基因突变敏感性分子开关检测β地中海贫血基因突变  

Detection of the gene mutations of β-thalassemia by gene mutation-sensitive molecular switch

作　　者：兰芬[1] 郭紫芬[1] 邓坤龙[1] 廖端芳[2] 李凯[3] 

机构地区：[1]南华大学药物药理研究所,湖南省衡阳市421001 [2]湖南中医药大学药学院中医诊断学系,410208 [3]苏州大学第二附属医院分子医学中心,215004

出　　处：《实用医学杂志》2012年第2期178-180,共3页The Journal of Practical Medicine

基　　金：国家高技术研究发展计划(编号:863计划2008AA02Z436)

摘　　要：目的:采用高保真DNA聚合酶介导的基因突变敏感性分子开关准确、快速检测β地中海贫血。方法:首先提取正常人血液基因组DNA,根据已知β珠蛋白基因热点突变CD41-42、IVS2-654区域序列,分别设计突变序列扩增引物,利用低保真酶进行引物延伸反应,将其PCR产物克隆到pMD19-T载体,得到β地中海贫血常见的两个突变位点的基因突变克隆:CD41-42、IVS2-654,然后以这两个突变位点为检测靶点,分别设计3′末端与野生型基因位点或突变基因位点配对的引物,硫化磷酸修饰3′末端并偶联高保真DNA聚合酶构成的基因突变敏感性分子开关,对含有相应突变的阳性质粒模板及正常人基因组DNA模板进行引物延伸反应,并通过凝胶成像系统对其进行分析。结果:当引物与模板完全配对时,引物被延伸,有PCR产物;当引物与模板不完全配对时,引物不被延伸,无PCR产物。结论:高保真DNA聚合酶介导的基因突变敏感性分子开关能准确、快速筛查β地中海贫血CD41-42、IVS2-654突变,提示其在单基因遗传病的诊断中具有广阔的应用前景。Objective To detect β-thalassemia by the approach of gene mutation-sensitive molecular switch. Methods Genomic DNA was extracted from normal blood. According to the sequences of known hot spot mutation region of CD41-42 and IVS2-654 inβ-globin gene, the mutant sequence primer was designed, and then primer extension reaction was performed with low-fidelity enzyme, and the PCR products was cloned into pMD19-T vector. Two kinds of clones of 13-thalassemia mutation gene （CD41-42 and IVS2-654） were obtained. Then these two mutation region were used as detection target, 3＇-terminal primer which was matched with wild type or mutation gene region was designed, and the 3＇-terminal was modified with sulfide phosphatethe and linked to gene mutation- sensitive molecular switch which was constructed by high-fidelity DNA polymerase. The positive plasmid template including the target mutation and normal genomic DNA template were administered by primer extension reaction, and were analyzed by gel-imagine system. Result When the primer was fully matched with the template, the primer was extended; When the primer was not fully matched with the template, the primer was not extended. Conclusion Gene mutation-sensitive molecular switch which was constructed by high-fidelity DNA polymerase could accurately and rapidly screened the mutation of CD41-42 and IVS2-654 of β-thalassemia, suggesting that it has broad application prospects in the diagnosis of single-gene genetic disease.

关 键 词：Β地中海贫血 高保真聚合酶 硫化修饰引物 

分 类 号：R556.9[医药卫生—血液循环系统疾病]