Monitoring Dissociation Kinetics during Electrophoretic Focusing to Enable 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. A single‐step kinetic homogenous (free solution) assay is presented in which free sequence‐specific probes are continuously separated from probe–target hybrids during electrophoretic sample focusing, allowing monitoring of dissociation kinetics. Under these conditions, the different kinetics of targets versus mismatches result in distinct patterns of the signal (for example, linear increase for target versus exponential decay for mismatch), allowing the detection of desired sequences even in the presence of high background nucleic acid content. Additionally, an analytical model provides insight into the underlying dynamics, and allows design of assays based on this mechanism.