DNA甲基化及其检测6.5讲课.ppt

致 谢 高效毛细管电泳（HPCE法） Resolution of nucleosides obtained from enzymatic hydrolysis of genomic DNA from human cancer cell line HT29 by HPCE. mC, 5-methylcytidine; A, adenosine; C, cytidine; G, guanosine; T, thymidine. 总体甲基化=100%×5mC/(5mC+5C） 生物质谱技术检测DNA总体甲基化水平 应用二：生物质谱技术 质谱分析法是通过对被测样品离子的质荷比的测定来进行分析的一种分析方法。被分析的样品首先要离子化，然后利用不同离子在电场或磁场的运动行为的不同，把离子按质荷比（m/z）分开而得到质量图谱，通过样品的质量图谱和相关信息，可以得到样品的定性定量结果。 质谱技术原理 质谱仪的示意图 (A), Nucleoside standards obtained under the optimized conditions. (B),Nucleosides from 2 ng genomic DNA of HCC tissues. Shown in the inset is the expanded chromatogram to reveal better the separation of 5-mdC, dC, C, dG, and 5-hmdC. Chen ML, Fan S, et al. Clinical Chemistry (2013)59:5; 全基因组DNA甲基化率 =100％×C5-mdC／(C5-dC+C5-mdC) 质谱法检测的优势： 需要DNA样本量多，1~2?g。 灵敏度准确性高。 操作较简便。 局限性： DNA样本要求纯度较高，提取过程中的小离子，未消化完全的蛋白，RNA对结果有干扰作用。 需用质谱仪。 Past and predicted methylome analysis milestones for current and potential future approaches. Beck S, et al. Trends Genet. 2008;24(5):231-7. DNA甲基化研究展望 * * * 同一个人，在不同的生活环境可能就会变成另外一个人，这种变化很有可能就是表观遗传的改变引起的，因为他们的基因序列并没有发生变化。 * * * * 相对于明确的DNA序列编码来说，表观遗传就想海底的冰山，更加多变，复杂，而且大多不为我们所知，有着更加重要的探索价值。 * * * Outcome and interpretation of MSAP analysis. Figure shows a schematic representation of banding patterns obtained by digestion of Apoe–/– or WT DNA with HpaII and EcoRI or MspI and EcoRI. Solid and dashed lines indicate present and absent bands, respectively. For clarity, only polymorphisms characterized by bands specifically present in WT DNA are shown. Flanking EcoRI (E) and HpaII/MspI (CCGG) sites are shown. The methylation status of the HpaII/MspI site is also indicated (hmC, hemimethylated cytosine, i.e. methylated on one strand only). Question marks indicate possible presence of a mutation. * * * Outcome and interpretation of MSAP analysis. Figure shows a schematic representation of banding patterns obtained by digestion of Apoe–/– or WT DNA with HpaII and EcoRI or MspI and EcoRI. Solid and dashed lines indicate pre