End labeled nucleotide probe

• US 5,082,830 A
• Filed: 02/26/1988
• Issued: 01/21/1992
• Est. Priority Date: 02/26/1988
• Status: Expired due to Term

• Current Assignee: Enzo Life Sciences Incorporated (Enzo Biochem Incorporated)
• Original Assignee: Enzo Biochem Incorporated
• Inventors: Brakel, Christine L., Cook, Alan F., Vuocolo, Edmund
• Primary Examiner(s): Rollins, John W.
• Application Number:
  US07/160,607
• Time in Patent Office: 1,425 Days
• Field of Search: 536/27, 514/44
• US Class Current: 435/6.16
• 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 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety

Abstract

An oligo- or polynucleotide having at least one biotin directly or indirectly attached to each of the 5'"'"' and 3'"'"' end nucleotides thereof and a nucleic acid hybridization assay system for use therewith. The biotins are optionally attached through linkage groups that do not interfere with hybridization. The biotins can further serve to attach a biotinylated polymer.

Claims

1. An oligo- or polynucleotide having at least one non-radioactive moiety directly or indirectly attached to each of the 5'"'"' and 3'"'"' end nucleotides thereof.

2. The oligo- or polynucleotide of claim 1 wherein said non-radioactive moiety comprises biotin or a biotin analogue.

3. The oligo- or polynucleotide of claim 2 comprising at least two molecules of said biotin or biotin analogue attached to at least one end thereof.

4. The oligo- or polynucleotide of claim 3 comprising from about two to about eighteen molecules of said biotin or biotin analogue attached to at least one end thereof.

5. The oligo- or polynucleotide of claim 2 comprising at least two molecules of said biotin or biotin analogue attached at each end thereof.

6. The oligo- or polynucleotide of claim 5 comprising from about two to about eighteen molecules of said biotin or biotin analogue at each end thereof.

7. The oligo- or polynucleotide of claim 2 wherein at least one molecule of said biotin or biotin analogue is attached to the terminal nucleotide through a non-interfering linkage group.

8. The oligo- or polynucleotide of claim 7 wherein said biotin attachment comprises biotin-11-dUMP.

9. The oligo- or polynucleotide of claim 7 wherein said biotin attachment comprises biotin-11-allylamine-dUMP.

10. The oligo- or polynucleotide of claim 2 wherein at least one of said end nucleotides is attached to a biotinylated polymer.

11. The oligo- or polynucleotide of claim 10 wherein said biotinylated polymer comprises poly biotinylated dextran.

14. A nucleic acid hybridization assay composition comprising an oligo- or polynucleotide of claims 1 or 12, and a preformed detectable molecular complex.

15. A nucleic acid hybridization assay composition comprising an oligo- or polynucleotide of claims 2, 3, 4, 5, 6, 7, 8, 9, 11 or 13, and a preformed avidin or streptavidin detectable molecular complex.

16. The composition of claim 15 wherein said detectable molecular complex is selected from a fluor, chromogen, enzyme, phosphor, electron dense reagent, or a combination of any of the foregoing.

17. The composition of claim 16 wherein said enzyme is selected from peroxidase, alkaline phosphatase, acid phosphatase, beta-galactosidase, and glucose oxidase, or a combination of any of the foregoing.

12. An oligo- or polynucleotide having at least one non-radioactive moiety directly or indirectly attached to each of the 5'"'"' and 3'"'"' terminal nucleotides external to a target hybridization region of said oligo- or polynucleotide.

13. The oligo- or polynucleotide of claim 12 wherein said non-radioactive moiety comprises biotin or a biotin analogue.

18. A method for detecting a target nucleic acid sequence in a sample comprising:

rendering the nucleic acid in said sample in single-stranded form;

contacting said single-stranded nucleic acid under hybridizing conditions with (i) an oligo- or polynucleotide probe having at least one non-radioactive moiety directly or indirectly attached to each of the 5'"'"' and 3'"'"' end nucleotides thereof, said probe being capable of hybridizing to said target nucleic acid sequence, and (ii) a preformed detectable molecular complex; and

detecting any hybridized complexes, thereby detecting the target nucleic acid sequence.

19. The method of claim 18 wherein the attachment of said non-radioactive moiety to the oligo- or polynucleotide probe is external to a target hybridization region of said probe.

20. The method of claim 18 wherein said non-radioactive moiety of the oligo- or polynucleotide probe comprises biotin or a biotin analogue.

21. The method of claim 20 wherein said oligo- or polynucleotide probe comprises at least two molecules of said biotin or biotin analogue attached to at least one end thereof.

22. The method of claim 21 wherein said probe comprises from about two to about eighteen molecules of said biotin or biotin analogue attached to at least one end thereof.

23. The method of claim 20 wherein said oligo- or polynucleotide probe comprises at least two molecules of said biotin or biotin analogue at each end thereof.

24. The method of claim 23 wherein said probe comprises from about two to about eighteen molecules of said biotin or biotin analogue at each end thereof.

25. The method of claim 20 wherein at least one of said biotin or biotin analogue molecules in said probe is attached to the terminal nucleotide through a non-interfering linkage group.

26. The method of claim 25 wherein said biotin attachment comprises biotin-11-dUMP.

27. The method of claim 25 wherein said biotin attachment comprises biotin-11-allylamine-dUMP.

28. The method of claim 18 wherein at least one of said end nucleotides in the probe is attached to a biotinylated polymer.

29. The method of claim 28 wherein said biotinylated polymer comprises poly biotinylated dextran.

30. The method of claim 20 wherein said detectable molecular complex comprises avidin or streptavidin.

31. The method of claims 18 or 30 wherein said detectable molecular complex is selected from a fluor, chromogen, enzyme, phosphor, electron dense reagent, or a combination of any of the foregoing.

32. The method of claim 31 wherein said enzyme is selected from peroxidase, alkaline phosphatase, acid phosphatase, beta-galactosidase, and glucose oxidase, or a combination of any of the foregoing.