[TaqMan probes based on oligonucleotide-hairpin minor groove ligand conjugates]

Hybridization of TaqMan probes derived from oligonucleotides containing fluorophores (fluorescein, FAM, or tetramethylrhodamine, (Tamra)), fluorescence quenchers (BHQ1 or BHQ2), and a conjugated hairpin ligand (MGB) composed of two tripyrrolcarboxamide residues connected through an aminobutyric acid residue were proposed for discrimination of point mutations using the real time PCR technique. Identification of point A/C substitution was shown to be highly specific for hepatitis C virus subtypes 1a and 1b with two variants of the probe (5'-3'): ATTGAGCGGGTTTAp-BHQ2-MGB for subtype 1a and FAM-ATTGAGCGGGTTGAp-BHQ1-MGB for subtype 1b. Perfect duplexes (A.T and G.C pairs) increase fluorescence in the process of amplification, whereas imperfect duplexes (A.G and T.C pairs) induce no fluorescence changes. This phenomenon enables simultaneous genotyping of hepatitis C virus subtypes 1a and 1b.