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

Authors

Nini Li, Jinding Liu, Jiaqi Zhang, Zhiyong Cheng, Yongqiang Cheng, Jiangyan Zhang

Published in

Analytica chimica acta. Volume 1424. Pages 346257. Nov 22, 2026. Epub Sep 10, 2026.

Abstract

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.

PMID:
42887997
Bibliographic data and abstract were imported from PubMed on 17 Oct 2026.

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