The Mechanism of DNA Phosphorylation

Phosphorylation is the process by which phosphate groups are added to a molecule by a kinase. The phosphorylation status of a fragment of DNA can influence its ability to proceed in reactions. Learn more about phosphorylation and kinases.

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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.

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