基于单分子免疫阵列的DNA甲基化关键分子5mC和5hmC超灵敏同步分析新方法的构建与评价

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.

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-甲基胞嘧啶（5-methylcytosine，5mC）和5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）是DNA甲基化的关键分子，其异常变化与癌症、心脑血管疾病和代谢性疾病等的发生发展密切相关。本项目将酶切、磁分离、免疫亲和、探针标记和单分子分析等技术有机结合在一起，成功构建特定基因区域5mC和5hmC的同步检测方法，测定结果和DNA测序分析具有可比性。首先，本项目以CYP24A1基因为研究对象，设计了特异性DNA捕获探针，结合限制性内切酶切和磁分离技术成功从基因组DNA中钓取目的片段，进而利用单链DNA外切酶Exonuclease I将其水解为单核苷酸，用于5mC和5hmC的测定。其次，依据5mC和5hmC的结构特征设计Biotin-tag-Phos修饰的功能性复合磁性纳米颗粒，采用EDC/Sulfo-NHS法合成5mC和5hmC抗体标记的核酸信号配基，实现5mC和5hmC的捕获、标记和检测。最后，对所构建新方法的线性范围、检出限、精密度和回收率等方法学指标进行评价，结果显示，5mC和5hmC的线性范围分别为 0.27 fmol/L~27000 fmol/L和2.7 fmol/L~27000 fmol/L；5mC和5hmC的检出限分别为2.7 amol/L和27 amol/L；5mC和5hmC在低、中、高三个浓度标准溶液的相对标准偏差范围分别为8.7 %~17.1 %和8.0 %~10.8 %；高浓度dATP、dTTP、dCTP和dGTP对5mC和5hmC的交叉反应率均小于0.05 %；5mC和5hmC的加标回收率分别为97.4%~103.5 %和98.0 %~104.0 %；测定结果和DNA测序分析具有可比性。因此，本项目成功构建一种可同步测定特定基因区域5mC和5hmC的方法，该方法检出限低、准确性好、特异性强，解决基于重亚硫酸盐修饰的常规方法不能同时分辨5mC和5hmC而导致分析结果无法细分的问题，有望进一步优化发展以应用于科研或临床的DNA甲基化检测分析。