LNA Thymidine Monomer Enables Differentiation of the Four Single-Nucleotide Variants by Melting Temperature

High-resolution melting (HRM) analysis of DNA is a closed-tube single-nucleotide polymorphism (SNP) detection method that has shown many advantages in point-of-care diagnostics and personalized medicine. While recently developed melting probes have demonstrated significantly improved discrimination of mismatched (mutant) alleles from matched (wild-type) alleles, no effort has been made to design a simple melting probe that can reliably distinguish all four SNP alleles in a single experiment. Such a new probe could facilitate the discovery of rare genetic mutations at lower cost. Here we demonstrate that a melting probe embedded with a single locked thymidine monomer (t_L) can reliably differentiate the four SNP alleles by four distinct melting temperatures (termed the "4T_m probe"). This enhanced discriminatory power comes from the decreased melting temperature of the t_L·C mismatched hybrid as compared to that of the t·C mismatched hybrid, while the melting temperatures of the t_L-A, t_L·G and t_L·T hybrids are increased or remain unchanged as compared to those of their canonical counterparts. This phenomenon is observed not only in the HRM experiments but also in the molecular dynamics simulations.