Method of preparing nucleotide probes using a bridging complement

• US 5,538,872 A
• Filed: 09/08/1994
• Issued: 07/23/1996
• Est. Priority Date: 12/21/1988
• Status: Expired due to Term

• Current Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Original Assignee: Ortho Diagnostic Systems, Inc. (The Carlyle Group LP (Corporate Private Equity))
• Inventors: Mendoza, Leopoldo, Bahl, Chander, McMahon, Frank J.
• Primary Examiner(s): Fleisher, Mindy
• Assistant Examiner(s): KETTER, JAMES S
• Application Number:
  US08/302,332
• Time in Patent Office: 684 Days
• Field of Search: 435/5, 435/6, 435/91.52, 536/24.3, 536/25.3, 536/23.7, 536/23.72, 536/24.32
• US Class Current: 435/91.52
• 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 1/6813 Hybridisation assays
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2561/125 Ligase Detection Reaction [LDR]

Abstract

The present invention provides labeled nucleotide probes and a procedure for synthesis of the labeled nucleotide probe by a coupling or joinder of two components of the probe, namely a target recognition sequence moiety and a signal generating moiety, through the use of a bridging complement that holds the two moieties in such a position as to allow formation of a sugar-phosphate chemical linkage between the 3'"'"' end of one moiety and the 5'"'"' end of the other moiety.

Claims

1. A method for the synthesis of a labeled nucleotide probe for detecting a target analyte sequence having a target recognition moiety and a signal generating moiety, each moiety having a 3'"'"' terminus and a 5'"'"' terminus, said method comprising the steps of:

a) providing a target recognition moiety having a specific end sequence of at least about 4 nucleotide bases;

b) providing a signal generating moiety that does not hybridize to the molecule which comprises said target analyte sequence, said signal generating moiety having multiple labels attached thereto and having a specific end sequence of at least about 4 nucleotide bases;

c) providing a bridging complement comprising a nucleotide sequence of about 8-25 nucleotides, at least about 4 of said nucleotides in said bridging complement being capable of hybridizing to said specific end sequence of said target recognition moiety and at least about 4 other nucleotides in said bridging complement capable of hybridizing to said specific end sequence of said signal generating moiety;

d) allowing, under appropriate hybridization conditions, hybridization of said bridging complement to at least part of said specific end sequence of said target recognition moiety and at least part of said specific end sequence of said signal generating moiety to form a hybridized complex of all three;

wherein the 3'"'"' terminus of one of said moieties is aligned with the 5'"'"' terminus of the other, said 3'"'"' terminus and said 5'"'"' terminus being positioned relative to one another in such a manner as to allow formation of a 3'"'"'-5'"'"' sugar-phosphate link between the target recognition and signal generating moieties; and

e) forming said 3'"'"'-5'"'"' sugar-phosphate link between said 3'"'"' terminus and said 5'"'"' terminus of said target recognition and signal generating moieties, to produce a nucleotide probe comprising said target recognition moiety and said signal generating moiety covalently attached to one another.

2. The method of claim 1 further comprising step (f) contacting said complex with an enzyme capable of removing said bridging complement from said complex.

3. The method of claim 1 wherein said nucleotide probe is labeled with a hapten.

4. The method of claim 3 wherein said hapten is biotin.

5. The method of claim 3 wherein said hapten is fluorescein.

6. The method of claim 1 wherein said bridging complement comprises about 14 nucleotide bases.

7. The method of claim 1 wherein the target recognition moiety is a DNA sequence.

8. The method of claim 1 wherein the target recognition moiety is a RNA sequence.

9. The method of claim 1 wherein the target recognition moiety is an oligonucleotide.

10. The method of claim 1 wherein the target recognition moiety is the sequence from a microorganism.

11. The method of claim 10 wherein the microorganism is a virus.

12. The method of claim 10 wherein the microorganism is a bacterium.

13. The method of claim 12 wherein the bacterium is a Neisseria.

14. The method of claim 12 wherein the bacterium is a Salmonella.

15. The method of claim 12 wherein the bacterium is a Shigella.

16. The method of claim 10 wherein the microorganism is Mycoplasma.

17. The method of claim 1 wherein the signal generating moiety is a polynucleotide.

18. The method of claim 1 wherein the signal generating moiety is nucleic acid.

19. The method of claim 18 wherein the nucleic acid is DNA.

20. The method of claim 18 wherein the nucleic acid is RNA.

21. The method of claim 1 wherein the signal generating moiety is an oligonucleotide.

22. The method of claim 1 wherein the signal generating moiety is a homopolynucleotide.

23. The method of claim 4 wherein the biotin is capable of binding to a conjugate containing an enzyme.

24. The method of claim 23 wherein the enzyme is a peroxidase.

25. The method of claim 23 wherein the enzyme is alkaline phosphatase.

26. The method of claim 1 wherein the labels on the signal generating moiety are fluorophores.

27. The method of claim 1 wherein the labels on the signal generating moiety are chemiluminescent.

28. The method of claim 1 wherein the labels on the signal generating moiety are catalysts.

29. The method of claim 1 wherein the labels on the signal generating moiety are dyes.

30. The method of claim 1 wherein the labels on the signal generating moiety are metal.

31. The method of claim 30 wherein the labels are colloidal gold.

32. A labeled nucleotide probe comprising:

a) a first target recognition moiety having a first specific end sequence of at least about 4 nucleotide bases;

b) a second signal generating moiety having multiple labels attached thereto and having a second specific end sequence of at least about 4 nucleotide bases; and

c) a bridging complement comprising a nucleotide sequence of about 8-25 nucleotides;

said specific end sequences of said moieties having termini aligned and chemically attached so that a 3'"'"' terminus of one of said moieties is aligned with a 5'"'"' terminus of the other, forming a 3'"'"'-5'"'"' sugar-phosphate chemical bond; and

said bridging complement hybridized at least in part to said specific end sequence only of each of said first moiety and said second moiety.