이 높게 나타나는 결과 메틸화된 DNA의 ΔCt>0 이고, ΔΔCt<0의 값을 가진다. 상기 공식에 의할 때, 메틸화 정도(X)는 0 내지 1 사이의 값을 나타낼 수 있다. 1에 가까울수록 메틸화 정도가 낮으며 0에 가까울수록 메틸화 정도가 높은 것을 의미한다. 비메틸화 컨트롤 DNA의 X값이 1이라는 절대 조건을 바탕으로 분석한 것이다.When amplification is inhibited, the result of high Ct value is ΔCt> 0 of the methylated DNA, and has a value of ΔΔCt <0. According to the above formula, the degree of methylation (X) may represent a value between 0 and 1. The closer to 1, the lower the degree of methylation, and the closer to 0, the higher the degree of methylation. The analysis was based on the absolute condition that the X value of the unmethylated control DNA was 1.

본 발명의 일 예에 따른 상기 리덕션 프로브는 올리고뉴클레오티드, LNA, PNA 및 이들의 혼합으로 구성된 군에서 선택되는 것일 수 있다.The reduction probe according to an example of the present invention may be selected from the group consisting of oligonucleotides, LNA, PNA and mixtures thereof.

본 발명의 일 예에 따른 상기 표적 유전자의 염기서열은 메틸화된 염기서열, 하이드록시 메틸화된 염기서열, 포르밀 메틸화된 염기서열, 카르복실 메틸화된 염기서열 중 어느 하나 또는 2이상의 조합으로 메틸화된 염기서열을 가지는 것일 수 있다.The base sequence of the target gene according to an embodiment of the present invention is a methylated base sequence, a hydroxy methylated base sequence, a formyl methylated base sequence, a carboxyl methylated base sequence or a combination of two or more of the methylated base sequence It may have a sequence.

본 발명의 일 예에 따른 ΔTm은 하기의 식 (1)을 만족하는 것을 특징으로 한다. ΔTm according to an example of the present invention is characterized by satisfying the following equation (1).

ΔTm=k×mC×n 식 (1)ΔTm = k × mC × n (1)

[상기 식에서 k는 상수, mC는 메틸화된 시토신이 1개일 경우의 Tm의 차이, n은 메틸화된 시토신의 개수이다.][In the above formula, k is a constant, mC is a difference in Tm when there is one methylated cytosine, n is the number of methylated cytosine.]

표적 DNA상에 메틸화된 시토신이 많을수록 수소결합력은 강하게 나타나므로 컨트롤 DNA와의 ΔTm은 커진다. 도 3에 나타난 그래프에서 이를 확인할 수 있다.The more methylated cytosine on the target DNA, the stronger the hydrogen-bonding power, so the ΔTm with the control DNA increases. This can be confirmed in the graph shown in FIG. 3.

본 발명의 일 예에 따른 상기 리덕션 프로브는 self-dimer 형성 가능한 서열을 포함할 수 있다. Self-dimer란 상보적인 염기서열을 내부에 가지고 있어 self-folding이 되는 경우를 가리킨다. 도 4는 비메틸화된 DNA의 경우 PNA 리덕션 프로브는 self-folding을 하여 self dimer를 형성하게 되고, PCR 증폭의 저해 작용이 나타나지 않아 Ct값이 메틸화된 DNA의 경우에 비해 큰 값을 나타내는 것을 보여주고 있다. Self dimer 형성 가능한 PNA 리덕션 프로브(이하, sdPNA라 함)의 염기서열은 다음의 표 2에 나타내었으나, 이에 한정하지 않고 내부에 상보적인 염기서열을 가진 것이라면 제한없이 사용 가능하다.The reduction probe according to an example of the present invention may include a self-dimer-formable sequence. Self-dimer refers to the case of self-folding because it has a complementary base sequence inside. FIG. 4 shows that in the case of non-methylated DNA, the PNA reduction probe self-folds to form a self dimer, and the inhibitory effect of PCR amplification is not exhibited, so that the Ct value is higher than that of the methylated DNA. have. The base sequence of the PNA reduction probe (hereinafter referred to as sdPNA) capable of forming a self dimer is shown in Table 2 below, but is not limited thereto, and any base sequence complementary thereto may be used without limitation.

Self dimer 형성 가능한 PNA 리덕션 프로브의 염기서열의 예Example of the base sequence of a PNA reduction probe capable of forming a self dimer 서열번호Sequence number 이름name 서열(5' ---->3')Sequence (5 '----> 3') 1One PNA_2mCPNA_2mC Dabcyl-ACC CCGCGGTCA-O-K(FAM)Dabcyl-ACC CCGCGG TCA-OK (FAM) 22 PNA_2mC_2PNA_2mC_2 Dabcyl-AC CCCGCGGGCA-O-K(FAM)Dabcyl-AC CCCGCGGG CA-OK (FAM) 33 PNA_2mC_3PNA_2mC_3 Dabcyl-AC ACCGCGGTCA-O-K(FAM)Dabcyl-AC ACCGCGGT CA-OK (FAM) 44 PNA_3mCPNA_3mC Dabcyl- CCGCGGTCAACGC-O-K(FAM)Dabcyl- CCGCGG TCAACGC-OK (FAM) 55 PNA_3mC_2PNA_3mC_2 Dabcyl- ACCGCGGTCAACGC-O-K(FAM)Dabcyl- ACCGCGGT CAACGC-OK (FAM) 66 PNA_3mC_3PNA_3mC_3 Dabcyl- GACCGCGGTCAACGC-O-K(FAM)Dabcyl- GACCGCGGTC AACGC -OK (FAM) 77 PNA_4mCPNA_4mC Dabcyl- CGCGGTCAA CGCGC-O-K(FAM)Dabcyl- CGCG GTCAA CGCG C-OK (FAM) 88 PNA_4mC_2PNA_4mC_2 Dabcyl- CGCGTTCA ACGCGC-O-K(FAM)Dabcyl- CGCG TTCA ACGCG C-OK (FAM) 99 PNA_4mC_3PNA_4mC_3 Dabcyl- CGCGGTCAC CGCGC-O-K(FAM)Dabcyl- CGCGG TCAC CGCG C-OK (FAM) 1010 PNA_5mCPNA_5mC Dabcyl-TTTCGGA CTTCGAAG-O-K (FAM)Dabcyl-TTTCGGA CTTCGAAG -OK (FAM) 1111 PNA_6mCPNA_6mC Dabcyl-ATGT CGGACC CCG-O-K (FAM)Dabcyl-ATGT CGG ACC CCG -OK (FAM) 1212 PNA_7mCPNA_7mC Dabcyl- GCGATTTCGGACT TCGC-O-K (FAM)Dabcyl- GCGA TTTCGGACT TCGC -OK (FAM)

sdPNA와 메틸화된 표적 DNA 사이의 Tm값은 PNA self dimer를 형성한 Tm보다 높게 나타나고, PNA self dimer를 형성한 Tm은 sdPNA와 비메틸화된 DNA와의 Tm보다 높거나 같게 나타난다. The Tm value between the sdPNA and the methylated target DNA is higher than the Tm that formed the PNA self dimer, and the Tm that formed the PNA self dimer appears to be higher than or equal to the Tm of the sdPNA and the unmethylated DNA.

본 발명의 일 예에 따르면 상기 ΔTm은 메틸화된 시토신이 1개일 경우 2℃ 이상 5℃ 이하로 나타나고, sdPNA를 사용한 경우 3℃이상이며, 바람직하게는 4℃이상 나타난다.According to an example of the present invention, ΔTm is 2 ° C or more and 5 ° C or less when one methylated cytosine is used, and 3 ° C or more when sdPNA is used, and preferably 4 ° C or more.

본 발명의 일 예에 따른 상기 메틸화 검출 방법은 유전자의 증폭 없이, 상기 비메틸화 또는 메틸화된 유전자와 리덕션 프로브의 ΔTm을 측정하여 메틸화 여부 및 메틸화 정도를 분석하는 것일 수 있다.The method for detecting methylation according to an embodiment of the present invention may be to analyze whether methylation and the degree of methylation by measuring ΔTm of the non-methylated or methylated gene and the reduction probe without amplification of the gene.

본 발명의 일 예에 따른 상기 검출 방법은 표준 PCR, 실시간 PCR(Real Time PCR), 디지털 PCR, 등온 PCR(Isothermal PCR), 정량 PCR, DNA 칩, DNA FISH (Fluorescence in situ hybridization)에서 선택되는 어느 하나일 수 있다.The detection method according to an embodiment of the present invention is selected from standard PCR, real time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), quantitative PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization) It can be one.

또는 상기 검출 방법은 유전자 증폭 효율 또는 PCR 산물을 검출할 수 있는 방법을 포함하는 것일 수 있다. 바람직하게는 실시간 PCR을 사용하는 것일 수 있으나 반드시 이에 한정되는 것은 아니며, 상기 PCR은 이 기술 분야에서 사용되는 것이라면 제한 없이 사용될 수 있다. Alternatively, the detection method may include a method for detecting gene amplification efficiency or PCR products. Preferably, real-time PCR may be used, but is not limited thereto, and the PCR may be used without limitation as long as it is used in the art.

실시간 PCR은 quantitative polymerase chain reaction (qPCR) 이라고도 불리는데, 타켓 유전자를 증폭시킴과 동시에 정량 분석하는 것이 가능하다. Real-time PCR is also called quantitative polymerase chain reaction (qPCR), and it is possible to amplify the target gene and perform quantitative analysis at the same time.

또한, 본 발명은 메틸화가 일어날 수 있는 유전자의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브를 포함하는 유전자의 메틸화 검출 방법에 사용하기 위한 키트를 제공한다.In addition, the present invention provides a kit for use in a method for detecting methylation of a gene including a reduction probe capable of specifically binding to a nucleotide sequence of a gene capable of methylation.

본 발명은 리덕션 프로브를 이용한 실시간 PCR을 수행하는 장치에 있어서, ΔTm 정보를 획득하는 Tm 분석부; ΔΔCt 정보를 획득하는 Ct 분석부; 사전에 설정된 ΔTm 및 ΔΔCt 정보를 바탕으로 상기 획득한 ΔTm 및 ΔΔCt값으로부터 유전자의 메틸화 여부 또는 메틸화 정도(X)를 판단하는 제어부; 및 상기 제어부에서 판단된 메틸화 여부, 메틸화 정도(X), 상기 ΔTm 및 ΔΔCt 중 적어도 하나에 기초한 정보를 사용자에게 전달하는 출력부; 를 포함하는 유전자 메틸화 검출용 PCR 장치를 제공한다.The present invention provides an apparatus for performing real-time PCR using a reduction probe, comprising: a Tm analysis unit for obtaining ΔTm information; Ct analysis unit for obtaining ΔΔCt information; A control unit for determining whether a gene is methylated or a degree of methylation (X) based on the obtained ΔTm and ΔΔCt values based on preset ΔTm and ΔΔCt information; And an output unit that transmits information based on at least one of methylation, methylation degree (X), and ΔTm and ΔΔCt determined by the controller to the user. It provides a PCR device for detecting gene methylation comprising a.

상기 출력부는 디스플레이를 통하여 사용자에게 시각적 전달 또는 오디오를 통한 음성 전달 방식일 수 있다.The output unit may be a visual transmission method to a user through a display or a voice transmission method through audio.

이하 실시예를 통하여 본 발명을 더욱 자세히 설명한다. 하기 실시예는 본 발명을 보다 구체적으로 설명하기 위한 하나의 예시에 해당하는 것으로서 본 발명의 범위가 실시예에 의해 한정되는 것은 아니다.The present invention will be described in more detail through the following examples. The following examples correspond to one example for explaining the present invention in more detail, and the scope of the present invention is not limited by the examples.

실시예Example 1.  One. PNA 리덕션PNA reduction 프로브와With probe 비메틸화Unmethylated 또는 메틸화된  Or methylated DNA타겟의DNA target 결합 온도 분석. Bond temperature analysis.

<1-1. 메틸화 또는 비메틸화된 시토신을 포함한 표적 DNA 및 DNA 형광 프로브 또는 PNA 리덕션 프로브의 결합 온도 비교 분석> <1-1. Comparative analysis of target DNA and methyl fluorescence probes containing methylated or unmethylated cytosine or binding temperature of PNA reduction probes>

본 발명에서 사용한 PNA 리덕션 프로브(FAM-labeled, Dabcyl)는 파나진(Panagene, 한국)에서 합성한 것이며, DNA 올리고머(Integrated DNA technologies, 미국)를 사용하였다. PNA 리덕션 프로브 서열 번호 16 또는 DNA 형광 프로브 서열번호 15 1μM, 비메틸화 서열번호 13 또는 메틸화된 서열번호 14 타켓 DNA 올리고머 1μM와 PCR 증폭용액(엔지노믹스, 한국)을 넣고 혼합한 뒤 실시간 유전자 증폭기(Real Time PCR machine, CFX96 TM Real Time PCR System, 바이로라드, 미국)를 이용하여 95℃에서 5분 동안 반응 후, 30℃까지 낮춘 뒤에, 30℃부터 90℃까지 1℃씩 상승시키며 형광을 측정하는 용해곡선 분석을 수행하였다. 상기 실험에 사용된 서열은 하기 표 3와 같다.The PNA reduction probe (FAM-labeled, Dabcyl) used in the present invention was synthesized from Panagene (Korea), and DNA oligomer (Integrated DNA technologies, USA) was used. PNA Reduction Probe SEQ ID NO: 16 or DNA Fluorescence Probe SEQ ID NO: 15 1 μM, unmethylated SEQ ID NO: 13 or methylated SEQ ID NO: 14 Target DNA oligomer 1 μM and mixed with PCR amplification solution (ENGNOMICS, Korea) and mixed with real-time gene amplifier (Real Time PCR machine, CFX96 TM Real Time PCR System, Bylarad, USA) reacts at 95 ° C for 5 minutes, lowers it to 30 ° C, and then increases fluorescence by increasing the temperature by 1 ° C from 30 ° C to 90 ° C. Dissolution curve analysis was performed. The sequences used in the experiment are shown in Table 3 below.

서열번호Sequence number 이름name 서열(N→C)Sequence (N → C) 1313 비메틸화 DNA 타켓Unmethylated DNA target GTTGAC[ C]G[ C]GGGGTCTGTTGAC [ C ] G [ C ] GGGGTCT 1414 메틸화 DNA 타켓Methylated DNA target GTTGAC[ iMe-dC]G[ iMe-dC]GGGGTCTGTTGAC [ iMe-dC ] G [ iMe-dC ] GGGGTCT 1515 DNA 형광 프로브DNA fluorescence probe Dabcyl-ACCCCGCGGTCA- (FAM)Dabcyl-ACCCCGCGGTCA- (FAM) 1616 PNA 리덕션 프로브PNA reduction probe Dabcyl-ACCCCGCGGTCA- (FAM)Dabcyl-ACCCCGCGGTCA- (FAM) [C]: 비메틸 시토신[iMe-dC]: 메틸 시토신[C]: Bimethyl cytosine [iMe-dC]: methyl cytosine

도 1은 상기 실험에 대한 결과를 그래프로 나타낸 것으로서, DNA 형광 프로브와 비메틸화 DNA 또는 메틸 시토신을 2개 포함한 DNA 타겟 간의 ΔTm은 1℃, PNA 리덕션 프로브와 비메틸화 DNA 또는 메틸 시토신 2개를 포함한 DNA 타겟 간의 ΔTm은 4℃로 확인되었다.1 is a graph showing the results of the above experiment, ΔTm between a DNA fluorescent probe and a DNA target containing two unmethylated DNA or methyl cytosine is 1 ° C., PNA reduction probe and two unmethylated DNA or methyl cytosine ΔTm between DNA targets was confirmed to be 4 ° C.

<1-2. 메틸화 또는 비메틸화된 시토신을 포함한 표적 DNA 및 일반 PNA 프로브 또는 sdPNA 리덕션 프로브의 결합 온도 비교 분석><1-2. Comparative analysis of the target DNA containing methylated or unmethylated cytosine and the binding temperature of a general PNA probe or sdPNA reduction probe>

메틸 시토신 2개를 포함한 DNA 타겟 서열번호 14와 self-dimer PNA 리덕션 프로브 서열번호 16 또는 일반 PNA 프로브 서열번호 17 간의 Tm을 확인한 결과 PNA 리덕션 프로브가 self-dimer 형태를 가질 수 있는 특정 염기서열을 포함하는 경우 PNA 리덕션 프로브와 메틸화 또는 비메틸화된 DNA간의 Tm값 차이가 더욱 높아지는 것을 확인하였다. 실험 방법은 실시예 1-1와 같으며 실험 결과는 도 2에 도시되어있다.As a result of confirming the Tm between the DNA target SEQ ID NO: 14 including two methyl cytosines and the self-dimer PNA reduction probe SEQ ID NO: 16 or the general PNA probe SEQ ID NO: 17, the PNA reduction probe contains a specific nucleotide sequence capable of having a self-dimer form. When it was confirmed that the difference in Tm value between the PNA reduction probe and methylated or unmethylated DNA was further increased. The experimental method is the same as in Example 1-1, and the experimental results are shown in FIG. 2.

서열번호Sequence number 이름name 서열(N→C)Sequence (N → C) 1717 PNA_2mC_1PNA_2mC_1 Dabcyl-ACCCCGCGATCA- (FAM)Dabcyl-ACCCCGCGATCA- (FAM) 1818 메틸화 DNA 타켓Methylated DNA target G[iMe-dC]GTTGAC[iMe-dC]G[iMe-dC]GGG [iMe-dC] GTTGAC [iMe-dC] G [iMe-dC] GG 1919 메틸화 DNA 타켓Methylated DNA target G[iMe-dC]G[iMe-dC]GTTGAC[iMe-dC]G[iMe-dC]GG [iMe-dC] G [iMe-dC] GTTGAC [iMe-dC] G [iMe-dC] G [iMe-dC]: 메틸 시토신 [iMe-dC]: methyl cytosine

<1-3. 메틸화 시토신 개수에 따른 표적 DNA와 PNA 리덕션 프로브 간의 Tm 비교 분석><1-3. Comparison of Tm between target DNA and PNA reduction probe according to the number of methylated cytosine>

메틸 시토신 2개, 3개 또는 4개를 포함한 DNA 타겟 (서열번호 14, 18, 19)와 self-dimer PNA 리덕션 프로브 (서열번호 16) 간의 Tm을 확인한 결과 표적 DNA를 포함하는 메틸 시토신 개수가 많을수록 PNA 리덕션 프로브 간의 Tm 차이가 더 높아지는 것을 확인하였다. 실험 방법은 실시예 1-1와 같으며 실험 결과는 도 3에 도시되어있다.As a result of confirming the Tm between a DNA target (SEQ ID NO: 14, 18, 19) containing 2, 3 or 4 methyl cytosine and a self-dimer PNA reduction probe (SEQ ID NO: 16), the more methyl cytosine containing the target DNA, the more It was confirmed that the Tm difference between the PNA reduction probes was higher. The experimental method is the same as in Example 1-1, and the experimental results are shown in FIG. 3.

실시예Example 2.  2. sdPNAsdPNA 프로브를Probe 이용한 메틸화 검출 방법의 검증 Verification of the methylation detection method used

<2-1. DNA 메틸화 확인을 위한 pyrosequencing 분석><2-1. Pyrosequencing analysis to confirm DNA methylation>

비메틸화 컨트롤 DNA로 septin 9 유전자의 CpG 섬의 해당 염기서열 부분이 메틸화 되지 않다고 알려져 있는 Jurkat 세포주 (thermofisher사, 미국)에서 추출한 DNA (Timothy Robert Church et al., 2013)와 메틸화된 DNA 시료로 상기 유전자가 메틸화 되어 있는 대표적인 세포주 대장암 세포주 SW480에서 (Tao Peng et al., 2017) 추출 한 DNA를 이용하여 실험을 진행하였다. 상기 세포주에서 분리된 gDNA 각각 500ng을 EpiTect Fast Bisulfite Conversion Kits (Qiagen, 미국)를 이용하여 bisulfite 처리를 실시한 후, pyrosequencing 분석을 통해 상기 DNA 시료들의 Septin 9 유전자의 CpG 섬에 해당 시토신 염기서열의 메틸화 여부를 확인하였다. 증폭(서열번호 20, 21)과 pyrosequencing(서열번호 22)에 사용된 서열은 하기 표 5와 같다.DNA extracted from the Jurkat cell line (thermofisher, USA), which is known to not methylate the corresponding nucleotide sequence of the CpG island of the septin 9 gene as non-methylated control DNA (Timothy Robert Church et al., 2013) and methylated DNA samples. The experiment was conducted using DNA extracted from the representative cell line colon cancer cell line SW480 (Tao Peng et al., 2017) in which the gene is methylated. 500 g of each gDNA isolated from the cell line was subjected to bisulfite treatment using EpiTect Fast Bisulfite Conversion Kits (Qiagen, USA), followed by pyrosequencing analysis to methylate the cytosine sequence on the CpG island of Septin 9 gene of the DNA samples Was confirmed. Sequences used for amplification (SEQ ID NO: 20, 21) and pyrosequencing (SEQ ID NO: 22) are shown in Table 5 below.

서열번호Sequence number 이름name 서열(N→C)Sequence (N → C) 2020 sepF_BS1sepF_BS1 GTAGTAGTTAGTTTAGTATTTATTTTGTAGTAGTTAGTTTAGTATTTATTTT 2121 sepR_BS3sepR_BS3 Biotin - CTA CCC ACC AAC CAT CAT ATBiotin-CTA CCC ACC AAC CAT CAT AT 2222 sepS-S1sepS-S1 GTT AGA AAT GAT TTT ATT TAG TTG GTT AGA AAT GAT TTT ATT TAG TTG

bisulfite 처리된 gDNA 45ng을 표 5에 나열한 DNA 프라이머(서열번호 20, 21)를 각각 0.1 uM 와 PCR 증폭용액 (엔지노믹스, 한국)을 이용하여 PCR 증폭하였다. PCR 조건은 다음과 같다. 95 ℃ 에서 5분 처리 후, 95 ℃ 에서 30초, 58 ℃ 에서 30초, 72 ℃ 에서 30초로 총 25회 실시한 다음, 72 ℃ 에서 5분 동안 반응한다. 상기 PCR 산물은 표 5에 나열한 서열번호 22를 이용하여 pyrosequencing을 진행하였다 (지노믹트리, 대전).DNA primers (SEQ ID NOs: 20 and 21) listed in Table 5 of 45 ng of bisulfite-treated gDNA were amplified by PCR using 0.1 uM and PCR amplification solution (Engineomyx, Korea), respectively. PCR conditions are as follows. After treating at 95 ° C for 5 minutes, a total of 25 times were performed at 95 ° C for 30 seconds, 58 ° C for 30 seconds, and 72 ° C for 30 seconds, followed by reaction at 72 ° C for 5 minutes. The PCR product was subjected to pyrosequencing using SEQ ID NO: 22 listed in Table 5 (Genomic Tree, Daejeon).

<2-2. sdPNA 리덕션 프로브를 이용한 메틸화 검출법의 민감도 테스트><2-2. Sensitivity test of methylation detection method using sdPNA reduction probe>

상기 2-1의 실험에 따라, sdPNA의 표적 사이트의 시토신이 87%-91% 메틸화된 것으로 측정된 SW480 gDNA를 상기 표적 사이트의 시토신이 0% 메틸화된 것으로 측정된 대조 샘플인 jurkat gDNA와 1%, 5%, 20%, 40%, 75%로 혼합 또는 혼합하지 않은 gDNA를 표적으로 하여 PNA 리덕션 프로브를 이용하여 메틸화 분석 PCR(CFX-96 Real-time)을 실시하였다. PCR 증폭 조건은 다음과 같다. 95 ℃에서 5분 처리 후, 95 ℃ 에서 30초, 58 ℃에서 45초, 65 ℃에서 30초로 총 45회 수행하였으며, 단일가닥 표적핵산을 생성하기 위해 비대칭 PCR(asymmetric PCR)을 이용하였다. 비대칭 PCR의 조건은 다음과 같다. 10㎕ PCR 증폭용액 (엔지노믹스, 한국), 1uM forward primer 서열번호 22, 0.1 uM reverse primer 서열번호 23, 0.1uM sdPNA 서열번호 1, 45ng gDNA 를 혼합한 후, 총 부피가 20㎕ 되도록 증류수를 첨가하여 실시간 PCR을 실시하였다.According to the experiment of 2-1, SW480 gDNA, which was determined to have 87% -91% methylation of cytosine at the target site of sdPNA, was 1% with jurkat gDNA, a control sample, which was determined to have 0% methylation of cytosine at the target site. , 5%, 20%, 40%, 75% mixed or unmixed gDNA was targeted and subjected to methylation analysis PCR (CFX-96 Real-time) using a PNA reduction probe. PCR amplification conditions are as follows. After 5 minutes of treatment at 95 ° C, a total of 45 times were performed at 95 ° C for 30 seconds, 58 ° C for 45 seconds, and 65 ° C for 30 seconds, and asymmetric PCR was used to generate single-stranded target nucleic acids. The conditions of the asymmetric PCR are as follows. 10 μl PCR amplification solution (ENGNOMICS, Korea), 1 uM forward primer SEQ ID NO: 22, 0.1 uM reverse primer SEQ ID NO: 23, 0.1 uM sdPNA SEQ ID NO: 1, 45 ng gDNA is mixed, and distilled water is added so that the total volume is 20 μl Real-time PCR was performed.

서열번호Sequence number 이름name 서열(N→C)Sequence (N → C) 2323 Sep_FSep_F GCCGCAGCAGCCAGCCCAGCCGCAGCAGCCAGCCCA 2424 SepR_C2SepR_C2 ACCAGCCATCATGTCGGACCACCAGCCATCATGTCGGACC

도 5는 상기 실험에 대한 결과를 그래프로 나타낸 것으로서, Septin 9 유전자가 메틸화 되지 않은 표준 세포(Jurkat)와 Septin 9이 약 90% 메틸화된 SW480 세포에서 분리된 DNA의 비율별 조합을 통한 메틸화 검출 성능 및 민감도를 확인할 수 있다. Figure 5 is a graph showing the results of the experiment, the methylation detection performance through a combination of the ratio of DNA separated from standard cells (Jurkat) where Septin 9 gene is not methylated and SW480 cells where Septin 9 is methylated about 90% And sensitivity.

sdPNA 리덕션 프로브를 이용한 메틸화 검출법의 검증 및 본 발명에 따른 검출 방법의 민감도 테스트를 진행한 결과 본 분석법을 이용하여 5%이하의 메틸화된 DNA를 포함한 검체까지 검출이 가능하다는 것이 확인되었다. As a result of verifying the methylation detection method using the sdPNA reduction probe and conducting a sensitivity test of the detection method according to the present invention, it was confirmed that it is possible to detect samples containing less than 5% of methylated DNA using this analysis method.

<2-3. sdPNA 리덕션 프로브를 이용한 메틸화 검출법의 정확도 테스트><2-3. Accuracy test of methylation detection method using sdPNA reduction probe>

상기 실험 2-1에 따라, sdPNA의 표적 사이트 (septin 9 유전자)의 시토신이 메틸화 되지 않은 것으로 측정된 jurkat gDNA를 대조군으로 하고, 대장암으로 확정되어 표적 사이트 시토신이 메틸화 된 걸로 확인된 임상샘플(20개)을 이용하여, 본 발명에 따른 검출 방법의 정확도 테스트를 실시하였다. PCR(CFX-96 Real-time) 증폭 조건은 다음과 같다. 95 ℃에서 5분 처리 후, 95 ℃에서 30초, 58 ℃에서 45초, 65 ℃에서 30초로 총 45회 수행하였으며, 단일가닥 표적핵산을 생성하기 위해 비대칭 PCR (asymmetric PCR)을 이용하였다. 비대칭 PCR의 조건은 상기 실험 2-2와 같다.According to the experiment 2-1, the jurkat gDNA measured as cytosine of the target site (septin 9 gene) of sdPNA is not methylated is used as a control, and a clinical sample confirmed to be confirmed as colon cancer and the target site cytosine methylated ( 20), the accuracy test of the detection method according to the present invention was performed. PCR (CFX-96 Real-time) amplification conditions are as follows. After 5 minutes of treatment at 95 ° C, a total of 45 times were performed at 95 ° C for 30 seconds, 58 ° C for 45 seconds, and 65 ° C for 30 seconds, and asymmetric PCR was used to generate single-stranded target nucleic acids. The conditions of the asymmetric PCR are the same as in Experiment 2-2 above.

도 6은 Septin 9 유전자가 메틸화 되지 않은 표준 Jurkat gDNA 및 상기 유전자의 메틸화된 Hela, SW480, Lovo 세포(Tao peng et al., 2017) 또는 대장암 임상샘플 20개에서 뽑은 genomic DNA를 이용하여 정확도 테스트를 진행한 결과 본 발명에 따른 검출 방법을 이용하였을 때, 100%의 정확도를 나타낸다는 것을 보여준다.6 is an accuracy test using standard Jurkat gDNA in which Septin 9 gene is not methylated and genomic DNA extracted from 20 methylated Hela, SW480, Lovo cells (Tao peng et al., 2017) or colorectal cancer clinical samples of the gene. The results show that when using the detection method according to the present invention, it shows 100% accuracy.

Claims (11)

메틸화가 일어날 수 있는 유전자(표적 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재 하에, 상기 유전자에 대한 PCR을 수행하는 단계;Performing PCR on the gene in the presence of a reduction probe capable of specifically binding to a nucleotide sequence of a gene (target gene) capable of methylation; 메틸화 되지 않은 유전자(컨트롤 시료의 동일 유전자)의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브의 존재하에, 상기 메틸화 되지 않은 유전자에 대한 PCR을 수행하는 단계;Performing PCR on the unmethylated gene in the presence of a reduction probe capable of specifically binding the nucleotide sequence of the unmethylated gene (the same gene in the control sample); 상기 PCR에서의 ΔTm(melting temperature)과 ΔCt(cycle threshold) 중 적어도 하나 이상을 측정하는 단계; 및Measuring at least one of ΔTm (melting temperature) and ΔCt (cycle threshold) in the PCR; And ΔTm의 수치를 통해 유전자의 메틸화 여부를 판단 또는 ΔΔCt값을 특정하여 메틸화의 여부 및 메틸화 정도를 판단하는 단계; 를 포함하는 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법Determining whether a gene is methylated through a value of ΔTm or determining a ΔΔCt value to determine whether it is methylated and the degree of methylation; Method for detecting methylation of a gene using a reduction probe, comprising: [상기 ΔTm=(메틸화가 일어날 수 있는 유전자와 리덕션 프로브 간의 Tm)-(메틸화 되지 않은 동일 유전자와 리덕션 프로브 간의 Tm), [The ΔTm = (Tm between the gene and the reduction probe where methylation may occur)-(Tm between the same unmethylated gene and the reduction probe), ΔΔCt=(리덕션 프로브의 첨가 없는 유전자 시료의 ΔCt)-(리덕션 프로브를 첨가한 유전자 시료의 ΔCt), ΔΔCt = (ΔCt of the genetic sample without the addition of the reduction probe)-(ΔCt of the genetic sample to which the reduction probe is added), ΔCt=(메틸화되지 않은 유전자의 Ct)-(메틸화된 유전자의 Ct)이다].ΔCt = (Ct of unmethylated gene)-(Ct of methylated gene)]. 제 1항에 있어서, According to claim 1, 상기 리덕션 프로브는 올리고뉴클레오티드, LNA, PNA 및 이들의 혼합으로 구성된 군에서 선택되는 것을 특징으로 하는 유전자의 메틸화 검출 방법.The reduction probe is a method for detecting methylation of a gene, characterized in that it is selected from the group consisting of oligonucleotides, LNA, PNA and mixtures thereof. 제 1항에 있어서, According to claim 1, 상기 표적 유전자의 염기서열은 메틸화된 염기서열, 하이드록시 메틸화된 염기서열, 포르밀 메틸화된 염기서열, 카르복실 메틸화된 염기서열 중 어느 하나 또는 2이상의 조합으로 메틸화된 염기서열을 가지는 것을 특징으로 하는 PNA 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법.The nucleotide sequence of the target gene is characterized by having a nucleotide sequence methylated by any one or a combination of two or more of methylated nucleotide sequence, hydroxy methylated nucleotide sequence, formyl methylated nucleotide sequence, carboxyl methylated nucleotide sequence. Method for detecting methylation of gene using PNA reduction probe. 제 1항에 있어서, According to claim 1, ΔTm은 하기의 식 (1)을 만족하는 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법ΔTm is a method for detecting methylation of a gene using a reduction probe, characterized in that the following equation (1) is satisfied ΔTm=k×mC×n 식 (1)ΔTm = k × mC × n (1) [상기 식에서 k는 상수, mC는 메틸화된 시토신이 1개일 경우의 Tm의 차이, n은 메틸화된 시토신의 개수이다].[In the above formula, k is a constant, mC is a difference in Tm when there is one methylated cytosine, n is the number of methylated cytosine]. 제 1항에 있어서,According to claim 1, 상기 리덕션 프로브는 self-dimer 형성 가능한 서열을 포함하는 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법.The reduction probe is a method for detecting methylation of a gene using a reduction probe, characterized in that it comprises a self-dimer-forming sequence. 제 4항에 있어서, The method of claim 4, 상기 ΔTm은 n=1일 때, 2℃ 이상 5℃ 이하인 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법.When ΔTm is n = 1, a method for detecting methylation of a gene using a reduction probe, characterized in that it is 2 ° C or more and 5 ° C or less. 제 1항에 있어서, According to claim 1, 상기 메틸화 검출 방법은 유전자의 증폭 없이, 상기 비메틸화 또는 메틸화된 유전자와 리덕션 프로브의 ΔTm 을 측정하여 메틸화 여부 및 메틸화 정도를 분석하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법.The methylation detection method is a method for detecting methylation of a gene using a reduction probe that analyzes the degree of methylation and the degree of methylation by measuring ΔTm of the non-methylated or methylated gene and the reduction probe without amplification of the gene. 제 1항에 있어서,According to claim 1, 상기 검출 방법은 표준 PCR, 실시간 PCR(Real Time PCR), 디지털 PCR, 등온 PCR(Isothermal PCR), 절량 PCR, DNA 칩, DNA FISH (Fluorescence in situ hybridization)에서 선택되는 어느 하나인 것을 특징으로 하는 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법.The detection method is any one selected from standard PCR, real-time PCR (Real Time PCR), digital PCR, isothermal PCR (Isothermal PCR), sequencing PCR, DNA chip, DNA FISH (Fluorescence in situ hybridization). Method for detecting methylation of a gene using a probe. 제 1항에 있어서,According to claim 1, 상기 검출 방법은 유전자 증폭 효율 또는 PCR 산물을 검출할 수 있는 방법을 포함하는 유전자의 메틸화 검출 방법.The detection method is a method for detecting methylation of a gene including a method for detecting gene amplification efficiency or PCR products. 메틸화가 일어날 수 있는 유전자의 염기서열과 특이적으로 결합할 수 있는 리덕션 프로브를 포함하는, 제1항 내지 제9항 중 어느 한 항에 따른 유전자의 메틸화 검출 방법에 사용하기 위한 키트.A kit for use in a method for detecting methylation of a gene according to any one of claims 1 to 9, comprising a reduction probe capable of specifically binding a nucleotide sequence of a gene capable of methylation. 리덕션 프로브를 이용한 실시간 PCR을 수행하는 장치에 있어서, In the apparatus for performing a real-time PCR using a reduction probe, 제 1항에 따른 ΔTm 정보를 획득하는 Tm 분석부;Tm analysis unit for obtaining the ΔTm information according to claim 1; 제 1항에 따른 ΔΔCt 정보를 획득하는 Ct 분석부;Ct analysis unit for obtaining the ΔΔCt information according to claim 1; 사전에 설정된 ΔTm 및 ΔΔCt 정보를 바탕으로 상기 획득한 ΔTm 및 ΔΔCt값으로부터 유전자의 메틸화 여부를 판단하는 제어부; 및A control unit for determining whether a gene is methylated from the obtained ΔTm and ΔΔCt values based on preset ΔTm and ΔΔCt information; And 상기 제어부에서 판단된 메틸화 여부, 메틸화 정도(X), 상기 ΔTm 및 ΔΔCt 중 적어도 하나에 기초한 정보를 사용자에게 전달하는 출력부;An output unit that transmits information based on at least one of the methylation degree, the degree of methylation (X), and the ΔTm and ΔΔCt determined by the controller; 를 포함하는 유전자 메틸화 검출용 PCR 장치.PCR device for gene methylation detection comprising a.
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