Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer
Methylated-CpG island recovery assay: a new technique for the rapid detection of methylated-CpG islands in cancer
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DOI：
10.1038/labinvest.3700311
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发表时间：
2005-09-01
影响因子：
5
通讯作者：
Pfeifer, GP
中科院分区：
文献类型：
--
作者：
Rauch, T;Pfeifer, GP
CpG岛的高甲基化是人类肿瘤发生和发展过程中常见的现象。在容易获得的生物材料如血清中检测甲基化CpG岛具有用于癌症的早期诊断的潜力。目前用于检测甲基化CpG岛的大多数方法是基于基因组DNA的亚硫酸氢钠转化，然后进行PCR反应。在这里，我们描述了一种方法，甲基化CpG岛恢复测定（MIRA），不依赖于使用亚硫酸氢钠，但具有类似的灵敏度和特异性，亚硫酸氢盐为基础的方法。甲基-CpG结合结构域蛋白，如甲基-CpG结合结构域蛋白-2（MBD 2），具有特异性结合甲基化DNA序列的能力。在MIRA程序中，将从细胞或组织中分离的超声处理的基因组DNA与含有谷胱甘肽-S-转移酶-MBD 2b的基质在甲基-CpG结合结构域蛋白3-样-1（MBD 2的结合配偶体，其增加MBD 2对甲基化DNA的亲和力）的存在下孵育。从基质中洗脱特异性结合的DNA，并进行基因特异性PCR反应以检测CpG岛甲基化。甲基化可以使用1 ng DNA或3000个细胞检测。MIRA是一种特异性和敏感性，但不费力的技术，可用于临床检测和诊断任何DNA甲基化相关疾病，包括癌症。
Hypermethylation of CpG islands is a phenomenon commonly observed during the development and progression of human tumors. Detection of methylated-CpG islands in easily accessible biological materials such as serum has the potential to be useful for the early diagnosis of cancer. Most currently used methods for detecting methylated-CpG islands are based on sodium bisulfite conversion of genomic DNA, followed by PCR reactions. Here we describe a method, methylated-CpG island recovery assay (MIRA) that does not depend on the use of sodium bisulfite but has similar sensitivity and specificity as bisulfite-based approaches. Methyl-CpG-binding domain proteins, such as methyl-CpG-binding domain protein-2 (MBD2), have the capacity to bind specifically to methylated DNA sequences. In the MIRA procedure, sonicated genomic DNA isolated from cells or tissue is incubated with a matrix containing glutathione-S-transferase-MBD2b in the presence of methyl-CpG- binding domain protein 3-like-1, a binding partner of MBD2 that increases the affinity of MBD2 for methylated DNA. Specifically bound DNA is eluted from the matrix and gene-specific PCR reactions are performed to detect CpG island methylation. Methylation can be detected using 1 ng of DNA or 3000 cells. MIRA is a specific and sensitive, but not laborious, technique that can be clinically useful in the detection and diagnosis of any DNA methylation-associated disease, including cancer.