5-甲基胞嘧啶（5mC）和5-羟甲基胞嘧啶（5hmC）是人类成熟体细胞DNA甲基化和去甲基化过程中的关键分子，在基因表达调控以及疾病的发生发展中具有重要作用。目前基于PCR或DNA测序的甲基化分析技术不能同时区分5mC和5hmC，而液质联用等技术手段受灵敏度限制不能实现二者痕量检测，故亟需建立一种超灵敏同时快速分析特定基因区域痕量5mC和5hmC的新方法。本项目以CYP24A1基因为研究对象，应用限制性内切酶BfmI、特异性DNA探针和磁分离技术从基因组DNA中钓取CYP24A1基因启动子区域目的片段；进而利用单链DNA外切酶Exonuclease I将目的片段水解为单核苷酸；依据单核苷酸的结构特征设计复合型功能化纳米颗粒，运用磁性荧光编码微球和单分子免疫阵列技术，实现DNA甲基化关键分子5mC和5hmC的超灵敏同步分析，为研究DNA甲基化在疾病发生发展中的作用提供技术支撑。

5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the key molecules in the process of DNA methylation and demethylation. They play an important role in the regulation of gene expression, occurrence and development of diseases. In view of the fact that 5hmC can not be distinguished from 5mC by the current methylation analysis based on PCR or DNA sequencing, as well as sensitivity limitation of other analytical techniques such as HPLC-MS, a new method is needed for ultra-sensitive and simultaneous analysis of trace 5mC and 5hmC in specific gene region. In this project, gene CYP24A1 is selected as the studying object. Restriction endonucleases BfmI, specific DNA probe and magnetic separation technique will be applied to fish the promotor region of CYP24A1 from genomic DNA. Subsequently, a single-stranded DNA exonuclease, Exonuclease I, is employed to hydrolyze the target fragment into single nucleotide. Then, a functional nanoparticle composite is designed and prepared according to the structural characteristics of single nucleotide. With the help of barcoded fluorescent microbeads and single molecular array, an ultra-sensitive and simultaneous analysis for trace 5mC and 5hmC will be achieved, which provides technical support for investigation of the role of DNA methylation in disease development.