Krenková P, Norambuena P, Stambergová A, Macek M
Charles University in Prague, 2nd Faculty of Medicine and University Hospital Motol, Department of Biology and Medical Genetics, Cystic Fibrosis Centre, Prague, Czech Republic.
Folia Biol (Praha). 2009;55(6):238-42.
Hereby we present evaluation of high-resolution melting for mutation scanning applied to the cystic fibrosis transmembrane conductance regulator gene. High resolution melting was used for mutation scanning of selected samples derived from cystic fibrosis patients with a known cystic fibrosis transmembrane conductance regulator genotype. We tested 19 different disease-causing cystic fibrosis transmembrane conductance regulator mutant genotypes located within six exons of the cystic fibrosis transmembrane conductance regulator gene (4, 7, 10, 11, 14b and 22). Normalized melting curves of tested samples were compared to sequenced-verified wildtype samples. Determined mutations are as follows: p.F508del, p.I507del, p.G551D, p.R347P, c.1717- 1G>A, c.621+1G>T, p.Y122X, p.I336K, p.R553X, c.2789+5G>A, c.574delA, c.1811+1G>C, p.L1335F, p.L1335P, p.L1324P and p.M470V and represent minimally 76.5 % of all cystic fibrosis alleles detected in the Czech cystic fibrosis population. All analysed samples with mutant genotypes were unambiguously distinguished from wild-type samples. High-resolution melting analysis enabled reliable detection of all single-nucleotide polymorphism classes and 1- or 3- base pair deletions. We examined the specificity, sensitivity and precision of this methodology. High-resolution melting analysis is an economical, sensitive and specific close-tube method and has a high utility for the detection of unknown mutations in cystic fibrosis DNA diagnostics.
在此，我们展示了应用于囊性纤维化跨膜传导调节因子基因的高分辨率熔解用于突变扫描的评估。高分辨率熔解用于对来自已知囊性纤维化跨膜传导调节因子基因型的囊性纤维化患者的选定样本进行突变扫描。我们测试了位于囊性纤维化跨膜传导调节因子基因六个外显子（4、7、10、11、14b和22）内的19种不同的致病囊性纤维化跨膜传导调节因子突变基因型。将测试样本的标准化熔解曲线与经测序验证的野生型样本进行比较。确定的突变如下：p.F508del、p.I507del、p.G551D、p.R347P、c.1717-1G>A、c.621+1G>T、p.Y122X、p.I336K、p.R553X、c.2789+5G>A、c.574delA、c.1811+1G>C、p.L1335F、p.Ll335P、p.L1324P和p.M470V，并且至少占捷克囊性纤维化人群中检测到的所有囊性纤维化等位基因的76.5%。所有分析的具有突变基因型的样本都能与野生型样本明确区分。高分辨率熔解分析能够可靠地检测所有单核苷酸多态性类别以及1或3个碱基对的缺失。我们研究了该方法的特异性、敏感性和精密度。高分辨率熔解分析是一种经济、灵敏且特异的闭管方法，在囊性纤维化DNA诊断中检测未知突变具有很高的实用性。
