Hi-Meth:特定位点DNA甲基化高通量检测平台  

Hi-Meth:a platform for high-throughput detection of site-specific DNA methylation

作　　者：李慧颖 刘庆 郭旻 王克剑 严长杰[1] 王春 LI Huiying;LIU Qing;GUO Min;WANG Kejian;YAN Changjie;WANG Chun(Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding,Jiangsu Key Laboratory of Crop Genetics and Physiology/Key Laboratory of Plant Functional Genomics of the Ministry of Education,Agricultural college of Yangzhou University,Yangzhou University,Yangzhou 225009,Jiangsu,China;State Key Laboratory of Rice Biology,China National Rice Research Institute,Chinese Academy of Agricultural Sciences,Hangzhou 310006,Zhejiang,China)

机构地区：[1]扬州大学农学院江苏省粮食作物现代产业技术协同创新中心教育部植物功能基因组学重点实验室江苏省作物基因组学与分子育种实验室,江苏扬州225009 [2]中国农业科学院中国水稻研究所水稻生物学重点实验室,浙江杭州310006

出　　处：《生物工程学报》2022年第8期3049-3061,共13页Chinese Journal of Biotechnology

基　　金：国家自然科学基金(31871703,31970519);中央级公益性科研院所基本科研业务费专项(CPSIBRF-CNRRI-202203)。

摘　　要：胞嘧啶甲基化是DNA表观遗传修饰的主要类型之一,在维持正常细胞功能和调控基因表达中具有重要作用。重亚硫酸盐测序法(bisulfite sequencing PCR,BSP)是特异性位点DNA甲基化检测的通用方法,能明确目的片段中每一个CpG位点的甲基化状态,但此方法需要大量的单克隆测序,操作过程较繁琐、成本昂贵。因此,开发准确、高效、便捷的DNA甲基化检测技术对提升表观遗传研究效率具有重要意义。基于本课题组开发的高通量突变类型检测平台Hi-TOM(high-throughput tracking of mutations),我们进一步建立了特定位点DNA甲基化高通量检测平台Hi-Meth(high-throughput detection of DNA methylation)。DNA样品通过重亚硫酸盐处理之后,仅需一轮PCR扩增即可通过Hi-Meth平台获得特定位点DNA甲基化分析结果。利用Hi-Meth平台,对水稻不同基因启动子区域进行了DNA甲基化检测分析,并与基于BSP方法获得的结果进行了比较。结果表明,Hi-Meth策略与BSP策略检测结果基本一致。而且通过Hi-Meth平台可以更准确、便捷地获得特异性位点DNA甲基化分析结果。综上所述,Hi-Meth为特定DNA区域提供了重要的甲基化检测平台,对表观遗传研究具有重要意义。Cytosine methylation is one of the major types of DNA epigenetic modifications and plays an important role in maintaining normal cell function and regulating gene expression.Bisulfite sequencing PCR(BSP)based cloning and sequencing is a general method for detecting DNA methylation at specific sites,which can clarify the methylation status of each CpG site in the target fragment.However,this method requires large amounts of single-clonal sequencing,which is complicated to operate,time consuming and expensive.Therefore,the development of an accurate,efficient and convenient DNA methylation detection technology is of great significance to improve the efficiency of epigenetic research.Based on the high-throughput mutation detection platform Hi-TOM(high-throughput tracking of mutations)developed by our group,we further established a site-specific DNA methylation high-throughput detection platform Hi-Meth(High-throughput Detection of DNA Methylation).After bisulfite treatment of DNA samples,the specific site-specific DNA methylation analysis results could be obtained through the Hi-Meth platform by performing only one round of PCR amplification.Using the Hi-Meth platform,the DNA methylation status of two promoter regions of rice were detected.The DNA methylation results from Hi-Meth were consistent with the results from BSP-based method.Thus,site-specific DNA methylation analysis results could be obtained accurately and conveniently through the Hi-Meth platform.In conclusion,Hi-Meth provides an important methylation detection platform for specific DNA regions,which has important significance for epigenetic research.

关 键 词：5-甲基胞嘧啶 甲基化特异位点检测 高通量测序平台 Hi-Meth 

分 类 号：Q75[生物学—分子生物学]