Methods and apparatus for detecting variants utilizing base stacking

• US 6,753,148 B2
• Filed: 03/25/2002
• Issued: 06/22/2004
• Est. Priority Date: 02/25/1998
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V.
• Original Assignee: Nanogen, Inc.
• Inventors: Sosnowski, Ronald G., Tu, Eugene
• Primary Examiner(s): Siew, Jeffrey
• Application Number:
  US10/108,280
• Publication Number:
  US 20020115098A1
• Time in Patent Office: 820 Days
• Field of Search: 435/6, 435/7.1, 435/91.1, 435/91.2, 435/287.2, 536/22.1, 536/23.1, 536/24.3, 536/24-1-, 536/25.32
• US Class Current: 435/6.12
• CPC Class Codes: B01J 2219/00653 the compounds being bound to electrodes embedded in or on the solid supports
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: C12Q 1/6825 Nucleic acid detection involving sensors
• CPC Class Codes: C12Q 1/6827 for detection of mutation or polymorphism
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2537/125 Sandwich assay format
• CPC Class Codes: C12Q 2565/518 characterised by the immobilisation of the nucleic acid sample or target
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: H01L 29/6656 using multiple spacer layers, e.g. multiple sidewall spacers

Abstract

Methods and apparatus are provided for the analysis and determination of the nature of repeat units in a genetic target. In one method of this invention, the nature of the repeat units in the genetic target is determined by the steps of providing a plurality of hybridization complex assays arrayed on a plurality of test sites, where the hybridization complex assay includes at least a nucleic acid target containing a simple repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n=0,1,2 . . . , or fractions thereof, being complementary to the target sequence, and a reporter probe having a selected sequence complementary to the same target sequence strand wherein the selected sequence of the reporter includes a second unique flanking sequence and m repeat units, where m=0,1,2 . . . , or fractions thereof, but where the sum of repeat units in the capture probe plus reporter probe is greater than 0 (n+m>0). Concordance and discordance among the hybridization complex assays at the test sites is determined at least in part by hybridization stability. Electronic stringency control may be utilized. Applications include paternity testing, forensic use, and disease diagnostics, such as for the identification of the existence of a clonal tumor.

Claims

1. A method for determining the nature of a genetic variant in a nucleic acid target, comprising the steps of:

providing a plurality of hybridization complex assays on a test device, where the hybridization complex comprises;

a nucleic acid target containing a genetic variant nucleic acid sequence,

a capture probe having a selected sequence complementary to a first target sequence, and

a reporter probe having a selected sequence complementary to a different portion of the same target strand,

wherein the reporter probe and capture probe form a hybridization complex with the target such that the termini of the capture and reporter are juxtaposed, and

selecting the capture probe and reporter probe from a plurality of options, such that the juxtaposed termini are stabilized through base stacking and the energy difference between a concordant assay and a discordant assay is increased.

2. The method of claim 1 such that the base-stacking stabilization between the juxtaposed termini is maximized within a concordant hybridization complex.

3. The method of claim 1 such that the base-stacking stabilization between the juxtaposed termini is minimized within a discordant hybridization complex.

4. The method of claim 1 such that the differential base-stacking stabilization between the juxtaposed termini is maximized for a discordant and concordant hybridization complex.

5. The method of claim 1 wherein the genetic variant is immediately adjacent the termini.

6. The method of claim 1 wherein the genetic variant is one base removed from the termini.

7. The method of claim 1 wherein the genetic variant is two or more bases removed from the termini.

8. The method of claim 1 wherein the base-stacking is maximized by selection of the terminal bases.

9. The method of claim 1 wherein the base-stacking is maximized by selection of the position of the genetic variant relative to the juxtaposed termini.

10. The method of claim 1 wherein the base-stacking is maximized by selection of complementary sequences.

11. The method of claim 1 wherein the base-stacking is maximized by selection of the terminal bases and complementary sequences.

12. The method of claim 1 wherein the base-stacking is maximized by selection of the terminal bases, complementary sequences and position of the genetic variant relative to the juxtaposed termini.

13. The method of claim 1 for determining the nature of a genetic variant wherein the test device includes a plurality of test sites.

14. The method of claim 13 for determining the nature of a genetic variant wherein a test site includes a permeation layer disposed adjacent the microelectrode.

15. The method of claim 1 or determining the nature of a genetic variant wherein two different hybridization complexes are distinguished at a single site with distinguishable reporters.

16. The method of claim 1 for determining the nature of a genetic variant wherein two different hybridization complexes are distinguished at multiple sites with different reporters.

17. The method of claim 1 or determining the nature of a genetic variant wherein the variant constitutes the number of repetitive DNA sequences.

18. The method of claim 1 wherein the genetic variants include deletions.

19. The method of claim 1 wherein the genetic variants include insertions.

20. The method of claim 1 wherein the genetic variants include transitions.

21. The method of claim 1 wherein the genetic variants include transversions.

22. The method of claim 1 wherein the genetic variants include single nucleotide variants.

23. The method of claim 1 wherein the genetic variants include mutations.

24. The method of claim 1 wherein the genetic variants include point mutations.

25. The method of claim 1 wherein the genetic variant affects a single base.

26. The method of claim 24 wherein the genetic variant affects more than a single base.

27. The method of claim 1 wherein multiple test locations indicate the nature of the target sequence by the detection of genetic variants in the target sequence.

28. The method of claim 1 wherein distinguishable reporters at a single test site location indicate the nature of the target sequence by the detection of genetic variants in the target sequence.

29. The method of claim 1 wherein the plurality of hybridization complex assays form an array.

30. The method of claim 29 wherein the location of the test site in the array indicates the nature of the genetic variant.

31. The method of claim 1 wherein a test site is determined to be concordant when the hybridization complex present there is stable relative to other test sites for the same locus.

32. The method of claim 1 wherein the stability is enhanced by complementary match of bases in the hybridization complex including the capture, reporter and target.

33. The method of claim 1 wherein the hybridization complex assay is determined to be concordant when a distinguishable reporter present there is stable relative to other distinguishable reporters for the same locus.

34. The method of claim 1 wherein the base stacked pair is 5′

GpC3′

.

35. The method of claim 1 wherein the base stacked pair is 5′

TpA3′

.

36. The method of claim 1 further including modifications of terminal nucleotides which increase base stacking.

37. The method of claim 1 wherein stability is enhanced with ligation techniques.

38. The method of claim 1 wherein discordance includes a genetic variant.

39. The method of claim 38 wherein the genetic variant is in the target sequence.

40. The method of claim 38 wherein the genetic variant in the target sequence is selected from the group comprising:

insertions, deletions, transitions and transversions.

41. The method of claim 1 wherein discordance includes genetic variations which are greater than a single nucleotide.

42. The method of claim 1 wherein the number of loci analyzed at one time is one.

43. The method of claim 1 wherein the number of loci analyzed at one time is three.

44. The method of claim 1 wherein the number of loci analyzed at one time is less than five.

45. The method of claim 1 wherein the number of loci analyzed at one time is less than ten.

46. The method of claim 1 wherein the number of loci analyzed at one time is greater than ten.

47. The method of claim 1 wherein the number of loci analyzed at one time is less than one hundred.

48. The method of claim 1 wherein the number of loci analyzed at one time is greater than one hundred.

49. The method of claim 1 wherein the disruption of the juxtaposed termini results in destabilization of the hybridization complex.

50. The method of claim 1 wherein the disruption of complementary sequences results in destabilization of the hybridization complex.

51. The method of claim 1 wherein the disruption of the juxtaposed termini and complementary sequences results in destabilization of the hybridization complex.

52. The method of claim 1 wherein the hybridization stability is determined, at least in part, by thermal regulation of stringency.

53. The method of claim 1 wherein the hybridization stability is determined, at least in part, by chemical regulation of stringency.

54. The method of claim 1 wherein initial hybridization step occurs in 10 minutes or less.

55. The method of claim 1 wherein initial hybridization step occurs in 5 minutes or less.

56. The method of claim 1 wherein initial hybridization step occurs in 1 minute or less.

57. The method of claim 1 wherein the hybridization complex is labeled.

58. The method of claim 57, further including the step of detecting the amounts of labeled hybridization complex at the test sites.

59. The method of claim 58 wherein the detecting is imaging.

60. The method of claim 59 wherein the imaging is optical imaging.

61. The method of claim 59 wherein the imaging is electronic imaging.

62. The method of claim 59 wherein the imaging is CCD imaging.

63. The method of claim 59 wherein the imaging is integrated optical imaging.

64. The method of claim 59 wherein the imaging detection is quantified.

65. The method of claim 1 further including a statistical analysis step.

66. The method of claim 1 wherein the labeled portion of complex is the target.

67. The method of claim 1 wherein the labeled portion of complex is the capture.

68. The method of claim 1 wherein the labeled portion of complex is the reporter.

69. The method of claim 1 wherein the labeling is by fluorescent labeling.

70. The method of claim 69 wherein the fluorescent labeling is with Bodipy Texas Red.

71. The method of claim 69 wherein the fluorescent labeling is with Bodipy 630/650.

72. The method of claim 69 wherein the fluorescent labeling is with Lucifer Yellow.

73. The method of claim 57 wherein the labeling is by colormetric labeling.

74. The method of claim 57 wherein the labeling is by chemiluminescent labeling.

75. The method of claim 1 further including energy transfer between molecules in the hybridization complex.

76. The method of claim 75 wherein the energy transfer includes quenching.

77. The method of claim 1 further including a redundant assay.

78. The method of claim 77 further including the step of repeating the redundant assay until a statistically significant result is obtained.

79. The method of claim 77 wherein the redundant assay includes multiple arrays.

80. The method of claim 1 wherein the target DNA is purified.

81. The method of claim 1 wherein the target is unamplified.

82. The method of claim 1 wherein the target is amplified.

83. The method of claim 1 wherein the target is applied to a reduction of test sites necessary to identify the number of repeat units in the target DNA.

84. The method of claim 83 wherein the reduction increases the statistical significance of results.

85. The method of claim 1 wherein the target material constitutes homozygous allele for a locus.

86. The method of claim 1 wherein the target material constitutes heterozygous allele for a locus.

87. The method of claim 1 wherein the target material constitutes more than one allele per locus for a mixed sample.

88. The method of claim 87 wherein the mixed sample further includes sample from more than one individual.

89. The method of claim 88 wherein the mixed sample further includes tumor tissue mixed with normal tissue.

90. The method of claim 1 wherein the capture probe and reporter probe are selected from a plurality of options by varying at least the position of the juxtaposed termini relative to the genetic variant.

91. The method of claim 1 wherein the genetic variant is at an end of the reporter probe.

92. The method of claim 1 wherein the genetic variant is at an end of the capture probe.