Luo Junfeng, Zheng Wenli, Wang Yan, Wu Zhixiang, Bai Yunfei, Lu Zuhong
State Key Laboratory of Bioelectronics, Department of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China.
Anal Biochem. 2009 Apr 15;387(2):143-9. doi: 10.1016/j.ab.2008.11.020. Epub 2008 Nov 24.
A method for determining methylation density of target CpG islands has been established. In the method, DNA microarray was prepared by spotting a set of PCR products amplified from bisulfite-converted sample DNAs. The PCR products on the microarray were treated by SssI methyltransferase and labeled with TAMRA fluorescence. A recombinant, antibody-like methyl-CpG-binding protein labeled with Cy5 fluorescence was used to identify symmetrical methyl-CpG dinucleotide of the PCR products on the microarray. By use of a standard curve with control mixtures, the ratio of two fluorescence signals can be converted into percentage values to assess methylation density of targeted fragments. We obtained the methylation density of six CpG islands on the two tumor suppressor genes of CDK2A and CDK2B from seven cancer cell line samples and two normal blood samples. The validity of this method was tested by bisulfite sequencing. This method not only allows the quantitative analysis of regional methylation density of a set of given genes but also could provide information of methylation density for a large amount of clinical samples.
一种确定目标CpG岛甲基化密度的方法已经建立。在该方法中，通过点样从亚硫酸氢盐转化的样品DNA中扩增得到的一组PCR产物来制备DNA微阵列。微阵列上的PCR产物用SssI甲基转移酶处理并用TAMRA荧光标记。一种用Cy5荧光标记的重组抗体样甲基-CpG结合蛋白用于识别微阵列上PCR产物的对称甲基-CpG二核苷酸。通过使用对照混合物的标准曲线，两个荧光信号的比率可以转换为百分比值，以评估靶向片段的甲基化密度。我们从七个癌细胞系样品和两个正常血液样品中获得了CDK2A和CDK2B两个肿瘤抑制基因上六个CpG岛的甲基化密度。通过亚硫酸氢盐测序对该方法的有效性进行了测试。该方法不仅允许对一组给定基因的区域甲基化密度进行定量分析，而且还可以为大量临床样品提供甲基化密度信息。
