Tag reagent and assay method

• US 6,576,426 B2
• Filed: 12/06/2001
• Issued: 06/10/2003
• Est. Priority Date: 07/30/1993
• Status: Expired due to Fees

• Current Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Original Assignee: Oxford Gene Technology Limited (Sysmex Corporation)
• Inventors: Cummins, William Jonathan, Southern, Edwin
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/003,830
• Publication Number:
  US 20020115091A1
• Time in Patent Office: 551 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/22.1, 536/23.1, 536/24.3, 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 one or more reporter groups suitable for detection by mass spectrometry after being cleaved from the reagent, 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. A regent as claimed in claim 1, wherein there is provided a linker group to which is attached the analyte moiety and the tag moiety.

3. A reagent as claimed in claim 1, wherein the analyte moiety is a chain of n analyte residues, and the tag moiety is a chain of up to n reporter groups, the reporter group at each position of the tag chain being chosen to designate the analyte residue at a corresponding position of the analyte chain.

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

5. A 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 the linker, R is a chain of up to n reporter groups constituting the tag moiety, and n is 2-20, wherein the tag moiety contains information defining the location of analyte residues in the analyte moiety.

6. A reagent as claimed in claim 2, wherein the linker comprises an aromatic group carrying a hydroxy, amino or sulphydryl group for analyte moiety synthesis, and a reactive group for tag moiety synthesis.

7. A reagent as claimed in claim 6, wherein the aromatic group carrying a hydroxy, amino or sulphydryl group for analyte moiety synthesis, also carries an o-nitro group for photocleavage.

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

9. A reagent as claimed in claim 1, wherein the analyte moiety is a peptide chain.

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

11. A library of the reagents as claimed in claim 1, wherein the library consists of a plurality of reagents each comprising a different analyte moiety.

12. A library as claimed in claim 11, wherein the library consists of 4ⁿ reagents each comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotides.

13. A library as claimed in claim 12, wherein the reagents are mixed together in solution.

14. An assay method which comprises the steps of

providing a target substance;

incubating the target substance with the library of reagents claimed in claim 11 under conditions to cause at least one reagent to bind to the target substance;

removing non-bound reagents;

recovering the tag moieties of the or each bound reagent; and

analysing the recovered tag moieties as an indication of the nature of the analyte moieties bound to the target substance.

15. An assay method as claimed in claim 14, wherein the target substance is an organism or tissue or group of cells.

16. A method of sequencing a target nucleic acid, which method comprises the steps of:

a) providing an oligonucleotide immobilised on a support,

b) hybridising the target nucleic acid with the immobilised oligonucleotide,

c) incubating the hybrid from b) with the library claimed in claim 13, so that an oligonucleotide chain of a first reagent of the library becomes hybridised to the target nucleic acid adjacent the immobilised oligonucleotide,

d) ligating the adjacent oligonucleotides, thus forming a ligated first reagent,

e) removing other non-ligated reagents, and

f) recovering and analysing the tag moiety of the ligated first reagent as an indication of the sequence of a first part of the target nucleic acid.

17. A method as claimed in claim 16, comprising the additional steps of

ci) incubating the hybrid from f) with a library consisting of 4ⁿ reagents each comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotides mixed together in solution, so that an oligonucleotide chain of a second reagent of the library becomes hybridised to the target nucleic acid adjacent the oligonucleotide chain of the first reagent,

di) ligating the adjacent oligonucleotides, thus forming a ligated second reagent,

ei) removing other non-ligated reagents, and

fi) recovering and analysing the tag moiety of the ligated second reagent as an indication of the sequence of a second part of the target nucleic acid.

18. A method as claimed in claim 16, wherein:

in step a) the oligonucleotide is immobilised on the ends of a series of pins as the support;

in step b) an individual clone of target DNA is hybridised to the oligonucleotide immobilised on each individual pin;

in steps c) and d) there are formed a series of ligated reagents, with different pins carrying different ligated reagents; and

in step f) the tag moiety of each ligated reagent is recovered and analysed as an indication of the sequence of a part of the target DNA.

19. A method as claimed in claim 16, wherein:

in step b) each individual clone of target DNA is hybridised to the oligonucleotide immobilised at an individual spaced location of the support;

in steps c) and d) there are provided a series of ligated reagents with different spaced locations of the support carrying different ligated reagents; and

in step 1) the tag moiety of each ligated reagent is recovered and analysed as an indication of the sequence of a part of the target DNA.

20. A method as claimed in claim 16, wherein the method comprises the steps of:

a) providing an array of oligonucleotides immobilised at spaced locations on a support, an oligonucleotide at one location being different from oligonucleotides at other locations,

b) incubating the target nucleic acid with the array of immobilised oligonucleotides, so as to form hybrids at one or more spaced locations on the support,

c) incubating the hybrids from b) with a library consisting of 4ⁿ reagents each comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotides mixed together in solution, so that an oligonucleotide chain of a reagent of the library becomes hybridised to the target nucleic acid adjacent each immobilised oligonucleotide,

d) ligating adjacent oligonucleotides, thus forming ligated reagents at the one or more spaced locations on the support,

e) removing other non-ligated reagents, and

f) recovering and analysing the tag moiety of each ligated reagent as an indication of the sequence of a part of the target nucleic acid.

21. A method as claimed in claim 20, wherein the sequence is known of the oligonucleotide immobilised by a covalent bond at each spaced location on the support.

22. A method of analysing a target DNA, which method comprises the steps of:

i) providing the target DNA immobilised on a support,

ii) incubating the immobilised target DNA from i) with a plurality of the reagents claimed claim 10, so that the oligonucleotide chains of different reagents become hybridised to the target DNA on the support,

iii) removing non-hybridised reagents, and

iv) recovering and analysing the tag moiety of each reagent as an indication of the sequence of a part of the target DNA.

23. A method as claimed in claim 22, comprising the additional steps of:

iia) incubating the hybrid from iv) with a library of reagents consisting of 4ⁿ reagents each comprising a different analyte moiety which is a different oligonucleotide chain of n nucleotides mixed together in solution, so that oligonucleotide chains of different reagents become hybridised to the target DNA,

iiia) ligating adjacent oligonucleotides hybridised to the target DNA and removing non-ligated reagents, and

iva) recovering and analysing the tag moiety of each ligated reagent as an indication of the sequence of part to the target DNA.

24. A method as claimed in claim 22, wherein individual clones of the target nucleic acid are immobilised at spaced locations on the support, whereby in step ii) the oligonucleotide chains of different reagents become hybridised to the target nucleic acid at different spaced locations on the support.

25. A method as claimed in claim 14, wherein each tag moiety is recovered by photocleavage from its associated reagent.

26. A method as claimed in claim 14, wherein the tag moiety is analysed by mass spectrometry.

27. Assay equipment comprising:

a support having two or more spaced locations thereon;

individual clones of a target nucleic acid immobilised at the spaced locations on the support;

and different reagents according to claim 10 hybridised to the individual clones of the target nucleic acid at the spaced locations on the support.