Cloning strategies: Blunt-end cloning
Blunt-end cloning overview
Blunt-end cloning involves ligating dsDNA into a plasmid where both the insert and linearized plasmid have no overhanging bases at their termini. It does not benefit from the hydrogen bond stabilization associated with the complementary overhanging bases used in cohesive-end cloning, but the transient associations of the available 5’ phosphate and 3’ hydroxyl groups are sufficient to produce successful clones in the presence of T4 ligase [1]. An illustration of a basic blunt-end cloning experiment is shown in Figure 1.
Blunt-end cloning is also one of the easiest and most versatile methods for cloning dsDNA into plasmid vectors. It is easy because the blunt-ended insert requires little to no preparation—avoiding the enzymatic digestion and subsequent purification needed for cohesive-end cloning. It is versatile because insert and vector have fewer sequence limitations than other methods. This means that blunt-end cloning has some unique advantages, and also disadvantages, over cohesive-end cloning and isothermal assembly methods.