Multiplex detection compositions, methods, and kits

• US 20050074774A1
• Filed: 08/29/2003
• Published: 04/07/2005
• Est. Priority Date: 08/29/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Hyldig-Nielsen, Jens J., Woudenberg, Timothy M., Sharaf, Muhammad A., Bahatt, Dar, Liu, Timothy Z., Ermakov, Serguei, Connell, Charles R.
• Granted Patent: US 7,198,900 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2563/185 Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
• CPC Class Codes: C12Q 2565/1025 labels being on the same oligonucleotide

Abstract

The present invention generally relates to the detection of analytes, particularly biomolecules in samples. The invention also relates to compositions, methods, and kits for detecting the presence of analytes, typically in multiplex detection formats. The invention also relates to methods for determining the presence of at least one analyte in a sample, the methods employing employ single molecule detection techniques to individually detect at least one molecular complex or at least part of a molecular complex.

Claims

1. A method for determining the presence of at least one analyte in a sample, comprising:

forming at least one molecular complex comprising (a) the at least one analyte and (b) at least one first probe comprising at least one reaction portion and at least one identity portion comprising at least one coded molecular tag; and

individually detecting the at least one molecular complex or at least part of the at least one molecular complex to determine the presence of the at least one analyte in the sample.

2. The method of claim 1, wherein the molecular complex further comprises at least one second probe comprising at least one reaction portion and at least one analytical portion.

3. The method of claim 1, wherein the at least one identity portion comprises a multiplicity of fluorescent reporter groups.

4. The method of claim 3, wherein the at least one identity portion comprises at least one peptide nucleic acid (PNA), at least one pseudocomplementary peptide nucleic acid (pcPNA), or combinations thereof.

5. The method of claim 1, wherein the at least one identity portion comprises at least one affinity tag, at least one mobility modifier, or at least one affinity tag and at least one mobility modifier.

6. The method of claim 1, wherein the at least one identity portion is within, coextensive with, or overlaps at least part of the reaction portion.

7. The method of claim 2, wherein the at least one analytical portion is within, coextensive with, or overlaps at least part of the reaction portion.

8. The method of claim 2, wherein the at least one analytical portion comprises at least one PNA, at least one pcPNA, at least one dendrimer, at least one reporter group, or combinations thereof.

9. The method of claim 2, wherein the at least one analytical portion comprises at least one fluorophore, at least one mobility modifier, at least affinity tag, or combinations thereof.

10. The method of claim 2, wherein at least part of the reaction portion of the at least one first probe and at least part of the reaction portion of the at least one second probe hybridize to complementary sequences on the same strand of the at least one analyte.

11. The method of claim 10, wherein the at least part of the reaction portion of the at least one first probe and the at least part of the reaction portion of the at least one second probe hybridize adjacent to one another.

12. The method of claim 11, further comprising at least one ligation agent.

13. The method of claim 12, wherein the at least one ligation agent comprises at least one DNA ligase, at least one RNA ligase, or combinations thereof.

14. The method of claim 13, wherein the at least one DNA ligase, the at least one RNA ligase, or both the at least one DNA ligase and the at least one RNA ligase comprises at least one thermostable ligase.

15. The method of claim 2, wherein the at least one analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, or at least one ribonucleotide and at least one deoxyribonucleotide.

16. The method of claim 15, wherein the at least one analyte comprises at least one nucleic acid sequence comprising at least one heritable mutation, at least one somatic mutation, at least one single nucleotide polymorphism (SNP), at least one point mutation, at least one deletion mutation, at least one insertion mutation, at least one chromosomal translocation, or combinations thereof.

17. The method of claim 1, wherein the at least one analyte comprises at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, at least one peptide, at least one nucleotide, at least one glycosidic bond, or combinations thereof.

18. The method of claim 2, wherein the at least one analyte comprises at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, at least one peptide, at least one nucleotide, at least one glycosidic bond, or combinations thereof.

19. The method of claim 1, wherein the individually detecting comprises at least one scanning probe microscopy technique, at least one applied optical spectroscopy technique, or both at least one scanning probe microscopy technique and at least one applied optical spectroscopy technique.

20. The method of claim 2, wherein the individually detecting comprises at least one scanning probe microscopy technique, at least one applied optical spectroscopy technique, or both at least one scanning probe microscopy technique and at least one applied optical spectroscopy technique.

21. The method of claim 20, wherein the at least one scanning probe microscopy technique comprises atomic force microscopy (AFM), magnetic resonance force microscopy (MRFM), scanning electrochemical microscopy (SECM), scanning tunneling microscopy, or combinations thereof.

22. The method of claim 20, wherein at least one applied optical spectroscopy technique comprises fluorescence correlation spectroscopy (FCS), evanescent wave induced fluorescence spectroscopy (EWIFS), scanning near-field optical microscopy (SNOM), surface enhances raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS), surface plasmon resonance (SPR), laser-confocal microscopy, or combinations thereof.

23. The method of claim 20, wherein the individually detecting comprises attaching at least one molecular complex or at least part of a molecular complex, tethering at least one molecular complex or at least part of a molecular complex, or both attaching and tethering at least one molecular complex or at least part of a molecular complex directly or indirectly to a substrate.

24. The method of claim 23, wherein the substrate comprises at least one capture moiety.

25. The method of claim 24, wherein the attaching, the tethering, or both the binding and the tethering comprises at least one first capture moiety and at least one second capture moiety, wherein the at least one first capture moiety and the at least one second capture moiety are spatially separated from each other on the substrate.

26. The method of claim 3, wherein individually detecting comprises attaching at least one molecular complex or at least part of a molecular complex, tethering at least one molecular complex or at least part of a molecular complex, or both attaching and tethering at least one molecular complex or at least part of a molecular complex to a substrate and identifying the order of the fluorescent reporter groups using laser-confocal microscopy;

and wherein the analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, or at least one ribonucleotide and at least one deoxyribonucleotide;

at least one heritable mutation, at least one somatic mutation, at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, or combinations thereof.

27. The method of claim 3, wherein individually detecting comprises identifying the order of reporter group species in a molecular complex or at least part of a molecular complex in solution using laser-confocal microscopy;

and wherein the analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, or at least one ribonucleotide and at least one deoxyribonucleotide;

at least one heritable mutation, at least one somatic mutation, at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, or combinations thereof.

28. The method of claim 2, further comprising amplifying the molecular complex.

29. A method for determining the presence of at least one analyte in a sample, comprising:

amplifying at least one analyte to form at least one analyte surrogate,

forming at least one molecular complex comprising (a) the at least one analyte, at least one analyte surrogate, or at least one analyte and at least one analyte surrogate and (b) at least one first probe comprising at least one reaction portion and at least one identity portion comprising at least one coded molecular tag; and

individually detecting the at least one molecular complex or at least part of at least one molecular complex to determine the presence of the at least one analyte in the sample.

30. The method of claim 29, wherein the molecular complex further comprises at least one second probe comprising at least one reaction portion and at least one analytical portion.

31. The method of claim 29, wherein the at least one identity portion comprises a multiplicity of fluorescent reporter groups.

32. The method of claim 31, wherein the at least one identity portion comprises at least one peptide nucleic acid (PNA), at least one pseudocomplementary peptide nucleic acid (pcPNA), at least one dendrimer, or combinations thereof.

33. The method of claim 30, wherein the at least one identity portion further comprises at least one affinity tag, at least one mobility modifier, or at least one affinity tag and at least one mobility modifier.

34. The method of claim 30, wherein the at least one identity portion is within, coextensive with, or overlaps at least part of the reaction portion.

35. The method of claim 30, wherein the at least one analytical portion is within, coextensive with, or overlaps at least part of the reaction portion.

36. The method of claim 30, wherein the at least one analytical portion comprises at least one PNA, at least one pcPNA, at least one reporter group, or combinations thereof.

37. The method of claim 36, wherein the at least one analytical portion comprises at least one fluorophore, at least one mobility modifier, at least affinity tag, or combinations thereof.

38. The method of claim 30, wherein at least part of the reaction portion of the at least one first probe and at least part of the reaction portion of the at least one second probe hybridize to complementary sequences on the same strand of the at least one analyte, the at least one analyte surrogate and the at least one analyte or at least one analyte surrogate.

39. The method of claim 38, wherein the at least part of the reaction portion of the at least one first probe and the at least part of the reaction portion of the at least one second probe hybridize adjacent to one another.

40. The method of claim 39, further comprising at least one ligation agent.

41. The method of claim 40, wherein the at least one ligation agent comprises at least one DNA ligase, at least one RNA ligase, or combinations thereof.

42. The method of claim 41, wherein the at least one DNA ligase, the at least one RNA ligase, or both the at least one DNA ligase and the at least one RNA ligase comprises at least one thermostable ligase.

43. The method of claim 30, wherein the at least one analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, or at least one ribonucleotide and at least one deoxyribonucleotide.

44. The method of claim 43, wherein the at least one analyte comprises at least one nucleic acid sequence comprising at least one heritable mutation, at least one somatic mutation, or at least one heritable mutation and at least one somatic mutation.

45. The method of claim 30, wherein the individually detecting comprises at least one scanning probe microscopy technique, at least one applied optical spectroscopy technique, or both at least one scanning probe microscopy technique and at least one applied optical spectroscopy technique.

46. The method of claim 45, wherein the at least one scanning probe microscopy technique comprises atomic force microscopy (AFM), magnetic resonance force microscopy (MRFM), scanning electrochemical microscopy (SECM), scanning tunneling microscopy, or combinations thereof.

47. The method of claim 45, wherein at least one applied optical spectroscopy technique comprises fluorescence correlation spectroscopy (FCS), evanescent wave induced fluorescence spectroscopy (EWIFS), scanning near-field optical microscopy (SNOM), surface enhances raman spectroscopy (SERS), surface enhanced resonant raman spectroscopy (SERRS), surface plasmon resonance (SPR), laser-confocal microscopy, or combinations thereof.

48. The method of claim 46, wherein the individually detecting comprises attaching at least one molecular complex or at least part of a molecular complex, tethering at least one molecular complex or at least part of a molecular complex, or both attaching and tethering at least one molecular complex or at least part of a molecular complex directly or indirectly to a substrate.

49. The method of claim 48, wherein the substrate comprises at least one capture moiety.

50. The method of claim 49, wherein the attaching, the tethering, or both the binding and the tethering comprises at least one first capture moiety and at least one second capture moiety, wherein the at least one first capture moiety and the at least one second capture moiety are spatially separated from each other on the substrate.

51. The method of claim 30, wherein individually detecting comprises attaching at least one molecular complex or at least part of a molecular complex, tethering at least one molecular complex or at least part of a molecular complex, or both attaching and tethering at least one molecular complex or at least part of a molecular complex directly or indirectly to a substrate and individually detecting the at least one molecular complex or at least part of a molecular complex using laser-confocal microscopy;

wherein at least one molecular complex comprises a multiplicity of fluorescent reporter group species; and

wherein the analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, at least one heritable mutation, at least one somatic mutation, at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, or combinations thereof.

52. The method of claim 31, wherein the at least one molecular complex or at least part of a molecular complex is individually detected in solution using laser-confocal microscopy;

and wherein the analyte comprises at least one nucleic acid sequence comprising at least one ribonucleotide, at least one deoxyribonucleotide, at least one heritable mutation, at least one somatic mutation, at least one diagnostic indicator, at least one foreign antigen, at least one drug, at least one metabolite, at least one small molecule, or combinations thereof.

53. A kit for determining the presence of at least one analyte in a sample comprising at least one probe set comprising (a) at least one first probe comprising at least one first reaction portion and (b) at least one second probe comprising at least one second reaction portion, wherein at least one probe in the at least one probe set further comprises at least one identity portion, and wherein the at least one first probe and the at least one second probe are suitable for forming a molecular complex in the presence of the at least one analyte.

54. The kit of claim 53, further comprising at least one ligase, at least one polymerase, or at least one ligase and at least one polymerase.

55. The kit of claim 54, wherein the at least one polymerase comprises at least one DNA polymerase, at least one RNA polymerase, at least one reverse transcriptase, or combinations thereof.

56. The kit of claim 53, further comprising at least one dendrimer, at least one PNA, at least one pcPNA, at least one intercalating substance, at least one antibody, or combinations thereof.

57. The kit of claim 53, wherein at least one probe in the at least one probe set further comprises at least one analytical portion.

58. The kit of claim 53, further comprising at least one reporter group.

59. The kit of claim 53, further comprising biotin, a derivative of biotin, avidin, a derivative of avidin, strepavidin, a derivative of streptavidin, or combinations thereof.

60. The kit of claim 59, further comprising at least one substrate.