Minor groove binder-conjugated DNA probes for quantitative DNA detection by hybridization-triggered fluorescence.

Abstract

Here we describe the properties of a novel class of oligonucleotide probes capable of sensitive hybridization-triggered fluorescence. These fluorogenic probes, known commercially as MGB Eclipse probes, are characterized by having a conjugated minor groove binder (MGB) ligand at the 5'-end and a fluorophore at the 3'-end. Additionally, they have an efficient quencher moiety at the 5'-end that is useful with a wide variety of fluorescent dyes. Fluorescence of the single-stranded MGB Eclipse probe is efficiently quenched by the interaction of the terminal dye and quencher groups when not hybridized. Upon hybridization to a complementary target, the MGB molecule folds into duplex and hyper-stabilizes it, allowing the use of shorter, more specific probe sequences. The 5'-MGB-quencher group also prevents nuclease digestion by Taq DNA polymerase during PCR. Because of the hybridization-triggered fluorescence and the excellent specificity imparted by the MGB, these 5'-MGB Eclipse probes have great versatility for real-time PCR applications. The high sensitivity and specificity are illustrated using single nucleotide polymorphism detection, viral load determination, and gene expression analysis.