Monitoring dissociation kinetics during electrophoretic focusing enables high-specificity nucleic acid detection

A wide range of medical conditions can be diagnosed through sequence-specific analysis of nucleic acids. However, a major challenge remains in detecting a specific target in samples containing a high concentration of mismatching sequences. We present a single-step kinetic homogenous (free solution) assay, in which free sequence-specific probes are continuously separated from probe-target hybrids during electrophoretic sample focusing, allowing monitoring of dissociation kinetics. We show that under these conditions, the different kinetics of targets vs. mismatches result in distinct patterns of the signal (e.g. linear increase for target vs. exponential decay for mismatch), allowing for the detection of desired sequences even in the presence of high background nucleic acid content. Additionally, we present an analytical model that provides insight into the underlying dynamics, and allows design of assays based on this mechanism.