Molecular Beacons for DNA-Photography
The present invention refers to a detection method for analytes using the principle of black-and-white photography and to reagent kits for performing the method, furthermore applied this new technology to detect a biologically relevant sequence in the nanomolar range (femtomoles) in an application circumventing the necessity of a PCR. There are still numerous ways to optimize this methodology that is suitable for a large variety of applications in the genomic diagnostics and proteomics areas.
1 - 23 . (canceled)
24 . A method for detecting an analyte in a sample comprising the steps:
(i) providing a sample;
(ii) providing a reporter molecule comprising a photosensitizer group or a handle group for introducing a photosensitizer group and a quencher group wherein the photosensitizer group is quenched in the absence of the analyte to be detected;
(iii) contacting the sample with the reporter molecule under conditions wherein the quenching of the photosensitizer group is at least partially reduced or terminated in the presence of the analyte;
(iv) reacting the handle group with a reaction partner comprising a photosensitizer group;
(v) irradiating said reporter molecule in contact with a photosensitive medium under conditions wherein marker groups are formed in said photosensitive medium in the presence of unquenched photosensitizer groups in said reporter molecule; and
(vi) detecting said marker groups.
25 . The method of claim 24 wherein the analyte is selected from the group consisting of nucleic acids and nucleoside-, nucleotide- or nucleic acid-binding molecules.
26 . The method of claim 24 , wherein the analyte to be detected is a nucleic acid selected from the group consisting of DNA and RNA.
27 . The method of claim 24 , wherein the sample is a biological sample.
28 . The method of claim 24 , wherein the sample is an agricultural sample, nutritional sample or a clinical sample.
29 . The method of claim 24 , wherein the detection is carried out directly without amplification.
30 . The method of claim 24 , wherein the detection is carried out in combination with an amplification step.
31 . The method of claim 24 , wherein reporter molecule is a nucleic acid molecule.
32 . The method of claim 24 , wherein the handle group is selected from azide and alkyne groups.
33 . The method of claim 32 , wherein said azide groups are reacted by performing a Click reaction with a an alkyne group of a reaction partner comprising a photosensitizer group.
34 . The method of claim 32 , wherein said alkyne group is reacted by performing a Click reaction with an azide group of a reaction partner comprising a photosensitizer group.
35 . The method of claim 24 , wherein the photosensitizer group is selected from fluorescent dye groups.
36 . The method of claim 35 wherein the photosensitizer group is selected from cyanine-based indoline groups and quinoline groups.
37 . The method of claim 24 , wherein the photosensitive medium comprises metal atoms or ions capable of forming metal nuclei.
38 . The method of claim 37 , wherein the metal is Ag.
39 . The method of claim 24 , wherein the photosensitive medium is a light sensitive paper selected from the group consisting of photographic paper, a light sensitive emulsion and a gel on a supportive material.
40 . The method of claim 24 , wherein the irradiating step (v) is carried out with long wave visible light and/or with infrared light.
41 . A reagent kit for detecting an analyte in a sample comprising:
(a) a reporter molecule comprising a photosensitizer group or a handle group for introducing a photosensitizer group and a quencher group wherein the photosensitizer group is quenched in the absence of the analyte to be detected,
(b) optionally a reaction partner for the handle group comprising a photosensitizer group and
(c) a photosensitive medium which forms marker groups upon irradiation of unquenched photosensitizer groups.
42 . The kit of claim 41 , wherein the reporter molecule is present as reagent impregnated on the photosensitive medium.
43 . The method of claim 41 , wherein the kit is used for an application selected from the group consisting of agricultural applications, medical applications, diagnostic applications, forensic applications, detection function and/or expression of genes, for brand protection, nutritional applications, and feed applications.
44 . The method of claim 28 , wherein the method is used for an application selected from the group consisting of agricultural applications, medical applications, diagnostic applications, forensic applications, detection function and/or expression of genes, for brand protection, nutritional applications, and feed applications.
45 . The method of claim 24 , for detecting an analyte which has been modified by genetic engineering.
46 . The method of claim 24 , for detecting an analyte which is a product of a genetically modified organism.