Center for Medical Genetics Ghent, Ghent University, Ghent, 9000, Belgium.
Cancer Research Institute Ghent (CRIG), Ghent University, Ghent, 9000, Belgium.
Sci Rep. 2019 Feb 15;9(1):2150. doi: 10.1038/s41598-019-38581-z.
For a wide range of diseases, SNPs in the genome are the underlying mechanism of dysfunction. Therefore, targeted detection of these variations is of high importance for early diagnosis and (familial) screenings. While allele-specific PCR has been around for many years, its adoption for SNP genotyping or somatic mutation detection has been hampered by its low discriminating power and high costs. To tackle this, we developed a cost-effective qPCR based method, able to detect SNPs in a robust and specific manner. This study describes how to combine the basic principles of allele-specific PCR (the combination of a wild type and variant primer) with the straightforward readout of DNA-binding dye based qPCR technology. To enhance the robustness and discriminating power, an artificial mismatch in the allele-specific primer was introduced. The resulting method, called double-mismatch allele-specific qPCR (DMAS-qPCR), was successfully validated using 12 SNPs and 15 clinically relevant somatic mutations on 48 cancer cell lines. It is easy to use, does not require labeled probes and is characterized by high analytical sensitivity and specificity. DMAS-qPCR comes with a complimentary online assay design tool, available for the whole scientific community, enabling researchers to design custom assays and implement those as a diagnostic test.
对于广泛的疾病而言，基因组中的 SNP 是功能障碍的潜在机制。因此，针对这些变异的靶向检测对于早期诊断和（家族性）筛查非常重要。虽然等位基因特异性 PCR 已经存在多年，但由于其鉴别能力低和成本高，其在 SNP 基因分型或体细胞突变检测中的应用受到了阻碍。为了解决这个问题，我们开发了一种具有成本效益的基于 qPCR 的方法，能够以稳健和特异的方式检测 SNP。本研究描述了如何将等位基因特异性 PCR 的基本原理（野生型和变异型引物的组合）与基于 DNA 结合染料的 qPCR 技术的简单读数相结合。为了提高稳健性和鉴别能力，在等位基因特异性引物中引入了一个人工错配。由此产生的方法称为双错配等位基因特异性 qPCR（DMAS-qPCR），在 48 种癌细胞系上使用 12 个 SNP 和 15 个临床相关的体细胞突变进行了成功验证。它易于使用，不需要标记探针，具有高分析灵敏度和特异性。DMAS-qPCR 附带一个免费的在线检测设计工具，可供整个科学界使用，使研究人员能够设计定制的检测，并将其用作诊断测试。
