It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5′ end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

中文翻译：

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甲基化位点显示（MSD）-AFLP，一种灵敏且价格合理的CpG甲基化分布图分析方法。

已经指出，环境因素或化学物质可能导致起源于疾病的疾病。为了检测DNA甲基化中异常的表观遗传学改变，这种研究需要方便且具有成本效益的方法，其中同时处理多个样品。我们在这里介绍了甲基化位点展示（MSD），这是一种制备DNA文库的独特技术。通过将其与扩增片段长度多态性（AFLP）分析相结合，我们开发了一种新方法MSD-AFLP。甲基化位点展示库仅包含衍生自原始基因组DNA样本中5'端CpG甲基化的DNA片段的DNA。为了测试此方法的有效性，我们在肝脏，肾脏，比较小鼠的海马组织和海马组织，以检查MSD-AFLP是否可以检测组织特异性差异甲基化CpGs水平的细微差异。结果，发现了许多怀疑是组织特异性差异甲基化的CpG位点。确定了与这些甲基-CpG位点相邻的核苷酸序列，并通过甲基化敏感的限制性内切核酸酶（MSRE）-PCR分析确定了甲基化水平，以确认AFLP分析的准确性。在这些方法中，组织之间的甲基化水平差异几乎相同。通过MSD-AFLP分析，我们检测到许多CpG表现出低于5％的统计学显着组织特异性差异和低于10％的变异度。此外，MSD-AFLP分析可用于鉴定其他生物（包括人类）中的CpG甲基化位点。MSD-AFLP分析可潜在地用于测量CpG甲基化水平的轻微变化。关于MSD-AFLP分析研究的卓越准确性，灵敏度和通量，该方法在基于表观遗传学的各种研究中将是有利的。