Hybridization assay signal enhancement

• US 6,103,474 A
• Filed: 10/30/1998
• Issued: 08/15/2000
• Est. Priority Date: 10/21/1996
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Ach, Robert A., Dellinger, Douglas J., Dahm, SueAnn C., Ilsley, Diane D., Troll, Mark A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/183,619
• Time in Patent Office: 655 Days
• Field of Search: 435/6, 536/27.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/6.14
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: C12Q 2565/102 Multiple non-interacting labels

Abstract

A signal amplification method for detecting a target nucleic acid analyte having a homopolymeric region and a target sequence includes steps of: contacting an analyte under hybridizing conditions with a multiplicity of reporter probes, each reporter probe including a signal region and an oligonucleotide sequence which is complementary to and capable of forming a stable hybrid with the analyte homopolymeric region to form an analyte:reporter probe hybrid; and forming an analyte:capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions. The analyte:reporter probe hybrid may formed prior to contacting the analyte target sequence with the capture probe, so the result of contacting the analyte target sequence with the capture probe results in formation of an analyte:reporter probe:capture probe complex. The analyte:capture probe hybrid may be immobilized on a solid generally planar surface in an array format. Multiple reporter probes may form triple helix structures for further signal enhancement. Multiply-labeled hairpin reporter probes may be employed according to the invention. Also, a kit for carrying out the invention includes one or more capture probes immobilized on a surface, reporter probes each having a signal region and a sequence for binding analyte homopolymer regions, and, optionally, reagents for addition of homopolymer regions to nucleic acid analytes and for carrying out the hybridization reaction.

Claims

1. A method comprising steps of

adding a homopolymeric region to a target nucleic acid analyte, which target nucleic acid analyte has a target sequence;

contacting the analyte under hybridizing conditions with a multiplicity of reporter probes, said probes comprising a signal region and an oligonucleotide sequence, said oligonucleotide sequence being complementary to said analyte homopolymeric region, so as to form a stable hybrid of multiple reporter probes with said analyte homopolymeric region; and

forming an analyte;

capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions.

2. The method of claim 1 wherein the step of contacting the analyte with the reporter probes is performed in solution and the step of forming the analyte:

capture probe hybrid is carried out with an immobilized capture probe.

3. The method of claim 1 wherein said reporter probes hybridize with said analyte homopolymeric region to form a double helix.

4. The method of claim 1 wherein said reporter probes hybridize with said analyte homopolymeric region to form a triple helix.

5. The method of claim 1 wherein said reporter probe comprises a fluorescent polymer.

6. The method of claim 5 wherein said fluorescent polymer is selected from the group consisting of protein, oligonucleotide and polysaccharide.

7. The method of claim 6 wherein said fluorescent polymer comprises a fluorophore, an oligonucleotide and a linker for joining said oligonucleotide and said fluorophore.

8. The method of claim 7 wherein:

said fluorescent oligonucleotide comprises a linear polymer comprising (dT)_n, (U)_n or (U'"'"')_n, where n is an integer between 8 and 30; and

the 3'"'"' terminus of the target nucleic acid analyte has a homopolymeric region comprising poly A.

9. The method of claim 7 wherein said fluorophore is linked to said polymer by a phosphoramidite linker.

10. The method of claim 7 wherein said linker has length and rigidity characteristics sufficient to maintain fluorophores apart by 30 Angstroms or more when said reporter probes are hybridized to said analyte homopolymeric region.

11. The method of claim 10 wherein said linker is selected from the group consisting of alkyl, alkene, alkyne, aryl and polyethylene glycol chains.

12. The method of claim 11 wherein said linker is an alkene or alkyne group from 2 to about 200 carbons in length.

13. The method of claim 11 wherein said linker is a polyethylene glycol of the formula H(OCH₂ CH₂)_n OH, where n is an integer from 4 to about 200.

14. The method of claim 7 wherein:

said oligonucleotide comprises a stable hairpin structure linked to multiple fluorophores;

said hairpin structure is covalently linked at its 5'"'"' or 3'"'"' strand terminus to (dT)_n, (U)_n, or (U'"'"')_n, where n is an integer from about 8 to about 30; and

said homopolymeric region of said target nucleic acid analyte comprises poly A.

15. The method of claim 7 wherein:

said oligonucleotide comprises a circular molecule having self-complementary homopolymeric domains, and said circular molecule is linked to multiple fluorophores;

wherein;

under hybridizing conditions said domains interact with one another to form a stable hybrid thereby to convert said circular molecule into a loop-stem-loop structure and said homopolymeric region of said target nucleic acid analyte binds to said oligonucleotide stem region to form a triple helix.

16. The method of claim 6 wherein said polymer is a fluorescent protein.

17. The method of claim 16 wherein said fluorescent protein is a phycoerythrin.

19. A kit for carrying out a signal amplification method according to claim 1 comprising a capture probe immobilized on a surface, a reporter probe and a reagent for the addition of a homopolymeric region to the analyte wherein said reagent comprises poly(A) polymerase.

20. A kit according to claim 19 wherein the poly(A) polymerase comprises bacterial poly(A) polymerase.

21. A kit according to claim 20 wherein the poly(A) polymerase comprises yeast poly(A) polymerase.

22. A kit according to claim 21 wherein the reagent adds a poly(U) region to the analyte.

23. A kit according to claim 20 wherein the reagent adds a poly(iso-G) region to the analyte.

24. A method according to claim 1 wherein the analyte:

capture probe hybrid is formed prior to forming the hybrid of multiple reporter probes with said analyte homopolymeric region.

18. A method comprising steps of

adding a 3'"'"'-poly A region to a target mRNA analyte in a biological sample;

contacting the sample under hybridization conditions with a capture probe immobilized on a solid surface and a multiplicity of reporter probes, said probes comprising a signal region linked to a homopolymeric sequence comprising (dT)_n (U)_n or (U'"'"')_n, where n is an integer between 8 and 30, wherein said signal region is comprised of fluorescently-labeled linear or hairpin oligonucleotides, so as to form a stable hybrid of multiple reporter probes with said analyte poly A region; and

detecting a signal from said signal region.