Process for detecting a nucleic acid of interest using an oligo- or polynucleotide probe and intercalators

• US 6,566,068 B2
• Filed: 03/23/2001
• Issued: 05/20/2003
• Est. Priority Date: 02/24/1989
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Inventors: Hurley, Ian, Rabbani, Elazar
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US09/815,649
• Publication Number:
  US 20010026921A1
• Time in Patent Office: 788 Days
• Field of Search: 435/6, 435/7.1, 436/94, 436/501
• US Class Current: 435/6.12
• 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: C07H 23/00 Compounds containing boron, silicon, or a metal, e.g. chelates, vitamin B12 esters with inorganic acids C07H11/00; metal salts, see parent compounds
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 2563/137 Metal/ion, e.g. metal label
• CPC Class Codes: C12Q 2563/173 staining/intercalating agent, e.g. ethidium bromide

Abstract

Disclosed is a nucleic acid hybridization assay composition for detecting the presence or absence of a target oligo- or polynucleotide in a sample. The composition comprises: a solid matrix having at least one surface which is substituted with a first intercalator capable of binding dsDNA, dsRNA, or DNA-RNA hybrids; a second intercalator, which may or may not comprise at least one fluorophore, said intercalator or said fluorophore, each acting as either an energy donor or an energy acceptor: and an oligo- or polynucleotide probe which is specifically hybridizable with the target oligo- or polynucleotide and has directly or indirectly bound thereto, at least one lanthanide metal chelate or at least one fluorophore, each acting as either an energy donor or an energy acceptor. Also disclosed are a method and kit for its use.

Claims

1. A process for detecting the presence of a nucleic acid sequence of interest comprising:

(a) providing;

(i) an oligo- or polynucleotide which is specifically hybridizable with said nucleic acid sequence of interest, said oligo or polynucleotide comprising at least one lanthanide metal or at least one fluorophore, each acting as either an energy donor or an energy acceptor;

(ii) a solid matrix having at least one surface to which is fixed or immobilized a first intercalator capable of capturing double-stranded nucleic acid, and

(iii) a second intercalator, which may or may not comprise at least one fluorophore, said second intercalator or said fluorophore, each acting as either an energy donor or an energy acceptor;

(b) forming a complex comprising;

said oligo- or polynucleotide (i),

said solid matrix (ii),

said second intercalator (iii), and

said nucleic acid sequence of interest if contained in said sample; and

(c) detecting the presence of said nucleic acid sequence of interest by detecting any energy emitted from said energy acceptor.

2. The process according to claim 1, wherein in said providing step (a), the nucleic acid sequence of interest is present in single-stranded form or has been rendered at least partially single-stranded.

3. The process according to claim 1, wherein in said providing step (a), the double-stranded nucleic acid capturable by said first intercalator fixed or immobilized to the solid matrix (ii) is selected from the group consisting of double-stranded DNA, double-stranded RNA and DNA-RNA hybrids.

4. The process according to claim 1 wherein said forming step further comprises the step of separating any unhybridized oligo- or polynucleotide from said mixture.

5. The process according to claim 1, wherein in said providing step (a), the lanthanide metal (i) is selected from the group consisting of europium and terbium.

6. The process according to claim 5 wherein said lanthanide metal is chelated.

7. The process of claim 6 wherein said lanthanide chelate is bound to said oligo- or polynucleotide through a linkage group.

8. The process according to claim 1, wherein in said providing step (a), the first intercalator fixed or immobilized to the solid matrix (ii) is selected from the group consisting of phenanthridines and psoralenenamines.

9. A process for detecting the presence of a nucleic acid sequence of interest in a sample comprising:

(a) providing a solid matrix having at least one surface to which is attached a first intercalator capable of capturing double-stranded nucleic acid;

(b) contacting under hybridizable conditions said solid matrix with the sample suspected of containing said nucleic acid sequence of interest and an oligo- or polynucleotide which is specifically hybridizable therewith, said oligo or polynucleotide comprising at least one lanthanide metal or at least one fluorophore, each acting as either an energy donor or an energy acceptor, to form a complex which is captured by said solid matrix;

(c) contacting said captured complex with a second intercalator, which may or may not comprise at least one fluorophore, said second intercalator or said fluorophore, each acting as either an energy donor or an energy acceptor, and

(d) detecting the presence of said nucleic acid sequence of interest by detecting any energy emitted from said energy acceptor.

10. The process according to claim 9 wherein said nucleic acid sequence of interest is present in single-stranded form or has been rendered at least partially single-stranded.

11. The process according to claim 9 wherein the double-stranded nucleic acid capturable by said first intercalator is selected from the group consisting of double-stranded DNA, double-stranded RNA and DNA-RNA hybrids.

12. The process according to claim 9 further comprising the step of separating any unhybridized oligo- or polynucleotide from said complex.

13. The process according to claim 9 wherein said lanthanide metal is selected from the group consisting of europium and terbium.

14. The process according to claim 13 wherein said lanthanide metal is chelated.

15. The process of claim 14 wherein said lanthanide chelate is bound to said oligo- or polynucleotide through a linkage group.

16. The process of claim 9 wherein said first intercalator is selected from the group consisting of phenanthridines and psoralenamines.

17. A process for detecting the presence of a nucleic acid sequence of interest comprising:

(a) contacting a sample suspected of containing said nucleic acid sequence of interest with an oligo- or polynucleotide hybridizable therewith to form a hybrid, said oligo- or polynucleotide having directly or indirectly bound thereto a lanthanide metal or a fluorophore, each independently acting as either an energy donor or an energy acceptor;

(b) contacting said hybrid with a solid matrix having at least one surface to which is attached a first intercalator capable of capturing double-stranded nucleic acid;

(c) permitting capture of said hybrid to form a complex;

(d) contacting said complex with a second intercalator, which may or may not comprise at least one fluorophore, said second intercalator or said fluorophore, each acting as either an energy donor or an energy acceptor; and

(e) detecting the presence of said nucleic acid sequence of interest by detecting any energy emitted from said energy acceptor.

18. The process according to claim 17, wherein said nucleic acid sequence of interest is present in single-stranded form or has been rendered at least partially single-stranded.

19. The process according to claim 17, wherein the double-stranded nucleic acid capturable by the first intercalator in said contacting step (b) is selected from the group consisting of double-stranded DNA, double-stranded RNA and DNA-RNA hybrids.

20. The process according to claim 17, further comprising the step of separating prior to said detecting step (e) any unhybridized oligo- or polynucleotide from any complex that is formed.

21. The process according to claim 17, wherein said lanthanide metal in step (a) is selected from the group consisting of europium and terbium.

22. The process according to claim 21, wherein said lanthanide metal in step (a) is chelated.

23. The process of claim 22, wherein said lanthanide chelate is bound to said oligo- or polynucleotide through a linkage group.

24. The process of claim 17, wherein said first intercalator attached to the solid matrix in said contacting step (b) is selected from the group consisting of phenanthridines and psoralenamines.