Department of Medical Genetics, University of Athens, Athens, Greece.
Mol Biotechnol. 2013 Sep;55(1):1-9. doi: 10.1007/s12033-007-0062-9. Epub 2007 Aug 14.
Many human diseases are caused by small alterations in the genes and in the majority of cases sophisticated protocols are required for their detection. In this study we estimated the efficacy of an enzymatic protocol, which using a new mismatch-specific DNA plant endonuclease from celery (CEL family) recognizes and cleaves mismatched alleles between mutant and normal PCR products. The protocol was standardized on a variety of known mutations, in 11 patients with cystic fibrosis (CF), Fabry's disease (FD), steroid 21-hydroxylase deficiency (21-HD), and Duchenne/Becker muscular dystrophy (DMD/BMD). The method does not require special equipment, labeling or standardization for every PCR product, since conditions of heteroduplex formation and enzyme digestion are universal for all products. The results showed that the method is rapid, effective, safe, reliable, and very simple, as the mutations are visualized on agarose or nusieve/agarose gels. The protocol was furthermore evaluated in three DMD patients with the detection of three alterations, which after sequencing, were characterized as disease causative mutations. The proposed assay, which was applied for the first time in a variety of monogenic disorders, indicates that point mutation identification is feasible in any conventional molecular lab even for cases where other techniques have failed.
许多人类疾病是由基因的微小改变引起的，在大多数情况下，需要复杂的方案来检测这些改变。在这项研究中，我们评估了一种酶促方案的效果，该方案使用一种来自芹菜的新型错配特异性 DNA 植物内切酶（CEL 家族），可以识别和切割突变型和正常 PCR 产物之间的错配等位基因。该方案在 11 名囊性纤维化 (CF)、法布里病 (FD)、类固醇 21-羟化酶缺乏症 (21-HD) 和杜氏/贝克肌营养不良症 (DMD/BMD) 患者的各种已知突变体上进行了标准化。该方法不需要特殊设备、对每个 PCR 产物进行标记或标准化，因为异源双链体形成和酶消化的条件对所有产物都是通用的。结果表明，该方法快速、有效、安全、可靠，非常简单，因为突变可以在琼脂糖或 nusieve/琼脂糖凝胶上可视化。该方案还在 3 名 DMD 患者中进行了评估，检测到了 3 种改变，经测序后，这些改变被确定为疾病致病突变。该检测方案首次应用于多种单基因疾病，表明即使在其他技术失败的情况下，点突变的识别在任何常规分子实验室都是可行的。
