DNA甲基化检测中亚硫酸氢盐修饰方法的改进与评价
发布时间：2018-11-29 07:41
【摘要】：目的:建立一种快速便捷的亚硫酸氢盐修饰DNA方法用于DNA甲基化检测。方法:提高亚硫酸氢盐浓度及修饰温度、缩短修饰时间并用玻璃奶回收DNA改进DNA修饰方法,用亚硫酸氢盐测序法分析MAGE-A3基因和DAP-K基因目的片段中胞嘧啶转化为胸腺嘧啶的效率,评价改进方法与传统方法、试剂盒方法对DNA的修饰效果。结果:改进的方法可将操作过程缩短到3 h左右;非甲基化的胞嘧啶到胸腺嘧啶的转化率超过99%,与传统方法、试剂盒方法相比差异无统计学意义(χ~2=0.0564,P0.05);只针对非甲基化的胞嘧啶进行转化修饰,而在对甲基化胞嘧啶过度修饰为胸腺嘧啶方面与传统方法无统计学差异(χ~2=0.0149,P0.05)。结论:改进的亚硫酸氢盐DNA修饰方法修饰效率高,对甲基化的胞嘧啶过度修饰影响不显著,且具有节省时间,操作简便等优点。
[Abstract]:Objective: to establish a rapid and convenient method for the detection of DNA methylation by hydrogen sulfite modified DNA. Methods: the concentration and temperature of hydrogen sulfite were increased, the modification time was shortened, and the DNA modification method was improved by recovering DNA from glass milk. The conversion efficiency of cytosine to thymine in the target fragment of MAGE-A3 gene and DAP-K gene was analyzed by bisulfite sequencing. The modified method, traditional method and kit method were evaluated for the modification effect of DNA. Results: the improved method could shorten the operation process to about 3 h, and the conversion rate of unmethylated cytosine to thymine was more than 99%, there was no significant difference compared with the traditional method and kit method (蠂 ~ 2 ~ 2, 0.0564g, P0.05). Only the unmethylated cytosine was modified by transformation, but there was no significant difference between the traditional method and the hypermethylated cytosine (蠂 ~ 2) 0.0149 (P0.05). Conclusion: the modified DNA modification method has the advantages of high modification efficiency, little effect on methylated cytosine overmodification, time saving, simple operation and so on.
【作者单位】： 南阳理工学院张仲景国医学院;南阳市第一人民医院神经内科;中山大学达安基因股份有限公司;
【分类号】：R440
，
本文编号：2364356
[Abstract]:Objective: to establish a rapid and convenient method for the detection of DNA methylation by hydrogen sulfite modified DNA. Methods: the concentration and temperature of hydrogen sulfite were increased, the modification time was shortened, and the DNA modification method was improved by recovering DNA from glass milk. The conversion efficiency of cytosine to thymine in the target fragment of MAGE-A3 gene and DAP-K gene was analyzed by bisulfite sequencing. The modified method, traditional method and kit method were evaluated for the modification effect of DNA. Results: the improved method could shorten the operation process to about 3 h, and the conversion rate of unmethylated cytosine to thymine was more than 99%, there was no significant difference compared with the traditional method and kit method (蠂 ~ 2 ~ 2, 0.0564g, P0.05). Only the unmethylated cytosine was modified by transformation, but there was no significant difference between the traditional method and the hypermethylated cytosine (蠂 ~ 2) 0.0149 (P0.05). Conclusion: the modified DNA modification method has the advantages of high modification efficiency, little effect on methylated cytosine overmodification, time saving, simple operation and so on.
【作者单位】： 南阳理工学院张仲景国医学院;南阳市第一人民医院神经内科;中山大学达安基因股份有限公司;
【分类号】：R440
，
本文编号：2364356
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