Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR
Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR
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DOI：
10.1038/s41598-019-38581-z
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发表时间：
2019-02-15
影响因子：
4.6
通讯作者：
Vandesompele, Jo
中科院分区：
文献类型：
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作者：
Lefever, Steve;Rihani, Ali;Vandesompele, Jo
对于多种疾病，基因组中的 SNP 是功能障碍的潜在机制。因此，有针对性地检测这些变异对于早期诊断和（家族）筛查非常重要。虽然等位基因特异性 PCR 已存在多年，但其在 SNP 基因分型或体细胞突变检测中的应用因其低区分能力和高成本而受到阻碍。为了解决这个问题，我们开发了一种经济高效的基于 qPCR 的方法，能够以稳健且特异的方式检测 SNP。本研究描述了如何将等位基因特异性 PCR（野生型和变异引物的组合）的基本原理与基于 DNA 结合染料的 qPCR 技术的直接读数相结合。为了增强稳健性和区分能力，在等位基因特异性引物中引入了人为错配。由此产生的方法称为双错配等位基因特异性 qPCR (DMAS-qPCR)，使用 48 个癌细胞系的 12 个 SNP 和 15 个临床相关体细胞突变成功进行了验证。它使用方便，不需要标记探针，具有分析灵敏度和特异性高的特点。 DMAS-qPCR 附带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检测并将其实施为诊断测试。
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.