Method for the detection of an analyte by means of a nucleic acid reporter

• US 20020051986A1
• Filed: 05/16/2001
• Published: 05/02/2002
• Est. Priority Date: 06/13/2000
• Status: Active Grant

• Current Assignee: Olink Proteomics AB (Thermo Fisher Scientific Incorporated)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Ebersole, Richard C., Baez, Luis, Perry, Michael P., Neelkantan, Neel, Hendrickson, Edwin R.
• Granted Patent: US 6,511,809 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

A process is disclosed for the detection of an analyte utilizing a nucleic acid label as a reporter. The analyte is detected by the binding of at least two reporter conjugates, each conjugate comprising a member of a binding pair and a nucleic acid label. The binding of the reporter conjugates to the analyte facilitates the juxtaposition of the nucleic acid labels, forming a single nucleic acid amplicon. The amplicon may then be detected directly, or may be used as a template of the generation of amplification products. Detection of the analyte by this process significantly reduces assay background caused by non-specific reporter conjugate binding.

Claims

1. A method for the detection of an analyte comprising:

(i) contacting at least one analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising;

a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte;

b) a nucleic acid label;

wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;

(ii) contacting said analyte dependent reporter complex with a enzyme composition wherein the nucleic acid labels on said reporter conjugates are joined to form an analyte specific amplicon;

(iii) contacting the analyte dependent amplicon with an replication composition wherein amplification products are produced; and

(iv) detecting said amplification products.

2. A method according to claim 1 wherein said analyte at step (i) is optionally immobilized on a solid support.

3. A method according to claim 1 wherein said enzyme composition comprises a DNA polymerase and wherein said nucleic acid labels on said reporter conjugates are joined by an overlap at each 3′

end.

4. A method according to claim 1 wherein said enzyme composition comprises a DNA ligase and wherein said nucleic acid labels on said reporter conjugates are enzymatically joined by means of a ligation linker comprising a replication inhibitory moiety.

5. A method according to claim 3 wherein said overlap comprises from about 10 bases to about 30 bases.

6. A method according to claim 2 wherein said solid support is comprised of materials selected from the group consisting of polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), glass, ceramics, metal and metal oxides.

7. A method according to claim 1 wherein said one member of a binding pair is selected from the group consisting of an antigen, antibody, hapten, nucleic acid, a nucleic acid aptamer, biotin, streptavidin, avidin, folic acid, folate binding protein, protein A protein G, immunoglobulins, epoxide, malaimide and sulfhydryl reactive groups.

8. A method according to claim 1 wherein the at least two reporter conjugates have different specificites for different reporter conjugate binding sites on said analyte.

9. A method according to claim 1 wherein said nucleic acid labels are of different lengths.

10. A method according to claim 1 wherein said nucleic acid labels are of different nucleotide sequence.

11. A method according to claim 1 wherein said nucleic acid labels are from about 30 bases to about 1000 bases in length.

13. A method according to claim 12 wherein at step (iii) a ligation linker comprising a 3′

replication inhibitory moiety is optionally added together with said DNA ligase.

14. A method according to claim 13 wherein said replication inhibitory moiety is selected from the group consisting of dideoxynuleotides, a sequence of mismatched nucleotides, 3′

phosphate and cordycepin.

15. A method according to claim 12 wherein said solid support is comprised of materials selected from the group consisting of polyethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene terephthalate), nylon, poly(vinyl butyrate), glass, ceramics, metal and metal oxides.

16. A method according to claim 12 wherein said one member of a binding pair is selected from the group consisting of an antigen, antibody, hapten, nucleic acid, a nucleic acid aptamer, biotin, streptavidin, avidin, folic acid, folate binding protein, protein A protein G, immunoglobulins, epoxide, malaimide and sulfhydryl reactive groups.

17. A method according to claim 12 wherein the at least two reporter conjugates have different specificities for different reporter conjugate binding sites on said analyte.

18. A method according to claim 12 wherein said nucleic acid labels are of different lengths.

19. A method according to claim 12 wherein said nucleic acid labels are of different nucleotide sequence.

20. A method according to claim 12 wherein said nucleic acid labels are from about 25 bases to about 1000 bases in length.

12. A method for the detection of an analyte comprising:

(i) immobilizing at least one analyte on a solid support, said analyte having at least two reporter conjugate binding sites;

(ii) contacting said analyte with at least one reporter conjugate pair, said reporter conjugate pair comprising a first reporter conjugate and a second reporter conjugate, each of said first and second reporter conjugates further comprising;

a) one member of a binding pair having an affinity for at least one reporter conjugate biding site on said analyte;

b) a nucleic acid label;

 

wherein said nucleic acid label of said first reporter conjugate comprises a 3′

hydroxyl group and wherein said nucleic acid label of said second reporter conjugate comprises a 5′

phosphoryl group and wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;

(iii) contacting said analyte dependent reporter complex with a DNA ligase,

 

wherein said first and second nucleic acid labels are ligated to form an analyte dependent amplicon;

(iv) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and

(v) detecting said amplification products.

21. A method for the detection of an analyte comprising:

(i) contacting at least one analyte with at least one reporter conjugate pair, said reporter conjugate pair comprising a first reporter conjugate and a second reporter conjugate, each of said first and second reported conjugates further comprising;

a) one member of a binding pair having an affinity for at least one reporter conjugate biding site on said analyte;

b) a nucleic acid label;

 

wherein said nucleic acid label of said first reporter conjugate comprises a 3′

hydroxyl group and wherein said nucleic acid label of said second reporter conjugate comprises a 5′

phosphoryl group and wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;

(ii) contacting said analyte dependent reporter complex with a DNA ligase,

 

wherein said first and second nucleic acid labels are ligated to form an analyte dependent amplicon;

(iii) contacting said analyte specific amplicon with a replication composition wherein said amplicon is amplified forming amplification products; and

(iv) detecting said amplification products.

22. A method for the detection of an analyte comprising:

(i) contacting at least one analyte having at least two reporter conjugate binding sites with at least two reporter conjugates, said reporter conjugates each comprising;

a) one member of a binding pair having specificity for at least one reporter conjugate binding site on said analyte;

b) a nucleic acid label;

 

wherein said analyte binds to said reporter conjugate forming an analyte dependent reporter complex;

(ii) contacting said analyte dependent reporter complex with;

a) an enzyme composition; and

b) a nucleic acid reporting label selected from the group consisting of fluorescent moieties, chemiluminescent moieties, particles, enzymes, radioactive tags, light emitting moieties and intercalating dyes;

 

wherein the nucleic acid labels on said reporter conjugates are joined to form an analyte specific amplicon and wherein said nucleic acid reporting label is incorporated into said amplicon; and

(iii) detecting said labeled amplicon.

23. A method according to claim 22 wherein said enzyme composition comprises a DNA polymerase and wherein said nucleic acid labels on said reporter conjugates are joined by an overlap at each 3′

end.

24. A method according to claim 22 wherein said enzyme composition comprises a DNA ligase and wherein said nucleic acid labels on said reporter conjugates are enzymatically joined by means of a ligation linker comprising a replication inhibitory moiety.

25. A method according to claim 22 wherein said analyte of step (i) is optionally immobilized on a solid support.