A real-time PCR assay for DNA-methylation using methylation-specific blockers
A real-time PCR assay for DNA-methylation using methylation-specific blockers
复制标题
DOI：
10.1093/nar/gnh008
复制
发表时间：
2004-01-01
影响因子：
14.9
通讯作者：
Berlin, K
中科院分区：
文献类型：
--
作者：
Cottrell, SE;Distler, J;Berlin, K
基于DNA甲基化的生物标记物已经被发现，可以通过检测体液中循环的肿瘤衍生DNA来潜在地用于癌症的诊断。任何适用于这些样本的甲基化检测方法都必须能够在背景正常DNA存在的情况下检测到少量的肿瘤DNA。我们已经开发了一种称为HeavyMethyl的实时PCR检测方法，非常适合这一应用。HeavyMethyl使用甲基化特异的寡核苷酸阻断剂和甲基化特异的探针来实现甲基化特异的扩增和检测。我们对未甲基化和人工甲基化的DNA进行了检测，以确定检测下限。经过仔细的优化，我们的谷胱甘肽-S-转移酶Pi1和降钙素方法分别可以扩增30和60pg的甲基化DNA，两种方法都不能扩增未甲基化的DNA。降钙素检测显示正常结肠癌和结肠腺癌之间甲基化差异非常显著，结肠癌患者的血清DNA中也检测到甲基化。这些分析表明，HeavyMetyl技术可以成功地用于分析极低浓度的甲基化DNA，例如在肿瘤患者的血清中。
DNA methylation-based biomarkers have been discovered that could potentially be used for the diagnosis of cancer by detection of circulating, tumor-derived DNA in bodily fluids. Any methylation detection assay that would be applied to these samples must be capable of detecting small amounts of tumor DNA in the presence of background normal DNA. We have developed a real-time PCR assay, called HeavyMethyl, that is well suited for this application. HeavyMethyl uses methylation-specific oligonucleotide blockers and a methylation-specific probe to achieve methylation-specific amplification and detection. We tested the assays on unmethylated and artificially methylated DNA in order to determine the limit of detection. After careful optimization, our glutathione-S-transferase pi1 and Calcitonin assays can amplify as little as 30 and 60 pg of methylated DNA, respectively, and neither assay amplifies unmethylated DNA. The Calcitonin assay showed a highly significant methylation difference between normal colon and colon adenocarcinomas, and methylation was also detected in serum DNA from colon cancer patients. These assays show that HeavyMethyl technology can be successfully employed for the analysis of very low concentrations of methylated DNA, e.g. in serum of patients with tumors.