微阵列定量检测急性白血病患者E—cadherin基因启动子区的甲基化

Quantitative microarray-based DNA methylation analysis of E-cadherin gene promoter in acute leukemia

目的利用微阵列定量检测急性白血病患者E-cadherin基因启动子区CpG岛的甲基化改变，初步探讨基因芯片在肿瘤甲基化检测中的作用。方法用亚硫酸氢盐处理基因组DNA，并以此为模板进行PCR扩增。目的序列中未甲基化的CpG位点被翻转成TpG，而甲基化的CpG位点保持不变。设计5组探针构建一种检测急性白血病E—cadherin基因甲基化改变的微阵列。通过绘制标准曲线，定量检测样本的甲基化改变。结果利用微阵列定量检测了5例急性白血病患者的样本E．cadherin基因的甲基化改变，5个检测区域不同程度发生了甲基化，且发生高甲基化的区域一致。检测结果经过基因测序验证。结论基因芯片能够作为一种定量检测基因甲基化改变的有效工具，可用于白血病甲基化的定量研究。其较基因测序省时省力，且能实现定量和高通量检测。

Objective To quantitatively detect the methylation of E-cadherin gene 5＇-CpG islands in acute leukemia by microarray-based DNA analysis and to briefly discuss the role of microarry for detection of methylation in tumors. Methods Bisulfite-modified DNA was used as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. Five sets of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of E-cadherin gene CpG islands in acute leukemia. By drawing a standard curve to assess the levels of changes in methylation detected in the examined samples. Results Microarray assay was successfully used to quantitatively detect methylation changes of E-cadherin gene in 5 acute leukemia samples. Varying degree of methylation was detected in five regions and the hypermethylation region was the same. The result was validated by gene sequencing. Conclusion Microarray assay may be applied as an useful tool for mapping methylation changes in multiple CpG loci and for leukemia research. It is more timesaving and labor-saving than gene sequencing and can be used to quantitatively detect changes in methylation with high throughput.