Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids
Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids
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DOI：
10.4161/epi.8833
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发表时间：
2009-05-16
期刊：
影响因子：
3.7
通讯作者：
Herceg, Zdenko
中科院分区：
文献类型：
--
作者：
Vaissiere, Thomas;Cuenin, Cyrille;Herceg, Zdenko
从患有癌症的个体的血浆中分离的无细胞循环DNA已显示出在DNA甲基化中具有癌症相关的变化，因此它代表了生物标志物发现的有吸引力的靶标。然而，体液中DNA甲基化变化的可靠检测已被证明是技术上的挑战。在这里，我们描述了一种新的方法组合，允许定量和灵敏地检测存在于体液中的微量DNA中的DNA甲基化（全双亚硫基扩增后微量DNA量的定量甲基化分析，qMAMBA）。该方法涉及亚硫酸氢盐修饰的DNA模板的全基因组扩增，然后使用焦磷酸测序进行定量甲基化检测，并允许从少量起始DNA分析多个基因。为了验证我们的方法，我们使用qMAMBA检测四个基因和LINE1重复序列结合血浆DNA样本作为模型系统。qMAMBA在分析具有极少量DNA和低浓度甲基化等位基因的血浆样品中的甲基化水平和模式方面具有很高的功效。因此，qMAMBA将促进甲基化研究，旨在发现表观遗传生物标志物，并应证明特别有价值的分析一个大样本系列的体液从分子流行病学研究，以及在跟踪疾病的早期诊断。
Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method, we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.