Competitive homogeneous assay

• US 5,928,862 A
• Filed: 07/30/1990
• Issued: 07/27/1999
• Est. Priority Date: 01/10/1986
• Status: Expired due to Term

• Current Assignee: Amoco Corporation (BP PLC)
• Original Assignee: Amoco Corporation (BP PLC)
• Inventors: Morrison, Larry E.
• Primary Examiner(s): Nucker, Christine M.
• Assistant Examiner(s): SCHEINER, LAURIE A
• Application Number:
  US07/559,315
• Time in Patent Office: 3,284 Days
• Field of Search: 435/6, 935/78, 436/537, 436/546, 436/800, 436/805
• US Class Current: 435/6.18
• 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: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 2537/161 A competitive reaction step
• CPC Class Codes: C12Q 2565/107 Alteration in the property of hybridised versus free label oligonucleotides
• CPC Class Codes: C12Q 2565/525 characterised by the capture oligonucleotide being double stranded
• CPC Class Codes: G01N 33/582 with fluorescent label

Abstract

Methods and compositions for performing assays for target polynucleotide strands include contacting a sample with a reagent which includes a first and a second polynucleotide probe. The first and second probes are capable of assuming a first position wherein the probes are bound to each other and a second position wherein the probes are bound to a target. The probes include label moieties capable of interacting to produce a signal indicative of the probes being in one of the two positions.

Claims

1. A method for assaying a sample for target polynucleotides comprising:

(a) contacting sample with reagent under binding conditions wherein said reagent includes a first polynucleotide probe and a second polynucleotide probe, said first and second probes capable of assuming a first position wherein said first and second probes are bound to each other and at least one of said probes capable of assuming a second position wherein said probe strand is bound to said target, said first probe and second probe including a first label moiety associated with one of said probes and including a second label moiety associated with said opposite probe, said first and second label moieties capable of interacting via energy transfer when said first and second probes are bound to each other to produce a signal capable of detection characteristic of the probe in one of said two positions, said first label moiety located at the 3'"'"'-terminus of one of said probes and said second label moiety located at the 5'"'"'-terminus of said other probe; and

(b) monitoring said sample for said signal, the presence of which is related to the presence of target in said sample.

2. The method of claim 1 wherein each probe has a plurality of label moieties.

3. The method of claim 2 wherein each such label moiety is located at the termini of said probes.

4. The method of claim 2 wherein said first label moiety is located at the 3'"'"'-terminus of one of said probes and said second label moiety is located at the 5'"'"'-terminus of said opposite probe.

5. The method of claim 4 wherein said label moiety at the 3'"'"' terminus is a fluorescent derivative of a nucleotide.

6. The method of claim 5 wherein the label moiety is 1-N⁶ -ethenoadenosine-5'"'"'-monophosphate.

7. The method of claim 1 wherein said first label moiety is associated to said probe by an aminoalkyl derivative of the terminal nucleotide.

8. The method of claim 7 wherein said derivative is an aminoalkyl derivative of adenine.

9. The method of claim 7 wherein said derivative is an aminohexyl derivative of adenine.

10. The method of claim 7 wherein said derivative is 8-(6-aminohexyl)-aminoadenosine-5'"'"'-monophosphate.

11. The method of claim 1 further comprising the additional steps of:

splicing polynucleotide segments having base sequences substantially identical to said target sequence into amplification means to form multiple copies of said polynucleotide segments, isolating said segments, and associating said first and second label moieties to the termini of said segments to form probes.

12. The method of claim 11 wherein amplication means include plasmids and phage particles.

13. The method of claim 11 wherein said segments, after isolation, are subjected to restriction digestion to form further subsegments, and said subsections are associated with said label moieties to form probes.

14. A kit for assaying a sample for target polynucleotides comprising reagent, wherein said reagent includes a first polynucleotide probe and a second polynucleotide probe, said first and second probes capable of assuming a first position wherein said first and second probe are bound to each other and at least one of said probes capable of assuming a second position wherein said probe strand is bound to said target, said first probe and second probe including a first label moiety associated with one of said probes and including a second label moiety associated with said opposite probe, said first and second label moieties capable of interacting via energy transfer when said first and second probes are bound to each other to produce a signal capable of detection which is characteristic of the probe in one of said two positions, said first label moiety located at the 3'"'"'-terminus of one of said probes and said second label moiety located at the 5'"'"'-terminus of said other probe.

15. The kit of claim 14 wherein each probe has a plurality of label moieties.

16. The kit of claim 15 wherein each such label moiety is located at the termini of said probes.

17. The kit of claim 15 wherein said first label moiety is located at the 3'"'"'-terminus and said second label moiety is located at the 5'"'"'-terminus.

18. A method for assaying a sample for target nucleic acid comprising;

(a) contacting sample with reagent under binding conditions wherein said reagent includes a first nucleic acid probe and a second nucleic acid probe, said first and second probes capable of assuming a first position wherein said first and second probe are bound to each other and at least one of said probes capable of assuming a second position wherein said probe strand is bound to said target, said first probe and second probe including a first fluorophore label moiety associated with one of said probes and including a second fluorophore label moiety associated with said opposite probe, said first and second label moieties capable of interacting via energy transfer when said first and second probes are bound to each other to produce a signal capable of detection characteristic of the probe in one of said two positions, said first label moiety located at the 3'"'"'-terminus of one of said probes and said second label moiety located at the 5'"'"'-terminus of said other probe; and

(b) monitoring said sample for said signal, the presence of which is related to the presence of target in said sample.