Cuppens H, Buyse I, Baens M, Marynen P, Cassiman J J
Center for Human Genetics, University of Leuven, Belgium.
Mol Cell Probes. 1992 Feb;6(1):33-9. doi: 10.1016/0890-8508(92)90069-a.
An assay is described in which 11 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene can be screened simultaneously. Six different exons of the CFTR gene are amplified in a single multiplex amplification. Biotinylated dUTP is incorporated into the different fragments during the amplification process. A sample of this mixture is then hybridized to 21 different poly-dT tailed oligonucleotide probes which are bound to a nylon membrane. In order to screen the different mutations in a single step hybridization, the length of the different oligonucleotides and the amount used in the assay were optimized. The detection is performed by binding avidin-alkaline phosphatase to the biotin, followed by a chemiluminescent reaction. By means of this fast and sensitive assay, about 85% of all the cystic fibrosis mutations in the Belgian population can be detected.
本文描述了一种检测方法，可同时筛查囊性纤维化跨膜传导调节因子（CFTR）基因中的11种突变。在一次多重扩增中对CFTR基因的六个不同外显子进行扩增。在扩增过程中，生物素化的dUTP被掺入到不同的片段中。然后将该混合物的样品与21种不同的聚dT尾寡核苷酸探针杂交，这些探针固定在尼龙膜上。为了在一步杂交中筛查不同的突变，对不同寡核苷酸的长度和检测中使用的量进行了优化。通过将抗生物素蛋白-碱性磷酸酶与生物素结合，然后进行化学发光反应来进行检测。通过这种快速灵敏的检测方法，可以检测出比利时人群中约85%的所有囊性纤维化突变。
