Tag reagent and assay method

• US 6,218,111 B1
• Filed: 12/10/1997
• Issued: 04/17/2001
• Est. Priority Date: 07/30/1993
• Status: Expired due to Fees

• Current Assignee: Oxford Gene Technology IP Limited (Sysmex Corporation)
• Original Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Inventors: Southern, Edwin, Cummins, William Jonathan
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US08/988,384
• Time in Patent Office: 1,224 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/25.3, 536/25.31, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• 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: C12N 15/1075 by coupling phenotype to genotype, not provided for in other groups of this subclass
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6872 involving mass spectrometry
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2563/167 Mass label
• CPC Class Codes: C12Q 2565/627 being a mass spectrometer
• CPC Class Codes: Y10S 435/912 Absidia

Abstract

This invention provides reagents, libraries and sets of the reagents, and assay methods using the reagents, the reagents comprising an analyte moiety and a tag moiety, wherein the tag moiety contains information defining the identify and location of the analyte residues of the analyte moiety which is detectable by mass spectrometry.

Claims

1. A reagent comprising

a) an analyte moiety comprising at least two analyte residues, and linked to

b) a tag moiety comprising at least two reporter groups suitable for detection by mass spectrometry, excluding oligonucleotides,

wherein a reporter group identifies an analyte residue, and its position in the analyte moiety, and

wherein the analyte moiety has a known predetermined sequence which can be specifically identified by mass spectrometry detection of the tag moiety.

2. The reagent as claimed in claim 1, wherein there is provided a linker group to which is attached the analyte moiety and the tag moiety.

3. The reagent as claimed in claim 1, wherein the analyte moiety is a chain of n analyte residues, and the tag moiety comprises n reporter groups, each reporter group being chosen to identify an analyte residue and its position in the analyte chain, wherein n is at least 2.

4. The reagent as claimed in claim 1, wherein the analyte moiety is linked to the tag moiety by a photocleavable link.

5. The reagent as claimed in claim 1, having a formula A—

L—

R, where A is a chain of n analyte residues constituting the analyte moiety, L is a linker, R is the tag moiety, and n is 2-20, wherein the tag moiety contains information defining the identity and the location of analyte residues in the analyte moiety.

6. The reagent as claimed in claim 5, wherein R comprises a chain of n reporter groups.

7. The reagent as claimed in claim 2, wherein the linker comprises an aromatic group.

8. The reagent as claimed in claim 7, wherein the linker is joined to the analyte moiety by an ether, amino or thioether bond.

9. The reagent as claimed in claim 7, wherein the linker carries an o-nitro group for photocleavage.

10. The reagent as claimed in claim 1, wherein there is present a charged group for analysis by mass spectrometry.

11. The reagent as claimed in claim 1, wherein the tag moiety is cleavable from the rest of the molecule.

12. The reagent as claimed in claim 1, wherein the analyte moiety is an oligonucleotide chain.

13. A library of reagents wherein each reagent comprises:

a) an analyte moiety comprising at least two analyte residues, and linked to

b) a tag moiety comprising at least two reporter groups suitable for detection by mass spectrometry, excluding oligonucleotides,

wherein each reporter group identifies an analyte residue and its position in the analyte moiety,

and wherein the library consists of at least two of the reagents each comprising a different analyte moiety.

14. The library as claimed in claim 13, wherein the different analyte moieties are different oligonucleotides representing complements of known sequences of a predetermined target nucleic acid.

15. The library as claimed in claim 13, wherein the library consists of 4ⁿ reagents, each reagent comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotide residues.

16. The library as claimed in claim 13, wherein the reagents are mixed together in solution.

17. An assay method which comprises the steps of:

providing a target substance;

incubating the target substance with one or more reagents, wherein the or each reagent comprises

a) an analyte moiety comprising at least two analyte residues, and linked to

b) a tag moiety comprising at least two reporter groups suitable for detection by mass spectrometry, excluding oligonucleotides,

wherein each reporter group identifies an analyte residue, and its position in the analyte moiety,

and wherein the incubation is conducted under conditions to cause at least one reagent to bind to the target substance;

removing non-bound reagents; and

analyzing recovered tag moieties as an indication of the nature and/or the position of analyte moieties bound to the target substance.

18. The assay method as claimed in claim 17, wherein the target substance is incubated with a library of reagents each of which comprises a different analyte moiety.

19. The assay method as claimed in claim 17, wherein the target substance is an organism or tissue or group of cells.

20. A set of reagents, each reagent comprising:

a) an analyte moiety comprising a chain of n analyte residues, and linked to

b) a tag moiety comprising one or more reporter groups,

wherein a reporter group identifies an analyte residue and its position in the analyte moiety,

wherein, in said set of reagents;

a first reagent has a first reporter group which designates a first analyte residue; and

an n^th reagent has an n^th reporter group which designates an n^th analyte residue;

wherein n is at least 2; and

wherein the analyte moiety has a known predetermined sequence which can be specifically identified by mass spectrometry detection of the tag moiety.

21. The set of reagents as claimed in claim 20, wherein the reporter groups are suitable for detection by mass spectrometry.

22. The set of reagents as claimed in claim 20, wherein each reagent comprises a linker group to which is attached the analyte moiety and the tag moiety.

23. The set of reagents as claimed in claim 20, wherein in each reagent, the analyte moiety is linked to the tag moiety by a photocleavable link.

24. The set of reagents as claimed in claim 20, wherein each reagent has the formula A—

L—

R, where A is the chain of n analyte residues constituting the analyte moiety, L is a linker, R is the tag moiety, and n is 2-20, wherein the tag moiety contains information defining the identity and the location of analyte residues in the analyte moiety.

25. The set of reagents as claimed in claim 24, wherein R comprises a chain of up to n reporter groups.

26. The set of reagents as claimed in claim 22, wherein for each reagent, the linker comprises an aromatic group.

27. The set of reagents as claimed in claim 26, wherein the linker is joined to the analyte moiety by an ether, amine or thioether bond.

28. The set of reagents as claimed in claim 26, wherein for each reagent, the linker carries an o-nitro group for photocleavage.

29. The set of reagents as claimed in claim 20, wherein in each reagent, there is present a charged group for analysis by mass spectrometry.

30. The set of reagents as claimed in claim 20, wherein in each reagent, the tag moiety is cleavable from the rest of the molecule.

31. The set of reagents as claimed in claim 20, wherein in each reagent, the analyte moiety is an oligonucleotide chain.

32. A library of at least two sets of reagent, in which each set of reagents comprises a different analyte moiety, wherein in each set of reagents, each reagent comprises:

a) an analyte moiety comprising a chain of n analyte residues, and linked to

b) a tag moiety comprising one or more reporter groups,

wherein a reporter group identifies an analyte residue and its position in the analyte moiety,

and wherein, in each said set of reagents;

a first reagent has a first reporter group which designates a first analyte residue; and

an n^th reagent has an n^th reporter group which designates an n^th analyte residue;

where n is at least 2.

33. The library as claimed in claim 32, wherein the different analyte moieties are different oligonucleotides representing complements of known sequences of a predetermined target nucleic acid.

34. The library as claimed in claim 32, wherein the library consists of 4ⁿ sets of reagents, the reagents of each said set comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotide residues.

35. The library as claimed in claim 32, wherein the reagents are mixed together in solution.

36. An assay method which comprises the steps of:

providing a target substance;

incubating the target substance with one or more sets of reagents, in which the or each set of reagents comprises a different analyte moiety, wherein in the or each set of reagents, each reagent comprises;

a) an analyte moiety comprising a chain of n analyte residues, and linked to

b) a tag moiety comprising one or more reporter groups,

wherein a reporter group identifies an analyte residue and its position in the analyte moiety,

wherein in the or each said set of reagents;

a first reagent has a first reporter group which designates a first analyte residue; and

an n^th reagent has an n^th reporter group which designates an n^th analyte residue;

where n is at least 2;

and wherein the incubation is conducted under conditions to cause at least one set of reagents to bind to the target substance;

removing non-bound reagents; and

analyzing recovered tag moieties as an indication of the nature and/or the position of analyte moieties bound to the target substance.

37. The assay method as claimed in claim 36, wherein the target sequence is incubated with a library of a plurality of the sets of reagents.

38. The assay method as claimed in claim 36, wherein the target substance is an organism or tissue or group of cells.

39. A reagent comprising an analyte moiety comprising at least two analyte residues connected via a linkage group to a tag moiety comprising at least one reporter group suitable for detection by mass spectrometry, excluding oligonucleotides, wherein a reporter group identifies an analyte residue and its position in the analyte moiety, and wherein the analyte moiety has a known predetermined sequence which can be specifically identified by mass spectrometry detection of the tag moiety.

40. The reagent as claimed in claim 39, wherein the linkage group is cleavable.

41. The reagent as claimed in claim 40, wherein the linkage group is photocleavable.

42. The reagent as claimed in claim 39, wherein the analyte moiety is a chain of n analyte residues, and a tag moiety comprises up to n reporter groups, each reporter group being chosen to identify an analyte residue and its position in the analyte chain, wherein n is at least 2.

43. The reagent as claimed in claim 39, having a formula A—

L—

R, where A is a chain of n analyte residues constituting the analyte moiety, L is a linkage group, R is the tag moiety, and n is 2-20, where the tag moiety contains information defining the identity and the location of analyte residues in the analyte moiety.

45. The reagent as claimed in claim 39, wherein the linkage group is joined to the analyte moiety by an ether, amino, or thioether bond.

46. The reagent of claim 45, wherein the linkage group contains an aromatic group.

47. The reagent as claimed in claim 45, wherein the linkage group also carries an o-nitro group for photocleavage.

48. The reagent as claimed in claim 39, wherein there is present a charged group for analysis by mass spectrometry.

49. The reagent as claimed in claim 39, wherein the tag moiety is cleavable from the rest of the molecule.

50. The reagent as claimed in claim 39, wherein the analyte moiety is an oligonucleotide chain.

44. The reagent as claimed in claim 70, wherein the tag moiety R comprises a chain of up to n reporter groups, each reporter group being chosen to identify an analyte residue and its position in the analyte chain.

51. A library of reagents wherein each reagent comprises an analyte moiety comprising at least two analyte residues connected via a linkage group to a tag moiety comprising at least one reporter group suitable for detection by mass spectrometry, excluding oligonucleotides, wherein a reporter group identifies an analyte residue and its position in the analyte moiety, and wherein the library consists of at least two of the reagents each comprising a different analyte moiety.

52. The library as claimed in claim 51, wherein the different analyte moieties are different oligonucleotides representing complements of known sequences of a predetermined target nucleic acid.

53. The library as claimed in claim 51, wherein the reagents are mixed together in solution.

54. An assay method which comprises the steps of:

providing a target substance;

incubating the target substance with one or more reagents, wherein the or each reagent comprises an analyte moiety comprising at least two analyte residues connected via a linkage group to a tag moiety comprising at least one reporter group suitable for detection by mass spectrometry, excluding oligonucleotides, wherein a reporter group identifies an analyte residue and its position in the analyte moiety, and wherein the incubation is conducted under conditions to cause at least one reagent to bind to the target substance;

removing non-bound reagents; and

analyzing one or more recovered tag moieties as in indication of the nature and/or position of one or more analyte moieties bound to the target substance.

55. The assay method as claimed in claim 54, wherein the target substance is an organism or tissue or group of cells.

56. The assay method as claimed in claim 54, wherein the target substance is incubated with a library of the said reagents.