Homogeneous amplification and detection of nucleic acids

• US 5,914,230 A
• Filed: 12/20/1996
• Issued: 06/22/1999
• Est. Priority Date: 12/22/1995
• Status: Expired due to Fees

• Current Assignee: Siemens Healthcare Diagnostics Products GmbH (Siemens AG)
• Original Assignee: Dade Behring, Inc. (Siemens AG)
• Inventors: Patel, Rajesh D., Lin, Claire, Kurn, Nurith, Ullman, Edwin F., Rose, Samuel J., Liu, Yen Ping
• Primary Examiner(s): Degen, Nancy
• Assistant Examiner(s): Yucel, Irem
• Application Number:
  US08/771,624
• Time in Patent Office: 914 Days
• Field of Search: 536/24.3, 536/24.5, 435/6, 435/91.2
• US Class Current: 435/6.12
• 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 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2537/101 Homogeneous assay format, e.g. one pot reaction
• CPC Class Codes: C12Q 2563/103 luminescence
• CPC Class Codes: C12Q 2563/131 the label being a member of a cognate binding pair, i.e. extends to antibodies, haptens, avidin
• CPC Class Codes: C12Q 2563/149 Particles, e.g. beads
• CPC Class Codes: C12Q 2565/113 based on agglutination/precipitation

Abstract

The present invention relates to a method for detecting or amplifying and detecting a target polynucleotide sequence. The method comprises providing in combination (i) a medium suspected of containing the target polynucleotide sequence, (ii) all reagents required for conducting an amplification of the target polynucleotide sequence when amplification is desired, and (iii) two oligonucleotide probes capable of binding to a single strand of the product of the amplification. At least one of the probes has two sequences that either (i) are non-contiguous and bind to contiguous or non-contiguous sites on the single strand or (ii) can bind to non-contiguous sites on the single strand. Each probe may contain a label. The combination is subjected to conditions for amplifying the target polynucleotide sequence. Next, the combination is subjected to conditions under which both of the probes hybridize to one of the strands to form a termolecular complex, which is detected by means of the label.

Claims

1. A method for amplifying and detecting a target polynucleotide sequence, which comprises:

(a) providing in combination (i) a medium suspected of containing said target polynucleotide sequence, (ii) all reagents required for conducting an amplification of said target polynucleotide sequence, and (iii) two oligonucleotide probes that bind to a single strand of the product of said amplification wherein at least one of said probes has two sequences, which are each about 8 to 25 nucleotides in length and which either (i) are non-contiguous and can bind to contiguous or noncontiguous sites on said single strand or (ii) can bind to non-contiguous sites on said single strand,

(b) subjecting said combination to conditions for amplifying said target polynucleotide sequence,

(c) after step (b) subjecting said combination to conditions under which both of said probes hybridize to one of said strands to form a termolecular complex and

(d) detecting said complex.

2. The method of claim 1 wherein said reagents comprise two oligonucleotides that bind to said target polynucleotide sequence and an enzyme capable of modifying at least one of said oligonucleotides as a function of the presence of said target polynucleotide sequence.

3. The method of claim 1 wherein said reagents comprise an oligonucleotide primer that binds to and is extended along said target polynucleotide sequence, a nucleotide polymerase and nucleotide triphosphates.

4. The method of claim 1 wherein said reagents comprise (i) two oligonucleotide primers that each bind to and are extended along said target polynucleotide sequence and a sequence complementary to said target polynucleotide sequence, respectively, (ii) a nucleotide polymerase and (iii) nucleotide triphosphates.

5. The method of claim 1 wherein each probe contains a label and said combination further comprises particles that bind to said labels and said detecting comprises detecting agglutination of said particles.

6. A method for amplifying and detecting a target polynucleotide sequence of a polynucleotide analyte, which comprises:

(a) providing in combination a sample suspected of containing a polynucleotide analyte having said target polynucleotide sequence, reagents for amplifying said polynucleotide analyte to produce copies of said target polynucleotide sequence, a first oligonucleotide probe having nucleotide sequences S1 and S2 and a second oligonucleotide probe having sequences S3 and S4 wherein each of S1, S2, S3 and S4 is about 8 to 25 nucleotides in length and wherein the sequences comprising at least one of the probes are linked such that either (i) they are non-contiguous and can bind to contiguous or non-contiguous sites on one of the strands of said copies or (ii) the sites to which they hybridize on one of the strands of said copies are noncontiguous and wherein said probes (i) do not hybridize to said copies during said amplifying and (ii) subsequent to said amplifying, both of said probes can hybridize to one of the strands of said copies, and (iv) each of said probes is comprised of a label that facilitates detection of said probes hybridized to said strands,

(b) subjecting said combination to conditions for amplifying said polynucleotide analyte,

(c) after step (b) subjecting said combination to conditions under which both of said probes hybridize to one of said strands to form a termolecular complex, and

(d) detecting said complex.

7. The method of claim 6 wherein the 3'"'"'-terminus of said S1 is linked to the 5'"'"'-terminus of said S2 by a linking group comprising a chain of 1 to 200 atoms.

8. The method of claim 7 wherein said chain comprises from 1 to 40 nucleotides or nucleotide analogs.

9. The method of claim 7 wherein at least a portion of said inking group is an oligonucleotide label.

10. The method of claim 6 wherein the 3'"'"'-terminus of said S1 is linked to the 5'"'"'-terminus of said S2 by a linking group and the 3'"'"'-terminus of said S3 is linked to the 5'"'"'-terminus of said S4 by a linking group, each of said linking groups comprising from 0 to 40 nucleotides or nucleotide analogs.

11. The method of claim 6 wherein detection of said complex comprises association of said complex with particles.

12. The method of claim 11 wherein said detection comprises detecting agglutination of said particles.

13. The method of claim 6 wherein said label is an oligonucleotide label.

14. The method of claim 6 wherein said S1 hybridizes to a site on said copies that lies 5'"'"' of the hybridization site of said S2 on said copy wherein the 3'"'"'-end of said S1 is linked to the 5'"'"'-end of said S2.

15. The method of claim 6 wherein S1 and S2 are linked by a nucleotide linking group and S3 and S4 are linked by a nucleotide linking group.

16. The method of claim 15 wherein each of said linking groups comprises an oligonucleotide label.

17. The method of claim 16 wherein said combination comprises oligonucleotides N1 and N2 that are each respectivley complementary to one of said oligonucleotide labels wherein said N1 and N2 are each labeled with a reporter group.

18. The method of claim 17 wherein said N1 is associated with a particle having a photosensitizer associated therewith and said N2 is associated with a particle having a chemiluminescent compound associated therewith.

19. A method of detecting a target polynucleotide containing a target polynucleotide sequence wherein all reagents required for said method are first combined with said target polynucleotide, said method comprising:

(a) dissociating said target polynucleotide sequence into single strands when said target polynucleotide sequence is double stranded,

(b) hybridizing an oligonucleotide primer to the 3'"'"'-end of each of said single strands,

(c) extending said primer hybridized to each of said single strands along the single strand to produce a copy of said target polynucleotide sequence,

(d) dissociating said copy into single strands,

(e) hybridizing two oligonucleotide probes to one of said single strands wherein at least one of said probes is comprised of two sequences that hybridize with one of said single strands, wherein said sequences either (i) are non-contiguous and can bind to contiguous or non-contiguous sites on said strand or (ii) the sites on said strand to which said sequences hybridize are non-contiguous,

(f) detecting the binding of both of said probes to said single strand, the presence thereof being related to the presence of said target polynucleotide.

20. The method of claim 19 wherein each of said probes is comprised of a label.

21. The method of claim 19 wherein at least one of said probes is bound to or can become bound to a particle.

22. The method of claim 21 wherein said particle is comprised of a photosensitizer.

23. The method of claim 21 wherein said particle is comprised of a luminescent compound.

24. The method of claim 19 wherein each of said probes is comprised of two sequences that hybridize with said single strand, said sequences and/or said sites on said strand to which said sequences hybridize being non-contiguous.

25. The method of claim 19 wherein each of said probes is comprised of an oligonucleotide label.

26. The method of claim 25 wherein said detecting comprises binding of nucleotides N1 and N2, respectively, to said oligonucleotide label of each of said probes wherein said N1 is bound to a first label and said N2 is bound to a second label.

27. The method of claim 26 wherein said first label is comprised of a photosensitizer.

28. A method for detecting a target sequence of a target polynucleotide ("target sequence"), said method comprising:

(a) amplifying said target sequence by a method comprising;

(i) hybridizing to the 3'"'"'-end of said target sequence a first oligonucleotide primer ("first primer"),

(ii) extending, in the presence of a polymerase and nucleotide triphosphates, said first primer along at least said target sequence to produce an extended first primer, said first primer hybridizing to, and being extended along, (1) said extended first primer or (2) an extended second oligonucleotide primer ("second primer") wherein said extended second primer results from the extension of a second primer that hybridizes to and is extended along a polynucleotide that is complementary (complementary polynucleotide) to said target sequence,

(iii) dissociating said extended first primer from said target sequence,

(iv) hybridizing, to the 3'"'"'-end of said extended first primer, said first or said second primer,

(v) extending said first or said second primer along said extended first primer,

(vi) dissociating said extended first primer or said extended second primer from said extended first primer,

(vii) hybridizing, to the 3'"'"'-end of said extended first or said extended second primer, said first primer, and

(viii) repeating steps (v)-(vii), and

(b) detecting said extended first primer and/or said extended second primer by means of a first oligonucleotide probe having nucleotide sequences S1 and S2 and a second oligonucleotide probe having sequences S3 and S4, wherein S1, S2, S3 and S4 are each about 10 to 20 nucleotides in length wherein the sequences comprising at least one of the probes are linked such that its two sequences either (i) are non-contiguous and can bind to contiguous or non-contiguous sites on one of said extended primers or (ii) hybridize to sites on one of said extended primers that are noncontiguous and wherein said probes (A) are present during said amplifying, (B) do not hybridize to said extended first and/or second primers during said amplifying of step (a) and (C) do not interfere with said amplifying of step (a) and (D) subsequent to said amplifying of step (a), both of said probes can hybridize to one of said extended first and/or said extended second primers to form a termolecular complex, and (E) one or both of said probes contains a label that facilitates detection of said probes hybridized to said extended first and/or said extended second primers.

29. The method of claim 28 wherein the repeating of steps (v)-(vii) is achieved by repeated temperature cycling.

30. The method of claim 28 wherein said target polynucleotide is DNA.

31. The method of claim 28 wherein only said first primer is used and said target sequence contains at its 5'"'"'- end at least a 10-base sequence hybridizable with a sequence at the 3'"'"' end of said target sequence to which said first primer hybridizes.

32. The method of claim 28 wherein said first and said second primers are different and said extended first primer is a template for said second primer and said extended second primer is a template for said first primer.

33. The method of claim 28 wherein the 3'"'"'-terminus of said S1 is linked to the 5'"'"'-terminus of said S2 by a linking group comprising from 0 to 40 nucleotides or nucleotide analogs.

34. The method of claim 28 wherein the 3'"'"'-terminus of said S1 is linked to the 5'"'"'-terminus of said S2 by a linking group and the 3'"'"'-terminus of said S3 is linked to the 5'"'"'-terminus of said S4 by a linking group, each of said linking groups comprising from 0 to 40 nucleotides or nucleotide analogs.

35. The method of claim 28 wherein detection of said complex comprises association of said complex with particles.

36. The method of claim 35 wherein said detection comprises detecting agglutination of said particles.

37. The method of claim 28 wherein said label is an oligonucleotide label.

38. The method of claim 28 wherein said sequences comprising at least one of the probes are linked by a linking group, at least a portion of said linking group comprising an oligonucleotide label.

39. The method of claim 38 wherein each of said probes comprises an oligonucleotide label and said detecting of step (b) comprises the use of nucleotides N1 and N2, each respectively complementary to one of said oligonucleotide labels, wherein said N1 and N2 are each labeled with a reporter molecule.

40. The method of claim 39 wherein said N1 is associated with a particle having a photosensitizer associated therewith and said N2 is associated with a particle having a chemiluminescent compound associated therewith.

41. A kit for use in an amplification and detection of a target polynucleotide sequence of a target polynucleotide, said kit comprising in packaged combination:

(a) an oligonucleotide primer which hybridizes to said target polynucleotide and is extended along said target polynucleotide sequence to produce extended oligonucleotide primer,

(b) nucleoside triphosphates,

(c) a nucleotide polymerase,

(d) a first oligonucleotide probe having nucleotide sequences S1 and S2, and

(e) a second oligonucleotide probe having sequences S3 and S4,

wherein S1, S2, S3 and S4 are each about 10 to 20 nucleotides in length and

wherein the sequences comprising at least one of said first or said second oligonucleotide probes are linked such that either (i) they are non-contiguous and can bind to contiguous or non-contiguous sites on said extended polynucleotide primer or a complementary sequence thereto or (ii) the sites to which they hybridize on said extended polynucleotide primer or a complementary sequence thereto are noncontiguous, and

wherein said probes have the characteristics that they (i) do not hybridize to said extended oligonucleotide primer during said amplification and (ii) subsequent to said amplification, both of said first and second oligonucleotide probes can hybridize to said extended oligonucleotide primer or said complementary sequence, and (iii) one or both of said probes contain a label that facilitates detection of said probes hybridized to said extended oligonucleotide primer or said complementary sequence.

42. The kit of claim 41 wherein the 3'"'"'-terminus of said S1 is linked to the 5'"'"'-terminus of said S2 by a nucleotide linking group.

43. The kit of claim 42 wherein at least a portion of said nucleotide linking group is an oligonucleotide label.

44. The kit of claim 41 wherein one of said probes is associated with a particle.

45. The kit of claim 44 wherein said particle is bound to or binds to said probe.

46. The kit of claim 41 wherein said label is an oligonucleotide label.

47. The kit of claim 41 wherein S1 and S2 are linked by a nucleotide linking group and S3 and S4 are linked by a nucleotide linking group.

48. The kit of claim 47 wherein said linking groups comprise oligonucleotide labels.

49. The kit of claim 48 comprising nucleotide N1 and N2 each complementary to one of said oligonucleotide labels wherein said N1 and N2 are each labeled with a reporter molecule.

50. The kit of claim 49 wherein said N1 is associated with a particle having a photosensitizer associated therewith and said N2 is associated with a particle having a chemiluminescent compound associated therewith.

51. The kit of claim 41 comprising a second oligonucleotide primer.

52. A kit for detection of a target polynucleotide sequence, said kit comprising in packaged combination reagents for conducting an amplification of said target polynucleotide sequence and two labeled oligonucleotide probes that bind to the product of said amplification of said target polynucleotide sequence wherein at least one of said probes has two sequences that are each about 10 to 20 nucleotides in length and that are non-contiguous and can bind to contiguous or non-contiguous sites on a single strand of said product.

53. A method for amplifying and detecting a target polynucleotide sequence, which comprises:

(a) providing in combination a sample suspected of containing a target polynucleotide having said target polynucleotide sequence, reagents for amplifying said target polynucleotide sequence to produce copies thereof, a first oligonucleotide probe and a second oligonucleotide probe wherein said probes (i) do not hybridize to said copies during said amplifying and (ii) do not interfere with said amplifying and (iii) subsequent to said amplifying, both of said probes can hybridize to one the strands of said copies, and wherein at least one said probes is associated with a particle,

(b) subjecting said combination to conditions for amplifying said target polynucleotide sequence to produce said copies,

(c) thereafter subjecting said combination to conditions under which both of said probes hybridize to one of said strands and result in agglutination of said particles, and

(d) detecting said agglutination wherein the presence of agglutination indicates the presence of said target polynucleotide sequence.