Signal enhancement method and kit

• US 5,853,993 A
• Filed: 10/21/1996
• Issued: 12/29/1998
• Est. Priority Date: 10/21/1996
• Status: Expired due to Fees

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Hewlett-Packard Company (HP Inc.)
• Inventors: Dellinger, Douglas J., Dahm, SueAnn C., Troll, Mark A.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/735,381
• Time in Patent Office: 799 Days
• Field of Search: 435/6, 435/810, 935/76, 935/77, 935/78, 536/23.1, 536/24.3
• 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

The invention discloses and claims a signal amplification method for detecting a target nucleic acid analyte having a homopolymeric region and a target sequence. The method comprises (a) contacting an analyte under hybridizing conditions with a multiplicity of reporter probes, each 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, whereby the hybridization of multiple reporter probes to the homopolymeric region provides for signal amplification; and (b) forming an analyte:capture probe hybrid by contacting the analyte target sequence with a capture probe under hybridizing conditions.

Claims

1. A signal amplification method for detecting a target nucleic acid analyte having a homopolymeric region and a target sequence, wherein said homopolymeric region is poly A at the 3'"'"' terminus of said target nucleic acid analyte comprising the steps of:

(a) contacting said analyte under hybridizing conditions with a multiplicity of reporter probes to form a triple helix, said probes comprising;

(i) a signal region; and

(ii) an oligonucleotide sequence, wherein said oligonucleotide sequence is complementary to said analyte homopolymeric region for forming a stable hybrid therewith, and

wherein said reporter probes hybridize with said analyte homopolymeric region to form a triple helix and wherein the hybridization of multiple reporter probes to said analyte homopolymeric region provides for amplification of the detection signal and;

(b) forming an analyte;

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

2. The method of claim 1, wherein step (a) is performed in solution and step (b) is carried out with an immobilized capture probe.

3. The method of claim 1, wherein steps (a) and (b) are performed in solution, and said analyte:

capture probe hybrid formed in step (b) is subsequently bound to a surface for detection thereupon.

4. A signal amplification method for detecting a target nucleic acid analyte having a 3'"'"' terminus having a polyA region and a target sequence, comprising the steps of:

(a) contacting said analyte under hybridizing conditions with a multiplicity of reporter probes, said probes comprising a fluorescent polymer comprising an oligonucleotide linked to a fluorophore, wherein said oligonucleotide is a linear polymer comprising (dT)n, or (U)n, where n is an integer between 8 and 30; and

wherein the hybridization of multiple reporter probes to said analyte polyA region provides for amplification of the detection signal; and

(b) forming an analyte;

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

5. The method of claim 4, wherein said fluorophore is linked to said polymer by a phosphoramidite linker.

6. The method of claim 4, wherein said linker has length and rigidity characteristics sufficient to minimize the occurrence of fluorescence quenching of fluorophores of said fluorescent polymer when said reporter probes are hybridized to said analyte poly A region.

7. The method of claim 6, wherein said linker is selected from the group consisting of alkyl, alkene, alkyne, aryl and polyethylene glycol chains.

8. The method of claim 7, wherein said linker is an alkene or alkyne group from about 2 to about 200 carbons in length.

9. The method of claim 7, 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.

10. The method of claim 4, wherein:

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

(b) said hairpin structure is covalently linked at its 5'"'"' or 3'"'"' terminus.

11. The method of claim 4, 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 poly A of said target nucleic acid analyte binds to said oligonucleotide stem region to form a triple helix.

13. The method of claim 4, wherein step (a) is performed in solution and step (b) is carried out with an immobilized capture probe.

14. The method of claim 4, wherein steps (a) and (b) are performed in solution, and said analyte:

capture probe hybrid formed in step (b) is subsequently bound to a surface for detection thereupon.

15. The method of claim 4, wherein said reporter probes hybridize with said analyte poly A region to form a double helix.

16. The method of claim 4, wherein said reporter probes hybridize with said analyte poly A region to form a triple helix.

12. A method for detecting a target mRNA analyte in a biological sample, said mRNA having a 3'"'"'-poly A region, comprising the steps of:

(a) contacting the sample under hybridization conditions with a capture probe immobilized on a solid surface thereby forming a mRNA;

capture probe hybrid, and with a multiplicity of reporter probes, said reporter 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; and

(b) detecting the fluorescence emitted by the reporter probes hybridized to the 3'"'"' poly A region of the mRNA;

capture probe hybrid.