Department of Animal & Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK, Current address: Sugadiva Montane, Experimental Center, University of Tsukuba, Ueda, 386-2204, Japan.
Mol Ecol Resour. 2008 Nov;8(6):1230-8. doi: 10.1111/j.1755-0998.2008.02190.x.
We describe a convenient, cost-effective and flexible medium-throughput single nucleotide polymorphism (SNP) genotyping method, Multiplex SNP-SCALE, which enables the simultaneous amplification by polymerase chain reaction (PCR) of up to 25 (or potentially more) loci followed by electrophoresis in an automated DNA sequencer. We extended the original SNP-SCALE method to include (i) use of a commercial multiplex PCR kit, (ii) a four-dye system, (iii) much-reduced (2-µL) reaction volumes, (iv) drying down of template DNA before PCR, (v) use of pig-tailed primers, (vi) a PCR product weighting system, (vii) a standard optimized touchdown PCR thermocycling programme, and (viii) software (SNP-SCALE Primer Designer) that automatically designs suitable SNP-SCALE primers for a batch of loci. This new protocol was validated for different types of SNPs. The method is cost- and time-effective for medium-scale evolutionary and ecological projects involving 10s to 100s of loci.
我们描述了一种方便、经济且灵活的高通量单核苷酸多态性（SNP）基因分型方法，即多重 SNP-SCALE，该方法能够通过聚合酶链反应（PCR）同时扩增多达 25 个（或可能更多）位点，然后在自动 DNA 测序仪中进行电泳。我们对原始的 SNP-SCALE 方法进行了扩展，包括：(i) 使用商业的多重 PCR 试剂盒；(ii) 四色系统；(iii) 大大减少（2µL）的反应体积；(iv) 在 PCR 之前将模板 DNA 干燥；(v) 使用猪尾引物；(vi) PCR 产物重量系统；(vii) 标准优化的降落式 PCR 热循环方案；以及 (viii) 软件（SNP-SCALE Primer Designer），该软件能够自动为一批位点设计合适的 SNP-SCALE 引物。该新方案已针对不同类型的 SNP 进行了验证。对于涉及 10 到 100 多个位点的中等规模的进化和生态项目，该方法具有成本效益和时间效益。
