Unexpected transformation of black hole quenchers in electrophoretic purification of the fluorescein-containing TaqMan probes

The fluorescence quenchers BHQ1 and BHQ2 can be modified by trace amounts of ammonium persulfate, used for initiating gel polymerization, in electrophoretic purification of TaqMan probes using a denaturing polyacrylamide gel. The case study of BHQ1 quencher has demonstrated that a Boyland-Sims reaction proceeds in the presence of ammonium persulfate to give the corresponding sulfate. The absorption maximum of the resulting quencher shifts to the short-wavelength region relative to the absorption maximum of the initial BHQ1. The TaqMan probe containing such a quencher is less efficient as compared with the probe carrying an unmodified BHQ1. The presence of fluorescein in TaqMan probe plays decisive role in this transformation: the quencher modification proceeds at a considerably lower rate when the fluorescein is absent or replaced with a rhodamine dye (for example, R6G). It is assumed that the observed reaction can take place in two ways-both in darkness and in the reaction of the quencher in an excited state due to energy transfer from the fluorophore irradiated by light.