Restriction Enzyme-Free Method for Generating PCR Product Sticky Ends in Recombinant Vector Construction for Cloning

Abstract In biological research, particularly in genetics, one crucial aspect is the transfer of gene fragments or their derivatives into living organisms. These fragments are typically carried by plasmids to ensure stability and observable effects. Conventional cloning methods rely on restriction enzymes to create sticky ends for introducing these fragments into biological vectors. However, due to the limitations of these enzymes in efficiently cutting DNA insert fragments, as well as their associated costs and time-consuming nature, alternative methods for creating sticky ends and transferring DNA into plasmids have been explored. This study proposes a method for conducting cloning processes with minimal reliance on restriction enzymes. The approach involves separately amplifying each Watson and Crick strand in PCR tubes using primers with specific sticky end sequences at their 5' ends. During the high cycles of the PCR process, numerous Watson or Crick single strands are generated in each tube, which are then combined to produce a double-stranded product containing sticky sites for easy insertion into the plasmid. To validate this method, the creation of a plasmid containing the microRNA hsa-miR-21 precursor was examined. Upon its transfer into HEK 293 cells, a significant 12.5-fold increase in microRNA hsa-miR-21 concentration compared to the control was observed using real-time PCR. This novel, cost-effective, and time-saving method holds potential for various applications in genetics, biotechnology, and biology.