甲基化特异性PCR检测FMR1和XIST基因甲基化实验方法的建立  

Establishment of Methylation-specific PCR of FMR1 and XIST Genes Detection

作　　者：杨芳[1] 赵新[2] 张文红[1] 薛丽[1] 王琰[1] 白玉杰[1] 

机构地区：[1]第四军医大学基因诊断技术应用研究所 [2]第四军医大学唐都医院,西安710032

出　　处：《中国生物化学与分子生物学报》2006年第11期914-918,共5页Chinese Journal of Biochemistry and Molecular Biology

摘　　要：建立一种快速、灵敏的检测脆性X智障基因(fragile X mental retardation,FMR1)、X染色体失活基因(Xchromosome inactivation,XIST)甲基化的方法.用亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰.以修饰后的DNA为模板,用两套不同的引物对:1对甲基化特异性引物和1对非甲基化特异性引物扩增FMR1基因(CGG)n重复序列区、FMR1和XIST基因的启动子区.PCR产物进一步克隆、测序.以亚硫酸氢钠和对苯二酚脱氨基修饰后的DNA为模板进行PCR扩增后的产物与预期基因目的基因片段大小相符合,无非特异性扩增产物.测序结果表明,FMR1、XIST基因中的非甲基化的C碱基转变为U碱基,而CpG岛被甲基化的C碱基不改变.成功地建立了检测FMR1、XIST甲基化的方法,为实验室诊断脆性X综合征提供了新的方法.To establish time-efficient and sensitive method for detection of the methylation of FMR1 （fragile X mental retardation） and XIST （X chromosome inactivation） genes,genomic DNA was deaminated by sodium bisulfite. The two sets of PCR primers for unmenthylated and methylated DNA, respectively, were used to amplify the CGG-repeat and the promotors of the FMR1 and XIST genes. The allelic methylation of the latter serves as an internal control. The PCR products were further cloned and sequenced. The sequence of PCR products was identified by sequencing. Unmethylated cytosine residues （C） converse to uracil （U）, while methylcytosine （mc） was not modified under the condition used. Among the 40 clinical cases, 31 cases have methylated XIST gene, while 9 cases are heterozygosis of unmethylated and methylated XIST gene. The PCR products of CGG repeat region are ranging between 76 to 445 bp in unmethylated FMR1 ,while it is 1 000 bp in one case withmethylated FMR1. The evaluated repeat number is 4 to 120 in cases carrying unmethylated FMR1 and is 300 in one with methylated FMR1. In the present study, a new method of detection of the methylation of FMR1 and XIST genes was successfully established;it may apply a new technique for clinic diagnosis for fragile X syndrome.

关 键 词：脆性X智障基因 X染色体失活基因 甲基化特异性PCR 甲基化检测 

分 类 号：Q75[生物学—分子生物学]