Department of Molecular Biology and Genetics, Koc University, 34450 Istanbul, Turkey.
Anal Biochem. 2013 Oct 15;441(2):225-31. doi: 10.1016/j.ab.2013.07.007. Epub 2013 Jul 16.
Single-nucleotide polymorphism (SNP) genotyping is widely used in genetic association studies to characterize genetic factors underlying inherited traits. Despite many recent advances in high-throughput SNP genotyping, inexpensive and flexible methods with reasonable throughput levels are still needed. Real-time PCR methods for discovering and genotyping SNPs are becoming increasingly important in various fields of biology. In this study, we introduce a new, single-tube strategy that combines the tetra-primer ARMS PCR assay, SYBR Green I-based real-time PCR, and melting-point analysis with primer design strategies to detect the SNP of interest. This assay, T-Plex real-time PCR, is based on the T(m) discrimination of the amplified allele-specific amplicons in a single tube. The specificity, sensitivity, and robustness of the assay were evaluated for common mutations in the FV, PII, MTHFR, and FGFR3 genes. We believe that T-Plex real-time PCR would be a useful alternative for either individual genotyping requests or large epidemiological studies.
单核苷酸多态性（SNP）基因分型广泛用于遗传关联研究，以描述遗传特征的遗传因素。尽管最近在高通量 SNP 基因分型方面取得了许多进展，但仍需要具有合理通量水平的廉价且灵活的方法。用于发现和 SNP 基因分型的实时 PCR 方法在生物学的各个领域变得越来越重要。在这项研究中，我们介绍了一种新的单管策略，该策略将四引物 ARMS-PCR 测定法、基于 SYBR Green I 的实时 PCR 和熔解曲线分析与引物设计策略相结合，以检测感兴趣的 SNP。该测定法，T-Plex 实时 PCR，基于在单个管中扩增的等位基因特异性扩增子的 T(m)区分。评估了该测定法对 FV、PII、MTHFR 和 FGFR3 基因中常见突变的特异性、灵敏度和稳健性。我们相信 T-Plex 实时 PCR 将是个体基因分型请求或大型流行病学研究的有用替代方法。
