摘要
目的:建立焦磷酸测序技术联合甲基化敏感性内切酶高通量定量分析全基因组DNA甲基化水平的方法(PME)。方法:以Lambda DNA为标准品,取>10倍浓度梯度DNA(125、250、500、1 000和2 000ng)进行双酶切后焦磷酸测序,检测甲基化程度,分析测序峰值与模板量相关性;以经甲基化酶(M.SssⅠ)处理的Lambda DNA为阳性对照品,与阴性对照品(未经甲基化酶处理的阴性Lamb-da DNA)混合(混合比例分别为0、25%、50%、75%和100%),进行甲基化程度相关性分析;以该实验方法对8例健康体检者和6例非小细胞肺癌(NSCLC)组织DNA进行分析。结果:测序峰值与模板量呈线性相关,在125～2 000ng,实验重复性良好;阳性对照品甲基化程度为100%,阴性对照品为0,甲基化水平呈线性相关,相关系数为0.999。健康人甲基化水平平均值为0.446 64,NSCLC患者为0.489 150,差异明显。结论:成功构建了焦磷酸测序技术联合甲基化敏感性内切酶分析全基因组DNA甲基化水平的方法。该方法适合于样本的高通量分析,为临床检测全基因组DNA甲基化状态、肿瘤早期诊断提供了新思路。
OBJECTIVE： To establish a high throughput method for the detection of the genomic-wide DNA methylation levels by pyrosequencing joint methylation-sensitive restriction enzymes（PME）. METHODS： Different concentration Lambda DNA with 125,250,500,1 000 and Z 000 ng were digested by Hpa Ⅱ＋EcoR Ⅰ and Msp Ⅰ ＋EcoRⅠ ,analyzed by PME; Lambda DNA was treated by M. Sss I as a standard sample. Methylated and unmethylated Lambda DNA was mixed in differ- ent proportions with 0.25 %,50%,75% and 100%, analyzed by PME; Eight people and 6 NSCLC patients were detected with PME assay. RESULTS： The linearity of the PME assay was veri- fied by methylation Lambda DNA in vitro. For all the concentra- tions included, the method showed a good linearity from 125 to 2 000 ng. The degree of methylation level was 100% in methylated lambda DNA and 0 in unmethylated lambda DNA. By detecting mixed samples, a methylation-linear relationship between the frequency of complex was found, with the linear correlation greater than 0. 999. Genomic DNA was analyzed by PME assay,the average methylation level in normal people （0. 446 64 ）was lower than NSCLC patients （0. 489 150）. CONCLUSIONS.. The high throughput method using PME assay to detect the whole-genomic DNA methylation level is successfully established. The PME assay may provide a useful method for genomic DNA methylation study and tumor early diagnostics.
出处
《中华肿瘤防治杂志》
CAS
2011年第21期1672-1675,共4页
Chinese Journal of Cancer Prevention and Treatment
