Chemical phosphorylation is a cost-effective alternative to enzymatic methods, allowing efficient introduction of terminal phosphate groups. Oligonucleotides containing a 5’-phosphate group have various applications, e.g. as linkers and adapters, in cloning and gene construction, and in the ligase chain reaction.
Phosphorylation of the 5’-terminus on oligonucleotides is routinely achieved using the Chemical Phosphorylation Reagent (CPR). Aside from its inherent convenience, CPR also has the advantage over enzymatic methods in allowing determination of the phosphorylation efficiency due to the presence of the DMTr protecting group. However, the trityl group cannot be used as a purification handle. If left on the oligonucleotide, it is eliminated along with the sulphonyl ethyl group to produce the 5’-phosphate during the ammonium hydroxide deprotection.
In an improvement to the original reagent, solidCPR™ has been developed. This product is a stable solid and is much easier to handle and dissolve. It is supplied with a DMTr group side-chain that is stable under basic conditions. This feature gives the option of leaving the DMTr group in placing during cleavage of the oligo from the support for "trityl-on" purification. The DMTr group is subsequently removed, and the side-chain eliminated, using standard methods.
Although CPR can be used in 3’-phosphorylation, 3’-Phosphate SynBase™ CPG 1000/110 allows direct preparation of oligonucleotides with a 3’-phosphate group (the analogous 3000Å version is also available).