Homogeneous assay system using the nuclease activity of a nucleic acid polymerase

• US 5,210,015 A
• Filed: 08/06/1990
• Issued: 05/11/1993
• Est. Priority Date: 08/06/1990
• Status: Expired due to Term

• Current Assignee: Roche Molecular Systems Inc (Roche Holding AG)
• Original Assignee: Hoffmann-La Roche Incorporated (Roche Holding AG)
• Inventors: Holland, Pamela M., Gelfand, David H., Saiki, Randall K., Watson, Robert M.
• Primary Examiner(s): Moskowitz, Margaret
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/563,758
• Time in Patent Office: 1,009 Days
• Field of Search: 435/6, 435/18, 435/91, 435/196, 435/805, 436/501, 436/63, 436/815, 536/27, 935/17, 935/77, 935/78, 935/88
• US Class Current: 435/6.11
• 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 1/6823 Release of bound markers
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/703 Viruses associated with AIDS
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2521/325 Single stranded exonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2535/131 Allele specific probes
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2561/101 Taqman
• CPC Class Codes: C12Q 2565/1015 labels being on the same oligonucleotide
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit
• CPC Class Codes: Y10S 436/815 Test for named compound or class of compounds

Abstract

The present invention is directed to a process of detecting a target nucleic acid using labeled oligonucleotides. This process uses the 5'"'"' to 3'"'"' nuclease activity of a nucleic acid polymerase to cleave annealed labeled oligonucleotide from hybridized duplexes and release labeled oligonucleotide fragments for detection. This process is easily incorporated into a PCR amplification assay.

Claims

1. A process for the detection of a target nucleic acid sequence in a sample, said process comprising:

(a) contacting a sample comprising single-stranded nucleic acids with an oligonucleotide containing a sequence complementary to a region of the target nucleic acid and a labeled oligonucleotide containing a sequence complementary to a second region of the same target nucleic acid sequence strand, but not including the nucleic acid sequence defined by the first oligonucleotide, to create a mixture of duplexes during hybridization conditions, wherein the duplexes comprise the target nucleic acid annealed to the first oligonucleotide and to the labeled oligonucleotide such that the 3'"'"' end of the first oligonucleotide is upstream of the 5'"'"' end of the labeled oligonucleotide;

(b) maintaining the mixture of step (a) with a template-dependent nucleic acid polymerase having a 5'"'"' to 3'"'"' nuclease activity under conditions sufficient to permit the 5'"'"' to 3'"'"' nuclease activity of the polymerase to cleave the annealed, labeled oligonucleotide and release labeled fragments; and

(c) detecting and/or measuring the release of labeled fragments.

2. The process of claim 1 wherein the 3'"'"' end of the first oligonucleotide in the annealed duplex of step (a) is within about 20 nucleotides of the 5'"'"' end of an annealed, labeled oligonucleotide, thereby having spacing effective to permit the release of labeled fragments in the absence of nucleic acid polymerization.

3. The process of claim 1 wherein the oligonucleotides comprise deoxyribonucleotides.

4. The process of claim 1 wherein the nucleic acid polymerase is a DNA polymerase having a 5'"'"' to 3'"'"' nuclease activity.

5. The process of claim 1 wherein a nucleotide within the label oligonucleotide is modified to control nuclease cleavage specificity.

6. The process of claim 1 wherein said labeled oligonucleotide comprises at least one label.

7. The process of claim 1 wherein the labeled oligonucleotide comprises first and second labels wherein the first label is separated from the second label by a nuclease susceptible cleavage site.

8. The process of claim 6 wherein the labeled oligonucleotide is labeled at the 5'"'"' terminus.

9. The process of claim 7 wherein the labeled oligonucleotide further comprises a tail of non-nucleic acids or a sequence of nucleotides which is non-complementary to the target nucleic acid sequence.

10. The process of claim 9 wherein the label is attached to a nucleotide in the tail or non-complementary sequence.

11. The process of claim 10 wherein the label is at the 5'"'"' terminus and is separated from the sequence complementary to the target nucleic acid sequence by the tail or non complementary sequence.

12. The process of claim 1 performed under conditions sufficient to promote nucleic acid polymerization, wherein the release of labeled fragments occurs during extension of the first oligonucleotide.

13. A polymerase chain reaction (PCR) amplification process for detecting a target nucleic acid sequence in a sample, said process comprising:

(a) providing to a PCR assay containing said sample, at least one labeled oligonucleotide containing a sequence complementary to a region of the target nucleic acid, wherein said labeled oligonucleotide anneals within the target nucleic acid sequence bounded by the oligonucleotide primers of step (b);

(b) providing a set of oligonucleotide primers, wherein a first primer contains a sequence complementary to a region in one strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand, and a second primer contains a sequence complementary to a region in a second strand of the target nucleic acid sequence and primes the synthesis of a complementary DNA strand; and

wherein each oligonucleotide primer is selected to anneal to its complementary template upstream of any labeled oligonucleotide annealed to the same nucleic acid strand;

(c) amplifying the target nucleic acid sequence employing a nucleic acid polymerase having 5'"'"' to 3'"'"' nuclease activity as a template-dependent polymerizing agent under conditions which are permissive for PCR cycling steps of (i) annealing of primers and labeled oligonucleotide to a template nucleic acid sequence contained within the target sequence, and (ii) extending the primer wherein said nucleic acid polymerase synthesizes a primer extension product while the 5'"'"' to 3'"'"' nuclease activity of the nucleic acid polymerase simultaneously releases labeled fragments from the annealed duplexes comprising labeled oligonucleotide and its complementary template nucleic acid sequences, thereby creating detectable labeled fragments; and

(d) detecting and/or measuring the release of labeled fragments to determine the presence or absence of the target sequence in the sample.

14. The PCR process of claim 13 wherein said nucleic acid polymerase is a thermostable enzyme.

15. The PCR process of claim 14 wherein said thermostable enzyme is the DNA polymerase from a Thermus species.

16. The PCR process of claim 13 wherein the 3'"'"' end of an annealed oligonucleotide primer is within about 20 nucleotides the 5'"'"' end of the labeled oligonucleotide annealed to the same nucleic acid strand.

17. The PCR process of claim 16 wherein said labeled oligonucleotide has a blocked 3'"'"' terminus to prevent extension by the nucleic acid polymerase.

18. The PCR process of claim 16 wherein the labeled oligonucleotide further comprises a sequence of one to about ten nucleotides which sequence is substantially non-complementary to the target nucleic acid sequence.

19. The PCR process of claim 13 wherein the labeled oligonucleotide comprises first and second labels wherein the first label is separated from the second label by a nuclease susceptible cleavage site.

20. The PCR process of claim 13 wherein a pair of labeled oligonucleotides probes are provided in step (a).

21. The PCR process of claim 20 wherein said pair of labeled probes anneal to different, non-overlapping regions of the same complementary nucleic acid strand, wherein the 5'"'"' end of the second labeled probe is adjacent the 3'"'"' end of the first labeled probe.

22. The PCR process of claim 18 wherein the label is attached to a nucleotide in the non complementary sequence.

23. The PCR process of claim 22 wherein the label is at the 5'"'"' terminus and is separated from the complementary probe sequence by the non-complementary sequence.

24. The PCR process of claim 13 wherein the oligonucleotide is labeled at the 5'"'"' terminus.

25. The PCR process of claim 17 wherein the oligonucleotide is labeled at the blocked 3'"'"' terminus.

26. The PCR process of claim 13 wherein the label is attached to an internal sequence of the oligonucleotide.

27. The PCR process of claim 13 wherein the label provides a signal proportional to the number of target nucleic acid sequences amplified.

28. The PCR process of claim 13 wherein the label is a deoxyribonucleoside analog having signal-generating properties.

29. The PCR process of claim 13 wherein the labeled oligonucleotide comprises a pair of interactive signal-generating labels effectively positioned on the oligonucleotide to quench the generation of detectable signal, said labels being separated by a site within the oligonucleotide susceptible to nuclease cleavage, thereby allowing, during primer extension, the 5'"'"' to 3'"'"' nuclease activity of the nucleic acid polymerase to separate the first interactive signal generating label from the second interactive signal generating label by cleaving at the susceptible site thereby yielding a detectable signal.

30. The PCR process of claim 29 wherein said first label is a chemiluminescent substrate and said second label is a fluorophore which interacts therewith.

31. The PCR process of claim 13 wherein the label of said oligonucleotide is attached through a spacer arm of sufficient length to permit the 5'"'"' to 3'"'"' nuclease activity of the nucleic acid polymerase to release labeled fragments.

32. The PCR process of claim 13 wherein the melting temperature (T_m) differential between the labeled oligonucleotide and its'"'"' associated upstream oligonucleotide primer is effective to provide preferential binding of the labeled oligonucleotide during the annealing step of PCR cycles.

33. The PCR process of claim 32 wherein the T_m of the labeled oligonucleotide is as great as 40°

C. higher than the T_m of the upstream oligonucleotide primer.

34. The PCR process of claim 13 wherein the labeled oligonucleotide fragments comprise a mixture of mono-, di and larger nucleotide fragments.

35. The PCR process of claim 13 which further comprises separating labeled oligonucleotide fragments from other components in the PCR mixture prior to detection of labeled fragments.

36. The PCR process of claim 35 wherein the separation step uses size exclusion chromatography.

37. The PCR process of claim 35 wherein the labeled fragments are separated from the PCR mixture by solid phase extraction.

38. The PCR process of 37 wherein avidin or streptavidin is attached to the solid phase and the labeled oligonucleotide further comprises a bound biotin molecule separated from the label by a nuclease susceptible cleavage site.