One-pot ligation-PCR for universal RNA biomarker detection

Abstract

In the detection of RNA biomarkers, conventional PCR-based workflows such as reverse transcription-PCR (RT-PCR) and ligation-PCR each exhibit inherent limitations. RT-PCR requires cDNA synthesis and often struggles with targets of high sequence similarity or atypical length and structure. Ligation-PCR improves sequence discrimination by enzymatically joining adjacent probes only when perfectly matched, yet conventional two-step formats—where ligation and amplification are performed separately—introduce workflow complexity, increased handling, and contamination risk. To overcome these challenges, we developed a glyoxal-assisted one-pot ligation-PCR assay that integrates probe ligation and PCR amplification within a single closed-tube system. The method employs thermally responsive glyoxal-caged primers that remain inactive during the ligation phase and are gradually activated during PCR cycling, thereby preventing premature extension and minimizing nonspecific amplification. Validated primarily on mRNA splice variants, the assay achieved sensitivity comparable to conventional two-step ligation-PCR while providing markedly improved discrimination among closely related splice isoforms. Additional experiments demonstrate the feasibility of extending this strategy to microRNA detection. This streamlined one-tube strategy simplifies operation, reduces contamination risk, and establishes a robust and efficient one-pot ligation-PCR framework that is readily adaptable to different RNA targets for precise RNA biomarker detection.