Development and validation of a tail-extension PCR protocol for improved sanger sequencing of a short miR-16-5p-derived amplicon

Abstract Background MicroRNA-16-5p (miR-16-5p) plays an important role in gene regulation and has been implicated in several human diseases. Improved sequence verification of short miRNA-derived amplicons is important in assay development and control validation. Because stem-loop RT-PCR products derived from mature miRNAs are often very short, they may yield poor-quality Sanger chromatograms and complicate sequence verification. Methods A thermodynamic-guided tailed primer strategy was developed to improve sequencing performance. Primer sequences were redesigned to minimize secondary structure formation and primer–dimer interactions. A tail-extension approach was applied to increase the cDNA product length and facilitate stable Sanger sequencing. PCR conditions were optimized to enhance amplification specificity. Results The optimized strategy successfully extended the amplification product from 58 bp to 115 bp, enabling reliable Sanger sequencing and reliable identification of the miR-16-5p-derived target sequence within the full-length tail-extended amplicon. Specific amplification was consistently obtained in the synthetic template and all nine biological samples tested. Sequencing confirmed 100% identity in the expected miR-16-5p-derived target region and verified the presence of the engineered tail sequence within the tail-extended amplicon. The extended product length contributed to improved signal clarity and reduced sequencing noise. Conclusion This method provides a practical approach for sequence verification of short miRNA-derived amplicons generated by stem-loop RT-PCR.