A real-time gap ligase chain reaction assay enabling mutation detection and single-tube mutation fraction analysis

Accurate mutation analysis requires not only highly specific mutant recognition but also reliable mutation fraction analysis. Although gap ligase chain reaction (Gap-LCR) possesses excellent single-base discrimination capability, its integration with real-time fluorescence transduction and direct mutation fraction analysis remains insufficiently explored. Herein, we developed a real-time Gap-LCR platform by coupling Gap-LCR amplification with multicomponent nucleic acid enzyme (MNAzyme)-mediated fluorescence transduction. Using JAK2 V617F as a model target, the proposed assay enabled highly sensitive and specific real-time mutation detection over a concentration range from 10 aM to 100 fM. To enable direct mutation fraction analysis, a reference-locus-assisted dual-locus design was introduced to independently quantify the mutant target and the corresponding reference locus within a single closed-tube reaction. The calculated mutation fractions showed good agreement with predefined values down to 0.05% in synthetic DNA mixtures, and the assay further demonstrated overall agreement with reference NGS results in clinical samples. Overall, this work extends the analytical capability of Gap-LCR from mutation detection to single-tube mutation fraction analysis, providing a practical strategy for quantitative molecular diagnostics.