Cost-effective and robust genotyping using double-mismatch allele-specific quantitative PCR

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 genotypi...

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Bibliographic Details
Published in:Scientific reports 2019-02, Vol.9 (1), p.2150-2150, Article 2150
Main Authors: Lefever, Steve, Rihani, Ali, Van der Meulen, Joni, Pattyn, Filip, Van Maerken, Tom, Van Dorpe, Jo, Hellemans, Jan, Vandesompele, Jo
Format: Article
Language:English
Subjects:
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Alleles
Cancer
DNA probes
Genomes
Genotyping
Humanities and Social Sciences
multidisciplinary
Mutation
Polymerase chain reaction
Science
Science (multidisciplinary)
Single-nucleotide polymorphism
Tumor cell lines
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Summary: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.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-38581-z