Molecular Plant Breeding Co-operative Research Centre and School of Agriculture, Food and Wine, The University of Adelaide, Glen Osmond, SA 5064, Australia.
BMC Genomics. 2009 Dec 3;10:580. doi: 10.1186/1471-2164-10-580.
Many research and diagnostic applications rely upon the assay of individual single nucleotide polymorphisms (SNPs). Thus, methods to improve the speed and efficiency for single-marker SNP genotyping are highly desirable. Here, we describe the method of temperature-switch PCR (TSP), a biphasic four-primer PCR system with a universal primer design that permits amplification of the target locus in the first phase of thermal cycling before switching to the detection of the alleles. TSP can simplify assay design for a range of commonly used single-marker SNP genotyping methods, and reduce the requirement for individual assay optimization and operator expertise in the deployment of SNP assays.
We demonstrate the utility of TSP for the rapid construction of robust and convenient endpoint SNP genotyping assays based on allele-specific PCR and high resolution melt analysis by generating a total of 11,232 data points. The TSP assays were performed under standardised reaction conditions, requiring minimal optimization of individual assays. High genotyping accuracy was verified by 100% concordance of TSP genotypes in a blinded study with an independent genotyping method.
Theoretically, TSP can be directly incorporated into the design of assays for most current single-marker SNP genotyping methods. TSP provides several technological advances for single-marker SNP genotyping including simplified assay design and development, increased assay specificity and genotyping accuracy, and opportunities for assay automation. By reducing the requirement for operator expertise, TSP provides opportunities to deploy a wider range of single-marker SNP genotyping methods in the laboratory. TSP has broad applications and can be deployed in any animal and plant species.
许多研究和诊断应用都依赖于对个体单核苷酸多态性（SNP）的检测。因此，提高单标记 SNP 基因分型速度和效率的方法是非常需要的。在这里，我们描述了温度切换 PCR（TSP）的方法，这是一种双相四引物 PCR 系统，具有通用引物设计，可在热循环的第一阶段扩增目标基因座，然后切换到等位基因检测。TSP 可以简化一系列常用的单标记 SNP 基因分型方法的检测设计，并减少 SNP 检测应用中对个体检测优化和操作人员专业知识的需求。
我们通过生成总共 11232 个数据点，证明了 TSP 在基于等位基因特异性 PCR 和高分辨率熔解分析的快速构建稳健且方便的终点 SNP 基因分型检测中的应用。TSP 检测在标准化反应条件下进行，需要对个别检测进行最小优化。通过与独立基因分型方法进行的盲法研究，TSP 基因型的 100%一致性验证了其基因分型的准确性。
从理论上讲，TSP 可以直接纳入大多数当前单标记 SNP 基因分型方法的检测设计中。TSP 为单标记 SNP 基因分型提供了多项技术进步，包括简化检测设计和开发、提高检测特异性和基因分型准确性，以及检测自动化的机会。通过减少对操作人员专业知识的需求，TSP 为在实验室中更广泛地应用单标记 SNP 基因分型方法提供了机会。TSP 具有广泛的应用，可以应用于任何动植物物种。
