Seamless cloning is a new, simple, rapid, and restriction enzyme site-independent technology. This technology depends on homologous sequence-based gene cloning technology, utilizing homologous DNA sequences that can recombine any DNA fragment containing overlapping regions of the vector ends into a linearized vector. It is not limited by restriction sites, and has a low background of vector self-ligation. With advantages such as high flexibility, efficiency, and time savings, it has become an important technical means in modern molecular biology experiments. It is widely used in gene engineering, protein expression, cell biology, and other fields. The seamless cloning kits were developed by scientists based on this technology.
The classic method of cloning vector construction using restriction enzyme digestion and ligation can only select specific restriction sites on the vector. Therefore, when constructing multiple insertion fragments, it often requires multiple rounds of digestion and ligation, which is not only cumbersome but also introduces extra bases. It is a relatively inefficient way of vector construction. The seamless cloning technology, based on the principle of recombination, relies on the recombination of homologous sequences of 15~25 nt at the ends of the insertion fragments and linearized vectors, eliminating the relatively complex steps of restriction enzyme digestion and ligation. Theoretically, it can clone the insertion fragments into any site of any linear vector, with high recombination efficiency and extremely low background of vector self-ligation. It is a simple, rapid, and high-efficiency DNA directional cloning technology.
Seamless cloning kits are mainly used in frontier fields such as gene editing, synthetic biology, protein engineering, and vaccine development. In gene editing, these kits help researchers quickly construct specific gene combinations for functional validation. In synthetic biology, seamless cloning technology supports the design and synthesis of complex gene circuits, promoting the development of new biological systems. In the field of protein engineering, seamless cloning kits make the optimization and functional modification of protein structures more efficient. During vaccine development, this technology can accelerate the construction of vaccine candidates and improve research and development efficiency.
Design: First, design and synthesize DNA fragments with appropriate overlapping regions according to the experimental requirements. Ensure that the overlapping regions between the fragments can effectively undergo seamless splicing to ensure the accuracy of cloning.
Prepare the reaction mixture: Mix the designed DNA fragments with Gibson Assembly Master Mix. This mixture contains the enzymes and other reagents required for connecting the DNA fragments, enabling effective splicing of these fragments in the reaction.
Reaction: Incubate at 50°C for a certain period, allowing the DNA fragments to complete seamless splicing through the overlapping regions. During this process, the enzymes in the kit will handle all the necessary chemical reactions for the splicing process.
Transformation: Transform the reaction products into host cells for subsequent screening and analysis. The host cells will carry the cloned DNA for proliferation and functional validation.
