PCR-based SNP genotyping: A comprehensive comparison of methods for affordable and accurate detection of class IV mutations

Single nucleotide polymorphisms (SNPs) influence gene function and impact health and disease. Identifying and genotyping SNPs is crucial in various areas of research and applications. This study compared five PCR-based methods for detecting a challenging T-to-A SNP, rs9939609: ARMS-PCR, PIRA-PCR, TaqMan qPCR, CADMA with HRM, and HRM, using snapback primers. Sanger sequencing served as the gold standard. Five assays were designed and compared for genotyping rs9939609. Performance was evaluated based on affordability, ease of use, robustness, and sensitivity. Melting curve analysis was used for the CADMA and snapback primer HRM assays. All methods successfully genotyped the rs9939609 variant. ARMS-PCR was the simplest and most cost-effective method but was potentially less sensitive. PIRA-PCR offered increased sensitivity but required specific restriction enzymes, increasing cost and complexity. TaqMan qPCR was fast and sensitive but expensive due to probe requirements. The combination of CADMA with HRM balanced affordability and speed with good sensitivity and applicability to standard qPCR platforms. The snapback primer HRM offered high sensitivity but required longer assay times and careful optimization. CADMA emerged as the most balanced method, combining affordability with sensitivity and comparable to that of Sanger sequencing and TaqMan qPCR. Its effectiveness for challenging mutations and compatibility with standard qPCR platforms make it a practical choice for various laboratories. Each method offers trade-offs in cost, sensitivity, and complexity, catering to specific research and diagnostic needs. Future advancements may further refine these techniques for broader application.