【作者】 陈丹萍； 戴宗； 邹小勇；

【Author】 Danping Chen;Zong Dai;Xiaoyong Zou;School of Chemistry, University of Sun Yat-Sen;

【机构】 中山大学化学学院；

【摘要】 DNA甲基化是重要的表观遗传修饰方式之一,其中5m C、5hmC、5fC、5caC在体内含量十分低。传统PCR方法无法很好实现对这些位点的检测,此外,基于质谱的检测方法过于依赖仪器的灵敏性且操作复杂。因此,设计一种基于荧光定量PCR仪的高通量检测方法成功实现了5fC、5caC与C、5mC、5hmC的初步区分,并对5fC上的醛基进行化学衍生化,进一步实现了5fC与其他胞嘧啶衍生物的区分及检测。同时,本方法适用于全基因组5fC含量的定量检测,通过选择5fC含量相对丰富的基因组DNA作为全基因组DNA研究样本,建立了实际样品检测标准曲线。最后对C57新生小鼠脑组织、成年小鼠脑组织及成人脑组织基因组DNA进行检测,实验数据显示5fC的含量占DNA百分比分别为0.106%、0.069%和0.144%。该方法简单可行,是一种高通量的研究手段,可以同时满足对多个样本的检测要求。更多还原

【Abstract】 5-formylcytosine(5fC) is one of the important epigenetic marks, and its content is extremely low in genome as well. Polymerase chain reaction(PCR)-based technology usually loses the intrinsical epigenetic information after several cycles of replication, leading undistinguished. MS method has a limitation of low throughput and dependent on instrument-sensitive. Herein, we develop a facile method enabling high-throughput quantify fC in genome-wide directly on commercial qPCR instrument. Based on an isothermal amplification reaction, the fluorescence responses of 5hmC, 5fC and 5caC were discriminated from those of C and 5mC. Further adduction of a bulky of PHPA into 5fC would discriminate the fluorescence of 5fC from others specifically. The method was successfully applied to quantify the genetic 5fC level in several biologic samples including human brain(h-AB), New-born(m-NB) and adult(m-AB) in C57 mouse brain tissues. The results were well in line with those from strand MS-based methods.更多还原