| .改进亚硫酸盐测序技术研究拟南芥DNA甲基化水平[J].农业环境科学学报,2013,32(5):. |
| 改进亚硫酸盐测序技术研究拟南芥DNA甲基化水平 |
| An Improved Bisulfite Sequencing Technique for Analysis of DNA Methylation Level in Arabidopsis thaliana |
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| DOI：10.11654/jaes.2013.05.002 |
| 中文关键词: 亚硫酸盐测序 DNA甲基化 MLH1 |
| 英文关键词: bisulfite sequencing DNA methylation MLH1 |
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| 摘要点击次数: 2104 |
| 全文下载次数: 1956 |
| 中文摘要: |
| DNA胞嘧啶甲基化是一种重要的表观遗传修饰形式，在细胞增殖、分化、发育、基因组印迹、基因表达调控等过程中起着重要作用。随着对DNA甲基化研究的深入，各种DNA甲基化检测方法被开发出来满足不同类型的研究需求。通过实验建立的一种改进亚硫酸盐测序技术，将拟南芥基因组DNA经酶切纯化后，用亚硫酸盐修饰液修饰，将PCR产物克隆到pEASY-T1载体上，每个样品随机挑取15个阳性克隆，测序分析，研究拟南芥错配修复基因MutL-homologue 1（MLH1）启动子区域的甲基化水平。结果表明：与传统亚硫酸盐测序法相比，改进方法有利于DNA完全修饰，既减少了修饰时间，又提高了PCR目的片段的特异性、稳定性和重复性；MLH1基因启动子区域甲基化比率为27.3%，而不是45.6%，避免了非甲基化位点的错认。为植物DNA甲基化分析提供了一种更为理想的研究手段。 |
| 英文摘要: |
| DNA cytosine methylation is a central epigenetic modification that has essential roles in the control of many critically important biological processes, including cell proliferation, differentiation, development, genomic imprinting and regulation of gene expression. With progresses in study on DNA methylation, detection techniques of DNA methylation have been developed to meet requirements of various researches.This article has established an improved bisulfite sequencing technique for analysis of MutL-homologue 1（MLH1） promoter methylation level of mismatch repair gene in Arabidopsis thaliana. After the genomic DNA in Arabidopsis thaliana was digested, purified, and then modified with bisulfite liquid, the PCR products were cloned into the pEASY-T1 vector. 15 positive clones were randomly selected from each sample and then sequenced. The results showed that compared with the traditional method of methylation analysis,the improved method modified the digested DNA completely, reduced the modification time greatly, and improved the specificity, stability and repeatability of the PCR products. MLH1 promoter methylation level was 27.3% instead of 45.6%. The improved bisulfite sequencing technique avoided misidentification at non-methylated sites, and would provide a better means of research for analysis of DNA methylation in plant. |
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