Method of amplifying dna and rna mismatch cleavage products

Details Method of amplifying dna and rna mismatch cleavage products Method of amplifying dna and rna mismatch cleavage products

C - Chemistry, Metallurgy

12

Q

C12Q 1/68 (2006.01) C07H 21/04 (2006.01)

Patent

CA 2303206

Detection of probe fragment products of basepair mismatch cleavage indicate the presence and sequence of target DNA. Detection of the target is enhanced by amplification through recycling targets by maintaining an assay temperature between the melting point of the target/probe DNA duplex and that of the target/product complex, in the presence of an amplifier comprising ammonium acetate or an amine derivative (for example, diethylamine, piperidine or ammonium carbonate). Cleavage reduces the size of the duplex, thus lowering its melting point. The amplifier releases the target from the complex, thereby permitting further catalysis of cleavage and effectively amplifying the signal to be detected. The figure is a diagram of the theoretical mechanism for mismatch cleavage and amplification by ammonium acetate or an amine derivative.

La présente invention concerne la détection de produits de fragment d'échantillon de clivage de mésappariement de paires de bases, détection qui indique la présence et la séquence d'un ADN cible. La détection de la cible est améliorée par amplification par recyclage de cibles avec maintien d'une température de dosage comprise entre le point de fusion du duplex cible/ADN échantillon et celui du complexe cible/produit, en présence d'un amplificateur comprenant de l'acétate d'ammonium ou un dérivé d'amine (par exemple, de la diéthylamine, de la pipéridine, ou du carbonate d'ammonium). Le clivage réduit la taille du duplex et abaisse ainsi le point de fusion. L'amplificateur libère la cible du complexe, permettant ainsi une catalyse ultérieure de clivage et amplifiant efficacement le signal à détecter. La figure présente un schéma du mécanisme théorique de clivage de mésappariement et d'amplification à l'aide d'acétate d'ammonium ou d'un dérivé d'amine.

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