WO2015192644A1 - 检测dna甲基化的方法和试剂盒 - Google Patents

检测dna甲基化的方法和试剂盒 Download PDF

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WO2015192644A1
WO2015192644A1 PCT/CN2015/000361 CN2015000361W WO2015192644A1 WO 2015192644 A1 WO2015192644 A1 WO 2015192644A1 CN 2015000361 W CN2015000361 W CN 2015000361W WO 2015192644 A1 WO2015192644 A1 WO 2015192644A1
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antibody
dna
kit
methylation
concentration
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陈钊
莫敏俐
李晖
李隽�
丁凤
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北京雅康博生物科技有限公司
陈钊
莫敏俐
李晖
李隽�
丁凤
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Publication of WO2015192644A1 publication Critical patent/WO2015192644A1/zh

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor

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  • the present invention relates to methods and kits for detecting DNA methylation.
  • the invention relates to methods and kits for detecting DNA methylation by enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • DNA methylation occurs on the 5' carbon atom of cytosine in CpG dinucleotides and is widely found in human cancers. It is an important subject of epigenetics [1].
  • DNA methyltransferase (DNMT) is responsible for the methyl covalent modification of cytosine [2].
  • DNMT1 is primarily responsible for maintaining DNA methylation, such as methylation of the nascent DNA strand during DNA replication [3]; DNMT3a and DNMT3b are responsible for methylation of DNA from scratch. Modification [4, 5].
  • DNA methylation information can be stably transmitted during cell division, and plays an important role in organ development and function, X chromosome inactivation, genomic imprinting, silencing of transposons, and moderate expression of genes. Role [6].
  • the genome as a whole shows a decrease in the degree of methylation [7]; while in certain regions, it shows an increase in methylation, which is related to the transcriptional silencing of some genes [8].
  • the degree of DNA methylation has become a new generation of tumor markers, which can be used in principle for early detection, prognosis assessment, and efficacy monitoring of cancer [9].
  • HPLC high performance liquid chromatography
  • the invention adopts an enzyme-linked immunosorbent assay (ELISA) method to measure the degree of overall methylation of DNA derived from cells, tissues and serum, and has simple operation, good repeatability and low DNA usage (detectable to ng level). It is convenient for quantitative calculation and can achieve high-throughput detection.
  • ELISA enzyme-linked immunosorbent assay
  • the invention relates to a method of detecting DNA methylation comprising:
  • the restriction endonuclease is MseI; the blocking solution is PBS + 3% BSA + 0.05% Triton X-100; the anti-methylcytosine antibody is Santa Cruz# Sc-56615, the concentration is 1 to 10 ⁇ g / mL, preferably 5 ⁇ g / mL; the anti-single-strand DNA antibody is IBL code #18731, the concentration is 0.1 ⁇ 1 ⁇ g / mL, preferably 0.5 ⁇ g / mL; Is an anti-rabbit-derived IgG antibody conjugated to horseradish peroxidase (HRP); the TMB substrate is PIERCE #34028.
  • the invention also relates to a kit for detecting DNA methylation, comprising:
  • the blocking solution is PBS + 3% BSA + 0.05% Triton X-100;
  • the anti-methylcytosine antibody is Santa Cruz #sc-56615 at a concentration of 1 to 10 ⁇ g/mL, preferably 5 ⁇ g/mL;
  • the anti-single-strand DNA antibody is IBL code #18731, the concentration thereof It is 0.1 to 1 ⁇ g/mL, preferably 0.5 ⁇ g/mL;
  • the secondary antibody is an anti-rabbit-derived IgG antibody conjugated to horseradish peroxidase (HRP);
  • the TMB substrate is PIERCE #34028.
  • the kit of the present invention is used for predicting the therapeutic effect of cancer tissues, and is preferably used for detecting serum.
  • Figure 1 shows that there is no significant correlation between the degree of DNA methylation and DNA concentration in DNA derived from different samples.
  • Figure 2 shows a significant decrease in the degree of DNA methylation after treatment with a methyltransferase inhibitor.
  • Figure 3 shows the saturation curve measured relative to the degree of methylation.
  • Figure 4 shows a standard curve measured relative to the degree of methylation.
  • Figure 5 shows a significant decrease in the relative methylation level of DNA in lung cancer tissues relative to adjacent normal tissues (unpaired test).
  • Figure 6 shows a significant decrease in the relative methylation of DNA in lung cancer tissues relative to adjacent normal tissues (paired test).
  • Figure 7 shows that the degree of relative methylation of cancerous tissues in lung cancer patients decreases with age.
  • Figure 8 shows that the relative degree of methylation of cancerous tissues in lung cancer patients has a decreasing trend in smoking patients.
  • Figure 9 shows that the degree of relative methylation of cancerous tissues in lung cancer patients decreased significantly with increasing cancer stage.
  • Figure 10 shows a receiver operating characteristic curve (ROC) analysis of the relative degree of methylation of lung cancer patients (stages II, III, and IV) relative to normal lung tissue.
  • ROC receiver operating characteristic curve
  • Figure 11 shows the effect of the relative degree of methylation of cancerous tissue on overall survival of patients with lung cancer receiving chemotherapy.
  • Figure 12 shows the effect of chemotherapy on overall survival in patients with high levels of methylation.
  • Figure 13 shows a significant increase in the relative methylation level of DNA in serum of cancer patients relative to healthy controls.
  • Figure 14 shows that the degree of DNA methylation in serum of cancer patients is not significantly different from age. Relevance.
  • Figure 15 shows that there is no significant correlation between the degree of DNA methylation in serum of cancer patients and gender.
  • Figure 16 shows a comparison of the degree of DNA methylation in serum between healthy controls and cancer patients.
  • Figure 17 shows that the degree of DNA methylation in the serum of cancer patients increases significantly with increasing cancer stage.
  • Figure 18 shows receiver operating characteristic curve (ROC) analysis of the relative methylation level of DNA in serum of cancer patients relative to healthy controls.
  • ROC receiver operating characteristic curve
  • Figure 19 shows receiver operating characteristic curve (ROC) analysis of the relative degree of DNA methylation in serum of male cancer patients relative to male healthy controls.
  • ROC receiver operating characteristic curve
  • Figure 20 shows receiver operating characteristic curve (ROC) analysis of the relative methylation level of DNA in serum of female cancer patients relative to female healthy controls.
  • ROC receiver operating characteristic curve
  • Tissue genomic DNA extraction was performed using the QIAamp DNA Mini Kit (Qiagen). After the concentration was measured, the DNA was purified using a DNA purification kit (Tiangen Biochemical Technology Co., Ltd., Beijing) after treatment with MseI restriction enzyme (at 2 ° C for 2 hours). After measuring the DNA concentration, the DNA was diluted to 10 to 50 ng/ ⁇ L, denatured at 95 ° C for 10 minutes, and immediately placed on ice. 1 ⁇ L was mixed with 49 ⁇ L of 1 ⁇ blocking solution (PBS + 3% BSA + 0.05% Triton X-100) and placed on ice.
  • 1 ⁇ blocking solution PBS + 3% BSA + 0.05% Triton X-100
  • Serum/plasma 200 ⁇ L genomic DNA extraction was performed using a QIAamp DNA Blood Kit (Qiagen) eluting with 45 ⁇ L of water. Denatured at 95 ° C for 10 minutes and immediately placed on ice. Add 5 ⁇ L of 10 ⁇ blocking solution and place on ice.
  • ELISA assay plates were coated with anti-methylcytosine antibody (Santa Cruz #sc-56615, 5 ⁇ g/mL), 50 ⁇ L per well. Cover the plate and place at 4 ° C overnight. The coated antibody was discarded the next day and washed twice with PBS solution (0.2 g/L KH 2 PO 4 , 2.16 g/L Na 2 HPO 4 ⁇ 7H 2 O, 0.2 g/L KCl, 8.0 g/L NaCl). Use 200 ⁇ L per well and pat dry. 200 ⁇ L of blocking solution was added to each well and allowed to stand at room temperature for 2 hours. The blocking solution is then discarded.
  • PBS solution 0.2 g/L KH 2 PO 4 , 2.16 g/L Na 2 HPO 4 ⁇ 7H 2 O, 0.2 g/L KCl, 8.0 g/L NaCl
  • Premixed 49 ⁇ L blocking solution and 1 ⁇ L DNA sample were added to each well. Leave at 4 ° C for at least 2 hours. Wash twice with PBS and pat dry.
  • Anti-single-stranded DNA antibody (IBL code #18731, 0.5 ⁇ g/mL) was added to the wells of a 96-well ELISA assay plate, and 100 ⁇ L was added to each well. Cover the plate and leave it at room temperature for 2 hours. Wash four times with PBS and pat dry. The secondary antibody (1:4000 dilution) was added to the wells of a 96-well ELISA assay plate, and 100 ⁇ L was added to each well. Cover the plate and leave it at room temperature for 1 to 2 hours.
  • TMB substrate solution PIERCE #34028
  • 2M sulfuric acid solution was added, and after mixing, the sample was read by a microplate reader (wavelength was selected to be 450 nm).
  • Relative methylation degree calculation The genomic DNA of human non-small cell lung cancer cell line A549 was extracted, and the DNA concentration was determined by MseI digestion and purification, and diluted to 5, 10, 20, 40 ng/ ⁇ L standard, and then according to the above steps.
  • ELISA test After reading the ELISA test, subtract the experimental well data (ie, the background) from the standard and sample absorbance (OD) data without any DNA sample, and use the standard to go to the background data to make a standard curve (DNA content is the abscissa, The background OD value is plotted on the ordinate.
  • the de-background data of the sample is substituted into the linear fitting formula of the standard curve to calculate the relative DNA methylation degree in each sample. For tissue samples, this value is also divided by the amount of DNA added to calculate the relative methylation level per ng of tissue DNA.
  • DAC 5-aza-2'-deoxycytidine
  • DMSO solvent dimethyl sulfoxide
  • the patients were divided into two groups: low methylation degree (less than 1.31) and high (greater than or equal to 1.31).
  • the degree of methylation was high and the overall survival of the two groups was low.
  • the body survival rate is high (see Figure 11).
  • P the relative methylation level of DNA in the serum of cancer patients relative to healthy controls was significantly increased, P was 0.0002 and 0.0003, respectively (see Figure 14).
  • the degree of chemistry was significantly increased, with P being ⁇ 0.0001 and 0.0023, respectively (see Figure 15).
  • the area under the curve (AUC) in Figure 18 is 0.7224, P ⁇ 0.0001.
  • the sensitivity and specificity can reach 70.9% and 65.2%, respectively, indicating that the degree of DNA relative methylation in serum distinguishes between cancer and normal. The diagnostic effect is higher.
  • the area under the curve (AUC) in Figure 19 is 0.7740, P ⁇ 0.0001, at the critical value, Sensitivity and specificity were 79.2% and 67.5%, respectively, indicating that the degree of relative methylation of DNA in serum is more effective in distinguishing between cancer and normal in men.
  • AUC area under the curve
  • P 0.0022.
  • the sensitivity and specificity can reach 63.5% and 65.3%, respectively, indicating that the degree of DNA methylation in serum is differentiated among women. Cancer and normal diagnosis are more effective.

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Abstract

一种检测DNA甲基化的方法和试剂盒。具体地说,本发明的方法采用酶联免疫吸附测定(ELISA),对来源于细胞、组织、血清中DNA的整体甲基化程度进行测定,操作简单,重复性好,DNA用量少(可检测至ng级),定量计算方便,能够实现高通量检测。

Description

检测DNA甲基化的方法和试剂盒 技术领域
本发明涉及检测DNA甲基化的方法和试剂盒。具体地说,本发明涉及用酶联免疫吸附测定(ELISA)方法检测DNA甲基化的方法和试剂盒。
背景技术
DNA甲基化发生在CpG二核苷酸中胞嘧啶的5’碳原子上,在人类癌症中广泛存在,是表观遗传学的重要研究对象[1]。DNA甲基转移酶(DNMT)负责胞嘧啶的甲基共价修饰[2]。在DNMT家族中,DNMT1主要负责维持DNA甲基化程度,例如在DNA复制时,对新生DNA链进行甲基化修饰[3];DNMT3a和DNMT3b主要负责从无到有的对DNA进行甲基化修饰[4,5]。DNA甲基化的信息在细胞分裂过程中能够稳定的传递,对于器官的发育和行使功能、X染色体的失活、基因组印记(imprinting)、转座子等的沉默以及基因的适度表达等发挥重要作用[6]。
在癌细胞中,基因组整体表现出甲基化程度降低的现象[7];而在特定的区域表现出甲基化程度升高,同一些基因的转录沉默相关[8]。DNA甲基化程度已成为新一代的肿瘤标志物,原则上可以用于癌症的早期检测、预后评估、疗效监测等方面[9]。
在用于研究DNA整体甲基化程度的方法中,高效液相色谱(HPLC)是一种经典的方法[10],能够进行定量分析,可重复性强。然而这一方法所需的基因组DNA用量比较大,对DNA的质量要求也比较高,不适宜用于高通量分析。也有以亚硫酸氢钠处理为基础的方法,检测重复序列(例如Alu元件或LINE)的含量[11,12]。但是这类方法的操作步骤比较繁琐。
发明内容
本发明采用酶联免疫吸附测定(ELISA)方法,对来源于细胞、组织、血清中DNA的整体甲基化程度进行测定,操作简单,重复性好,DNA用量少(可检测至ng级),定量计算方便,能够实现高通量检测。
具体的说,本发明涉及一种检测DNA甲基化的方法,其包括:
(1)提取、限制性内切酶处理、纯化基因组DNA;
(2)将DNA变性后加入封闭液;
(3)用抗甲基胞嘧啶抗体包被ELISA实验板;
(4)弃去包被抗体,在实验板的每个孔中加入封闭液;
(5)弃去封闭液,加入预混的封闭液与DNA样品;
(6)用PBS溶液清洗,干燥后加入抗单链DNA抗体;
(7)用PBS溶液清洗,干燥后加入二抗;
(8)用PBS溶液清洗,干燥后加入TMB底物;
(9)加入硫酸溶液后用酶标仪读数;
(10)根据用基因组DNA标准品制得的标准曲线,计算出样本的相对DNA甲基化程度。
优选地,在本发明的方法中,所述限制性内切酶是MseI;所述封闭液是PBS+3%BSA+0.05%Triton X-100;所述抗甲基胞嘧啶抗体是Santa Cruz#sc-56615,其浓度是1~10μg/mL,优选5μg/mL;所述抗单链DNA抗体是IBL code#18731,其浓度是0.1~1μg/mL,优选0.5μg/mL;所述二抗是偶联辣根过氧化物酶(horseradish peroxidase,HRP)的抗兔源IgG的抗体;所述TMB底物是PIERCE#34028。
本发明还涉及一种检测DNA甲基化的试剂盒,其包括:
(1)封闭液;
(2)抗甲基胞嘧啶抗体;
(3)抗单链DNA抗体;
(4)二抗;
(5)TMB底物;
(6)2M硫酸;以及
(7)PBS溶液。
优选地,在本发明的试剂盒中,所述封闭液是PBS+3%BSA+0.05% Triton X-100;所述抗甲基胞嘧啶抗体是Santa Cruz#sc-56615,其浓度是1~10μg/mL,优选5μg/mL;所述抗单链DNA抗体是IBL code#18731,其浓度是0.1~1μg/mL,优选0.5μg/mL;所述二抗是偶联辣根过氧化物酶(horseradish peroxidase,HRP)的抗兔源IgG的抗体;所述TMB底物是PIERCE#34028。进一步优选地,本发明的试剂盒用于对癌组织进行化疗疗效预测,并且优选用于对血清进行检测。
附图说明
图1显示在来源于不同样本的DNA中,DNA相对甲基化程度与DNA浓度无显著相关性。
图2显示经甲基转移酶抑制剂处理后DNA相对甲基化程度显著降低。
图3显示相对甲基化程度测定的饱和曲线。
图4显示相对甲基化程度测定的标准曲线。
图5显示肺癌组织相对于癌旁正常组织的DNA相对甲基化程度显著下降(非成对检验)。
图6显示肺癌组织相对于癌旁正常组织的DNA相对甲基化程度显著下降(成对检验)。
图7显示肺癌患者癌组织相对甲基化程度随年龄增高而下降。
图8显示肺癌患者癌组织相对甲基化程度在吸烟患者中有下降趋势。
图9显示肺癌患者癌组织相对甲基化程度随癌症分期升高而显著下降。
图10显示肺癌患者(II、III、IV期)癌组织相对于正常肺组织的相对甲基化程度的受试者作业特征曲线(ROC)分析。
图11显示在接受化疗的肺癌患者中,癌组织的相对甲基化程度对患者总体生存率的影响。
图12显示在甲基化程度高的肺癌患者中,接受化疗与否对患者总体生存率的影响。
图13显示癌症患者相对于健康对照的血清中DNA相对甲基化程度显著升高。
图14显示癌症患者血清中DNA相对甲基化程度与年龄无显著相 关性。
图15显示癌症患者血清中DNA相对甲基化程度与性别无显著相关性。
图16显示健康对照与癌症患者血清中DNA相对甲基化程度比较。
图17显示癌症患者血清中DNA相对甲基化程度随癌症分期升高而显著上升。
图18显示癌症患者相对于健康对照的血清中DNA相对甲基化程度的受试者作业特征曲线(ROC)分析。
图19显示男性癌症患者相对于男性健康对照的血清中DNA相对甲基化程度的受试者作业特征曲线(ROC)分析。
图20显示女性癌症患者相对于女性健康对照的血清中DNA相对甲基化程度的受试者作业特征曲线(ROC)分析。
具体实施方式
实验步骤
样品处理:组织基因组DNA提取使用QIAamp DNA Mini Kit(Qiagen)。测定浓度后,使用MseI限制性内切酶处理(37℃放置2小时)后使用DNA纯化试剂盒(天根生化科技有限公司,北京)纯化DNA。测定DNA浓度后,将DNA稀释至10~50ng/μL,95℃变性10分钟,立即冰上放置。取1μL与49μL1×封闭液(PBS+3%BSA+0.05%Triton X-100)混合,冰上放置。
血清/血浆(200μL)基因组DNA提取使用QIAamp DNA Blood Kit(Qiagen),用45μL水洗脱。95℃变性10分钟,立即冰上放置。加入5μL 10×封闭液混合,冰上放置。
夹心法酶联免疫吸附测定(ELISA)实验步骤:用抗甲基胞嘧啶抗体(Santa Cruz#sc-56615,5μg/mL)包被ELISA实验板,每孔加入50μL。覆盖平板,4℃过夜放置。次日弃去包被抗体,用PBS溶液(0.2g/L KH2PO4,2.16g/L Na2HPO4·7H2O,0.2g/L KCl,8.0g/L NaCl)洗两遍,每次每孔使用200μL,拍干。每孔中加入200μL封闭液,室温放置2小时。随后弃去封闭液。每孔加入预混的49μL封闭液与 1μLDNA样品。4℃放置至少2小时。使用PBS洗两遍,拍干。将抗单链DNA抗体(IBL code#18731,0.5μg/mL)加入96孔ELISA实验板的孔中,每孔加入100μL。覆盖平板,室温放置2小时。使用PBS洗四遍,拍干。将二抗(1∶4000稀释)加入96孔ELISA实验板的孔中,每孔加入100μL。覆盖平板,室温放置1至2小时。使用PBS洗四遍,拍干。每孔加入100μL TMB底物溶液(PIERCE#34028)。3~10分钟后,加入2M硫酸溶液,混匀后用酶标仪读数(波长选择为450nm)。
相对甲基化程度计算:提取人非小细胞肺癌细胞系A549的基因组DNA,MseI酶切处理、纯化后测定DNA浓度,稀释为5、10、20、40ng/μL标准品,再按上述步骤进行后续实验。ELISA实验读数后,将标准品和样本吸光度(OD)数据减去不含任何DNA样品的实验孔数据(即去本底),利用标准品去本底数据作出标准曲线(DNA含量为横坐标,去本底OD值为纵坐标),将样本的去本底数据代入标准曲线的线性拟合公式中,计算出每个样本中相对DNA甲基化程度。对于组织样本,还需将该数值除以加样DNA含量,计算出每ng组织DNA中的相对甲基化程度。
实验数据:
1.选取多个不同的组织样本,测定其DNA浓度,之后测定等体积样本的相对甲基化程度,Pearson相关性系数r为0.1853,P值为0.6084>0.05,说明不同样本DNA的相对甲基化程度与其DNA浓度/含量无显著相关性(见图1)。
2.使用1μM甲基化酶抑制剂5-氮杂(aza)-2’-脱氧胞苷(DAC)或等体积溶剂二甲基亚砜(DMSO)处理A549细胞2天后,提取细胞DNA测定DNA相对甲基化程度/ng。DAC处理后细胞相对于DMSO处理后细胞的相对甲基化程度显著降低(P=0.0206)(见图2)。
3.将A549细胞基因组DNA稀释为不同浓度,加入实验体系中,使最终含量分别为0、4、8、16、32、64、80、120、160、200ng,将DNA含量作为横坐标,测得的相对甲基化程度作为纵坐标作图显示:曲线在100ng前后呈现饱和,而低至4ng依然可以检测出(见图3)。
4.将A549细胞基因组DNA稀释为不同浓度,加入实验体系中,使最终含量分别为0、4、8、16、32、64、80ng,将DNA含量作为横坐标,测得的相对甲基化程度作为纵坐标,作直线拟合,得到标准曲线。R2=0.9929,表明拟合直线的线性关系良好(见图4)。
5.检测46例肺癌组织样本和46例癌旁正常组织样本DNA的相对甲基化程度/ng,非成对数据检验结果显示癌组织相对于癌旁正常组织DNA相对甲基化程度显著下降(P=0.0263)(见图5)。
6.检测46例肺癌组织样本和各自对应的癌旁正常组织样本DNA的相对甲基化程度/ng,成对数据检验结果显示癌组织相对于癌旁正常组织DNA相对甲基化程度显著下降(P=0.0013)(见图6)。
7.肺癌患者癌组织相对甲基化程度与年龄无显著相关性(P=0.629);在低年龄组(60岁以下)与中年龄组(60~74岁)之间无显著性差异(P=0.1082>0.05),在中年龄组与高年龄组(74岁以上)之间有显著性差异(P=0.0271)(见图7)。
8.肺癌患者癌组织相对甲基化程度在吸烟患者中相对于不吸烟患者无显著性差异,但有下降趋势,其中男性中P=0.0581,女性中P=0.4329(见图8)。
9.I期肺癌患者癌组织相对甲基化程度相对正常肺组织无显著性差异(P=0.4033);II、III、IV期肺癌患者癌组织相对甲基化程度相对正常肺组织显著降低(P=0.0162);III、IV期肺癌患者癌组织相对甲基化程度相对正常肺组织显著降低(P=0.0316)(见图9)。
10.由于I期肺癌患者癌组织相对于正常肺组织的相对甲基化程度无显著差异,因此分析了非I期(II、III、IV期)肺癌患者癌组织相对于正常肺组织的相对甲基化程度的受试者作业特征曲线(ROC),曲线下面积(AUC)为0.7093,P=0.0323,在临界值下,灵敏度和特异性度可分别达到58.7%和80%,表明通过肺组织的相对甲基化程度区分非I期癌症患者和正常的诊断效力较高(见图10)。
11.按照相对甲基化程度数值分为甲基化程度低(小于1.31)、高(大于或等于1.31)两组,在接受化疗的肺癌患者中,甲基化程度高和低两组总体生存率存在显著性差异(P=0.0403<0.05),且在同一时间点,甲基化程度低的患者组相对于甲基化程度高的患者组,其总 体生存率较高(见图11)。
12.按照相对甲基化程度数值分为甲基化程度低(小于1.31)、高(大于或等于1.31)两组,在甲基化程度高的肺癌患者中,接受和不接受化疗两组总体生存率存在显著性差异(P=0.0377<0.05),且在同一时间点,未接受化疗患者组相对于接受化疗患者组,其总体生存率较高(见图12)。
13.检测103例癌症患者、89例健康对照的血清中(200μL)DNA相对甲基化程度,癌症患者相对于健康对照的血清中DNA相对甲基化程度显著升高(P<0.0001)(见图13)。
14.癌症患者血清中(200μL)DNA相对甲基化程度在低年龄组(60岁以下)与高年龄组(60岁以上)之间无显著性差异(P=0.1443),而不论低年龄组还是高年龄组,癌症患者相对于健康对照的血清中DNA相对甲基化程度均显著升高,P分别为0.0002和0.0003(见图14)。
15.癌症患者血清中(200μL)DNA相对甲基化程度在男性与女性之间无显著性差异(P=0.0564),而不论男性还是女性,癌症患者相对于健康对照的血清中DNA相对甲基化程度均显著升高,P分别为<0.0001和0.0023(见图15)。
16.多种癌症患者相对于健康对照的血清中(200μL)DNA相对甲基化程度显著升高,例如乳腺癌(P=0.0465)、食道癌(P=0.0002)、肝癌(P<0.0001)、肺癌(P<0.0001)、胃癌(P=0.0002)、消化系统癌症(P<0.0001)等(见图16)。
17.I、II期癌症患者相对健康对照的血清中(200μL)相对甲基化程度显著升高(P=0.0197);III期癌症患者相对健康对照的血清中(200μL)相对甲基化程度显著升高(P=0.0006);IV期癌症患者相对健康对照的血清中(200μL)相对甲基化程度显著升高(P<0.0001)(见图17)。
18.图18曲线下面积(AUC)为0.7224,P<0.0001,在临界值下,灵敏度和特异性度可分别达到70.9%和65.2%,表明通过血清中DNA相对甲基化程度区分癌症和正常的诊断效力较高。
19.图19曲线下面积(AUC)为0.7740,P<0.0001,在临界值下, 灵敏度和特异性度可分别达到79.2%和67.5%,表明通过血清中DNA相对甲基化程度在男性中区分癌症和正常的诊断效力较高。
20.图20曲线下面积(AUC)为0.6766,P=0.0022,在临界值下,灵敏度和特异性度可分别达到63.5%和65.3%,表明通过血清中DNA相对甲基化程度在女性中区分癌症和正常的诊断效力较高。
参考文献
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Claims (23)

  1. 一种检测DNA甲基化的方法,其包括:
    (1)提取、限制性内切酶处理、纯化基因组DNA;
    (2)将DNA变性后加入封闭液;
    (3)用抗甲基胞嘧啶抗体包被ELISA实验板;
    (4)弃去包被抗体,在实验板的每个孔中加入封闭液;
    (5)弃去封闭液,加入预混的封闭液与DNA样品;
    (6)用PBS溶液清洗,干燥后加入抗单链DNA抗体;
    (7)用PBS溶液清洗,干燥后加入二抗;
    (8)用PBS溶液清洗,干燥后加入TMB底物;
    (9)加入硫酸溶液后用酶标仪读数;
    (10)根据用基因组DNA标准品制得的标准曲线,计算出样本的相对DNA甲基化程度。
  2. 权利要求1的方法,其中所述限制性内切酶是MseI。
  3. 权利要求1的方法,其中所述封闭液是PBS+3%BSA+0.05%Triton X-100。
  4. 权利要求1的方法,其中所述抗甲基胞嘧啶抗体是Santa Cruz #sc-56615。
  5. 权利要求4的方法,其中所述抗甲基胞嘧啶抗体的浓度是1~10μg/mL。
  6. 权利要求5的方法,其中所述抗甲基胞嘧啶抗体的浓度是5μg/mL。
  7. 权利要求1的方法,其中所述抗单链DNA抗体是IBL code #18731。
  8. 权利要求7的方法,其中所述抗单链DNA抗体的浓度是0.1~1μg/mL。
  9. 权利要求8的方法,其中所述抗单链DNA抗体的浓度是0.5μg/mL。
  10. 权利要求1的方法,其中所述二抗是偶联辣根过氧化物酶的抗兔源IgG的抗体。
  11. 权利要求1的方法,其中所述TMB底物是PIERCE # 34028。
  12. 一种检测DNA甲基化的试剂盒,其包括:
    (1)封闭液;
    (2)抗甲基胞嘧啶抗体;
    (3)抗单链DNA抗体;
    (4)二抗;
    (5)TMB底物;
    (6)2M硫酸;以及
    (7)PBS溶液。
  13. 权利要求12的试剂盒,其中所述封闭液是PBS+3%BSA+0.05% Triton X-100。
  14. 权利要求12的试剂盒,其中所述抗甲基胞嘧啶抗体是Santa Cruz #sc-56615。
  15. 权利要求14的试剂盒,其中所述抗甲基胞嘧啶抗体的浓度是1~10μg/mL。
  16. 权利要求15的试剂盒,其中所述抗甲基胞嘧啶抗体的浓度是5μg/mL。
  17. 权利要求12的试剂盒,其中所述抗单链DNA抗体是IBL code # 18731。
  18. 权利要求17的试剂盒,其中所述抗单链DNA抗体的浓度是0.1~1μg/mL。
  19. 权利要求18的试剂盒,其中所述抗单链DNA抗体的浓度是0.5μg/mL。
  20. 权利要求12的试剂盒,其中所述二抗是偶联辣根过氧化物酶的抗兔源IgG的抗体。
  21. 权利要求12的试剂盒,其中所述TMB底物是PIERCE#34028。
  22. 权利要求12的试剂盒,其用于对癌组织进行化疗疗效预测。
  23. 权利要求12的试剂盒,其用于对血清进行检测。
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