Nucleic acid detection methods using universal priming

• US 20040224352A1
• Filed: 06/09/2004
• Published: 11/11/2004
• Est. Priority Date: 02/07/2000
• Status: Abandoned Application

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Fan, Jian-Bing, Chee, Mark S.
• Application Number:
  US10/864,935
• Publication Number:
  US 20040224352A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6834 Enzymatic or biochemical coupling of nucleic acids to a solid phase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 1/6862 Ligase chain reaction [LCR]
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• 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 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/501 being an array of oligonucleotides
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

The present invention is directed to providing sensitive and accurate assays for genotyping with a minimum or absence of target-specific amplification.

Claims

1. A method for determining the identification of a nucleotide at a detection position in a target sequence, comprising:

(a) hybridizing a target sequence with a probe set to form a hybridization complex, said probe set comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein said probe set further comprises at least one adapter sequence;

(b) contacting said hybridization complex with a ligase to form a ligated hybridization complex;

(c) removing non-hybridized probe sets;

(d) amplifying said ligated hybridization complex with a universal primer pair to form amplicons;

(e) immobilizing said amplicons to a solid support;

(f) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and

(g) determining a nucleotide at said detection position.

2. The method of claim 1, wherein said probe set further comprises a plurality of first and second probes.

3. The method of claim 1, wherein said first or second probe further comprises a partially double stranded nucleic acid.

4. The method of claim 1, wherein said first or second probe further comprises a linker.

5. The method of claim 1, wherein said first or second probe further comprises a nucleotide modification.

6. The method of claim 5, wherein said nucleotide modification comprises a nuclease inhibitor.

7. The method of claim 5, wherein said nucleotide modification comprises a nucleotide analog.

8. The method of claim 1, further comprising removing said non-hybridized probes by contacting said hybridization complex with a nuclease.

9. The method of claim 8, wherein said nuclease comprises an exonuclease.

10. The method of claim 1, wherein said removing of said non-hybridized probes further comprises the steps:

(a) immobilizing said ligated hybridization complex to a solid support, and

(b) washing said ligated hybridization complex-immobilized to said solid support.

11. The method of claim 1, wherein at least one primer of said universal primer pair further comprises biotin.

12. The method of claim 1, wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.

13. The method of claim 12, wherein said reaction vessel further comprises a microtiter well.

14. The method of claim 12, wherein said array further comprises a plurality of capture probes.

15. The method of claim 1, wherein said detection label further comprises a secondary label.

16. The method of claim 15, wherein said secondary label allows detection of said amplicons by separation.

17. The method of claim 1, wherein said detection label comprises a primer.

18. The method of claim 17, wherein said primer comprises a universal primer.

19. The method of claim 1, wherein said detection label comprises one or more nucleotides.

20. The method of claim 1, further comprising the step:

(a) extending one or more probes within said hybridization complex with a polymerase.

21. The method of claim 1, further comprising immobilizing said target sequence to a solid support.

22. The method of claim 1, further comprising amplifying said target sequence.

23. A method for determining the identification of nucleotides at detection positions in a plurality of target sequences, comprising:

(a) hybridizing a plurality of target sequences with a plurality of probe sets to form a plurality of hybridization complexes, each of said probe sets within said plurality comprising a first probe having a first portion containing an upstream universal priming site (UUP), a second portion containing a first target specific sequence and a detection position, and a second probe having a first portion containing a downsteam universal priming site (DUP) and a second portion containing a second target specific sequence, wherein each of said probe sets within said plurality of probe sets further comprises at least one adapter sequence;

(b) contacting said plurality of hybridization complexes with a ligase to form ligated hybridization complexes;

(c) removing non-hybridized probe sets;

(d) amplifying said plurality of ligated hybridization complexes with a plurality of universal primer pairs to form a plurality of amplicons;

(e) immobilizing said plurality of amplicons to a solid support;

(f) contacting said plurality of immobilized amplicons with a plurality of detection labels, wherein the mass of said detection labels is indicative of a nucleotide at said detection position, and

(g) determining a nucleotide at said detection position.

24. The method of claim 23, wherein said plurality of probe sets further comprise a plurality of first and second probes.

25. The method of claim 23, wherein said first or second probes further comprise a partially double stranded nucleic acid.

26. The method of claim 23, wherein said first or second probes further comprise a linker.

27. The method of claim 23, wherein said first or second probes further comprise a nucleotide modification.

28. The method of claim 27, wherein said nucleotide modification comprises a nuclease inhibitor.

29. The method of claim 27, wherein said nucleotide modification comprises a nucleotide analog.

30. The method of claim 23, further comprising removing said non-hybridized probe sets by contacting said ligated hybridization complexes with a nuclease.

31. The method of claim 30, wherein said nuclease comprises an exonuclease.

32. The method of claim 23, wherein said removing of said non-hybridized probe sets further comprises the steps:

(a) immobilizing said ligated hybridization complexes to a solid support, and

(b) washing said ligated hybridization complexes immobilized to said solid support.

33. The method of claim 23, wherein at least one primer of said universal primer pair further comprises biotin.

34. The method of claim 23, wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.

35. The method of claim 34, wherein said reaction vessel further comprises a microtiter well.

36. The method of claim 34, wherein said array further comprises a plurality of capture probes.

37. The method of claim 23, wherein said plurality of detection labels further comprise a plurality of secondary labels.

38. The method of claim 37, wherein said plurality of secondary labels allow detection of said plurality of amplicons by separation.

39. The method of claim 23, wherein said plurality of detection labels comprise a plurality of primers.

40. The method of claim 39, wherein said plurality of primers comprise a plurality of universal primers.

41. The method of claim 23, wherein said plurality of detection labels comprise one or more nucleotides.

42. The method of claim 23, further comprising the step:

(a) extending one or more probes within said plurality of hybridization complexes with a polymerase.

43. The method of claim 23, further comprising immobilizing said plurality of target sequences to a solid support.

44. The method of claim 23, further comprising amplifying said plurality of target sequences.

45. A method of determining the identification of a nucleotide at a detection position in a target sequence comprising a first target domain adjacent to said detection position and a second target domain comprising said detection position, wherein said method comprises:

(a) hybridizing a first ligation probe to said first target domain, said first ligation probe comprising;

(i) a first portion comprising an upstream universal priming site (UUP); and

(ii) a second portion comprising a first target-specific sequence comprising a first base at an interrogation position; and

(b) hybridizing a second ligation probe to said second target domain, said second ligation probe comprising;

(i) a third portion comprising a downstream universal priming site (DUP); and

(ii) a fourth portion comprising a second target-specific sequence;

wherein if said first base is complementary to said nucleotide at said detection position, a ligation complex is formed, and wherein at least one of said first and second ligation probes comprises a fifth portion comprising a distinct adapter sequence;

(c) providing a ligase that ligates said first and second ligation probes to form a ligated probe;

(d) removing non-hybridized probes;

(e) amplifying said ligated probe using said UUP and said DUP to generate a plurality of amplicons;

(f) immobilizing said amplicons to a solid support;

(g) contacting said immobilized amplicons with a detection label, wherein the mass of said detection label is indicative of said nucleotide at said detection position, and

(h) determining the nucleotide at said detection position.

46. The method of claim 45, wherein said first or second ligation probe further comprises a plurality of first or second probes.

47. The method of claim 45, wherein said first or second ligation probe further comprises a partially double stranded nucleic acid.

48. The method of claim 45, wherein said first or second ligation probe further comprises a linker.

49. The method of claim 45, wherein said first or second ligation probe further comprise a nucleotide modification.

50. The method of claim 49, wherein said nucleotide modification comprises a nuclease inhibitor.

51. The method of claim 49, wherein said nucleotide modification comprises a nucleotide analog.

52. The method of claim 45, further comprising removing said non-hybridized probes by contacting said hybridization complex with a nuclease.

53. The method of claim 52, wherein said nuclease comprises an exonuclease.

54. The method of claim 45, wherein said removing of said non-hybridized probes further comprises the steps:

(a) immobilizing said ligated probe to a solid support, and

(b) washing said ligated probe immobilized to said solid support.

55. The method of claim 45, wherein at least on primer of said universal primer pair further comprises biotin.

56. The method of claim 45, wherein said solid support is selected from the group consisting of a bead, a reaction vessel and an array.

57. The method of claim 56, wherein said reaction vessel further comprises a microtiter well.

58. The method of claim 56, wherein said array further comprises a plurality of capture probes.

59. The method of claim 45, wherein said detection label further comprises a secondary label.

60. The method of claim 59, wherein said secondary label allows detection of said amplicons by separation.

61. The method of claim 45, wherein said detection label comprises a primer.

62. The method of claim 61, wherein said primer comprises a universal primer.

63. The method of claim 45, wherein said detection label comprises one or more nucleotides.

64. The method of claim 45, further comprising the step:

(a) extending one or more probes within said ligation complex with a polymerase.

65. The method of claim 45, further comprising immobilizing said target sequence to a solid support.

66. The method of claim 45, further comprising amplifying said target sequence.