基因组DNA中 5-羟甲基胞嘧啶的系统分析及其生物功能研究

项目名称： 基因组DNA中 5-羟甲基胞嘧啶的系统分析及其生物功能研究

项目编号： No.21472120

项目类型： 面上项目

立项/批准年度： 2015

项目学科： 环境科学、安全科学

项目作者： 李正平

作者单位： 陕西师范大学

项目金额： 86万元

中文摘要： DNA中甲基化的胞嘧啶（mC）在生命过程中起着至关重要的调控作用。近年来发现mC可转化为羟甲基胞嘧啶（hmC），但其生物功能还不清楚。缺乏合适的hmC分析方法严重阻碍了hmC生物功能研究的进程。本项目拟利用β-葡萄糖转移酶使hmC与葡萄糖共价结合，基于硼酸基团与葡萄糖之间的特异性识别，以硼酸修饰的荧光分子及纳米粒子探针，研究建立染色体DNA中hmC原位荧光分析及基因组DNA中hmC的单分子分析方法；以硼酸修饰的磁性微球分离、富集含hmC的DNA片段，并进行高通量测序，绘制基因组DNA中5hmC的分布图谱；以连接PCR和连接酶链式反应建立能直接检测基因组DNA中hmC的高灵敏度分析方法。从而形成一个系统分析hmC的技术平台，促进hmC的生物功能研究的进展。同时探讨hmC在癌症发生过程中的作用，以及以hmC为新标志物进行癌症诊断分析的可能性。

中文关键词： 5-羟甲基胞嘧啶；荧光探针；原位分析；分布图谱；生物功能

英文摘要： The methylation of cytosine in DNA plays a crucial regulation role in life processes. Recently, it has been found that methylcytosine (mC) could be converted to hydroxymethylcytosine (hmC). The biological functions of hmC is now unclear. Lack of suitable analytical methods for hmC detection seriously hinders the process for the studies of hmC biological functions. The hmC can covalently bound glucose molecule with the catalysis of β-glucosyltransferase. In this project, we want to study and establish the in situ fluorescence analysis of hmC in chromosomes and hmC assay in single genomic DNA molecule by using fluorescent molecule probes or nanoparticle probes modified with boric acid groups based on the specific recognition between boric acid group and glucose molecule. We also plan to separate and enrich the DNA fragments containing hmC with magnetic microspheres modified with boric acid groups and to determine the sequences of the DNA fragments with massively parallel sequencing techniques in order to obtain the hmC distribution map in genomic DNA. Based on ligation-based PCR and ligase chain reaction, we will develop the highly sensitive methods for direct detection of hmC in genomic DNA. With this project, we can establish a systematic analysis platform for hmC detection, which can push the process of biological function studies of hmC in genomic DNA. The biological functions of hmC in cancer generation and development and the diagnosis of cancers by using hmC as new biomarkers will also be investigated in this project.

英文关键词： 5-hydroxymethylcytosine;fluorescence probe;in situ analysis;distribution map;biological function