title: Seven novel probe systems for real-time PCR provide absolute single-base discrimination, higher signaling, and generic components.
abstract: We have developed novel probe systems for real-time PCR that provide higher specificity, greater sensitivity, and lower cost relative to dual-labeled probes. The seven DNA Detection Switch (DDS)-probe systems reported here employ two interacting polynucleotide components: a fluorescently labeled probe and a quencher antiprobe. High-fidelity detection is achieved with three DDS designs: two internal probes (internal DDS and Flip probes) and a primer probe (ZIPR probe), wherein each probe is combined with a carefully engineered, slightly mismatched, error-checking antiprobe. The antiprobe blocks off-target detection over a wide range of temperatures and facilitates multiplexing. Other designs (Universal probe, Half-Universal probe, and MacMan probe) use generic components that enable low-cost detection. Finally, single-molecule G-Force probes employ guanine-mediated fluorescent quenching by forming a hairpin between adjacent C-rich and G-rich sequences. Examples provided show how these probe technologies discriminate drug-resistant Mycobacterium tuberculosis mutants, Escherichia coli O157:H7, oncogenic EGFR deletion mutations, hepatitis B virus, influenza A/B strains, and single-nucleotide polymorphisms in the human VKORC1 gene.
authors.affiliation: GeneTAG Technology, Inc., Atlanta, Georgia.
authors.forename: James L
authors.initials: JL
authors.lastname: Murray
authors.affiliation: GeneTAG Technology, Inc., Atlanta, Georgia; Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia.
authors.forename: Peixu
authors.initials: P
authors.lastname: Hu
authors.affiliation: GeneTAG Technology, Inc., Atlanta, Georgia; Department of Psychiatry and Behavioral Sciences, Emory University School of Medicine, Atlanta, Georgia. Electronic address: dshafer@genetagtechnology.com.
authors.forename: David A
authors.initials: DA
authors.lastname: Shafer
mesh_terms: D001483:Base Sequence; D017931:DNA Primers; D009696:Nucleic Acids / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015398:Signal Transduction
chemical_list: D017931:DNA Primers; D009696:Nucleic Acids