HyBeacon® probes for rapid DNA sequence detection and allele discrimination

David J. French*, David G. McDowell, Paul Debenham, Nittaya Gale, Tom Brown

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Title of host publicationMolecular beacons
Subtitle of host publicationsignalling nucleic acid probes, methods, and protocols
EditorsAndreas Marx, Oliver Seitz
Place of PublicationNew Jersey
PublisherHumana Press
Chapter12
Pages171-185
Number of pages15
ISBN (Electronic)9781603270403
ISBN (Print)9781588297006
DOIs
Publication statusPublished - 2008
Externally publishedYes

Publication series

NameMethods in Molecular Biology
PublisherHumana Press
Volume429

Keywords

  • Fluorescent probes
  • HyBeacon
  • LightCycler
  • single nucleotide polymorphisms

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