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dc.contributor.author江艳
dc.contributor.author王先良
dc.contributor.author王春晖
dc.contributor.author王小利
dc.contributor.author杨怡姝
dc.contributor.author段小丽
dc.contributor.author聂静
dc.contributor.author王菲菲
dc.contributor.author张金良
dc.date.accessioned2016-05-17T02:11:07Z
dc.date.available2016-05-17T02:11:07Z
dc.date.issued2010
dc.identifier.citation环境与健康杂志,2010,(5):24-27
dc.identifier.issn1001-5914
dc.identifier.otherHJYJ201005010
dc.identifier.urihttps://dspace.xmu.edu.cn/handle/2288/102191
dc.description.abstract目的改进亚硫酸氢钠测序法,并在CMV基因启动子甲基化检测中进行验证。方法提取PEgfP-C3质粒重组人肝癌细胞株HEPg2 dnA,亚硫酸氢钠化学修饰,针对修饰后质粒基因CMV启动子序列设计特异引物并结合梯度降落PCr扩增,T-A载体克隆、测序,目标区域甲基化定量。结果 PEgfP-C3质粒基因约600bP的CMV启动子区甲基化水平可以精确定量,检测结果与标准品一致,重复测量结果稳定。结论改进后亚硫酸氢钠测序法能明显减少非特异性扩增,提高PCr效率,更适于基因甲基化状态的检测。
dc.description.abstractObjective To improve the sodium bisulfite sequencing method and validate it in quantification of methylation in CMV promoter.Methods DNA was extracted from recombinant HepG2 hepatoma cell line with plasmid pEGFP-C3,and chemically modified with sodium bisulfite,the gene-specific primers were designed according to the modified CMV promoter sequence and conducted PCR amplification with gradient touch-down PCR,then the DNA methylation level in the target areas was quantitated after T-A cloning and sequencing.Results The DNA methylation level of the 600 bp CMV promoter within pEGFPC3 plasmid could be quantified accurately,and be consistent with that of the methylated DNA standards,repeated measurements indicated stable quantification result with this method.Conclusion The improved sodium bisulfite sequencing method can reduce non-specific amplification significantly and improve PCR efficiency,therefore has more potential for quantitative detection of gene methylation status.
dc.description.sponsorship国家自然科学基金资助项目(20907047);中央级公益性科研院所基本科研业务专项(2008KYYW05)
dc.language.isozh_CN
dc.subject基因
dc.subject亚硫酸氢钠测序法
dc.subject甲基化
dc.subject梯度降落PCR
dc.subjectCMV启动子
dc.subjectpEGFP-C3
dc.subjectGene
dc.subjectBisulfite sequencing
dc.subjectMethylation
dc.subjectGradient landing PCR
dc.subjectCMV promoter
dc.subjectpEGFP-C3
dc.titlepEGFP基因启动子区甲基化的梯度降落PCR定量检测法
dc.title.alternativeQuantification of Methylation at CMV Promoter of pEGFP-C3 Vector by Gradient Touch-Down PCR Sequencing
dc.typeArticle


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