Nonequilibrium Hybridization Enables Discrimination of a Point Mutation within 5-40 degrees C

Detection of point mutations and single nucleotide polymorphisms in DNA and RNA has a growing importance in biology, biotechnology, and medicine. For the application at hand, hybridization assays are often used. Traditionally, they differentiate point mutations only at elevated temperatures (>40 degrees C) and in narrow intervals (Delta T = 1-10 degrees C). The current study demonstrates that a specially designed multistranded DNA probe can differentiate point mutations in the range of 5-40 degrees C. This unprecedentedly broad ambient-temperature range is enabled by a controlled combination of (i) nonequilibrium hybridization conditions and (ii) a mismatch-induced increase of equilibration time in respect to that of a fully matched complex, which we dub "kinetic inversion". We thank Dr. Yulia V. Gerasimova for helpful discussions. D.M.K. was funded by NSF CCF 1423219 and NIAID R15AI10388001A1. J.H. received funding from the Research Fund - Flanders (FWO K226314N). A.B. was supported by the UCF startup funds. The computational time for the project was provided by the UCF STOKES cluster.