【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。更多还原

【Abstract】 Objective To detect the hotspot mutations of β-thalassemia by high fidelity DNA polymerase-mediated PCR. Methods CD41-42,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 TATAbox-28 mutations from the patients with β-thalassemia were detected by optimized multiplex PCR. Results For wild-type template,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 patients 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.更多还原