Phosphalink, a non-nucleosidic phosphoramidite, directly incorporates a phosphate at either the 3' or 5' end, or both, of chemically synthesized oligonucleotides. It provides an efficient alternative to labor-intensive enzymatic methods. Phosphalink is also scaleable, providing pure, phosphorylated oligonucleotides up to 10 umol, a quantity unobtainable by enzymatic means.
Only 5' phosphorylated oligonucleotides will ligate in the presence of T4 DNA ligase. Phosphalink extends the flexibility and control you get with synthetic DNA to ligation chain reaction (LCA), oligonucleotide ligation assay (OLA), gene construction, cDNA sequencing to the 5' end, and cloning experiments.
3' phosphorylation of an oligonucleotide prevents extension in the presence of Taq DNA Polymerase. Phosphalink allows you to design a 3'-phosphorylated probe that anneals to a PCR template without participating in the reaction itself. You can use such probes to monitor the progress of amplification.
5'- and 3'-phosphorylated oligonucleotides are susceptible to dephosphorylation by alkaline phosphatase, a ubiquitous environmental contaminant (from skin, bacteria, etc) and also a common reagent in the molecular biology lab. Take extreme care to protect solutions containing phosphorylated oligonucleotides from introduction of alkaline phosphatase.