The Mechanism of DNA Phosphorylation

In standard cloning protocols, a five-prime phosphate is required to serve as the donor in the ligation reaction. At a minimum, either the fragment ends or vector ends must be phosphorylated.

PCR products need to have a five-prime phosphate added before ligation is attempted with a non-phosphorylated vector. If the vector is phosphorylated or the PCR products regenerated with phosphorylated primers, no phosphorylation step is needed.

Similarly, digestion of DNA with a restriction enzyme will always produce a five-prime phosphate making a separate phosphorylation step unnecessary.

In cloning protocols, phosphorylation is typically accomplished by T4 polynucleotide kinase, which transfers the terminal gamma phosphate to a polynucleotide like DNA.

The phosphorylated DNA is now ready for ligation. Visit CloneWithNEB.com for the full list of products available for this application.