"Quantitative analysis of DNA methylation using sequence-specific, real-time loop-mediated isothermal amplification"

作者全名："Fu, Yao; Zhang, Li; Xing, Yu; Deng, Shixiong"

作者地址："[Fu, Yao; Zhang, Li; Xing, Yu; Deng, Shixiong] Chongqing Med Univ, Dept Forens Med, Chongqing 400016, Peoples R China"

通信作者："Deng, SX (通讯作者)，Chongqing Med Univ, Dept Forens Med, Chongqing 400016, Peoples R China."

来源：ANALYTICA CHIMICA ACTA

ESI学科分类：CHEMISTRY

WOS号：WOS:000891056800002

JCR分区：Q1

影响因子：6.2

年份：2022

卷号：1235

期号：　

开始页：　

结束页：　

文献类型：Article

关键词：DNA methylation; Loop-mediated isothermal amplification; Real-time fluorescence probes; Sequence specificity

摘要："A number of genetic diseases and in the majority of tumor diseases have been reported to be associated with anomalous methylation levels of DNA. Accurate determine of the methylation level is an essential prerequisite for epigenetic diagnostics. Although some studies have used probe with a quencher enable real-time monitor of loop-mediated isothermal amplification (LAMP), most of them lack systematic investigations of design principle and applicable ambient when applied in TAMP based methylation detection. Herein, a sequence-specific, real-time loop-mediated isothermal amplification was developed for the quantitative analysis of DNA methylation. An oligonucleotide dimer was designed to implement three functions at the same time, including primer, sense probe and signal probe (PSS probe). Systematic investigations were performed to optimize the performance of PSS probe. After that, our method can accurately determine as low as 0.1% synthetic methylated DNA in the presence of numerous unmethylated DNA. Moreover, a higher methylated level of kazrin periplakin interacting protein (KAZN) gene was detected in colorectal cancer cells with a stable housekeeping gene beta-actin used as an endogenous control. The developed PSS probe for LAMP provide an option to analyze DNA methylation levels in resource-limited environments, and hold great potential for the diagnosis of methylation-related diseases."

基金机构：Natural Science Foundation of Chongqing Province [cstc2021jcyj-msxmX0485]; Major Decision-Making Consulting Project of Chongqing [20182B-03]

基金资助正文："This work was sponsored by the Natural Science Foundation of Chongqing Province [grant number cstc2021jcyj-msxmX0485], Major Decision-Making Consulting Project of Chongqing [grant number 20182B-03]."