Qi X, Bakht S, Devos K M, Gale M D, Osbourn A
Sainsbury Laboratory and John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK.
Nucleic Acids Res. 2001 Nov 15;29(22):E116. doi: 10.1093/nar/29.22.e116.
A flexible, non-gel-based single nucleotide polymorphism (SNP) detection method is described. The method adopts thermostable ligation for allele discrimination and rolling circle amplification (RCA) for signal enhancement. Clear allelic discrimination was achieved after staining of the final reaction mixtures with Cybr-Gold and visualisation by UV illumination. The use of a compatible buffer system for all enzymes allows the reaction to be initiated and detected in the same tube or microplate well, so that the experiment can be scaled up easily for high-throughput detection. Only a small amount of DNA (i.e. 50 ng) is required per assay, and use of carefully designed short padlock probes coupled with generic primers and probes make the SNP detection cost effective. Biallelic assay by hybridisation of the RCA products with fluorescence dye-labelled probes is demonstrated, indicating that ligation-RCA (L-RCA) has potential for multiplexed assays.
本文描述了一种灵活的、非基于凝胶的单核苷酸多态性（SNP）检测方法。该方法采用热稳定连接进行等位基因鉴别，并采用滚环扩增（RCA）进行信号增强。用Cybr-Gold对最终反应混合物进行染色并通过紫外线照射进行可视化后，实现了清晰的等位基因鉴别。使用适用于所有酶的兼容缓冲系统可使反应在同一试管或微孔板孔中启动和检测，从而可轻松扩大实验规模以进行高通量检测。每次检测仅需少量DNA（即50 ng），并且使用精心设计的短锁式探针与通用引物和探针相结合，使得SNP检测具有成本效益。通过RCA产物与荧光染料标记探针杂交进行的双等位基因检测得到了证明，表明连接-RCA（L-RCA）具有用于多重检测的潜力。
