State Key Laboratory of Chemo/BioSensing and Chemometrics, College of Biology, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China.
Anal Chem. 2021 Jun 8;93(22):8077-8083. doi: 10.1021/acs.analchem.1c01510. Epub 2021 May 21.
The aberrant methylation of many genes has been reported to be associated with various carcinomas. Accurate detection of the methylation level could provide critical insights into the diagnostic analysis of diseases. Here, a sensitive HpaII-edited absolute droplet loop-mediated isothermal amplification (HEADLAMP) method based on methylation-sensitive restriction enzyme (MSRE) HpaII was developed for the digital quantification of DNA methylation. Methylation levels of the death-associated protein kinase 1 (DAPK1) gene that is associated with many cancers were studied using β-actin as an internal reference. DAPK1 (2.5 pM) with 0.01% methylation (250 aM) can be detected with the conventional HpaII-edited LAMP assay. Using HEADLAMP, as low as 1% methylation level can be distinguished with an estimated limit of detection of 5 aM (ca. 3 copies/μL). Moreover, HEADLAMP can detect low levels of methylated DAPK1 in normal L-02 cells, while the conventional assay cannot. Finally, HEADLAMP was applied to the detection of DAPK1 methylation in 20 clinical tissue samples, which revealed hypermethylated DAPK1 in cervical cancer patients. We envisage potential applications of this robust, specific, and sensitive HEADLAMP assay in epigenetic studies and early clinical diagnosis.
已有研究报道，许多基因的异常甲基化与各种癌症有关。准确检测甲基化水平可以为疾病的诊断分析提供重要的见解。在这里，我们开发了一种基于甲基化敏感限制性内切酶（MSRE）HpaII 的灵敏 HpaII 编辑绝对液滴环介导等温扩增（HEADLAMP）方法，用于 DNA 甲基化的数字定量。使用β-肌动蛋白作为内参，研究了与许多癌症相关的死亡相关蛋白激酶 1（DAPK1）基因的甲基化水平。使用常规 HpaII 编辑的 LAMP 测定法，可检测到 2.5 pM 的 DAPK1（250 aM）和 0.01%的甲基化（250 aM）。通过 HEADLAMP，即使是低至 1%的甲基化水平也可以区分，估计检测限为 5 aM（约 3 拷贝/μL）。此外，HEADLAMP 可以检测到正常 L-02 细胞中低水平的甲基化 DAPK1，而常规测定法则不能。最后，HEADLAMP 应用于 20 个临床组织样本中 DAPK1 甲基化的检测，结果显示宫颈癌患者的 DAPK1 发生了高甲基化。我们预计这种稳健、特异和灵敏的 HEADLAMP 测定法在表观遗传学研究和早期临床诊断中有潜在的应用。
