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

摘要

[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 alter functionalization and immobilization,with pore diameter of 55 nm,window size of 30 nm,and a high pore volume of 1.0 cm3/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.%目的 用功能化氧化硅泡沫负载甲基化CpG结合域(methyl-CpG binding domain,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(mesocellular silica foam,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55 nm,窗口约30 nm,空隙容积高达1.0 m3/g,MBD蛋白负载量达53 wt％.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500～550 mmol/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.