Improved quantification of DNA methylation using methylation-sensitive restriction enzymes and real-time PCR
Improved quantification of DNA methylation using methylation-sensitive restriction enzymes and real-time PCR
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DOI：
10.4161/epi.2.2.4203
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发表时间：
2007-04-01
期刊：
影响因子：
3.7
通讯作者：
Roach, Helmtrud I.
中科院分区：
文献类型：
--
作者：
Hashimoto, Ko;Kokubun, Shoichi;Roach, Helmtrud I.
当评估甲基化状态时，细胞在特定CpG位点处的DNA甲基化状态的异源性是一个问题。我们开发了一种简单的两步法，用于定量在特定基因启动子中特定CpG位点显示甲基化的细胞百分比。第一步是用相关的甲基化敏感性限制酶（最佳2单位）过夜消化基因组DNA（最佳浓度20 ng/5 μ l）。然后进行真实的时间PCR，使用SYBR（R）绿色方法，使用包含酶切割位点的引物。通过在每个PCR板中包括完全甲基化和完全非甲基化的DNA，可以测量由非特异性消化或不完全消化引起的误差，并用于调整原始结果，从而提高特异性。如果甲基化状态的差异超过10%，则该方法可以检测到这些差异。除了实时PCR系统之外，不需要专门的设备，并且该方法可以适用于53种市售的甲基化敏感性限制性内切酶中的任何一种。
Heterogeneity of cells with respect to the DNA methylation status at a specific CpG site is a problem when assessing methylation status. We have developed a simple two-step method for the quantification of the percent of cells that display methylation at a specific CpG site in the promoter of a specific gene. The first step is overnight digestion of genomic DNA (optimal conc. 20ng/5 mu l) with a relevant methylation-sensitive restriction enzyme (optimal 2 units). This is followed by real time PCR, using the SYBR (R) Green method, with primers that bracket the site cleaved by the enzyme. By including fully methylated and fully non-methylated DNA in each PCR plate, the errors caused by non-specific digestion or incomplete digestion can be measured and used to adjust the raw results and thus increase specificity. The method can detect differences in methylation status if these are more than 10%. No specialized equipment is required beyond the real-time PCR system and the method can be adapted for any of the 53 commercially available methylation-sensitive restriction enzymes.