ASSAY METHODS USING DNA BINDING PROTEINS

• US 20120214162A1
• Filed: 02/10/2012
• Published: 08/23/2012
• Est. Priority Date: 02/11/2011
• Status: Active Grant

• Current Assignee: NABsys, Inc.
• Original Assignee: NABsys, Inc.
• Inventors: Oliver, John S.
• Granted Patent: US 11,274,341 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2533/101 Primer extension
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: C12Q 2565/629 being a microfluidic device
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

Assay methods for preparing a biomolecule analyte includes hybridizing a sequence specific oligonucleotide probe to a biomolecule template and reacting the resulting analyte with a binding moiety.

Claims

1. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template;

b. hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, each probe having a 5′

end and a 3′

end, to thereby form an analyte having at least one single-stranded region and at least two duplex regions;

c. conducting a base extension reaction in the at least one single-stranded region from the 3′

end of a hybridized probe toward the 5′

end of an adjacent hybridized probe;

d. terminating the base-extension reaction such that there remains for each single-stranded region a single-stranded portion thereof adjacent to the 5′

end of each hybridized probe; and

e. reacting the resulting analyte with a binding moiety that selectively binds to the at least one single-stranded portion to thereby prepare the biomolecule analyte.

2. The method of claim 1, wherein the probes comprise single-stranded DNA.

3. The method of claim 1, wherein the probes comprise RNA.

4. The method of claim 1, wherein the base extension reaction is performed by a DNA or RNA polymerase.

5. The method of claim 1, wherein the binding moiety comprises a protein.

6. The method of claim 5, wherein said protein comprises one or more proteins selected from the group consisting of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.

7. The method of claim 1, wherein a length of each probe is selected from a range of 4 to 12 bases.

8. The method of claim 1, wherein the method comprises performing steps a-e sequentially.

9. The method of claim 1, further comprising repeating steps a-e sequentially by replacing said first plurality of probes with a subsequent plurality of different unique probes.

10. The method of claim 1, wherein at least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.

11. The method of claim 1, wherein the biomolecule analyte is configured for detection of positional information in a nanopore system.

12. The method of claim 11, further comprising:

monitoring changes in an electrical property across a nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

13. The method of claim 12, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.

14. The method of claim 1, wherein the biomolecule analyte is configured for detection of positional information in a fluidic channel system.

15. The method of claim 14, wherein the fluidic channel system comprises a micro-channel or a nano-channel.

16. The method of claim 14, further comprising:

monitoring changes in an electrical property across a fluidic channel as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

17. The method of claim 16, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.

18. The method of claim 13, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

19. The method of claim 1, wherein sequence specific oligonucleotide analog probes selected from the group consisting of LNAs, PNAs and 2′

-methoxy nucleotide analogs are substituted for the sequence specific oligonucleotide probes.

20. The method of claim 1, wherein the first plurality of identical, sequence specific oligonucleotide probes is replaced by a pool of sequence specific oligonucleotide probes comprising at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, wherein the probes of the second plurality are different from the probes of the first plurality.

76. The method of claim 17, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

21. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA template,

b. hybridizing a first plurality of identical, sequence specific RNA probes to the template, each probe having a 5′

end and a 3′

end, to thereby form an analyte having at least one single-stranded region and at least one duplex region,

c. conducting a base extension reaction in the at least one single-stranded region from the 3′

end of a hybridized probe,

d. allowing the base-extension reaction to fill each single-stranded region on the analyte,

e. removing the RNA probes to provide the analyte with at least one single-stranded segment in the region to which an RNA probe had been hybridized, and

f. reacting the resulting analyte with a binding moiety that selectively binds to the single-stranded segment, to thereby prepare the biomolecule analyte.

22. The method of claim 21, wherein the base extension reaction is performed by a DNA or RNA polymerase.

23. The method of claim 21, wherein the binding moiety comprises a protein.

24. The method of claim 23, wherein said protein comprises one or more proteins selected from the group consisting of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.

25. The method of claim 21, wherein a length of each probe is selected from a range of 4 to 12 bases.

26. The method of claim 21, wherein the method comprises performing steps a-f sequentially.

27. The method of claim 21, further comprising repeating steps a-f sequentially by replacing said first plurality of probes with a subsequent plurality of different unique probes.

28. The method of claim 21, wherein removing the RNA probes comprises reacting the analyte with a hydroxyl ion.

29. The method of claim 21, wherein the biomolecule analyte is configured for detection of positional information in a nanopore system.

30. The method of claim 29, further comprising:

monitoring changes in an electrical property across a nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

31. The method of claim 30, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.

32. The method of claim 21, wherein the biomolecule analyte is configured for detection of positional information in a fluidic channel system.

33. The method of claim 32, wherein the fluidic channel system comprises a micro-channel or a nano-channel.

34. The method of claim 32, further comprising:

monitoring changes in an electrical property across a fluidic channel as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

35. The method of claim 34, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.

36. The method of claim 31, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

37. The method of claim 21, wherein the first plurality of identical, sequence specific RNA probes is replaced by a pool of sequence specific RNA probes comprising at least a first plurality of identical, sequence specific RNA probes and a second plurality of identical, sequence specific RNA probes, wherein the probes of the second plurality are different from the probes of the first plurality.

77. The method of claim 35, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

38. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template,

b. hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, to thereby form an analyte having at least one single-stranded region and at least one duplex region, and

c. reacting the resulting analyte with a binding moiety that selectively binds to the at least one duplex region to thereby prepare the biomolecule analyte.

39. The method of claim 38, wherein the probes comprise single-stranded DNA.

40. The method of claim 38, wherein the probes comprise RNA.

41. The method of claim 38, wherein the binding moiety comprises a protein.

42. The method of claim 41, wherein said protein comprises one or more proteins selected from the group consisting of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.

43. The method of claim 38, wherein a length of each probe is selected from a range of 4 to 12 bases.

44. The method of claim 38, wherein the method comprises performing steps a-c sequentially.

45. The method of claim 38, wherein steps a-c are repeated sequentially by replacing said first plurality of probes with a subsequent plurality of different unique probes.

46. The method of claim 38, wherein at least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.

47. The method of claim 38, wherein the biomolecule analyte is configured for detection of positional information in a nanopore system.

48. The method of claim 47, further comprising:

monitoring changes in an electrical property across a nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

49. The method of claim 48, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the changes in the electrical property, to thereby determine binding moiety locations.

50. The method of claim 38, wherein the biomolecule analyte is configured for detection of positional information in a fluidic channel system.

51. The method of claim 50, wherein the fluidic channel system comprises a micro-channel or a nano-channel.

52. The method of claim 50, further comprising:

monitoring changes in an electrical property across a fluidic channel as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

53. The method of claim 52, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the detected changes in the electrical property, to thereby determine binding moiety locations.

54. The method of claim 49, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

55. The method of claim 38, wherein sequence specific oligonucleotide analog probes selected from the group consisting of LNAs, PNAs and 2′

-methoxy nucleotide analogs are substituted for the sequence specific oligonucleotide probes.

56. The method of claim 38, wherein the first plurality of identical, sequence specific oligonucleotide probes is replaced by a pool of sequence specific oligonucleotide probes comprising at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, wherein the probes of the second plurality are different from the probes of the first plurality.

78. The method of claim 53, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

57. A method for preparing a biomolecule analyte, the method comprising:

a. providing a single-stranded DNA or RNA template,

b. hybridizing a first plurality of identical, sequence specific oligonucleotide probes to the template, to thereby form an analyte having at least one single-stranded region and at least one duplex region, and

c. reacting the resulting analyte with a binding moiety that selectively binds to the at least one single-stranded region, to thereby prepare the biomolecule analyte.

58. The method of claim 57, wherein the probes comprise single-stranded DNA.

59. The method of claim 57, wherein the probes comprise RNA.

60. The method of claim 57, wherein the binding moiety comprises a protein.

61. The method of claim 60, wherein said protein comprises one or more proteins selected from the group consisting of RecA, T4 gene 32 protein, f1 geneV protein, human replication protein A, Pf3 single-stranded binding protein, adenovirus DNA binding protein, and E. coli single-stranded binding protein.

62. The method of claim 57, wherein a length of each probe is selected from a range of 4 to 12 bases.

63. The method of claim 57, wherein the method comprises performing steps a-c sequentially.

64. The method of claim 57, wherein steps a-c are repeated sequentially by replacing said first plurality of probes with a subsequent plurality of different unique probes.

65. The method of claim 57 wherein at least a portion of the probes in the first plurality of probes has attached thereto a detectable tag.

66. The method of claim 57, wherein the biomolecule analyte is configured for detection of positional information in a nanopore system.

67. The method of claim 66, further comprising:

monitoring changes in an electrical property across a nanopore as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

68. The method of claim 67, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the detected changes in the electrical property, to thereby determine binding moiety locations.

69. The method of claim 57, wherein the biomolecule analyte is configured for detection of positional information in a fluidic channel system.

70. The method of claim 69, wherein the fluidic channel system comprises a micro-channel or a nano-channel.

71. The method of claim 69, further comprising:

monitoring changes in an electrical property across a fluidic channel as the biomolecule analyte is translocated therethrough, the changes in the electrical property being indicative of regions including or lacking the binding moiety.

72. The method of claim 71, further comprising:

differentiating between regions of the biomolecule analyte including or lacking the binding moiety based, at least in part, on the detected changes in the electrical property, to thereby determine binding moiety locations.

73. The method of claim 68, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.

74. The method of claim 57, wherein sequence specific oligonucleotide analog probes selected from the group consisting of LNAs, PNAs and 2′

-methoxy nucleotide analogs are substituted for the sequence specific oligonucleotide probes.

75. The method of claim 57, wherein the first plurality of identical, sequence specific oligonucleotide probes is replaced by a pool of sequence specific oligonucleotide probes comprising at least a first plurality of identical, sequence specific oligonucleotide probes and a second plurality of identical, sequence specific oligonucleotide probes, wherein the probes of the second plurality are different from the probes of the first plurality.

79. The method of claim 71, further comprising:

determining a sequence of at least a portion of the single-stranded DNA or RNA template according to the determined binding moiety locations.