Molecular strategies applied in fluorescence energy transfer-based SNP assays. Amplified target sequences are shown in black and oligonucleotide probes and nucleotide triphosphates in blue, with the variable positions indicated by small white squares. In these examples, the green circles represent reporter fluorophores on reagents that match the target allele; the red circles represent fluorophores on the mismatched reagents. Jagged arrows indicate excitation of and emission from, as well as energy transfer between the fluorophores. Polymerases and ligases are represented by gray ovals. (A) In the TaqMan assay, pairs of fluorophores are conjugated to allele-specific oligonucleotides designed to hybridize downstream of one amplification primer. The donor (red and green fluorophores) fluorescence is quenched in the intact TaqMan probes as a result of FRET to the acceptor (white circle). Fluorescence can be detected when an allele-specific oligonucleotide hybridizes to a target molecule and is digested by the advancing polymerase during amplification. (B) Molecular beacon probes are hybridization probes that form hairpin–loop structures in the absence of the correct target sequence. When an allele-specific probe hybridizes to a target sequence, a fluorescent dye conjugated to the end of the probe is brought apart from a chromophore at the other (shown in black), and fluorescence is emitted. (C) In dye-labeled oligonucleotide ligation, the ligation of an oligonucleotide probe to another, allele-specific probe hybridizing downstream, results in energy transfer between fluorophores on the two oligonucleotides. (D) In FRET minisequencing, an allele-specific fluorophore-labeled nucleotide is added to a primer, resulting in energy transfer between a fluorophore present on the primer and that on the nucleotide.