Fluorescent intensity assay for duplex and triplex nucleic...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01)

Patent

CA 2394752

The invention provides a homogeneous assay for nucleic acid hybridization. The fluorescent intensity of a hybridization medium containing a probe, a target and an intercalating agent is a function of the hybridization efficiency of the probe with respect to the target. The assay can detect specific hybridization between single-stranded probes and non-denatured double-stranded targets to form triplexes, thus obviating the need to denature the targets. The assay can also detect duplex hybridization complexes. The assay can be used to identify accessible regions in folded nucleotide sequences, to determine the number of mismatched pairs in a hybridization complex, and to map genomes.

L'invention concerne un dosage homogène d'hybridation d'acides nucléiques. L'intensité fluorescente d'un milieu d'hybridation contient une sonde, une cible et un agent intercalant étant une fonction de l'efficacité d'hybridation de la sonde par rapport à la cible. Le dosage permet de détecter une hybridation spécifique entre des sondes simple brin et des cibles double brin non dénaturées, manifestant ainsi la nécessité de dénaturer les cibles. Le dosage permet également de détecter des complexes d'hybridation duplex, et peut être utilisé afin d'identifier des régions accessibles dans des séquences nucléotidiques pliées, de déterminer le nombre de paires présentant un défaut de complémentarité dans un complexe d'hybridation et de cartographier des génomes.

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