We disclosed a novel nucleic acid signal detection system based on the cleavage of a phosphodiester linkage in a dualdye-labeled oligonucleotide probe by the Endo IV enzyme from E.coli. The parameters influencing the activity of the Endo IV enzyme were optimized. This optimized Endo IV detection assay requires, in addition to an oligonucleotide probe substrate, a fluorophore attached through a relatively rigid linker to the oligonucleotide and a helper oligonucleotide. The rigid linker conveys the exquisite specificity of Endo IV cleavage of the phosphodiester linkage in the probe and shows high-specific/non-specific rate ratios. The synthesis of fluorophore analogs coupled to a rigid linker and solid support, used in the synthesis of labeled oligonucleotides, was also disclosed. Excellent mismatch discrimination is observed from positions 1 to 6 in a 12mer probe. The ability to put the mismatch in any one of positions 1-6, allows for flexibility in probe design for genotyping assays where sequence and secondary structural constraints are considered. This method allows the use of probes shorter than those typically used in other methods (28) contributing to the improved mismatch discrimination.