Schaaff F, Wedemann H, Schwinger E
Institute of Human Genetics, Medizinische Universität Lübeck, Germany.
Hum Genet. 1996 Aug;98(2):158-61. doi: 10.1007/s004390050180.
Reliable sex determination is an inevitable prerequisite in prenatal and preimplantation diagnosis of X-linked diseases. We report on an amelogenin-based nested polymerase chain reaction sexing method that simultaneously amplifies distinguishable fragments from both sex chromosomes. Primers matching a largely homologous region on both sex chromosomes are used that encompass a 177-bp deletion on the Y chromosome. Thus amplification results in X- and Y-specific fragments of different sizes that are resolved simply by agarose gel electrophoresis. We applied our sexing strategy to 102 single amniocytes previously subjected to primer extension preamplification. 95 showed successful amplification (93.14% sensitivity). The genotyping of all successful amplifications (from 42 male and 53 female amniocytes) was found to be correct (100% specificity). None of the media blanks showed amplification products (no false positives). Additional amplification of the locus of the most common cystic fibrosis mutation resulted in 95.1% success: 89 amniocytes (87.3%) showed no mutated allele and 7 (6.9%) were found to be heterozygous for the delta F508 mutation.
可靠的性别鉴定是X连锁疾病产前和植入前诊断中不可或缺的前提条件。我们报告了一种基于牙釉蛋白的巢式聚合酶链反应性别鉴定方法，该方法可同时从两条性染色体上扩增出可区分的片段。使用与两条性染色体上一个高度同源区域匹配的引物，该区域包含Y染色体上一个177bp的缺失。因此，扩增产生不同大小的X和Y特异性片段，只需通过琼脂糖凝胶电泳即可分辨。我们将我们的性别鉴定策略应用于102个先前经过引物延伸预扩增的单个羊水细胞。95个显示成功扩增（灵敏度为93.14%）。发现所有成功扩增（来自42个男性和53个女性羊水细胞）的基因分型都是正确的（特异性为100%）。所有培养基空白均未显示扩增产物（无假阳性）。对最常见囊性纤维化突变位点的额外扩增成功率为95.1%：89个羊水细胞（87.3%）未显示突变等位基因，7个（6.9%）被发现为ΔF508突变的杂合子。
