一种快速高效的全基因组甲基化CpG岛富集方法的建立

A Rapid and Efficient Method for Whole Genome Methyl-CpG Islands Enrichment

高甲基化的CpG岛所致基因表观遗传学转录失活已经成为肿瘤表观基因组学研究的重要内容,但尚未建立一种能快速在全基因组水平上进行甲基化CpG岛的富集方法。利用甲基化结合蛋白MBD2b具有特异性结合甲基化DNA的特性,建立了一种基于DNA免疫共沉淀技术的全基因组甲基化CpG岛的富集方法。在大肠杆菌中表达重组的GST-MBD2b蛋白,通过Glutathione Sepharose 4B对重组蛋白进行纯化,制备成亲和层析柱,利用在不同的盐离子强度下甲基化DNA和非甲基化DNA的结合能力不同,对甲基化DNA进行富集。用甲基化酶SssI处理过的DNA片段与非甲基化DNA片段进行富条件的摸索,发现0.5mol/L KCl的浓度是甲基化DNA片段和非甲基化DNA片段得以分开的临界条件。样品的富集效率用Real Time PCR进行检测。结果表明,这种方法能够实现对全基因组甲基化DNA的有效富集且最高的富集倍数可达到100多倍。富集到的甲基化DNA可以进行后续的定量PCR,DNA测序和全基因组芯片的分析等工作,为大规模分析全基因组CpG岛甲基化的改变奠定了基础。

Hypermethylation of CpG islands is an epigenetic modification and plays key role in tumorigenesis. Several methods for methyl-CpG islands detection have been established, but high through methyl- CpG islands enrich method for whole genome was not reported till now. A rapid and efficient genome-wide methylation enrichment method is reported, which is based on DNA immunoprecipitation for accumulating methyl -CpG islands. The GsT-MBD2b recombinant protein was expressed in E. Coli and purified by using Glutatione Sepharose 4B. The methylated CpG islands was enriched by the GST-MBD2b affinity column, which has the capability to bind methylated CpG islands at high salt concentration. The enrich efficiency was detected by real time PCR and the results indicated that the methylated CpG islands was enriched more than 100 folds. The enriched methylated CpG islands could be used further experiments for example real time quantitative PCR, DNA sequencing and whole genome CpG islands microarray analysis.