QUAL probes1 consist of two oligonucleotides, the first containing a nucleophilic group at the 3'-terminus, while the second has an electrophilic group at the 5'-terminus. When the probe pair finds the target, the oligos line up with the 3'-terminus of the first directly adjacent to the 5'-terminus of the second. An autoligation reaction then takes place to combine the two oligos into a single probe. As usual, the 3' nucleophilic group is the 3-thiophosphate, easily prepared using 3'-phosphate CPG with a sulfurizing step in the first cycle. In this case, the electrophilic group is a 5'-dabsyl group, which is an excellent leaving group as well as a fine quencher of fluorescence. The second oligo, therefore, contains a fluorophore which is quenched by the dabsyl group. A popular choice for fluorophore is fluorescein-dT but it is easy to imagine that a variety of fluorophores could be attached to any of the commercially available amino-modified nucleoside phosphoramidites.
Coupling: Monomers that allow for UltraMILD deprotection must be used. (dA: 10-1601-xx, dC: 10-1015-xx, dG: 10-1621-xx, dT: 10-1030-xx ). To avoid any exchange of the iPr-Pac group on the dG|with acetyl, use the UltraMild Cap Mix A (40-4210-xx/ 40-4212-xx).
Deprotection: UltraMILD deprotection: 0.05M Potassium Carbonate in Methanol, 4 hours at Room Temperature.