Guangdong Institute of Gastroenterology, Guangdong Provincial Key Laboratory of Colorectal and Pelvic Floor Disease, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Department of Colorectal Surgery, The Sixth Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China.
Clin Chem. 2019 May;65(5):664-673. doi: 10.1373/clinchem.2018.298570. Epub 2019 Feb 8.
The DNA methylation profile provides valuable biological information with potential clinical utility. Several methods, such as quantitative methylation-specific PCR (qMSP), have been developed to examine methylation of specific CpG sites. Existing qMSP-based techniques fail to examine the genomic methylation at a single-base resolution, particularly for loci in gene bodies or extensive CpG open seas lacking flanking CpGs. Therefore, we established a novel assay for quantitative analysis of single-base methylation.
To achieve a robust single-base specificity, we developed a PCR-based method using paired probes following bisulfite treatment. The 6-carboxyfluorescein- and 2'-chloro-7'phenyl-1,4-dichloro-6-carboxy-fluorescein-labeled probes conjugated with minor groove binder were designed to specifically bind to the methylated and unmethylated allele of targeted single CpGs at their 3' half regions, respectively. The methylation percentage was calculated by values of methylation / (methylation + unmethylation).
In the detection of single CpGs within promoters or bodies of 4 human genes, the quantitative analysis of the single-base methylation assay showed a detection capability in the 1 to 1:10000 dilution experiments with linearity over 4 orders of magnitude ( = 0.989-0.994; all < 0.001). In a cohort of 10 colorectal cancer samples, the assay showed a comparable detection performance with bisulfite pyrosequencing ( = 0.875-0.990; all < 0.001), which was better than conventional qMSP methods normalized by input control reaction ( = 0.841 vs 0.769; = 0.002 vs 0.009).
This assay is highly specific and sensitive for determining single-base methylation and, thus, is potentially useful for methylation-based panels in diagnostic and prognostic applications.
DNA 甲基化谱提供了有潜在临床应用价值的有价值的生物学信息。已经开发了几种方法，例如定量甲基化特异性 PCR(qMSP)，以检查特定 CpG 位点的甲基化。现有的基于 qMSP 的技术无法以单碱基分辨率检查基因组甲基化，特别是对于基因体中的基因座或缺乏侧翼 CpG 的广泛 CpG 开放海域。因此，我们建立了一种用于定量分析单碱基甲基化的新测定法。
为了实现稳健的单碱基特异性，我们开发了一种基于 PCR 的方法，该方法在亚硫酸氢盐处理后使用配对探针。6-羧基荧光素和 2'-氯-7'苯基-1,4-二氯-6-羧基荧光素标记的探针与小沟结合物缀合，设计用于分别特异性结合靶向单 CpG 的甲基化和未甲基化等位基因的 3'半区。甲基化百分比通过甲基化值/（甲基化+未甲基化）计算。
在检测 4 个人类基因启动子或体内的单个 CpG 时，单碱基甲基化测定的定量分析在 1 至 1:10000 稀释实验中具有线性度超过 4 个数量级（ = 0.989-0.994；均<0.001）。在 10 个结直肠癌样本的队列中，该测定与亚硫酸氢盐焦磷酸测序具有可比的检测性能（ = 0.875-0.990；均<0.001），优于通过输入对照反应归一化的常规 qMSP 方法（ = 0.841 与 0.769； = 0.002 与 0.009）。
该测定法对确定单碱基甲基化具有高度特异性和敏感性，因此在诊断和预后应用中可能对基于甲基化的面板有用。
