甲基化特异性熔点曲线分析检测FMR1基因CpG岛甲基化状态方法的建立  

Establishment of Methylation-specific Melting Curve Analysis Detection for the Methylation Profiling of the FMR1 Gene CpG Islands

作　　者：富显果[1] 廖娟[1] 张朵[1] 严爱贞[1] 郭小燕[1] 兰风华[1] 

机构地区：[1]南京军区福州总医院临床遗传与实验医学科,福州350025

出　　处：《福建医科大学学报》2012年第4期255-258,共4页Journal of Fujian Medical University

基　　金：福建省重点科技项目(2010Y0045)

摘　　要：目的建立一种可靠的检测脆性X智力障碍1基因(FMR1)CpG岛甲基化状态的方法。方法用亚硫酸氢钠对基因组DNA进行脱氨基修饰,以修饰后的DNA为模板,利用荧光定量PCR仪扩增FMR1基因CpG岛序列并进行熔点曲线分析,并对PCR产物进行克隆测序,验证其甲基化状态。结果分析包含10个CpG位点FMR1基因CpG岛105bp片段显示,非甲基化与甲基化引物扩增产物之间熔点温度相差2.5℃;克隆测序显示两者之间未被修饰的胞嘧啶相差7个。结论所建立甲基化特异性熔点曲线分析方法可以有效地检测FMR1基因CpG岛甲基化状态,为临床诊断脆性X综合征提供依据。Objective To establish a reliable methods for detection of CpG island methylation of fragile X mental retardation 1 gene（FMR1）.MethodsGenomic DNA was deaminated by sodium bisulfite.With the modified DNA,the FMR1 gene CpG island sequences were amplified and performed the melting curve analysis by using fluorescent quantitative PCR.The PCR products were then cloned and sequenced to verify the CpG island methylation status.ResultsAnalysis of a 105 bp sequence comprising 10 CpG sites at the FMR1 gene CpG island showed that the melting temperature（Tm） differed by 2.5 ℃ between unmethylated and methylated PCR products.Cloning and sequencing showed that the methylated cytosine differed by 7 between them.ConclusionThe methylation-specific melting curve analysis methods can effectively detect the CpG island methylation of the FMR1 gene,which could provide a support evidence for clinical diagnosis of Fragile X Syndrome.

关 键 词：脆性X综合征 CPG岛 甲基化 

分 类 号：R749.94[医药卫生—神经病学与精神病学]