Nucleic acid amplification using single primer

• US 6,124,090 A
• Filed: 05/09/1995
• Issued: 09/26/2000
• Est. Priority Date: 01/19/1989
• Status: Expired due to Fees

• Current Assignee: DADE Behring Beteiligungs GmbH (Siemens AG)
• Original Assignee: Behringwerke Aktiengesellschaft (Sanofi )
• Inventors: Rose, Samuel, Western, Linda M., Ullman, Edwin F., Becker, Martin, Goodman, Thomas C.
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US08/438,149
• Time in Patent Office: 1,967 Days
• Field of Search: 435/6, 435/91.2
• US Class Current: 435/6.12
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/301 Hairpin oligonucleotides

Abstract

A method is disclosed for determining the presence of a polynucleotide analyte in a sample suspected of containing the analyte. The method comprises (a) forming as a result of the presence of an analyte a single stranded polynucleotide comprising a target polynucleotide binding sequence flanked by first and second polynucleotide sequences that differ from the sequence of the analyte or a sequence complementary to the analyte sequence, (b) forming multiple copies of the single stranded polynucleotide, and (c) detecting the single stranded polynucleotide. Also disclosed is a method of producing at least one copy of a single stranded polynucleotide. The method comprises (a) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'"'"'-end of which has at least a 10 base sequence hybridizable with a second sequence flanking the 3'"'"'-end of the single stranded polynucleotide, the second sequence being partially or fully complementary with at least a 10 base first sequence flanking the 5'"'"' end of the single stranded polynucleotide, (b) dissociating the extended polynucleotide primer and the single stranded polynucleotide, (c) repeating step a and (d) dissociating the extended polynucleotide primer and the copy of the single stranded polynucleotide.

Claims

1. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises the steps of:

(a) forming as a result of the presence of said analyte a single stranded polynucleotide flanked by at least 50% complementary first and second flanking sequences wherein said second flanking sequence is 3'"'"' of said first flanking sequence,

(b) forming multiple copies of said single stranded polynucleotide and said flanking sequences in the presence of nucleoside triphosphates, template dependent polynucleotide polymerase and a polynucleotide primer that has at least a 10 base sequence hybridizable with said second flanking sequence, and

(c) detecting said single stranded polynucleotide, the presence thereof indicating the presence of said analyte.

2. The method of claim 1 wherein said first and second flanking sequences are comprised of complementary sequences of at least 10 bases.

3. The method of claim 2 wherein said multiple copies are formed by incubating said assay medium under conditions for either wholly or partially sequentially or concomitantly (1) hybridizing a single stranded polynucleotide primer at its 3'"'"'-end to the flanking sequence at the 3'"'"'-end of said single stranded polynucleotide, (2) extending said polynucleotide primer in the presence of nucleoside triphosphates and a polynucleotide polymerase to provide a first extended polynucleotide primer, (3) dissociating said first extended polynucleotide primer and said single stranded polynucleotide, (4) hybridizing said first extended polynucleotide primer with said polynucleotide primer, (5) extending said polynucleotide primer along said first extended polynucleotide primer to provide a second extended polynucleotide primer, (6) dissociating said second extended polynucleotide primer from said first extended polynucleotide primer, and (7) repeating steps (4)-(6) above.

4. The method of claim 1 wherein said polynucleotide analyte is DNA.

5. The method of claim 3 wherein said polynucleotide primer is 10 to 100 nucleotides in length.

6. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises the steps of:

(a) forming as a result of the presence of said analyte a single stranded polynucleotide flanked by at least 80% complementary first and second flanking sequences, each comprised of at least 15 bases,

(b) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'"'"'-end of which can hybridize with the flanking sequence at the 3'"'"'-end of said single stranded polynucleotide sequence,

wherein steps (a) and (b) can be performed wholly or partially sequentially or concomitantly, and

(c) detecting extended polynucleotide primer containing a sequence identical to and/or complementary with said polynucleotide sequence, the presence thereof indicating the presence of said analyte.

7. The method of claim 6 wherein said first and second flanking sequences are fully complementary.

8. The method of claim 6 wherein a portion of said polynucleotide primer is labeled with one reporter group and a portion is labeled with a second reporter group.

9. The method of claim 6 wherein said polynucleotide analyte is DNA.

10. The method of claim 6 wherein said polynucleotide primer is 20 to 100 nucleotides in length.

11. The method of claim 6 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

12. The method of claim 6 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said single stranded polynucleotide.

13. The method of claim 6 wherein step (b) is repeated such that the number of said copies of said extended polynucleotide primer formed is increased by at least a factor of three.

14. The method of claim 6 wherein said polynucleotide primer is labeled with a reporter group.

15. The method of claim 14 wherein said reporter group is selected from the group consisting of, fluorescers, chemiluminescers, catalysts, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, and small organic molecules.

16. The method of claim 6 wherein said polynucleotide primer is labeled with a ligand.

17. The method of claim 6 wherein said single stranded polynucleotide contains a sequence that when hybridized to its complementary sequence can be bound specifically by a receptor.

18. The method of claim 17 wherein said receptor is selected from the group consisting of repressors, activators, and restriction enzymes.

19. The method of claim 6 wherein said single stranded polynucleotide contains a sequence at its end that when hybridized to its complementary sequence, can be bound specifically by a receptor, and said extended polynucleotide primer is detected by binding said receptor to said extended polynucleotide primer.

20. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises;

(a) contacting said sample with first and second polynucleotide probes, said first probe comprising (i) a target polynucleotide binding sequence at its 3'"'"'-end complementary to a first portion of one strand of said analyte and (ii) a first flanking sequence and said second probe comprising (i) a target polynucleotide binding sequence at its 5'"'"' end complementary to a second portion of the same strand of said analyte and (ii) a second flanking sequence wherein said first and second flanking sequences are at least 65% complementary, under conditions for binding of said first and second probes with said analyte,

(b) providing conditions for ligating said first and second polynucleotide probes to one another only when bound to said analyte,

(c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'"'"'-end of which can hybridize with said second flanking sequence, and

(d) detecting extended polynucleotide primer containing a sequence complementary to said first probe, the presence thereof indicating the presence of said analyte.

21. The method of claim 20 wherein said polynucleotide analyte is DNA.

22. The method of claim 20 wherein said polynucleotide primer is 10 to 100 nucleotides in length.

23. The method of claim 20 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and DTTP.

24. The method of claim 20 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide analytes.

25. The method of claim 20 wherein step (c) is repeated such that the number of said copies of said extension polynucleotide primer formed is increased by at least a factor of three.

26. The method of claim 20 wherein said polynucleotide primer is labeled with a reporter group.

27. The method of claim 26 wherein said reporter group is selected from the group consisting of, fluorescers, chemiluminescers, catalysts, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, and small organic molecules.

28. The method of claim 26 wherein said first probe contains an operator sequence between said target polynucleotide binding sequence complementary to said analyte and said first flanking complementary sequence.

29. The method of claim 20 wherein said detection includes detection of a specific DNA sequence.

30. The method of claim 26 wherein separate molecules of polynucleotide primer are labeled with a group capable of becoming bound to a surface.

31. The method of claim 20 wherein said first and second polynucleotide probes when hybridized with said analyte are less than two nucleotides apart and conditions for ligating said first and second polynucleotide probes include addition of a ligase.

32. The method of claim 20 wherein said first and second polynucleotide probes when hybridized with said analyte are at least one nucleotide apart and conditions for ligating said first and second polynucleotide probes include

(a) adding nucleotide triphosphates and a polynucleotide polymerase to render the hybridized portions contiguous and

(b) adding ligase to ligate said hybridized portions.

33. A method for detecting the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

(a) hybridizing to said analyte a first polynucleotide probe and a second polynucleotide probe, said first polynucleotide probe having (i) a sequence S1 that is at least 10 nucleotides in length and at least 50% complementary to a sequence S2 of said second polynucleotide probe and (ii) a sequence S3 that is 3'"'"' of said S1 and having a free 3'"'"'-end, said second polynucleotide probe having a sequence S4 that is 5'"'"' of said S2, said S4 having a free 5'"'"'-end, wherein the 3'"'"'-end of said S3 and the 5'"'"'-end of said S4 are ligatable, said first polynucleotide probe hybridizing to said analyte at a portion thereof that is 3'"'"' of the portion to which said second polynucleotide probe hybridizes to said analyte,

(b) ligating said S3 and said S4 to form ligated first and second polynucleotide probes,

(c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least the 3'"'"'-end of which hybridizes with said S2 of said ligated first and second polynucleotide probes,

wherein steps (a) and (b) and (c) are performed wholly or partially sequentially of concomitantly and,

(d) detecting extended polynucleotide primer and/or a sequence identical to and/or complementary with at least that portion of said ligated first and second polynucleotide probes, the presence thereof indicating the presence of said analyte.

34. The method of claim 33 wherein said first and second flanking sequences are capable of forming an inverted repeat.

35. The method of claim 33 wherein said polynucleotide analyte is DNA.

36. The method of claim 33 wherein said S1 and S2 are fully complementary.

37. The method of claim 33 wherein said S3 and S4 are fully complementary to separate portions of said analyte.

38. The method of claim 33 wherein said polynucleotide primer is 15 to 100 nucleotides in length.

39. The method of claim 33 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

40. The method of claim 33 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide analyte.

41. The method of claim 33 wherein step (b) is repeated such that the number of said copies of said extended polynucleotide primer formed is increased by at least a factor of three.

42. The method of claim 33 wherein said polynucleotide primer is labeled with a reporter group.

43. The method of claim 42 wherein said reporter group is selected from the group consisting of fluorescers, chemiluminescers, catalysts, co-enzymes, radioactive substances, amplifiable polynucleotide sequences, and small organic molecules.

44. The method of claim 43 wherein said first polynucleotide probe contains an operator sequence.

45. The method of claim 33 wherein said detection includes detection of a specific DNA sequence.

46. The method of claim 33 wherein said S3 and S4 when hybridized with said analyte are less than two nucleotides apart and conditions for ligation of said S3 and S4 include addition of a ligase.

47. The method of claim 33 wherein said S3 and S4 when hybridized with said analyte are at least one nucleotide apart an ligation of said S3 and S4 includes:

(a) adding nucleoside triphosphates and a polynucleotide polymerase to render said S3 and S4 contiguous, and

(b) adding ligase to ligate said hybridized portions.

48. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises;

(a) combining in an assay medium said sample with a first polynucleotide probe and a second polynucleotide probe, said first probe comprising a sequence S1 at least 10 nucleotides in lenath and at least 80% complementary with a sequence S2 in said second probe, said first probe being attached at its 3'"'"'-end to a sequence having a portion S3 that is hybridizable with a first portion of a single strand of said analyte wherein said S2 is attached at is 5'"'"'-end to a sequence having a portion S4 that is hybridizable with a second portion of said single strand of said analyte,

(b) incubating said assay medium under conditions for binding of said first and second probes with said analyte, said first and second probes being ligatable, or capable of being rendered ligatable, to one another only when bound to said analyte,

(c) ligating said first and second probes to form ligated first and second probes with the proviso that, if necessary, where they are not otherwise ligatable, said first and second probes are rendered ligatable,

(d) combining said assay medium with a polynucleotide primer having a 3'"'"' terminal sequence complementary to said S2 in said second probe,

(e) incubating said assay medium under conditions for either wholly or partially sequentially or concomitantly (1) dissociating any internally hybridized ligated first and second probes, (2) hybridizing said polynucleotide primer with said ligated first and second probes, (3) extending said polynucleotide primer along said ligated first and second probes, (4) dissociating said hybridized extended polynucleotide primer and said ligated first and second probes, (5) hybridizing said extended polynucleotide primer with said polynucleotide primer, (6) extending said polynucleotide primer along said extended polynucleotide primer, (7) dissociating said hybridized extended primer, and (8) repeating steps (5)-(7) above,

wherein steps (a)-(d) are carried out wholly or partially sequentially or concomitantly followed by step (e), and

(f) detecting said extended primer, the presence thereof indicating the presence of said analyte.

49. The method of claim 48 wherein said polynucleotide analyte is DNA.

50. The method of claim 48 wherein said S1 is full complementary to said S2.

51. The method of claim 48 wherein said polynucleotide primer is 10 to 100 nucleotides in length.

52. The method of claim 48 wherein said template-dependent polynucleotide polymerase is a DNA polymerase and said nucleoside triphosphates are dATP, dGTP, dCTP, and dTTP.

53. The method of claim 48 wherein said method is carried out at a substantially excess concentration of said polynucleotide primer relative to the concentration of said polynucleotide analyte.

54. The method of claim 48 wherein the number of said copies of said extended polynucleotide primer formed is increased by at least a factor of three.

55. The method of claim 48 wherein said polynucleotide primer is labeled with a reporter group.

56. The method of claim 55 wherein said reporter group is selected from the group consisting of, fluorescers, chemiluminescers, radioactive substances, co-enzymes, catalysts, amplifiable polynucleotide sequences, and small organic molecules.

57. The method of claim 55 wherein one of said probes contains an operator or repressor binding sequence.

58. The method of claim 55 wherein separate molecules of polynucleotide primer are labelled with a group capable of becoming bound to a surface.

59. The method of claim 48 wherein said detection includes detection of a specific DNA sequence.

60. The method of claim 48 wherein a portion of said polynucleotide primer is labeled with fluorescein and another portion of said polynucleotide primer is labeled with biotin and said detecting includes

(a) adding a support to which avidin is bound,

(b) separating said support from said assay medium, and

(c) examining said support for the presence of fluorescein.

61. The method of claim 48 wherein said extending of said polynucleotide primer along said ligated first and second probes forms at least part of an operator and said detecting includes:

(a) adding a repressor bound to a solid support and

(b) detecting a sequence bound to said solid support that is identical or complementary to a sequence in said first polynucleotide probe.

62. The method of claim 61 wherein said sequence is detected by the binding of a labeled polynucleotide probe to said first polynucleotide probe sequence.

63. The method of claim 48 wherein said first and second polynucleotide probes when hybridized with said analyte are less than two nucleotides apart and conditions for ligating said first and second polynucleotide probes include addition of a ligase.

64. The method of claim 48 wherein said first and second polynucleotide probes when hybridized with said analyte are at least one nucleotide apart and conditions for ligating said first and second polynucleotide probes include

(a) adding nucleoside triphosphates and a polynucleotide polymerase to render the hybridized portions contiguous and

(b) adding ligase to ligate said hybridized portions.

65. A method for determining the presence of a polynucleotide analyte in a sample suspected of containing said analyte, which comprises:

(a) contacting said sample with a first polynucleotide probe and a second polynucleotide probe, said first probe comprising (1) a first sequence S1 that is at least 10 nucleotides in length and at least 80% complementary to a second sequence S2 in said second probe and (2) a sequence S3 complementary to a portion of said analyte other than a portion complementary to a sequence S4 of said second probe under conditions for binding of said first and second probes with said analyte,

(b) ligating said first and second probes to one another to form ligated first and second probes,

(c) forming in the presence of nucleoside triphosphates and template dependent polynucleotide polymerase an extension of a polynucleotide primer at least a portion of which is hybridized with said S1 or said S2 of said ligated first and second probes,

wherein steps (a) and (b) and (c) are carried out wholly or partially sequentially or concomitantly and

(d) detecting said extended polynucleotide primer and/or said ligated first and second probes, the presence thereof indicating the presence of said analyte.