Quenched Autoligation (QUAL) Probes

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.


(1) S. Sando and E.T. Kool, J Amer Chem Soc, 2002, 124, 2096-2097.

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