WO2020001416A1 - Method for determining dna methylation level at specific site in biological sample and application thereof - Google Patents

Method for determining dna methylation level at specific site in biological sample and application thereof Download PDF

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WO2020001416A1
WO2020001416A1 PCT/CN2019/092657 CN2019092657W WO2020001416A1 WO 2020001416 A1 WO2020001416 A1 WO 2020001416A1 CN 2019092657 W CN2019092657 W CN 2019092657W WO 2020001416 A1 WO2020001416 A1 WO 2020001416A1
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dna fragment
methylation
dna
tested
primer
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陈琦
梁昊原
谷东风
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深圳市圣必智科技开发有限公司
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    • 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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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the invention relates to the technical field of early detection of diseases, in particular to a method for measuring DNA methylation level of a specific site in a biological sample and application thereof.
  • DNA methylation is mainly expressed in the mammalian genome as 5-mC, 5-methyl-cytosine, and occurs mostly in two adjacent cytosines. (Cytosine, C) and Guanine (G) dinucleotide (CpG) on C. 60% to 90% of CpG in the mammalian genome are methylated. Many CpG clusters form CpG islands, which are mostly located in the core sequence of the structural gene promoter and the transcription initiation point. They participate in changes in methylation levels. Regulation of gene expression. Recent studies have found that the abnormality of DNA methylation is related to many major diseases such as tumors and mental diseases. The degree of methylation of CpG sites in some genes may have become a potential marker for the occurrence of these diseases. Rapid detection of the methylation status of CpG sites of these disease-related genes is very important for the timely detection and treatment of these diseases.
  • the main purpose of the present invention is to provide a simple and rapid method for measuring CpG methylation at specific sites, which aims to solve the technical problems of complicated operation in the prior art and inconvenience in widespread application in clinical and scientific research fields.
  • the present invention provides a method for determining the level of DNA methylation at a specific site in a biological sample.
  • the method includes the following steps:
  • the DNA fragment modified by sodium bisulfite is used as a template DNA fragment, and the first and second primers are used to amplify the template DNA fragment from both sides of the template DNA fragment, and the first primer and the second primer are amplified
  • the template DNA fragment includes the CpG site of the DNA fragment to be tested;
  • alkaline phosphatase to treat PCR products after PCR amplification, remove excess first primer, second primer and dNTPs, and purify and recover;
  • the methylation level of the DNA fragment to be tested was calculated according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested.
  • the step of performing a methylation-specific third primer-guided single-base extension labeling reaction in two separate reaction systems includes:
  • fluorescein dCTP is used to detect the methylation of the CpG site of the DNA fragment to be tested
  • luciferin dUTP is used to detect the demethylation of the CpG site of the DNA fragment to be tested
  • the step of calculating the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested includes:
  • the dCTP fluorescence polarization value and dUTP fluorescence polarization value were used to calculate the methylation level of the DNA fragment to be tested.
  • the distance between the first primer and the second primer and the CpG site of the DNA fragment to be detected is greater than the length of the third primer.
  • the length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%.
  • the sequence of the third primer specifically binds to a corresponding complementary sequence of a 20-30 bp sequence upstream of the CpG site of the DNA fragment to be tested.
  • the third primer is 22-25bp in length and does not contain a CpG site.
  • the test DNA fragment is derived from a blood sample, a pathological sample, an organ or a tissue cell sample, and a recombinant DNA or a synthetic DNA.
  • the pathological sample is a tumor tissue sample.
  • the tumor tissue sample is a lung cancer, gastric cancer or liver cancer tissue sample.
  • the invention also provides an application of the method for measuring DNA methylation level of a specific site in a biological sample to detecting target DNA methylation.
  • the method for measuring DNA methylation levels of specific sites in a biological sample and its application according to the present invention adopt the above technical scheme, and achieve the following technical effects: the fluorescence polarization amount of a marker is used to determine Determining the methylation status of the target CpG overcomes the shortcomings of existing DNA methylation detection technologies, such as the need for separation and purification in electrophoretic analysis, and the complexity of the operation. It is suitable for a large number of clinically valuable low-concentration DNA samples.
  • FIG. 1 is a schematic flow chart of a preferred embodiment of a method for measuring DNA methylation levels at specific sites in a biological sample according to the present invention.
  • FIG. 1 is a schematic flowchart of a preferred embodiment of a method for measuring DNA methylation level at a specific site in a biological sample according to the present invention.
  • the experimental methods in the following examples are conventional methods.
  • the LINE1 element in the human genome (NCBI sequence number: X58075, the specific sequence is shown in SEQ ID NO. 1) is used as the research object, and the methylation of the CpG site in the promoter region is detected.
  • the specific sequence of the LINE1 element in the human genome (SEQ ID NO.1) is:
  • the test DNA fragment may also be derived from a pathological sample, an organ or tissue cell sample, and recombinant DNA or synthetic DNA.
  • the pathological sample is preferably a tumor tissue sample, and the tumor tissue sample is preferably a lung cancer, gastric cancer, or liver cancer tissue sample.
  • the embodiment of the present invention is described by taking the application of genomic DNA methylation detection in human peripheral blood as an example.
  • the method includes the following steps:
  • Step S1 obtaining a biological sample, and extracting a DNA fragment to be tested in the biological sample;
  • human peripheral blood genomic DNA is extracted according to a conventional phenol / chloroform method.
  • Step S2 using sodium bisulfite to modify the DNA fragment to be tested, and deaminating the unmethylated cytosine in the DNA fragment to be converted into uracil;
  • 1 microgram of human peripheral blood genomic DNA is treated with sodium bisulfite, so that the unmethylated cytosine in the test DNA is deaminated into uracil and stored at -20 ° C.
  • reaction parameters of the test DNA treated with sodium bisulfite are:
  • test DNA 1 ⁇ g is incubated with 0.3M NaOH at 42 ° C for 20 minutes, then 95 ° C for 3 minutes and 0 ° C for 1 minute;
  • Step S3 using the DNA fragment modified by sodium bisulfite as a template DNA fragment, and using a first primer and a second primer to perform PCR amplification on the template DNA fragment from both sides of the template DNA fragment, the first primer and the second
  • the template DNA fragment amplified by the primer includes the CpG site of the DNA fragment to be tested;
  • Second primer CATCTCTAAAAAATACCAAACAA (SEQ ID NO. 3);
  • PCR reaction conditions 15 ⁇ l reaction system contains: 75nM first and second primers, 50nM dNTPs, 1X PCR buffer, and 1 unit of Taq DNA polymerase; after the mixture is placed at 95 ° C for 1 minute, The following 30 cycles were performed: heating at 95 ° C for 1 minute, heating at 60 ° C for 1 minute, and heating at 72 ° C for 1 minute.
  • Step S4 treating the PCR product amplified by PCR with alkaline phosphatase, removing excess first primers, second primers, and dNTPs, and purifying and recovering them;
  • the PCR product is digested with enzymes to remove unreacted primers and dNTPs.
  • the enzyme digestion system contains: 3.75 ⁇ l of 10 ⁇ alkaline phosphatase reaction buffer, 3.4 units of alkaline phosphatase, 2.3 units of exonuclease and 13 ⁇ l of PCR product, made up to 37.5 ⁇ l with deionized water. The mixture was left to react at 37 ° C for 45 minutes, and then heated at 85 ° C for 15 minutes to inactivate the enzyme.
  • Step S5 Take an equal amount of the purified PCR products separately, and perform methylation-specific third primer-guided single-base extension labeling reactions in two separate reaction systems to detect the methyl group at the CpG site of the DNA fragment to be tested. And demethylated fluorescence polarization values;
  • the total volume of the extension labeling reaction is 15 ⁇ l, containing 1.5 ⁇ l of the enzyme-digested PCR product, 0.75 unit Tag polymerase, 1X PCR Buffer, 3.75 ⁇ M third primer, 1.125 ⁇ M fluorescein dCTP or 0.5625 ⁇ M fluorescein dUTP.
  • Reaction conditions After denaturing at 95 ° C for 3 minutes, the following 30 cycles were performed: incubation at 95 ° C for 30 seconds, and incubation at 55 ° C for 30 seconds.
  • fluorescein dCTP is used to detect the methylation of the CpG site of the DNA fragment to be tested
  • luciferin dUTP is used to detect the demethylation of the CpG site of the DNA fragment to be tested.
  • Step S6 Calculate the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested.
  • the step of calculating the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization value of the CpG site of the DNA fragment to be tested includes: according to the dCTP fluorescence polarization value and dUTP fluorescence The polarization value calculates the methylation level of the DNA fragment to be tested. Immediately extend the labeled product to FP (Flourence polarization (fluorescence polarization) detector directly measured its fluorescence polarization value, the excitation wavelength is 544nm, the emission wavelength is 595nm.
  • FP Fluorescence polarization
  • the methylation level of the test site is estimated according to the following formula:
  • Methylation level% 100 ⁇ FP dCTP / (FP dCTP + FP dUTP );
  • the distance between the first primer and the second primer and the CpG site of the DNA fragment to be detected is greater than the length of the third primer.
  • the length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%.
  • the sequence of the third primer specifically binds to a corresponding complementary sequence of a 20-30 bp sequence upstream of the CpG site of the DNA fragment to be tested.
  • the third primer is 22-25bp in length and does not contain a CpG site.
  • the invention also provides an application of the method for measuring DNA methylation level of a specific site in a biological sample to detecting target DNA methylation.
  • the method for measuring the methylation level of specific sites in a biological sample uses the measurement of the fluorescence polarization amount of a marker to determine the methylation status of a target CpG, which overcomes existing DNA methylation detection techniques such as electrophoretic analysis It needs to be separated, purified, and complicated in operation. It has the advantages of simplicity, fastness, high throughput, low cost, and no need for separation and purification. It is suitable for a large number of clinically valuable low-concentration DNA samples.
  • the method for measuring DNA methylation levels of specific sites in a biological sample and its application according to the present invention adopt the above technical scheme, and achieve the following technical effects: the fluorescence polarization amount of a marker is used to determine Determining the methylation status of the target CpG overcomes the shortcomings of existing DNA methylation detection technologies, such as the need for separation and purification in electrophoretic analysis, and the complexity of the operation. It is suitable for a large number of clinically valuable low-concentration DNA samples.
  • tagtgagatg 250 The following is the case:

Abstract

A method for determining a DNA methylation level at a specific site in a biological sample and an application thereof. The method comprises steps of: acquiring a biological sample and extracting a DNA fragment to be tested from the biological sample; modifying the DNA fragment to be tested to deaminize unmethylated cytosine in the DNA fragment so as to convert the cytosine into uracil; using the modified DNA fragment as a template DNA fragment, and using a first primer and a second primer to perform PCR amplification on the template DNA fragment from both sides of the template DNA fragment, respectively; processing the PCR product amplified by PCR to remove excessive first primers, second primers, and dNTPs, and performing purification and recovery; detecting methylation and demethylation fluorescence polarization values of a CpG site of the DNA fragment; and calculating, according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested, the methylation level of the DNA fragment.

Description

测定生物样本中特异位点DNA甲基化水平的方法及其应用Method for measuring DNA methylation level of specific site in biological sample and its application 技术领域Technical field
本发明涉及疾病早期检测技术领域，尤其涉及一种测定生物样本中特异位点DNA甲基化水平的方法及其应用。The invention relates to the technical field of early detection of diseases, in particular to a method for measuring DNA methylation level of a specific site in a biological sample and application thereof.
背景技术Background technique
DNA甲基化作为一种重要的表观遗传修饰方式，在哺乳动物基因组中主要表现为5-甲基胞嘧啶(5mC,5-methyl-cytosine)，且多发生在两个相邻的胞嘧啶(Cytosine，C)和鸟嘌呤(Guanine，G)二核苷酸(CpG)的C上。哺乳动物基因组中60％～90％的CpG都被甲基化，很多个CpG成簇的组成CpG岛，多位于结构基因启动子的核心序列和转录起始点，通过甲基化水平的变化，参与基因的表达调控。近年来的研究发现DNA甲基化的异常与很多诸如肿瘤、精神疾病等重大疾病存在相关，一些基因CpG位点的甲基化程度高低可能已成为这些疾病发生的潜在标志物。快速检测这些与疾病相关基因的CpG位点的甲基化状态，对于及时发现和治疗这些疾病非常重要。DNA methylation, as an important epigenetic modification, is mainly expressed in the mammalian genome as 5-mC, 5-methyl-cytosine, and occurs mostly in two adjacent cytosines. (Cytosine, C) and Guanine (G) dinucleotide (CpG) on C. 60% to 90% of CpG in the mammalian genome are methylated. Many CpG clusters form CpG islands, which are mostly located in the core sequence of the structural gene promoter and the transcription initiation point. They participate in changes in methylation levels. Regulation of gene expression. Recent studies have found that the abnormality of DNA methylation is related to many major diseases such as tumors and mental diseases. The degree of methylation of CpG sites in some genes may have become a potential marker for the occurrence of these diseases. Rapid detection of the methylation status of CpG sites of these disease-related genes is very important for the timely detection and treatment of these diseases.
目前针对基因特异CpG位点甲基化检测多数是基于亚硫酸氢钠转换和PCR扩增基础上进行的。基因组DNA经亚硫酸氢纳处理后，甲基化的C保持不变，而非甲基化的C转换尿嘧啶(Uracil，U)并经PCR扩增后变成T。这样对于不完全甲基化的CpG位点C位置就变成了C/T的混合，对C的定量就可反映该CpG位点的甲基化水平。目前常用的定量方法多数是基于甲基化特异引物的单核苷酸延伸标记(Ms-SnuPE)，然后再依据不同的标记进行分离和定量，虽然可以测定多个位点的甲基化，但是操作复杂，需要较昂贵的仪器和设备，成本也较高；基于限制性内切酶方法或者基于同位素标记法需通过DNA胶分离，然后进行定量，虽然成本相对较低，但需要复杂的分离或转移步骤，造成定量结果偏差较大，亦或涉及放射性标记；基于生物素标记或荧光标记能量转移(FRET)等检测方法，虽然增加了灵敏度，但是需要标记、纯化和分离等步骤，增加了操作的复杂性；基于荧光探针的实时定量PCR技术，虽然提高了检测的灵敏度，但是由于探针长度和特异性的要求高，不能检测所有位点。现有方法存在的不足，限制了其在临床和科研领域的广泛应用。At present, the detection of gene-specific CpG site methylation is mostly based on sodium bisulfite conversion and PCR amplification. After genomic DNA was treated with sodium bisulfite, methylated C remained unchanged, while non-methylated C converted to uracil (Uracil, U) and amplified by PCR to become T. In this way, the C position of the incompletely methylated CpG site becomes a C / T mixture, and the quantification of C can reflect the methylation level of the CpG site. Most of the currently used quantitative methods are single nucleotide extension tags (Ms-SnuPE) based on methylation-specific primers, and then separated and quantified based on different tags. Although methylation can be measured at multiple sites, The operation is complicated, requiring more expensive instruments and equipment, and the cost is higher; the restriction enzyme method or the isotope labeling method need to be separated by DNA gel and then quantified. Although the cost is relatively low, complex separation or The transfer step causes large deviations in quantitative results or involves radiolabeling. Although detection methods based on biotin labeling or fluorescent labeling energy transfer (FRET) increase sensitivity, they require steps such as labeling, purification, and separation, and increase operations. Complexity; real-time quantitative PCR technology based on fluorescent probes, although the detection sensitivity is improved, but due to the high requirements on the length and specificity of the probes, all sites cannot be detected. The shortcomings of the existing methods limit their wide application in clinical and scientific research fields.
技术问题technical problem
本发明的主要目的在于提供一种简便、快速测定特异位点CpG甲基化的方法，旨在解决现有技术操作复杂，不方便在临床和科研领域的广泛应用的技术问题。The main purpose of the present invention is to provide a simple and rapid method for measuring CpG methylation at specific sites, which aims to solve the technical problems of complicated operation in the prior art and inconvenience in widespread application in clinical and scientific research fields.
技术解决方案Technical solutions
为实现上述目的，本发明提供了一种测定生物样本中特异位点DNA甲基化水平的方法，该方法包括如下步骤：In order to achieve the above object, the present invention provides a method for determining the level of DNA methylation at a specific site in a biological sample. The method includes the following steps:
获取生物样本，并提取生物样本中的待测DNA片段；Obtain a biological sample and extract the DNA fragments to be tested in the biological sample;
采用亚硫酸氢钠修饰待测DNA片段，将待测DNA片段中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶；Modifying the DNA fragment to be tested with sodium bisulfite to deaminate cytosine that is not methylated into uracil in the DNA fragment to be tested;
以亚硫酸氢钠修饰后的DNA片段为模板DNA片段，以第一引物和第二引物分别从模板DNA片段两侧对模板DNA片段进行PCR扩增，所述第一引物和第二引物扩增的模板DNA片段包括待测DNA片段的CpG位点；The DNA fragment modified by sodium bisulfite is used as a template DNA fragment, and the first and second primers are used to amplify the template DNA fragment from both sides of the template DNA fragment, and the first primer and the second primer are amplified The template DNA fragment includes the CpG site of the DNA fragment to be tested;
采用碱性磷酸酶处理经过PCR扩增后的PCR产物，去除多余的第一引物、第二引物和dNTPs，并纯化回收；Use alkaline phosphatase to treat PCR products after PCR amplification, remove excess first primer, second primer and dNTPs, and purify and recover;
分别取等量纯化后的PCR产物，在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应，检测待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值；Take equal amounts of purified PCR products, and perform methylation-specific third primer-guided single-base extension labeling reactions in two separate reaction systems to detect the methylation and demethylation of the CpG site of the test DNA fragment. Methylation fluorescence polarization value;
根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平。The methylation level of the DNA fragment to be tested was calculated according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested.
优选地，所述在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应的步骤包括：Preferably, the step of performing a methylation-specific third primer-guided single-base extension labeling reaction in two separate reaction systems includes:
反应一，采用荧光素dCTP检测待测DNA片段CpG位点的甲基化；In reaction one, fluorescein dCTP is used to detect the methylation of the CpG site of the DNA fragment to be tested;
反应二，采用荧光素dUTP检测待测DNA片段CpG位点的去甲基化；In reaction two, luciferin dUTP is used to detect the demethylation of the CpG site of the DNA fragment to be tested;
所述根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平的步骤包括：The step of calculating the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested includes:
根据dCTP荧光偏振值和dUTP荧光偏振值计算待测DNA片段的甲基化水平。The dCTP fluorescence polarization value and dUTP fluorescence polarization value were used to calculate the methylation level of the DNA fragment to be tested.
优选地，所述第一引物和第二引物与待测DNA片段CpG位点的距离大于第三引物的长度。Preferably, the distance between the first primer and the second primer and the CpG site of the DNA fragment to be detected is greater than the length of the third primer.
优选地，所述第一引物和第二引物的长度为20～30bp，GC含量为55-60％。Preferably, the length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%.
优选地，所述第三引物的序列与待测DNA片段CpG位点上游20～30bp序列的对应互补序列特异结合。Preferably, the sequence of the third primer specifically binds to a corresponding complementary sequence of a 20-30 bp sequence upstream of the CpG site of the DNA fragment to be tested.
优选地，所述第三引物的长度为22-25bp，且不含有CpG位点。Preferably, the third primer is 22-25bp in length and does not contain a CpG site.
优选地，所述待测DNA片段来源于血液样本、病理样本、器官或组织细胞样本，以及重组DNA或合成DNA。Preferably, the test DNA fragment is derived from a blood sample, a pathological sample, an organ or a tissue cell sample, and a recombinant DNA or a synthetic DNA.
优选地，所述病理样本为肿瘤组织样本。Preferably, the pathological sample is a tumor tissue sample.
优选地，所述肿瘤组织样本为肺癌、胃癌或肝癌组织样本。Preferably, the tumor tissue sample is a lung cancer, gastric cancer or liver cancer tissue sample.
本发明还提供一种所述测定生物样本中特异位点DNA甲基化水平的方法在检测目标DNA甲基化中的应用。The invention also provides an application of the method for measuring DNA methylation level of a specific site in a biological sample to detecting target DNA methylation.
有益效果Beneficial effect
相较于现有技术，本发明所述一种测定生物样本中特异位点DNA甲基化水平的方法及其应用采用上述技术方案，达到了如下技术效果：采用测定标记物的荧光偏振量来确定目标CpG的甲基化状态，克服了现有DNA甲基化检测技术诸如电泳分析中需要分离、纯化，操作复杂的不足，具有简便、快捷、高通量、低成本、无需分离纯化等优点，适合大量检测临床珍贵的低浓度DNA样本的甲基化。Compared with the prior art, the method for measuring DNA methylation levels of specific sites in a biological sample and its application according to the present invention adopt the above technical scheme, and achieve the following technical effects: the fluorescence polarization amount of a marker is used to determine Determining the methylation status of the target CpG overcomes the shortcomings of existing DNA methylation detection technologies, such as the need for separation and purification in electrophoretic analysis, and the complexity of the operation. It is suitable for a large number of clinically valuable low-concentration DNA samples.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是本发明测定生物样本中特异位点DNA甲基化水平的方法较佳实施例的流程示意图。FIG. 1 is a schematic flow chart of a preferred embodiment of a method for measuring DNA methylation levels at specific sites in a biological sample according to the present invention.
本发明目的实现、功能特点及优点将结合实施例，参照附图做进一步说明。The purpose, function characteristics and advantages of the present invention will be further described with reference to the embodiments and the accompanying drawings.
本发明的实施方式Embodiments of the invention
为更进一步阐述本发明为达成上述目的所采取的技术手段及功效，以下结合较佳实施例，对本发明的具体实施方式进行详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。In order to further explain the technical means and effects adopted by the present invention to achieve the above-mentioned objectives, specific implementations of the present invention will be described in detail below with reference to preferred embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.
参照图1所示，图1是本发明测定生物样本中特异位点DNA甲基化水平的方法较佳实施例的流程示意图。Referring to FIG. 1, FIG. 1 is a schematic flowchart of a preferred embodiment of a method for measuring DNA methylation level at a specific site in a biological sample according to the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中以人类基因组中的LINE1元件(NCBI序列号为X58075，具体序列如SEQ ID NO.1所示)为研究对象，对其启动子区部分CpG位点的甲基化进行检测。Unless otherwise specified, the experimental methods in the following examples are conventional methods. In the following examples, the LINE1 element in the human genome (NCBI sequence number: X58075, the specific sequence is shown in SEQ ID NO. 1) is used as the research object, and the methylation of the CpG site in the promoter region is detected.
人类基因组中的LINE1元件具体序列（SEQ ID NO.1）为：The specific sequence of the LINE1 element in the human genome (SEQ ID NO.1) is:
ttttttgagt taggtgtggg atatagtttc gtggtgcgtc gttttttaag tcggtttgaattttttgagt taggtgtggg atatagtttc gtggtgcgtc gttttttaag tcggtttgaa
aagcgtaata ttcgggtggg agtgattcga ttttttaggt gcgttcgtta tttttttttt aagcgtaata ttcgggtggg agtgattcga ttttttaggt gcgttcgtta tttttttttt
tgattcggaa agggaatttt ttgatttttt gcgtttttta ggtgaggtaa tgtttcgttt tgattcggaa agggaatttt ttgatttttt gcgtttttta ggtgaggtaa tgtttcgttt
tgtttcggtt cgcgtacggt gcgcgtatat agtggtttgc gtttattgtt tggtattttttgtttcggtt cgcgtacggt gcgcgtatat agtggtttgc gtttattgtt tggtattttt
tagtgagatgtagtgagatg
作为其他实施例，所述待测DNA片段还可以来源于病理样本、器官或组织细胞样本，以及重组DNA或合成DNA。病理样本优选为肿瘤组织样本，肿瘤组织样本优选为肺癌、胃癌或肝癌组织样本。As another embodiment, the test DNA fragment may also be derived from a pathological sample, an organ or tissue cell sample, and recombinant DNA or synthetic DNA. The pathological sample is preferably a tumor tissue sample, and the tumor tissue sample is preferably a lung cancer, gastric cancer, or liver cancer tissue sample.
本发明实施例以在人类外周血基因组DNA甲基化检测的应用为例展开说明，该方法包括如下步骤：The embodiment of the present invention is described by taking the application of genomic DNA methylation detection in human peripheral blood as an example. The method includes the following steps:
步骤S1：获取生物样本，并提取生物样本中的待测DNA片段；Step S1: obtaining a biological sample, and extracting a DNA fragment to be tested in the biological sample;
在本实施例中，按照传统苯酚/氯仿方法抽提人类外周血基因组DNA。In this embodiment, human peripheral blood genomic DNA is extracted according to a conventional phenol / chloroform method.
步骤S2：采用亚硫酸氢钠修饰待测DNA片段，将待测DNA片段中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶；Step S2: using sodium bisulfite to modify the DNA fragment to be tested, and deaminating the unmethylated cytosine in the DNA fragment to be converted into uracil;
在本实施例中，将1微克人类外周血基因组DNA采用亚硫酸氢钠处理，使所述待测DNA中未甲基化的胞嘧啶脱氨基变成尿嘧啶，放在-20℃保存。In this embodiment, 1 microgram of human peripheral blood genomic DNA is treated with sodium bisulfite, so that the unmethylated cytosine in the test DNA is deaminated into uracil and stored at -20 ° C.
所述的亚硫酸氢钠处理待测DNA的反应参数为：The reaction parameters of the test DNA treated with sodium bisulfite are:
(1)1μg待测DNA与0.3M NaOH一起在42℃孵育20分钟，然后95℃孵育3分钟和0℃孵育1分钟；(1) 1 μg of the test DNA is incubated with 0.3M NaOH at 42 ° C for 20 minutes, then 95 ° C for 3 minutes and 0 ° C for 1 minute;
(2)接下来同pH 5.0的亚硫酸氢纳(终浓度5.0M)和对苯二酚(终浓度0.5mM)在55℃孵育16小时，并保持避光；(2) Next incubate with sodium bisulfite (final concentration 5.0M) and hydroquinone (final concentration 0.5mM) at 55 ° C for 16 hours, and keep away from light;
(3)处理后DNA用Promega Wizard DNA Clean System进行纯化，并溶解在0.3M的NaOH中，37℃孵育15分钟后用3M的醋酸铵中和至pH7.0；(3) Promega Wizard for processed DNA The DNA Clean System was purified and dissolved in 0.3M NaOH. After incubation at 37 ° C for 15 minutes, it was neutralized with 3M ammonium acetate to pH 7.0;
(4)75％的乙醇沉淀中和产物，并溶解在20μl TE中，保存在-20℃。(4) The neutralized product was precipitated by 75% ethanol, and dissolved in 20 μl of TE, and stored at -20 ° C.
步骤S3：以亚硫酸氢钠修饰后的DNA片段为模板DNA片段，以第一引物和第二引物分别从模板DNA片段两侧对模板DNA片段进行PCR扩增，所述第一引物和第二引物扩增的模板DNA片段包括待测DNA片段的CpG位点；Step S3: using the DNA fragment modified by sodium bisulfite as a template DNA fragment, and using a first primer and a second primer to perform PCR amplification on the template DNA fragment from both sides of the template DNA fragment, the first primer and the second The template DNA fragment amplified by the primer includes the CpG site of the DNA fragment to be tested;
在本实施例中，In this embodiment,
第一引物：TTTTTTGAGTTAGGTGTGTGGG(SEQ ID NO.2)；First primer: TTTTTTGAGTTAGGTGTGTGGG (SEQ ID NO. 2);
第二引物：CATCTCTAAAAAATACCAAACAA(SEQ ID NO.3)；Second primer: CATCTCTAAAAAATACCAAACAA (SEQ ID NO. 3);
PCR反应条件：15微升反应体系包含：75nM的第一引物和第二引物，50nM的dNTPs，1X PCR缓冲液，和1个单位的Taq DNA聚合酶；混合物置于95℃反应1分钟后，进行以下30个循环：95℃加热1分钟，60℃加热1分钟，72℃加热1分钟。PCR reaction conditions: 15 μl reaction system contains: 75nM first and second primers, 50nM dNTPs, 1X PCR buffer, and 1 unit of Taq DNA polymerase; after the mixture is placed at 95 ° C for 1 minute, The following 30 cycles were performed: heating at 95 ° C for 1 minute, heating at 60 ° C for 1 minute, and heating at 72 ° C for 1 minute.
步骤S4：采用碱性磷酸酶处理经过PCR扩增后的PCR产物，去除多余的第一引物、第二引物和dNTPs，并纯化回收；Step S4: treating the PCR product amplified by PCR with alkaline phosphatase, removing excess first primers, second primers, and dNTPs, and purifying and recovering them;
在本实施例中，对PCR产物进行酶切消化处理以去除未反应的引物和dNTPs。酶切体系中含：3.75μl 10×碱性磷酸酶反应缓冲液,3.4单位碱性磷酸酶，2.3单位核酸外切酶和13μl PCR产物，用去离子水补至37.5μl。将混合物置于37℃反应45分钟，再于85℃加热15分钟使酶失活。In this embodiment, the PCR product is digested with enzymes to remove unreacted primers and dNTPs. The enzyme digestion system contains: 3.75 μl of 10 × alkaline phosphatase reaction buffer, 3.4 units of alkaline phosphatase, 2.3 units of exonuclease and 13 μl of PCR product, made up to 37.5 μl with deionized water. The mixture was left to react at 37 ° C for 45 minutes, and then heated at 85 ° C for 15 minutes to inactivate the enzyme.
步骤S5：分别取等量纯化后的PCR产物，在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应，检测待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值；Step S5: Take an equal amount of the purified PCR products separately, and perform methylation-specific third primer-guided single-base extension labeling reactions in two separate reaction systems to detect the methyl group at the CpG site of the DNA fragment to be tested. And demethylated fluorescence polarization values;
延伸标记反应总体积15μl，含1.5μl酶消化过的PCR产物，0.75单位Tag聚合酶，1XPCR Buffer，3.75μM第三引物，1.125μM荧光素dCTP或0.5625μM荧光素dUTP。反应条件：95℃变性3分钟后，进行以下30个循环：95℃温育30秒，55℃温育30秒。The total volume of the extension labeling reaction is 15 μl, containing 1.5 μl of the enzyme-digested PCR product, 0.75 unit Tag polymerase, 1X PCR Buffer, 3.75 μM third primer, 1.125 μM fluorescein dCTP or 0.5625 μM fluorescein dUTP. Reaction conditions: After denaturing at 95 ° C for 3 minutes, the following 30 cycles were performed: incubation at 95 ° C for 30 seconds, and incubation at 55 ° C for 30 seconds.
第三引物：GGTGGGAGTGATT(SEQ ID NO.4)。Third primer: GGTGGGAGTGATT (SEQ ID NO. 4).
在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应的步骤包括：The steps of performing methylation-specific third primer-guided single-base extension labeling reactions in two separate reaction systems include:
反应一，采用荧光素dCTP检测待测DNA片段CpG位点的甲基化；In reaction one, fluorescein dCTP is used to detect the methylation of the CpG site of the DNA fragment to be tested;
反应二，采用荧光素dUTP检测待测DNA片段CpG位点的去甲基化。In reaction two, luciferin dUTP is used to detect the demethylation of the CpG site of the DNA fragment to be tested.
步骤S6：根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平。Step S6: Calculate the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested.
在本实施例中，所述根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平的步骤包括：根据dCTP荧光偏振值和dUTP荧光偏振值计算待测DNA片段的甲基化水平。即将延伸标记后的产物置于FP（Flourence polarization，荧光偏振）检测仪上直接测定其荧光偏振值，激发波长为544nm，发射波长为595nm。In this embodiment, the step of calculating the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization value of the CpG site of the DNA fragment to be tested includes: according to the dCTP fluorescence polarization value and dUTP fluorescence The polarization value calculates the methylation level of the DNA fragment to be tested. Immediately extend the labeled product to FP (Flourence polarization (fluorescence polarization) detector directly measured its fluorescence polarization value, the excitation wavelength is 544nm, the emission wavelength is 595nm.
每一个DNA浓度点的FP值减去零浓度空白点FP值后，按照如下公式估算待测位点甲基化水平：After subtracting the zero concentration blank point FP value from the FP value of each DNA concentration point, the methylation level of the test site is estimated according to the following formula:
甲基化水平％＝100×FP dCTP/(FP dCTP+FP dUTP)； Methylation level% = 100 × FP dCTP / (FP dCTP + FP dUTP );
通过实验发现，20-100ng范围内，本发明利用第三引物对CpG1的甲基化水平检测表现出很好的稳定性，其值为：57.13±0.26％It was found through experiments that the methylation level detection of CpG1 using the third primer in the present invention showed good stability in the range of 20-100ng, and the value was: 57.13 ± 0.26%
作为较佳实施例，所述第一引物和第二引物与待测DNA片段CpG位点的距离大于第三引物的长度。所述第一引物和第二引物的长度为20～30bp，GC含量为55-60％。所述第三引物的序列与待测DNA片段CpG位点上游20～30bp序列的对应互补序列特异结合。第三引物的长度为22-25bp，且不含有CpG位点。As a preferred embodiment, the distance between the first primer and the second primer and the CpG site of the DNA fragment to be detected is greater than the length of the third primer. The length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%. The sequence of the third primer specifically binds to a corresponding complementary sequence of a 20-30 bp sequence upstream of the CpG site of the DNA fragment to be tested. The third primer is 22-25bp in length and does not contain a CpG site.
本发明还提供一种所述测定生物样本中特异位点DNA甲基化水平的方法在检测目标DNA甲基化中的应用。The invention also provides an application of the method for measuring DNA methylation level of a specific site in a biological sample to detecting target DNA methylation.
本发明提供的测定生物样本中特异位点DNA甲基化水平的方法，采用测定标记物的荧光偏振量来确定目标CpG的甲基化状态，克服了现有DNA甲基化检测技术诸如电泳分析中需要分离、纯化，操作复杂的不足，具有简便、快捷、高通量、低成本、无需分离纯化等优点，适合大量检测临床珍贵的低浓度DNA样本的甲基化。The method for measuring the methylation level of specific sites in a biological sample provided by the present invention uses the measurement of the fluorescence polarization amount of a marker to determine the methylation status of a target CpG, which overcomes existing DNA methylation detection techniques such as electrophoretic analysis It needs to be separated, purified, and complicated in operation. It has the advantages of simplicity, fastness, high throughput, low cost, and no need for separation and purification. It is suitable for a large number of clinically valuable low-concentration DNA samples.
以上仅为本发明的优选实施例，并非因此限制本发明的专利范围，凡是利用本发明说明书及附图内容所作的等效结构或等效功能变换，或直接或间接运用在其他相关的技术领域，均同理包括在本发明的专利保护范围内。The above are only preferred embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent functional transformation made by using the description and drawings of the present invention, or directly or indirectly used in other related technical fields All are included in the patent protection scope of the present invention.
工业实用性Industrial applicability
相较于现有技术，本发明所述一种测定生物样本中特异位点DNA甲基化水平的方法及其应用采用上述技术方案，达到了如下技术效果：采用测定标记物的荧光偏振量来确定目标CpG的甲基化状态，克服了现有DNA甲基化检测技术诸如电泳分析中需要分离、纯化，操作复杂的不足，具有简便、快捷、高通量、低成本、无需分离纯化等优点，适合大量检测临床珍贵的低浓度DNA样本的甲基化。Compared with the prior art, the method for measuring DNA methylation levels of specific sites in a biological sample and its application according to the present invention adopt the above technical scheme, and achieve the following technical effects: the fluorescence polarization amount of a marker is used to determine Determining the methylation status of the target CpG overcomes the shortcomings of existing DNA methylation detection technologies, such as the need for separation and purification in electrophoretic analysis, and the complexity of the operation. It is suitable for a large number of clinically valuable low-concentration DNA samples.
序列表自由内容Sequence Listing Free Content
<110> 深圳市圣必智科技开发有限公司<110> Shenzhen Shengbizhi Technology Development Co., Ltd.
<120>测定生物样本中特异位点DNA甲基化水平的方法及其应用<120> Method for measuring DNA methylation level at specific sites in biological samples and its application
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ttttttgagt taggtgtggg atatagtttc gtggtgcgtc gttttttaag tcggtttgaa       60ttttttgagt taggtgtggg atatagtttc gtggtgcgtc gttttttaag tcggtttgaa 60
aagcgtaata ttcgggtggg agtgattcga ttttttaggt gcgttcgtta tttttttttt        120aagcgtaata ttcgggtggg agtgattcga ttttttaggt gcgttcgtta tttttttttt 120
tgattcggaa agggaatttt ttgatttttt gcgtttttta ggtgaggtaa tgtttcgttt        180tgattcggaa agggaatttt ttgatttttt gcgtttttta ggtgaggtaa tgtttcgttt 180
tgtttcggtt cgcgtacggt gcgcgtatat agtggtttgc gtttattgtt tggtattttt       240tgtttcggtt cgcgtacggt gcgcgtatat agtggtttgc gtttattgtt tggtattttt 240
tagtgagatg                                               250tagtgagatg 250. The following is the case:
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<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial sequence (unknown)
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Ttttttgagttaggtgtgtggg          22Ttttttgagttaggtgtgtggg twenty two
 Zh
<210> 3<210> 3
<211> 23<211> 23
<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial sequence (unknown)
<400> 3<400> 3
catctctaaaaaataccaaacaa         23catctctaaaaaataccaaacaa twenty three
 Zh
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<211> 13<211> 13
<212> DNA<212> DNA
<213> 人工序列(未知)<213> Artificial sequence (unknown)
<400> 4<400> 4
ggtgggagtgatt            13ggtgggagtgatt 13 13

Claims (10)

  1. 一种测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，该方法包括如下步骤：A method for measuring the level of DNA methylation at a specific site in a biological sample, characterized in that the method includes the following steps:
    获取生物样本，并提取生物样本中的待测DNA片段；Obtain a biological sample and extract the DNA fragments to be tested in the biological sample;
    采用亚硫酸氢钠修饰待测DNA片段，将待测DNA片段中未发生甲基化的胞嘧啶脱氨基转变成尿嘧啶；Modifying the DNA fragment to be tested with sodium bisulfite to deaminate cytosine that is not methylated into uracil in the DNA fragment to be tested;
    以亚硫酸氢钠修饰后的DNA片段为模板DNA片段，以第一引物和第二引物分别从模板DNA片段两侧对模板DNA片段进行PCR扩增，所述第一引物和第二引物扩增的模板DNA片段包括待测DNA片段的CpG位点；The DNA fragment modified by sodium bisulfite is used as a template DNA fragment, and the first and second primers are used to amplify the template DNA fragment from both sides of the template DNA fragment, and the first primer and the second primer are amplified. The template DNA fragment includes the CpG site of the DNA fragment to be tested;
    采用碱性磷酸酶处理经过PCR扩增后的PCR产物，去除多余的第一引物、第二引物和dNTPs，并纯化回收；Use alkaline phosphatase to treat PCR products after PCR amplification, remove excess first primer, second primer and dNTPs, and purify and recover;
    分别取等量纯化后的PCR产物，在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应，检测待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值；Take equal amounts of purified PCR products, and perform methylation-specific third primer-guided single-base extension labeling reactions in two separate reaction systems to detect the methylation and demethylation of the CpG site of the DNA fragment to be tested. Methylation fluorescence polarization value;
    根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平。The methylation level of the DNA fragment to be tested was calculated according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested.
  2. 如权利要求1所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述在两个单独的反应体系中分别进行甲基化特异第三引物引导的单碱基延伸标记反应的步骤包括：The method for measuring DNA methylation level of a specific site in a biological sample according to claim 1, wherein the single bases guided by the methylation-specific third primer are separately performed in two separate reaction systems. The steps of the extended labeling reaction include:
    采用荧光素dCTP检测待测DNA片段CpG位点的甲基化；采用荧光素dUTP检测待测DNA片段CpG位点的去甲基化；Fluorescein dCTP was used to detect the methylation of CpG sites of the DNA fragments to be tested; fluorescein dUTP was used to detect the methylation of CpG sites of the DNA fragments to be tested;
    所述根据待测DNA片段CpG位点的甲基化和去甲基化荧光偏振值计算待测DNA片段的甲基化水平的步骤包括：The step of calculating the methylation level of the DNA fragment to be tested according to the methylation and demethylation fluorescence polarization values of the CpG site of the DNA fragment to be tested includes:
    根据dCTP荧光偏振值和dUTP荧光偏振值计算待测DNA片段的甲基化水平。The dCTP fluorescence polarization value and dUTP fluorescence polarization value were used to calculate the methylation level of the DNA fragment to be tested.
  3. 如权利要求1所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述第一引物和第二引物与待测DNA片段CpG位点的距离大于第三引物的长度。The method for measuring DNA methylation level of a specific site in a biological sample according to claim 1, wherein the distance between the first primer and the second primer and the CpG site of the test DNA fragment is greater than that of the third primer. length.
  4. 如权利要求3所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述第一引物和第二引物的长度为20～30bp，GC含量为55-60％。The method for measuring DNA methylation level of a specific site in a biological sample according to claim 3, wherein the length of the first primer and the second primer is 20-30 bp, and the GC content is 55-60%.
  5. 如权利要求3所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述第三引物的序列与待测DNA片段CpG位点上游20～30bp序列的对应互补序列特异结合。The method for determining DNA methylation level of a specific site in a biological sample according to claim 3, characterized in that the sequence of the third primer and the corresponding complementary sequence of the sequence of 20-30 bp upstream of the CpG site of the DNA fragment to be tested Specific binding.
  6. 如权利要求5所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述第三引物的长度为22-25bp，且不含有CpG位点。The method for measuring DNA methylation level of a specific site in a biological sample according to claim 5, wherein the third primer is 22-25bp in length and does not contain a CpG site.
  7. 如权利要求1所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述待测DNA片段来源于血液样本、病理样本、器官或组织细胞样本，以及重组DNA或合成DNA。The method for measuring DNA methylation level at a specific site in a biological sample according to claim 1, wherein the DNA fragment to be tested is derived from a blood sample, a pathological sample, an organ or tissue cell sample, and recombinant DNA or Synthetic DNA.
  8. 如权利要求7所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述病理样本为肿瘤组织样本。The method for measuring DNA methylation level of a specific site in a biological sample according to claim 7, wherein the pathological sample is a tumor tissue sample.
  9. 如权利要求8所述的测定生物样本中特异位点DNA甲基化水平的方法，其特征在于，所述肿瘤组织样本为肺癌、胃癌或肝癌组织样本。The method according to claim 8, wherein the tumor tissue sample is a lung cancer, gastric cancer or liver cancer tissue sample.
  10. 权利要求1至9任一所述的测定生物样本中特异位点DNA甲基化水平的方法在检测目标DNA甲基化中的应用。Application of the method for measuring DNA methylation level of a specific site in a biological sample according to any one of claims 1 to 9 in detecting target DNA methylation.
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