Affiliation:
1. Epoch Biosciences, Bothell, WA, and Synthetic Genetics, San Diego, CA, USA
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.
Subject
General Biochemistry, Genetics and Molecular Biology,Biotechnology
Cited by
111 articles.
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