A Novel Enzyme-Free Approach for Precursor microRNA Cloning and Expression

Abstract Background: Traditional cloning methods heavily depend on enzymatic digestion for generating sticky ends in DNA inserts, presenting challenges in efficiency and cost-effectiveness. MicroRNAs play crucial roles in biological processes and are integral to disease studies. Cloning microRNA precursors involves directional binding, necessitating meticulous techniques. Existing methods require multiple enzymes and suffer from limitations, urging the need for innovative, streamlined approaches.Methods: This study introduces a revolutionary technique eliminating the reliance on restriction enzymes for cloning pre-microRNA genes. Strategic manipulation of the expression vector's sequence and design of tailored primers enable consecutive PCR reactions, producing pre-microRNA sequences with compatible sticky ends. The engineered vector, incorporating BbsI recognition sites, undergoes a simplified cloning process, validated through colony PCR and sequencing. HEK293 cells transfected with the construct affirm method robustness, demonstrating microRNA-21 expression via real-time PCR.Results: The method yields pre-microRNA sequences with compatible ends for the expression vector, eliminating costly and time-consuming enzymatic digestion. Optimizing BbsI enzyme usage reduces the risk of re-ligation. The approach significantly accelerates cloning, completing the process in approximately 4 hours with high precision. Real-time PCR confirms a 12.5-fold increase in microRNA-21 expression compared to controls, validating successful transfection.Conclusion: This study presents an innovative, cost-effective, and rapid cloning method for microRNA precursor genes. By bypassing the limitations of traditional techniques, this approach accelerates molecular biology research, offering a dependable protocol for various pre-microRNAs and expression vectors. The method's simplicity, efficiency, and reliability mark a significant advancement, fostering progress in the field of molecular biology.