WO2020096394A1 - Procédé, kit et dispositif d'acp pour la détection de méthylation d'un gène à l'aide d'une sonde de réduction - Google Patents

Procédé, kit et dispositif d'acp pour la détection de méthylation d'un gène à l'aide d'une sonde de réduction 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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어칠 친바야바트
윤경주
전미향
이한우
박희경
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주식회사 시선바이오머티리얼스
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—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
    • C12Q2525/107—Modifications characterised by incorporating a peptide nucleic acid
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    • C12Q2527/00—Reactions demanding special reaction conditions
    • C12Q2527/107—Temperature of melting, i.e. Tm
    • C—CHEMISTRY; METALLURGY
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    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/113—PCR
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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
    • C12Q2537/10—Reactions characterised by the reaction format or use of a specific feature the purpose or use of
    • C12Q2537/165—Mathematical modelling, e.g. logarithm, ratio
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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

La présente invention concerne un procédé, un kit et un dispositif d'ACP pour détecter la méthylation d'un gène à l'aide d'une sonde de réduction qui peut détecter la méthylation d'un gène sans traiter le bisulfite ou une enzyme spécifique à la méthylation. Elle est ainsi simple et présente une sensibilité et une précision élevées dans la détection et est donc utile dans des domaines pour diagnostiquer diverses maladies, y compris le cancer.
PCT/KR2019/015112 2018-11-09 2019-11-08 Procédé, kit et dispositif d'acp pour la détection de méthylation d'un gène à l'aide d'une sonde de réduction WO2020096394A1 (fr)

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KR102019800B1 (ko) * 2018-11-09 2019-09-10 주식회사 시선바이오머티리얼스 리덕션 프로브를 이용한 유전자의 메틸화 검출 방법, 검출 키트 및 pcr 장치
KR20220091891A (ko) * 2020-12-24 2022-07-01 주식회사 시선바이오머티리얼스 폐암 조기 진단을 위한 유전자의 메틸화 검출 방법, 검출키트 및 검출용 핵산의 조성물
KR20220096382A (ko) * 2020-12-31 2022-07-07 주식회사 시선바이오머티리얼스 비메틸화 특이적 프로브를 이용한 유전자의 메틸화 검출 방법 및 검출 키트
KR102575618B1 (ko) 2021-03-22 2023-09-07 에이치엘비파나진 주식회사 가이드 프로브 및 클램핑 프로브를 이용한 표적핵산 증폭방법 및 이를 포함하는 표적핵산 증폭용 조성물
CN113981046A (zh) * 2021-11-05 2022-01-28 朱运峰 一种基于定量pcr技术dna甲基化检测方法及其试剂盒

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