Methods and reagents for detecting cpg methylation with a...

C - Chemistry – Metallurgy – 12 – Q

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C12Q 1/68 (2006.01) C07K 19/00 (2006.01) C12N 15/11 (2006.01)

Patent

CA 2724343

The present invention provides a simple and sensitive technology for the detection of CpG methylation in DNA without chemical modification of sample DNA by bisulfite treatment or PCR amplification. Signal generation is based on an Ab-scription (Abortive Transcription) technology in which DNA signal generators called Abortive Promoter Cassettes (APCs) are bound to target mCpG sites via mCpG target specific probes based on methyl binding polypeptides or methyl binding domains thereof. RNA polymerase produces uniform, short RNA molecules from synthetic promoters in APCs as signals of the presence of methylated CpGs. Detection of CpG methylation and hypermethylation of DNA targets such as CpG islands provides a convenient means for detecting and monitoring cancer in a subject.

L'invention concerne une technologie simple et sensible pour la détection d'une méthylation de CpG dans de l'ADN sans modification chimique de l'ADN d'échantillon par un traitement au bisulfite ou une amplification en chaîne par polymérase (PCR). Une création de signal a pour base une technologie d'abscription (transcription abortive) dans laquelle des générateurs de signaux d'ADN appelés cassettes de promoteur abortives (APC) sont liés à des sites de mCpG cibles via des sondes spécifiques pour cible mCpG sur la base de polypeptides de liaison à du méthyle ou de domaines de liaison à du méthyle de ceux-ci. La polymérase ARN produit de courtes molécules d'ARN uniformes à partir de promoteurs synthétiques dans des APC comme signaux de la présence de CpG méthylés. La détection d'une méthylation de CpG et d'une hyperméthylation de cibles ADN telles que des îlots CpG fournit un moyen commode de détection et de surveillance du cancer chez un sujet.

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