Designing a Restriction Enzyme-free Method to Construct a MicroRNA Precursor gene for microRNA Cloning

Abstract Common cloning strategies depend on the enzymatic digestion of the insert. In addition, the enzymatic digestion of PCR product ends by restriction enzymes is of low efficiency. These limitations are related to the need for enzymatic digestion to produce sticky ends in the insert sequence. Hence, in the present study, we aimed to present a new generation of pre-microRNA cloning method without using restriction enzymes for constructing pre-microRNA gene. In this strategy, by engineering an expression vector's sequence and designing two intelligent primer sets for two consecutive PCR reactions, the pre-microRNA sequence with appropriate restriction sites related to the expression vector was produced, without restriction enzymes. The recombinant expression vector was transfected into HEK293 cells, and microRNA-21 expression was assayed in these cells by real-time PCR, confirming the high efficacy of the presented cloning method. The present method can be an inexpensive and reliable method for microRNA precursor cloning by providing a high-performance protocol.