MethyLight: a high-throughput assay to measure DNA methylation
MethyLight: a high-throughput assay to measure DNA methylation
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DOI：
10.1093/nar/28.8.e32
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发表时间：
2000-04-15
影响因子：
14.9
通讯作者：
Laird, Peter W.
中科院分区：
文献类型：
--
作者：
Eads, Cindy A.;Danenberg, Kathleen D.;Laird, Peter W.
脊椎动物中，胞嘧啶 5 DNA 甲基化发生在 CpG 二核苷酸的背景下。人类肿瘤中 CpG 岛的异常甲基化已被证明会导致肿瘤抑制基因的转录沉默。大多数用于分析 5 号胞嘧啶甲基化模式的方法都需要繁琐的手动技术，包括凝胶电泳、限制性酶消化、放射性标记的 dNTP 或杂交探针。用于 DNA 甲基化分析的高通量技术的发展将显着扩展我们从临床样本中获取分子信息的能力。本研究描述了一种高通量定量甲基化测定，该测定利用基于荧光的实时 PCR (TaqMan (R)) 技术，在 PCR 步骤后无需进一步操作。 Methy-Light 是一种高度灵敏的检测方法，能够在未甲基化等位基因过量 10 000 倍的情况下检测甲基化等位基因。该测定也是高度定量的，可以非常准确地确定特定 DNA 甲基化模式的相对流行率。我们证明 MethyLight 可以区分人类结直肠肿瘤标本中 MLH1 错配修复基因的单等位基因和双等位基因甲基化。这项技术的发展将大大增强我们快速、准确地生成肿瘤样本表观遗传图谱的能力。
Cytosine-5 DNA methylation occurs in the context of CpG dinucleotides in vertebrates. Aberrant methylation of CpG islands in human tumors has been shown to cause transcriptional silencing of tumor-suppressor genes. Most methods used to analyze cytosine-5 methylation patterns require cumbersome manual techniques that employ gel electrophoresis, restriction enzyme digestion, radiolabeled dNTPs or hybridization probes. The development of high-throughput technology for the analysis of DNA methylation would significantly expand our ability to derive molecular information from clinical specimens. This study describes a high-throughput quantitative methylation assay that utilizes fluorescence-based real-time PCR (TaqMan (R)) technology that requires no further manipulations after the PCR step. Methy-Light is a highly sensitive assay, capable of detecting methylated alleles in the presence of a 10 000-fold excess of unmethylated alleles. The assay is also highly quantitative and can very accurately determine the relative prevalence of a particular pattern of DNA methylation. We show that MethyLight can distinguish between mono-allelic and bi-allelic methylation of the MLH1 mismatch repair gene in human colorectal tumor specimens. The development of this technique should considerably enhance our ability to rapidly and accurately generate epigenetic profiles of tumor samples.