化学
亚硫酸氢盐
DNA甲基化
亚硫酸氢盐测序
甲基化
分子生物学
甲基化DNA免疫沉淀
DNA
波形蛋白
连锁反应
限制性酶
生物化学
基因
生物
基因表达
免疫组织化学
光化学
免疫学
作者
Yunlong Liu,Xiaoming Wang,Yujiao Li,Haiping Wu
标识
DOI:10.1016/j.aca.2023.341001
摘要
The fragmentation and low concentration of cell-free DNA (cfDNA) pose higher challenges for the cfDNA methylation detection technologies. Conventional bisulfite conversion-based methods are inadequate for cfDNA methylation analysis due to cumbersome operation and exacerbating cfDNA degradation. Herein, we proposed temperature-programmed enzymatic reactions for cfDNA methylation analysis in a single tube. Endonuclease was used to mildly recognize DNA methylation to avoid the degradation of cfDNA. And two stages of amplification reactions significantly improved the detection sensitivity for GC-rich sequence. With vimentin as the target, the detection sensitivity was 10 copies of methylated DNA. Meanwhile, the proposed method can accurately quantify the methylation level of target sequence from 1000-fold of unmethylated DNA background. Further, the methylated vimentin gene in 20 clinical plasma samples was successfully detected. The results shown significant differences in methylation levels of the vimentin gene between healthy volunteers and colorectal cancer patients. These results lead us to believe that the proposed method has great application potential for DNA methylation analysis as a complement to bisulfite conversion-based methods.
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