用高保真DNA聚合酶介导的PCR检测β地中海贫血热点突变  

Detection of hotspot mutations of β-thalassemia by high fidelity DNA polymerase-mediated PCR

作　　者：郭紫芬[1] 兰芬[1] 周翠兰[1] 廖端芳[1,2] 李凯[1,3] 

机构地区：[1]南华大学药物药理研究所,湖南衡阳421001 [2]湖南中医药大学药学院,长沙410208 [3]苏州大学药学院,江苏苏州215123

出　　处：《临床检验杂志》2014年第4期245-248,共4页Chinese Journal of Clinical Laboratory Science

基　　金：国家自然科学基金(81102516);南华大学博士启动基金(2012XQD18)

摘　　要：目的用高保真DNA聚合酶介导的PCR检测β地中海贫血基因热点突变。方法选取已知中国人群β珠蛋白基因CD41-42、IVS-2nt654、TATAbox-28、CD17、CD71-72及CD26热点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型基因位点完全配对的引物,并用硫化磷酸修饰,进行高保真DNA聚合酶介导的引物延伸反应。用多重PCR反应体系检测临床已知分别含CD26和TATAbox-28突变热点患者的DNA标本。结果对野生模板而言,仅有野生型等位基因相关引物有产物产生,而突变型等位基因位点特异性引物无产物产生。反之,使用突变模板,仅突变型等位基因位点相关引物有产物产生,而野生型等位基因位点特异性引物无产物产生。高保真DNA聚合酶介导的多重引物延伸反应筛查含有CD26或TATAbox-28突变的患者DNA标本时显示,突变引物混合物只有相应突变位点的产物产生,而野生引物混合物则有除突变位点以外的其他5个位点的产物产生。结论建立了基于高保真DNA聚合酶的筛查β地中海贫血基因热点突变的PCR技术。Objective To detect the hotspot mutations of 13-thalassemia by high fidelity DNA polymerase-mediated PCR. Methods CIM1-42, IVS-2nt654, TATAbox-28, CD17, CD71-72 and CD26 hotspot mutational sites in β-globin gene were selected as detection targets. Then, allelic specific primers for these wild or mutational alleles were designed and modified with phosphorothioate. Next, high fidelity DNA polymerase-mediated primer extension reaction was carried out. Moreover, the DNA samples containing CD26 or TATAhox-28 mutations from the patients with β-thalassemia were detected by optimized multiplex PCR. Results For wild-type tem- plate, only allelic primers specific for wild-type allele could amplify the specific products, and similarly, only mutant template could be amplified by allelic primers specific for mutant allele. When the DNA samples containing CD26 or TATAbox-28 mutations from the pa- tients with β-thalassemia were detected by multiplex PCR, we found that primers mixture specific for mutant alleles could only amplify the specific products containing mutation sites, and that primers mixture specific for wide-type alleles could amplify all the specific products containing wide-type sites but not mutation sites. Conclusion The PCR technology based on high fidelity DNA polymerase to detect the hotspot mutations of β-thalassemia was successfully established.

关 键 词：高保真聚合酶 硫化修饰 Β地中海贫血 PCR 

分 类 号：Q341[生物学—遗传学]