Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.
Fluorescence energy transfer detection as a homogeneous DNA diagnostic method.
复制标题
DOI：
10.1073/pnas.94.20.10756
复制
发表时间：
1997-09
影响因子：
11.1
通讯作者：
Xiangning Chen;Barbara A. Zehnbauer;Andreas Gnirke;Pui-Yan Kwok
中科院分区：
文献类型：
--
作者：
Xiangning Chen;Barbara A. Zehnbauer;Andreas Gnirke;Pui-Yan Kwok
一种基于荧光共振能量转移检测的模板导向引物延伸的均相DNA诊断分析方法，被称为模板导向染料终止子掺入（TDI）分析，已被开发用于突变检测和高通量基因组分析。在此，我们报道了TDI分析在检测囊性纤维化跨膜传导调节因子（CFTR）基因、人类白细胞抗原H（HLA - H）基因以及受体酪氨酸激酶（RET）原癌基因中的突变的成功应用，这些基因分别与囊性纤维化、血色素沉着症和2型多发性内分泌肿瘤相关。从人类总DNA开始，样本先通过聚合酶链反应（PCR）扩增，然后在进行引物延伸反应之前对多余的引物和脱氧核糖核苷三磷酸进行酶促降解。所有这些标准化步骤都在同一试管中进行，并且实时监测荧光变化，这使其成为一种有用的临床DNA诊断方法。
A homogeneous DNA diagnostic assay based on template-directed primer extension detected by fluorescence resonance energy transfer, named template-directed dye-terminator incorporation (TDI) assay, has been developed for mutation detection and high throughput genome analysis. Here, we report the successful application of the TDI assay to detect mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the human leukocyte antigen H (HLA-H) gene, and the receptor tyrosin kinase (RET) protooncogene that are associated with cystic fibrosis, hemochromatosis, and multiple endocrine neoplasia, type 2, respectively. Starting with total human DNA, the samples are amplified by the PCR followed by enzymatic degradation of excess primers and deoxyribonucleoside triphosphates before the primer extension reaction is performed. All these standardized steps are performed in the same tube, and the fluorescence changes are monitored in real time, making it a useful clinical DNA diagnostic method.