3种 DNA 甲基化定量分析方法的比较  

Comparison of Three Different Methods for Quantitative Analysis of DNA Methylation

作　　者：路兆宁[1] 周在威[2] 马晴雯[2] 颜景斌[2] 

机构地区：[1] 上海交通大学附属儿童医院,上海市儿童医院,上海交通大学医学遗传研究所,上海市200040 [2]卫生部医学胚胎与分子生物学重点实验室 上海市胚胎与生殖工程重点实验室,上海市200040

出　　处：《医学分子生物学杂志》2015年第2期84-89,共6页Journal of Medical Molecular Biology

基　　金：国家重点基础研究发展规划项目（973计划）,国家自然科学基金,上海市科委基础研究重点项目（No.11 JC1411000）

摘　　要：目的：比较特定基因区域 DNA 甲基化检测方法的优缺点。方法采用焦磷酸测序、克隆测序和DNA 甲基化芯片等3种 DNA 甲基化检测方法，对7例唐氏综合征和5例正常对照样本的 PRDM8内含子7 CpG 岛中的部分序列进行甲基化检测。从准确度、重复性和精确度（分辨率）等方面来比较3种方法，探讨哪种方法更适合特定基因的 DNA 甲基化水平定量检测。结果①焦磷酸测序可在单碱基水平定量检测样本的甲基化水平，结果重复性好，准确度高，而且能发现样本中 CpG 位点甲基化水平变化的规律；②克隆测序也可在单碱基水平定量检测样本的甲基化水平，但结果重复性较差，无法发现 CpG 位点甲基化水平变化的规律；③甲基化芯片可通过所测区域的两个探针来判断样本间甲基化水平的差异，但无法在单碱基水平定量检测样本的甲基化水平；④上述3种方法检测结果之间存在一定的差异，但是样本间甲基化水平总体趋势基本一致。此外，唐氏综合征样本的甲基化水平高于正常对照样本。结论焦磷酸测序准确度高、重复性好、费用较低、操作较为简单快捷，更为适合在单碱基水平检测特定基因区域的 DNA 甲基化水平。Objective To compare the advantages and disadvantages of different methods for quantitative analysis of locus-specific DNA methylation. Methods Three different methods inclu-ding bisulfite pyrosequencing, bisulfite sequencing of clones and methylated DNA immunoprecipita-tion （MeDIP） -chip were used to identify the DNA methylation level at CpG island of PRDM8 in-tron 7 in 7 Down syndrome （DS） and 5 normal samples. To choose the suitable methods for quantita-tive analysis of locus-specific DNA methylation, the accuracy, repeatability and resolution of these methods were compared. Results DNA methylation was identified at single-base resolution by bisul-fite pyrosequencing with high accuracy and good reproducibility. More importantly, the variation tendency of methylation levels at different CpG sites was found with bisulfite pyrosequencing. However, these advantages were not revealed with bisulfite sequencing of clones, though it was a useful method for the detection of methylation at single-nucleotide resolution. In addi-tion, relative enrichment of methylated DNA was obtained by MeDIP-chip through the signals of two probes corresponding to the detection sequence. Nevertheless, it was not suitable for detection of＆amp;nbsp;methylation at single-nucleotide resolution. In this study, hypermethylation was observed at CpG is-land of PRDM8 intron 7 in DS, although some differences existed in three methods. Conclusion Bisulfite pyrosequencing is a suitable method for quantitative analysis of locus-specific DNA methyla-tion at single-nucleotide resolution with some prominent characteristics, such as higher accuracy, excellent reproducibility, lower costs and more convenient operation.

关 键 词：焦磷酸测序 克隆测序 DNA 甲基化芯片 甲基化修饰 

分 类 号：Q31[生物学—遗传学]