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HyBeacon probes are single-stranded oligonucleotides with one or more internal base(s) labeled with a fluorescent dye. When a probe forms a duplex with its target sequence, the level of fluorescence emission increases considerably. HyBeacons have been developed as new tools for rapid sequence detection and discrimination and have been employed in a wide variety of applications including infectious diagnostics and analysis of human polymorphisms. Single-labeled (FVG1) and dual-labeled (FVG11) probes were designed to analyze the factor V Leiden (R506Q) polymorphism which causes an increased risk of deep vein thrombosis and pulmonary embolism. Detection and identification of factor V alleles is performed by melting curve analysis and determination of probe melting temperature (T(m)). HyBeacon hybridization to the glutamine allele (Q) causes the formation of mismatched DNA duplexes that are detected through decreases in T(m). HyBeacon probes are included in homogeneous PCR assays to genotype samples with respect to the factor V polymorphism within 20 min, using purified DNAs and unpurified saliva/blood samples. This paper describes the preparation of homogeneous PCR assays, LightCycler target amplification, and subsequent melting curve analysis. This chapter also describes the use of homologous oligonucleotides and melting curve analysis as a method for probe evaluation.

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Transthyretin (TTR) is a 55 kD homotetrameric serum protein transporter of retinol binding protein charged with retinol and thyroxine (T4). The highly amyloidogenic human TTR variant in which leucine at position 55 is replaced by proline (L55P TTR) is responsible for aggressive fatal amyloidosis with peripheral and autonomic neuropathy, cardiomyopathy and nephropathy. Mice bearing one or two copies of a 19.2 kB human genomic fragment containing the entire coding sequence and the known control regions of the L55P TTR transgene, failed to develop TTR amyloidosis even though their sera contained mutant human TTR. The frequency of TTR tissue deposition was increased when the L55P TTR transgene was bred onto a murine TTR-null background. Denaturation of sera from the transgenic animals and murine TTR-knockouts expressing the human L55P TTR transgene revealed that the TTR tetramer was much more stable in the presence of the murine protein because the TTR circulates as hybrid human/murine heterotetramers. Intraperitoneal administration of diflunisal, a non-steroidal anti-inflammatory drug that binds to TTR in its T4-binding site and inhibits fibril formation in vitro, to human L55P TTR transgenic animals in which the murine TTR gene had been silenced, also stabilizes the circulating mutant protein to in vitro urea denaturation.