Assays using crosslinkable immobilized nucleic acids

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01)

Patent

CA 2340361

Improved methods for in situ hybridization assays of cellular and subcellular systems and tissue sections, and immobilization-based assay techniques such as Northern blotting, Southern blotting, dot blots, and the like, and assay techniques wherein the probes are bound to substrates are disclosed. The subject invention employs crosslinker containing hybridization probes capable of forming covalent bonds between the probe and the target nucleic acid. Upon activation, the crosslinker will, if the probe has hybridized with its essentially complementary target, form covalent bonds with the complementary strand to covalently crosslink the probe to the target. Subsequently, stringent wash conditions may be employed to reduce background signals due to non-specific absorption or probes or targets, while retaining all crosslinked probe/target hybrids. Also disclosed are diagnostic kits for use in clininical and diagnostic laboratories.

L'invention concerne des procédés améliorés destinés aux dosages d'hybridation in situ de systèmes cellulaires et sous-cellulaires et de parties de tissus, ainsi que des techniques de dosage fondées sur l'immobilisation telles que Northern blot, Southern blot et dot blot, de même que des techniques de dosage dans lesquelles les sondes sont liées aux substrats. L'invention utilise des sondes d'hybridation employant des agents de réticulation capables de former des liaisons covalentes entre la sonde et l'acide nucléique cible. Après activation, si la sonde s'est hybridée avec sa cible sensiblement complémentaire, l'agent de réticulation va former des liaisons covalentes avec le brin complémentaire pour réticuler de manière covalente la sonde et la cible. Par la suite, on peut recourir à des conditions strictes de lavage pour réduire les signaux d'arrière-plan dus à l'absorption non spécifique ou aux sondes ou encore aux cibles, et ce tout en préservant tous les hybrides réticulés sonde/cible. L'invention concerne aussi des kits diagnostiques utilisés dans des laboratoires cliniques et diagnostiques. -

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