After restriction enzyme digestion, the 5’ end of strand of DNA carries a phosphate group, while the 3’ end of a strand of DNA bears a hydroxyl group. It is frequently necessary to modify these ends in order to facilitate ease-of-joining with one’s construct. Depending on the enzyme used, the resulting DNA will have either blunt- or sticky-ends. Blunt-ended DNA is double-stranded and matched base-for-base and the terminus of the molecule has no overhang or unpaired nucleotides. DNA products are said to have sticky-ends when they possess a terminus with an unpaired overhang, either 3’ or 5’. When joining DNA fragments in cloning procedures, it is essential that the ends are compatible. Compatibility depends both on end architecture (blunt- or sticky-ended) and end phosphorylation.
- A 5’ phosphate group is essential for self-ligation of DNA ends. However, when preparing DNA for directed ligation, such as into a vector, the property of self-ligation can be counterproductive. Generally, it is a good idea to dephosphorylate DNA fragments until ligation is desired. This can be accomplished with a phosphatase.
- Proper end architecture is essential to maintaining the modularity of DNA fragments. While blunt ends are universally compatible, they are more difficult to ligate together and decrease yield where directional insertion is required. Sticky ends can confer directionality (with non-compatible ends).
- Once a fragment is compatible with its vector, the ends must be treated with a kinase to add a 5’ phosphate group, to ensure its ligation with the vector.