CAJ | 학술논문

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.
목적 용공능화양화규포말부재갑기화CpG결합역(methyl-CpG binding domain,MBD)건립일충용우대양본초사시부집갑기화DNA적방법.방법 장구유초대개공척촌적양화규포말(mesocellular silica foam,MCF)공능화병부재융합단백GST-MBD이결합갑기화DNA.결과 MCF공능화후잉보지교대적롱상공결구,공경약위55 nm,창구약30 nm,공극용적고체1.0 m3/g,MBD단백부재량체53 wt％.구유교고적결합갑기화DNA적활성화은정성.재완충액염농도위500～550 mmol/L시,MCF-MBD능교위전일지구분병부집혼합용액중적갑기화DNA.결론 해방법위고통량화자동화검측DNA갑기화제공료일개교호적선택화분자생물학의거,위림상응용화DNA갑기화상관질병적연구전정료기출.
[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.