Holding C, Bentley D, Roberts R, Bobrow M, Mathew C
Paediatric Research Unit, UMDS, Guy's Hospital, London, UK.
J Med Genet. 1993 Nov;30(11):903-9. doi: 10.1136/jmg.30.11.903.
In order to develop and validate methods for the preimplantation diagnosis of Duchenne muscular dystrophy (DMD), we have established and evaluated PCR assays for the analysis of four loci within the DMD gene and for two Y chromosome sequences in single cells. A model system using buccal cells picked from mouthwash samples has been used for an extensive evaluation of the sensitivity and specificity of the assays, and each assay has been tested in samples containing single cells, two cells, and three cells per tube. The four DMD and two Y assays have been combined in duplex and triplex reactions to enable simultaneous diagnosis of DMD and of fetal sex. One of the DMD markers is a highly polymorphic simple tandem repeat locus which produces a basic DNA profile, and provides a control for contamination by foreign DNA. Amplification of DMD or Y sequences was observed in 78 to 92% of single male cells, rising to 96% and 97% in tubes containing two or three male cells respectively. Coamplification of both a DMD and a Y sequence together occurred with a mean success of 74% in single male cells, increasing to 93% with two, and 95% with three cells per tube. With appropriate precautions, we believe that it is now possible to proceed to clinical application of these procedures.
为了开发和验证杜氏肌营养不良症（DMD）植入前诊断的方法，我们建立并评估了用于分析DMD基因内四个位点和单细胞中两个Y染色体序列的PCR检测方法。一个使用从漱口水样本中采集的颊细胞的模型系统已被用于广泛评估检测方法的敏感性和特异性，并且每个检测方法都已在每管含有单个细胞、两个细胞和三个细胞的样本中进行了测试。四个DMD检测方法和两个Y检测方法已在双重和三重反应中组合，以实现对DMD和胎儿性别的同时诊断。其中一个DMD标记是一个高度多态性的简单串联重复位点，它产生一个基本的DNA图谱，并可作为外源DNA污染的对照。在78%至92%的单个雄性细胞中观察到DMD或Y序列的扩增，在每管含有两个或三个雄性细胞的样本中分别升至96%和97%。DMD和Y序列的共同扩增在单个雄性细胞中的平均成功率为74%，在每管含有两个细胞时增至93%，含有三个细胞时增至95%。采取适当的预防措施后，我们认为现在有可能将这些程序应用于临床。
