Labelling strategies for the sensitive detection of analytes

• US 9,005,892 B2
• Filed: 03/02/2012
• Issued: 04/14/2015
• Est. Priority Date: 05/02/2005
• Status: Active Grant

• Current Assignee: Baseclick GmbH
• Original Assignee: Baseclick GmbH
• Inventors: Carell, Thomas, Burley, Glenn A., Gierlich, Johannes, Mofid, Mohammad Reza, Schwgler, Anja
• Primary Examiner(s): Gross, Christopher M
• Application Number:
  US13/411,021
• Publication Number:
  US 20120157327A1
• Time in Patent Office: 1,138 Days
• Field of Search: None
• US Class Current: 435/6.1
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide
• CPC Class Codes: G01N 33/5308 for analytes not provided for elsewhere, e.g. nucleic acids, uric acid, worms, mites
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

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.

Claims

1. A method for detecting an analyte selected from nucleic acids 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 a nucleobase of a nucleosidic or nucleotidic compound or an oligomer or polymer thereof wherein the functional group is attached directly or directly via a spacer to a nucleobase, under conditions wherein said compound forms a hybridization product with the analyte to be detected or the complement thereof,

(iii) contacting the hybridization 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 marker group or a marker precursor group,

(iv) if necessary, converting marker precursor groups to marker groups, and

(v) detecting said marker groups.

2. The method of claim 1, comprising a qualitative detection.

3. The method of claim 1, comprising a quantitative detection.

4. The method of claim 1, wherein the analyte to be detected is a nucleic acid selected from the group consisting of DNA and RNA.

5. The method of claim 1, wherein the sample is a biological sample.

6. The method of claim 5, wherein the sample is an agricultural or nutritional sample.

7. The method of claim 5, wherein the sample is a clinical sample.

8. The method of claim 1, wherein the detection is carried out directly without amplification.

9. The method of claim 1, wherein the detection is carried out in combination with an amplification step.

10. The method of claim 1, wherein a sequence-specific detection of a nucleic acid analyte is carried out.

11. The method of claim 10, wherein the forming of said association product comprises a sequence-specific hybridization reaction.

12. The method of claim 1, wherein the nucleobase is selected from the group consisting of naturally occurring and non-naturally occurring purine and pyrimidine bases.

13. The method of claim 12, wherein the functional group is attached to position 5 or 6 of a pyrimidine nucleobase or to positions 7 or 8 of a purine nucleobase.

14. The method of claim 13, wherein the functional group is attached to position 5 of the pyrimidine nucleobase.

15. The method of claim 13, wherein the functional group is attached to position 7 of the purine nucleobase.

16. The method of claim 1, wherein the functional group is attached to the compound via a direct bond or spacer.

17. The method of claim 14, wherein said spacer is a flexible spacer or an at least a partially rigid spacer.

18. The method of claim 17, wherein the spacer comprises at least one group selected from the group consisting of alkene, alkyne, cyclic groups, aromatic groups, heteroaromatic groups, and combinations thereof.

19. The method of claim 1, wherein said functionalized compound is selected from the group consisting of:

(i) a functionalized nucleic acid or nucleic acid analogue building block; and

(ii) a functionalized nucleic acid or nucleic acid analogue.

20. The method of claim 19, wherein the forming of said association product comprises incorporating said functionalized building block into a nucleic acid or nucleic acid analogue molecule.

21. The method of claim 20, wherein said functionalized nucleotide or nucleotide analogue is enzymatically incorporated into a nucleic acid molecule.

22. The method of claim 19, comprising a primer extension reaction optionally in combination with a nucleic acid amplification reaction.

23. The method of claim 22 comprising:

providing at least one primer molecule which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof and extending said primer, wherein at least one functionalized nucleotide or nucleotide analogue is incorporated into the extension product.

24. The method of claim 22 comprising:

providing at least one functionalized primer molecule which hybridizes under the assay conditions with the nucleic acid analyte to be detected or the complement thereof, extending said primer molecule, wherein an extended primer molecule is formed.

25. The method of claim 1, wherein the detection is carried out by optical means.

26. The method of claim 1, wherein the detection is carried out by electrical means.

27. The method of claim 1, comprising a parallel detection of a plurality of nucleic acids.

28. The method of claim 1, wherein the detection is carried out on a solid surface, e.g. on a microarray chip.

29. The method of claim 1, wherein the functional group is selected from the group consisting of azide groups or alkyne groups.

30. The method of claim 1, wherein the second reaction partner comprises a marker group selected from the group consisting of optically detectable marker groups, fluorescence marker groups, and redox active marker groups.

31. The method of claim 1, wherein the second reaction partner comprises a marker precursor group selected from the group consisting of aldehyde groups and protected aldehyde groups and aldehyde precursor groups.

32. The method of claim 31, wherein converting the marker precursor groups to marker groups comprises forming metal depositions around aldehyde groups.

33. The method of claim 1, wherein said sample is an agricultural sample.

34. The method of claim 33 nucleic acids are detected from plants, plant pathogens or plant pests.

35. The method of claim 33, wherein genetic variabilities are detected in plants, plant pathogens or plant pests.

36. The method of claim 33, wherein herbicide, fungicide or pesticide resistances, tolerances or intolerances in fungi, insects or plants are detected.

37. The method of claim 33, wherein species or strains of fungi, insects or plants are genotyped and/or differentiated.

38. The method of claim 33, wherein genetically modified organisms or strains of fungi, insects or plants are detected and/or differentiated.

39. The method of claim 1, wherein genetic variabilities in humans are detected.

40. The method of claim 39, wherein medicament resistance, tolerance or intolerance, or allergy is detected.

41. The method of claim 39, wherein genotyping is determined.

42. The method of claim 39, wherein a genetically modified organism or strain is detected.

43. The method of claim 39 genetic diseases, allergic diseases, autoimmune diseases or infectious diseases are detected.

44. The method of claim 1, wherein the function and/or expression of a gene is detected.

45. The method of claim 1, wherein specific information encoded in products is detected.

46. The method of claim 39, wherein specific information encoded in a product is detected.

47. The method of claim 39, wherein DNA methylation pattern is determined.