Newton C R, Graham A, Heptinstall L E, Powell S J, Summers C, Kalsheker N, Smith J C, Markham A F
ICI Diagnostics, Northwich, Cheshire, UK.
Nucleic Acids Res. 1989 Apr 11;17(7):2503-16. doi: 10.1093/nar/17.7.2503.
We have improved the "polymerase chain reaction" (PCR) to permit rapid analysis of any known mutation in genomic DNA. We demonstrate a system, ARMS (Amplification Refractory Mutation System), that allows genotyping solely by inspection of reaction mixtures after agarose gel electrophoresis. The system is simple, reliable and non-isotopic. It will clearly distinguish heterozygotes at a locus from homozygotes for either allele. The system requires neither restriction enzyme digestion, allele-specific oligonucleotides as conventionally applied, nor the sequence analysis of PCR products. The basis of the invention is that unexpectedly, oligonucleotides with a mismatched 3'-residue will not function as primers in the PCR under appropriate conditions. We have analysed DNA from patients with alpha 1-antitrypsin (AAT) deficiency, from carriers of the disease and from normal individuals. Our findings are in complete agreement with allele assignments derived by direct sequencing of PCR products.
我们改进了“聚合酶链反应”（PCR），以便能够快速分析基因组DNA中的任何已知突变。我们展示了一种系统，即ARMS（扩增不应性突变系统），该系统仅通过琼脂糖凝胶电泳后检查反应混合物就能进行基因分型。该系统简单、可靠且非同位素。它能清晰地区分某一位点上的杂合子与任一纯合子等位基因。该系统既不需要限制性酶切，也不需要像传统方法那样使用等位基因特异性寡核苷酸，也不需要对PCR产物进行序列分析。本发明的基础是，出乎意料的是，在适当条件下，3'-残基错配的寡核苷酸在PCR中不会作为引物起作用。我们分析了α1-抗胰蛋白酶（AAT）缺乏症患者、该疾病携带者和正常个体的DNA。我们的发现与通过PCR产物直接测序得出的等位基因分型完全一致。
