Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces
Rapid quantification of DNA methylation by measuring relative peak heights in direct bisulfite-PCR sequencing traces
复制标题
通过测量直接亚硫酸氢盐 PCR 测序迹线中的相对峰高来快速定量 DNA 甲基化
DOI：
10.1038/labinvest.2009.132
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发表时间：
2010-02-01
影响因子：
5
通讯作者：
Lu, Daru
中科院分区：
文献类型：
--
作者：
Jiang, Minghong;Zhang, Yuhao;Lu, Daru
目前有多种技术可用于量化DNA甲基化。然而，用于确定基因中CpG位点的DNA甲基化状态的快速和简单的方法仍然是难以捉摸的。在这份报告中，我们描述了一种新的方法，用于快速定量的CpG甲基化的基础上直接亚硫酸氢盐PCR测序法。根据亚硫酸氢盐-PCR的原理，将未甲基化的胞嘧啶转化为胸腺嘧啶，同时保持甲基化的胞嘧啶不变，我们将CpG位点视为SNP，并通过测量自动DNA测序迹线中胞嘧啶峰高与胞嘧啶和胸腺嘧啶峰高之和的比率来估计给定CG二核苷酸中胞嘧啶的甲基化状态。此外，我们采取了一些有效的措施来突破“瓶颈”的问题，使常规的亚硫酸氢盐测序方法不适合定量甲基化。与焦磷酸测序和亚硫酸氢盐克隆测序相比，我们的方法被证实是一种简单，高通量和成本效益的技术，用于确定特定基因的甲基化状态。因此，这种新方法预计是一种有效和经济的替代工具，用于快速定量甲基化模式，在筛选大量的临床样品跨多个基因。
Various technologies are currently available to quantify DNA methylation. However, rapid and simple methods for determining the DNA methylation status of CpG sites in genes still remain elusive. In this report, we describe a novel method for the rapid quantification of CpG methylation on the basis of direct bisulfite-PCR sequencing method. According to the principles of bisulfite-PCR, converting unmethylated cytosines to thymine while leaving methylated cytosines unchanged, we regard the CpG site as a SNP and estimate the methylation status of cytosines in the given CG dinucleotides by measuring the ratio of the cytosine peak height to the sum of cytosine and thymine peak heights in automated DNA sequencing traces. Furthermore, we take several effective measures to break through the 'bottleneck' problems that render the routine bisulfite sequencing method unsuitable for quantitative methylation. In comparison with pyrosequencing and bisulfite-cloning sequencing, our method is confirmed to be a simple, high-throughput and cost-effective technology for determining the methylation status of specific genes. Accordingly, this novel method is anticipated to be an efficient and economical alternative tool for rapid quantification of methylation patterns in screening large numbers of clinical samples across multiple genes.