应用降落PCR及克隆测序检测2型糖尿病外周血白细胞胰岛素样生长因子受体IGF1R基因甲基化

Applying Touchdown-PCR and Clone-based Sequencing to Detect the Methylation of IGF1R Gene Promoter in the Peripheral Leukocyte of Type 2 Diabetes Mellitus

目的:比较应用降落PCR(TD-PCR)及克隆测序检测2型糖尿病患者外周血白细胞中胰岛素样生长因子受体(IGF1R)基因启动子区DNA甲基化位点的方法。方法:提取2型糖尿病患者外周血白细胞DNA进行亚硫酸氢盐处理,设计引物扩增IGF1R基因启动子区富含CG位点序列,分别行普通PCR和TD-PCR扩增,对含目的条带产物进行直接测序和克隆后测序。结果:与普通PCR扩增结果相比,TD-PCR扩增条带清晰、产物量多、二聚体少;且TD-PCR减少了对退火温度不断摸索的过程。克隆后测序峰图清晰,碱基丢失错判频率减少。结论:推荐应用TD-PCR检测亚硫酸氢盐处理的DNA甲基化位点,并进行克隆后测序。

Objective To apply touchdown-PCR and clone-based sequencing detecting the methylation sites of IGF1R gene promoter in the peripheral leukocyte of type 2 diabetes mellitus.Methods After the bisulfite treatment of the DNA extracted from the peripheral leukocyte in patients with type 2 diabetes mellitus,the primer was design to amplify the target fragments by general PCR and touchdown-PCR,then the target fragments were used to direct sequencing and clone sequencing.Results Compared the amplify results betweem touchdown-PCR and general PCR,the former had clearer fragment,more products,less dimers and saved time to explore the annealing temperature.The results of clone sequencing displayed clearly and accurately of every base.Conclusion Touchdown-PCR were recommend to be used to detect the methylation status after the DNA underwent the bisulfite treatment,the clone sequencing is a advisable choice.