Labelling strategies for the sensitive detection of analytes
First Claim
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1. A method for detecting a nucleic acid analyte in a sample comprising the steps:
- (i) providing a sample;
(ii) contacting the sample with a functionalized compound comprising at least one alkyne functional group which is a first reaction partner for a Click reaction, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring, and wherein the functional group is attached to position 5 or 6 of a pyrimidine nucleobase or to position 7 of a 7-deazaadenine or 7-deazaguanine nucleobase or to position 8 of a purine nucleobase or an oligomer or polymer thereof, under conditions wherein said compound forms an association product which specifically binds by hybridization to the analyte to be detected or the complement thereof,(iii) contacting the association product with a second reaction partner for said Click reaction comprising a complementary azide group under conditions wherein a Click reaction between the first and second reaction partner occurs, wherein the second reaction partner further comprises a fluorescence marker group, and(iv) detecting said marker group.
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Abstract
The present invention relates to methods and reagents for detecting analytes, e.g. nucleic acids. The new methods and reagents allow a simple and sensitive detection even in complex biological samples.
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Citations
40 Claims
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1. A method for detecting a nucleic acid analyte in a sample comprising the steps:
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(i) providing a sample; (ii) contacting the sample with a functionalized compound comprising at least one alkyne functional group which is a first reaction partner for a Click reaction, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring, and wherein the functional group is attached to position 5 or 6 of a pyrimidine nucleobase or to position 7 of a 7-deazaadenine or 7-deazaguanine nucleobase or to position 8 of a purine nucleobase or an oligomer or polymer thereof, under conditions wherein said compound forms an association product which specifically binds by hybridization to the analyte to be detected or the complement thereof, (iii) contacting the association product with a second reaction partner for said Click reaction comprising a complementary azide group under conditions wherein a Click reaction between the first and second reaction partner occurs, wherein the second reaction partner further comprises a fluorescence marker group, and (iv) detecting said marker group. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method for detecting a nucleic acid analyte in a sample comprising the steps:
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(i) providing a sample; (ii) contacting the sample with a functionalized compound comprising at least one alkyne functional group which is a first reaction partner for a Click reaction, said Click reaction being a (3+2) cycloaddition reaction between azide and alkyne groups forming a 1,2,3-triazole ring, and wherein the functional group is attached to position 5 or 6 of a pyrimidine nucleobase or to positions 7 or 8 of a 7-deazaadenine or 7-deazaguanine nucleobase or to position 8 of a purine nucleobase or an oligomer or polymer thereof, under conditions wherein said compound forms an association product which specifically binds by hybridization to the analyte to be detected or the complement thereof, (iii) contacting the association product with a second reaction partner for said Click reaction comprising a complementary azide group under conditions wherein a Click reaction between the first and second reaction partner occurs, wherein the second reaction partner further comprises a fluorescence marker group, and (iv) detecting said marker group, wherein the (3+2) cycloaddition reaction between azide and alkyne groups is catalyzed by copper(I).
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Specification