应用功能化氧化硅泡沫负载甲基化结合结构域建立一种富集甲基化DNA的方法  

Development of a method for methylated DNA enrichment with functionalized mesocellular silica foams immobilized with methyl CpG binding domain

作　　者：陈雅婷[1] 韩路[2] 赵东元[2] 屠波[2] 马端[1] 

机构地区：[1]复旦大学上海医学院分子医学教育部重点实验室,上海200032 [2]复旦大学上海医学院分子医学教育部化学系,上海200032

出　　处：《中华医学遗传学杂志》2012年第3期284-288,共5页Chinese Journal of Medical Genetics

基　　金：基金项目：973项目（2009CB941704）;十一五国家科技支撑项目（2006BAI05A05）

摘　　要：目的用功能化氧化硅泡沫负载甲基化CpG结合域（methyl—CpGbindingdomain，MBD）建立一种用于大样本初筛时富集甲基化DNA的方法。方法将具有超大介孔尺寸的氧化硅泡沫（mesocellularsilicafoam，MCF）功能化并负载融合蛋白GST-MBD以结合甲基化DNA。结果MCF功能化后仍保持较大的笼状孔结构，孔径约为55Dm，窗口约30nm，空隙容积高达1．0m^3／g，MBD蛋白负载量达53wt％。具有较高的结合甲基化DNA的活性和稳定性。在缓冲液盐浓度为500～550mmol／L时，MCF-MBD能较为专一地区分并富集混合溶液中的甲基化DNA。结论该方法为高通量和自动化检测DNA甲基化提供了一个较好的选择和分子生物学依据，为临床应用和DNA甲基化相关疾病的研究奠定了基础。Objective To develop a method for enriching methylated DNA in clinical samples using mesocellular silica foams （MCFs） immobilized with methyl-CpG binding domain （MBD）. Methods MCFs with ultra-large pore size were synthesized, functionalized and immobilized with GST-MBD. Results The large cage-like pore structures of MCF materials was retained after functionalization and immobilization, with pore diameter of 55 nm, window size of 30 nm, and a high pore volume of 1.0 cm^3/g. The loading amount of MBD was as high as 53 wt%. Immobilized MBD showed high binding activity and stability. In a binding buffer with salt concentrations ranging 500 - 550 mmol/L, the MCF-MBD can selectively enrich methylated DNA from the mixed DNA solution. Conclusion The MCF-MBD method may offer a better choice for high-throughout DNA methylation screening, and has laid a foundation for clinical application, prenatal diagnosis and research on DNA methylation-related genetic diseases.

关 键 词：甲基化结合结构域 甲基化DNA 功能化氧化硅泡沫 富集 

分 类 号：R440[医药卫生—诊断学]