Process for the detection of nucleic acids

• US 5,344,757 A
• Filed: 01/09/1990
• Issued: 09/06/1994
• Est. Priority Date: 01/12/1988
• Status: Expired due to Term

• Current Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Original Assignee: Boehringer Mannheim GmbH (Roche Holding AG)
• Inventors: Scholer, Hans R., Kessler, Christoph, Seibl, Rudolf, Mattes, Ralf, Holtke, Hans-Joachim, Schmitz, Gudrun
• Primary Examiner(s): Schwartz, Richard A.
• Assistant Examiner(s): Carter, Philip
• Application Number:
  US07/415,307
• Time in Patent Office: 1,701 Days
• Field of Search: 435/6, 435/7.1, 435/7.92, 536/24.3, 536/24.31, 536/24.32, 536/25.32
• US Class Current: 435/6.1
• 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 1/6804 Nucleic acid analysis using immunogens immunoassay G01N33/53
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin

Abstract

For the detection of nucleic acids of definite sequence by hybridisation with a complementary nucleic acid probe which contains bound via a chemical bonding at least one hapten as labelling one uses, as hapten, a steroid which is bound via a bridge of at least 4 atoms length to at least one position of the nucleic acid probe which does not participate in hydrogen bridge formation and detects the hybridised probe via an in turn labelled anti-hapten antibody.

Claims

1. Method for detection of a first nucleic acid sequence comprising contacting said sequence with a complementary nucleic acid probe under conditions favoring hybridization between said sequence and said probe, wherein said probe is a second nucleic acid sequence having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said second nucleic acid sequence via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with said first sequence, contacting hybridized probe with a labelled antibody which specifically binds to said steroid hapten, and detecting said labelled antibody to detect said first nucleic acid sequence.

2. Method of claim 1 wherein said bridge is from 4 to 32 atoms in length.

3. Method of claim 1 wherein said bridge is from 11 to 16 atoms in length.

4. Method of claim 1 wherein said bridge contains hydrophilic groups.

5. Method of claim 1 wherein said bridge is linear.

6. Method of claim 1 wherein said bridge is branched and contains a hapten molecule at at least one chain end.

7. Method of claim 1 wherein said steroid hapten is bound to a nucleotide base via said bridge.

8. Method of claim 7 wherein said base is uracil or cytosine and said steroid hapten is bound to C5 via said bridge.

9. Method of claim 7 wherein said base is adenine or quanine and said steroid hapten is bound to C8 via said bridge.

10. Method of claim 1 wherein said steroid hapten is bound to a ribose via said bridge.

11. Method of claim 10 wherein said steroid hapten is bound to the 2'"'"' position of ribose via said bridge.

12. Method of claim 1 wherein said steroid hapten is bound to said bridge via an ester, amide, or ether bond.

13. Method of claim 1 wherein said antibody is labelled with an enzyme label, a radioactive label, a fluorescence label or a bioluminiscent label.

14. Method of claim 13 wherein said antibody is labelled with an enzyme.

15. Method of claim 14 wherein said enzyme is alkaline phosphatase, peroxidase or beta galactosidase.

16. Method of claim 13 comprising detecting said labelled antibody via a redox reaction system.

17. Method of claim 16 wherein said enzyme is alkaline phosphatase and said redox reaction system comprises X phosphate and nitroblue tetrazolium.

18. Method of claim 13 comprising detecting said labelled antibody via a system using a leuko indicator.

19. Method of claim 13 comprising detecting said label with an indigoid compound and a tetrazolium salt.

20. Method of claim 1 wherein said first nucleic acid sequence is in a cell sample fixed to a solid support.

21. Method of claim 1 wherein said first nucleic acid sequence is in a smear of tissue sample.

22. Method of claim 1, wherein said hapten has been incorporated into said probe enzymatically.

23. Method of claim 22, wherein said hapten is bound to a deoxy or ribonucleoside triphosphate moiety and said moiety has been incorporated into said probe enzymatically via an enzyme selected from the group consisting of a DNA polymerase, an RNA polymerase, a reverse transcriptase and a terminal transferase.

24. Method of claim 1, wherein said hapten has been incorporated into said probe photochemically.

25. Method of claim 24, wherein said hapten is incorporated in the form of a photo-hapten.

26. Method of claim 1, wherein said hapten has been incorporated into said probe chemically.

27. Method of claim 26, wherein said hapten has been incorporated into said probe by incorporating a modified nucleoside phosphoamidite having a substitutable amino functional group into said probe as a protective group, removing said protective group, activating an ester, amide, or ether group of said hapten and reacting the activated hapten with said nucleoside phosphoamidite.

28. Method for detection of a first nucleic acid sequence comprising:

(a) contacting said first nucleic acid with at least two oligonucleotide primers under conditions favoring hybridization between said first nucleic acid sequence and said primers, wherein the first primer comprises a first nucleotide sequence which is complementary to a part of a nucleic acid sequence of a DNA strand to be detected and wherein the second primer comprises a different second nucleotide sequence which is identical to a part of a nucleotide sequence of said DNA strand to be detected,

(b) treating said mixture from (a) with a polymerase and deoxyribonucleotides under conditions favoring nucleic acid polymerization and in the presence of at least one deoxyribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with any other nucleotide,

(c) subjecting said mixture of (b) at least once to a cycle of denaturing, hybridizing and polymerizing according to steps (a) and (b),

(d) contacting said mixture from (c) with a labelled antibody which specifically binds to said steroid hapten, and

(e) detecting said labelled antibody as a detection of said first nucleic acid sequence.

29. Method of claim 28 wherein said polymerase is a Thermus aquaticus DNA polymerase.

30. Method of claim 28, wherein at least one of said primers is immobilized on a solid phase.

31. Method for detection of a first nucleic acid sequence comprising:

(a) contacting said first nucleic acid with at least two oligonucleotide primers under conditions favoring hybridization between said first nucleic acid sequence and said primers, wherein the first primer comprises a first nucleotide sequence which is complementary to a part of a nucleic acid sequence to be detected and wherein the second primer comprises a different second nucleotide sequence which is identical to a part of said nucleotide sequence to be detected,

(b) treating said mixture from (a) with a polymerase and deoxyribonucleotides under conditions favoring nucleic acid polymerization,

(c) subjecting said mixture of (b) at least once to a cycle of denaturing, hybridizing and polymerizing according to steps (a) and (b),

(d) repeating the cycle according to step (c) in the presence of at least one deoxyribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide via a bridge of at least 4 atoms length at a position thereon which does not participate in formation of hydrogen bonds with any other nucleotide,

(e) contacting said mixture from (c) with a labelled antibody which specifically binds to said steroid hapten, and

(f) detecting said labelled antibody as a detection of said first nucleic acid sequence.

32. Method of claim 31 wherein said polymerase is a Thermus aquaticus DNA polymerase.

33. Method of claim 31, wherein at least one of said primers is immobilized on a solid phase.

34. A deoxyribonucleotide or ribonucleotide having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length at a position which does not participate in formation of hydrogen bonds with any other nucleotide.

35. A deoxyribonucleotide or ribonucleotide triphosphate having bound thereto at least one steroid hapten selected from the group consisting of digoxin and digoxigenin, said steroid hapten being bound to said deoxyribonucleotide or ribonucleotide via a bridge of at least 4 atoms length at a position which does not participate in formation of hydrogen bonds with any other nucleotide.