Double-labeled fluorescent probes for 5' nuclease assays: Purification and performance evaluation

An inexpensive method for the purification and evaluation of user-synthesized or crude commercially prepared double-labeled fluorescent probes is presented. These probes exhibit the characteristics required for use in 5′-nuclease assays, including efficient reporter dye quenching, target specificity and susceptibility to cleavage by Taq DNA polymerase during PCR amplification. The method is suitable for research laboratories that wish to develop 5′ nuclease assays for the detection of PCR-amplified target sequences to eliminate the requirement for agarose gels and to advance throughput.