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

Abstract
Although allele-specific PCR has been around for many years, its adoption for SNP genotyping has been hampered by its low discriminating power and the need for post-PCR gel electrophoretic analysis. In this study, we combine the basic principles of allele-specific PCR with the straightforward readout of SYBR Green I based qPCR technology, thereby eliminating the need for probes (either labeled or not) and reducing post-analysis time to a minimum. To further enhance the robustness and discriminating power of the method, an artificial mismatch in the allele-specific primer was introduced, resulting in a new type of assays coined double-mismatch allele-specific qPCR (DMAS-qPCR) assays. Our assays outperform hydrolysis probe based assays when looking at calling success rate (100 % vs. 96 % call rate, for 12 SNP tested on 48 samples) and can be used with sample input as low as 250 pg. The ease of use, the availability of online software for assay design, the absence of labeled probes and the high sensitivity of DMAS-qPCR are characteristic of a cost-efficient and powerful new way of SNP genotyping.