亚硫酸氢盐测序法DNA甲基化标记筛查技术的改进研究

An improved bisulfite genomic sequencing for DNA methylation marker scanning

目的改进DNA亚硫酸氢盐测序法（bisulfite genomic sequencing，BGS），建立一种简便快速的甲基化标记筛查技术。方法基因组DNA在亚硫酸氢盐处理、脱盐纯化后直接进入碱性的PCR体系，通过延长预变性时间完成修饰过程，然后进行常规亚硫酸氢盐PCR（bisulfite-PCR，BSP）扩增；首轮扩增产物再用5′端加有高GC标签序列的引物进行2次扩增，达到调整GC含量和直接测序的目的。同时用传统和改进BGS法检测3T3-L1细胞肿瘤坏死因子α（TNF-α）基因和Hela细胞雄激素受体基因启动子的甲基化情况，以评估改进方案的可行性。并用Alu序列实时定量BSP技术比较传统和改进BGS法的灵敏度。结果无论是高甲基化还是低甲基化片段，改进的BGS法均可实现BSP产物的直接测序，结果与传统法一致。改进法修饰的DNA转化率达到100％，特异度〉93．75％，灵敏度显著高于传统方法（t=2．9782，P〈0．05），并有良好的重复性。结论改进后的BGS方法简单灵敏，可用于甲基化标记的快速筛查。

Objective To develop a simplified bisulfite genomic sequencing （BGS） method for DNA methylation marker scanning. Methods According to modified BGS protocol, the desalt DNA treated with bisulfite were directly used for bisulfite-PCR （BSP） without alkali treatment. Complement of the bisulfite modification was accomplished by a prolonged pre-denaturation stage. After BSP, a second round PCR was performed with a pair of GC tagged primers to adjust the GC content of the amplicon for direct sequencing. To assess this improved protocol, promotor methylation of TNF-α gene in 3T3-L1 cell and androgen receptor （AR） gene in Hela cell was investigated. The real time BSP for Alu was also used to compare the sensitivity of the modified assay with traditional assay. Results Both the hypermethylated TNF-α promotor and hypomethylated AR promotor were successfully sequenced by improved BGS method, and the results were consistent with that of the traditional assay. The conversion rate reached 100%, while the conversion specificity was higher than 93.75% . The sensitivity of improved BGS method increased significantly （t = 2. 978 2, P 〈 0. 05 ） and showed good reproducibility. Conclusion The improved BGS method is simple and sensitive, facilitating more ambitious genomic methylation mapping studies.