Ng I S, Pace R, Richard M V, Kobayashi K, Kerem B, Tsui L C, Beaudet A L
Institute for Molecular Genetics, Baylor College of Medicine, Houston, TX 77030.
Hum Genet. 1991 Sep;87(5):613-7. doi: 10.1007/BF00209023.
A large number of mutations causing cystic fibrosis (CF) have been reported. In an attempt to improve methods for genetic diagnosis and for heterozygote screening, we evaluated methods for efficient analysis of the delta F508, G542X, G551D, R553X, and N1303K mutations. We found that multiple mutations can be analyzed simultaneously using hybridization with allele-specific oligonucleotides. Alternatively all of these mutations can be detected by amplification of DNA followed by restriction enzyme digestion and analysis on polyacrylamide gels. A previously reported method for use of modified primers for DNA amplification to allow detection of virtually any single-base change by restriction enzyme analysis proved particularly useful. The common delta F508 mutation and three mutations in exon 11 were analyzed using a multiplex amplification reaction followed by double digestion with restriction enzymes and electrophoresis in a single lane on a polyacrylamide gel. In a sample of 439 CF chromosomes from North American Caucasians, the frequencies of various mutations were as follows: delta F508 = 75.8%, G542X = 2.7%, G551D = 3.2%, R553X = 1.4%, and N1303K = 1.4% for a total of 84.5% detection of CF chromosomes by analysis for these five mutations.
已报道了大量导致囊性纤维化（CF）的突变。为了改进基因诊断和杂合子筛查方法，我们评估了对ΔF508、G542X、G551D、R553X和N1303K突变进行有效分析的方法。我们发现，使用等位基因特异性寡核苷酸杂交可同时分析多个突变。另外，所有这些突变都可通过DNA扩增，然后进行限制性内切酶消化并在聚丙烯酰胺凝胶上分析来检测。一种先前报道的使用修饰引物进行DNA扩增以便通过限制性酶切分析检测几乎任何单碱基变化的方法被证明特别有用。使用多重扩增反应，然后用限制性酶进行双酶切，并在聚丙烯酰胺凝胶的单泳道上进行电泳，对常见的ΔF508突变和第11外显子中的三个突变进行了分析。在来自北美白种人的439条CF染色体样本中，各种突变的频率如下：ΔF508 = 75.8%，G542X = 2.7%，G551D = 3.2%，R553X = 1.4%，N1303K = 1.4%，通过对这五个突变进行分析，总共检测到84.5%的CF染色体。
