Orou A, Fechner B, Utermann G, Menzel H J
Institute for Medical Biology and Human Genetics, University of Innsbruck, Austria.
Hum Mutat. 1995;6(2):163-9. doi: 10.1002/humu.1380060209.
We have developed a novel one-step pool screening PCR procedure which is based on the principles of amplification refractory mutation system (ARMS) and competitive oligonuleotide priming (COP) PCR. In addition to the usual primers, this approach uses two allele-specific competitive oligonucleotides, one of which is 3'-end labeled with a dideoxynucleotide and blocks amplification of the wild-type allele. An allele-specific product is generated only in the presence of the mutation. The introduction of an allele-specific competitive blocker oligonucleotide improves the specificity and robustness of ARMS-PCR. Further its sensitivity is dramatically increased, which allows detection of one mutant allele in a large excess of wild-type-bearing genomic DNA by electrophoresis in an ethidium bromide-stained agarose gel (up to 1 in 10(4) alleles). This makes the method ideal for nonradioactive pool screening. The successful application of the method has been demonstrated for four different point mutations, two in the apolipoprotein B gene (R3500Q, R3531C) which result in familial defective apolipoprotein B-100, one in the CFTR gene (R1162X), and one in the gene for lipoprotein lipase (G188E).
我们开发了一种基于扩增阻滞突变系统（ARMS）和竞争性寡核苷酸引物（COP）PCR原理的新型一步法混合样本筛查PCR程序。除了常用引物外，该方法还使用两种等位基因特异性竞争性寡核苷酸，其中一种在3'末端用双脱氧核苷酸标记，可阻断野生型等位基因的扩增。只有在存在突变的情况下才会产生等位基因特异性产物。等位基因特异性竞争性阻断寡核苷酸的引入提高了ARMS-PCR的特异性和稳健性。此外，其灵敏度显著提高，这使得通过在溴化乙锭染色的琼脂糖凝胶中进行电泳，能够在大量携带野生型的基因组DNA中检测到一个突变等位基因（高达1/10⁴个等位基因）。这使得该方法成为非放射性混合样本筛查的理想选择。该方法已成功应用于四种不同的点突变，其中两种位于载脂蛋白B基因（R3500Q、R3531C），可导致家族性载脂蛋白B-100缺陷；一种位于CFTR基因（R1162X）；还有一种位于脂蛋白脂肪酶基因（G188E）。
