Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements.
Genomic profiling of CpG methylation and allelic specificity using quantitative high-throughput mass spectrometry: critical evaluation and improvements.
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DOI：
10.1093/nar/gkm662
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发表时间：
2007
影响因子：
14.9
通讯作者：
Clark, Susan J
中科院分区：
文献类型：
--
作者：
Coolen, Marcel W;Statham, Aaron L;Gardiner-Garden, Margaret;Clark, Susan J
CpG 甲基化是表观基因组结构的关键组成部分，与不改变 DNA 序列的基因表达变化相关。自从第一份关于癌症等疾病中 DNA 甲基化失调的报告以及人类表观基因组计划的启动以来，人们越来越需要一种详细、高通量和定量的分析方法，以发现和验证大样本群体中正常和异常的 DNA 甲基化谱。在这里，我们提出了一种使用碱基特异性片段化和 MALDI-TOF 质谱法的改进方案，该方案能够实现灵敏且高通量的 DNA 甲基化分析方法，对每个信息性 CpG 残基定量至 5% 甲基化。我们确定了该方案的准确性、可变性和敏感性，对实验设计和数据解释进行了关键改进，并开发了一种新公式来准确测量 CpG 甲基化。现在的关键创新允许确定差异和等位基因特异性甲基化，例如癌症和印记中的甲基化。新方案非常适合对多个基因组区域和大样本群体进行详细的 DNA 甲基化分析，这对于正常和患病人类表观基因组的全面分析至关重要。
CpG methylation is a key component of the epigenome architecture that is associated with changes in gene expression without a change to the DNA sequence. Since the first reports on deregulation of DNA methylation, in diseases such as cancer, and the initiation of the Human Epigenome Project, an increasing need has arisen for a detailed, high-throughput and quantitative method of analysis to discover and validate normal and aberrant DNA methylation profiles in large sample cohorts. Here we present an improved protocol using base-specific fragmentation and MALDI-TOF mass spectrometry that enables a sensitive and high-throughput method of DNA methylation analysis, quantitative to 5% methylation for each informative CpG residue. We have determined the accuracy, variability and sensitivity of the protocol, implemented critical improvements in experimental design and interpretation of the data and developed a new formula to accurately measure CpG methylation. Key innovations now permit determination of differential and allele-specific methylation, such as in cancer and imprinting. The new protocol is ideally suitable for detailed DNA methylation analysis of multiple genomic regions and large sample cohorts that is critical for comprehensive profiling of normal and diseased human epigenomes.