Methods for determination of single nucleic acid polymorphisms using bioelectronic microchip

• US 6,468,742 B2
• Filed: 04/12/1999
• Issued: 10/22/2002
• Est. Priority Date: 11/01/1993
• Status: Expired due to Term

• Current Assignee: Gamida for Life B.V., Nanogen, Inc.
• Original Assignee: Nanogen, Inc.
• Inventors: Wang, Ling, Canter, David M., Radtkey, Ray R., Oʼconnell, James P., Sosnowski, Ronald G., Nerenberg, Michael I.
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US09/291,129
• Publication Number:
  US 20010014449A1
• Time in Patent Office: 1,289 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 436/501, 422/50, 422/68.1
• US Class Current: 435/6.12
• CPC Class Codes: B01J 19/0046 Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays synthesis methods per se C40B50/00
• CPC Class Codes: B01J 19/0093 Microreactors, e.g. miniaturised or microfabricated reactors laboratory containers with capillary fluid transport in microfabricated channels or chambers B01L3/5027
• CPC Class Codes: B01J 2219/00315 Microtiter plates
• CPC Class Codes: B01J 2219/00317 Microwell devices, i.e. having large numbers of wells
• CPC Class Codes: B01J 2219/00527 Sheets
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00605 the compounds being directly bound or immobilised to solid supports
• CPC Class Codes: B01J 2219/00621 by physical means, e.g. trenches, raised areas
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• 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: B01J 2219/00686 Automatic
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00713 Electrochemical synthesis
• CPC Class Codes: B01J 2219/0072 Organic compounds
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: B01J 2219/00731 Saccharides
• CPC Class Codes: B01L 2300/0636 : Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0645 : Electrodes
• CPC Class Codes: B01L 3/502761 : specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: B01L 3/5085 : for multiple samples, e.g. microtitration plates
• CPC Class Codes: B82Y 10/00 : Nanotechnology for information processing, storage or transmission, e.g. quantum computing or single electron logic
• CPC Class Codes: B82Y 5/00 : Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• CPC Class Codes: C07H 21/00 : Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/04 : on carriers C07K1/003, C07K1/006 take precedence
• CPC Class Codes: C07K 1/045 : using devices to improve synthesis, e.g. reactors, special vessels
• CPC Class Codes: C07K 1/047 : Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6844 : Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2565/513 : characterised by the pattern of the arrayed oligonucleotides
• 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: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 : Libraries containing peptides or polypeptides, or derivatives thereof
• CPC Class Codes: C40B 40/12 : Libraries containing saccharides or polysaccharides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G11C 13/0014 : comprising cells based on organic memory material
• CPC Class Codes: G11C 13/0019 : comprising bio-molecules
• CPC Class Codes: G11C 13/04 : using optical elements ; using other beam accessed elements, e.g. electron or ion beam
• CPC Class Codes: G11C 19/00 : Digital stores in which the information is moved stepwise, e.g. shift registers
• CPC Class Codes: H01L 25/50 : Multistep manufacturing processes of assemblies consisting of devices, each device being of a type provided for in group H01L27/00 or H01L29/00 H01L21/50 takes precedence
• CPC Class Codes: H01L 2924/00 : Indexing scheme for arrangements or methods for connecting or disconnecting semiconductor or solid-state bodies as covered by H01L24/00
• CPC Class Codes: H01L 2924/0002 : Not covered by any one of groups H01L24/00, H01L24/00 and H01L2224/00

Abstract

Methods are provided for the analysis and determination of the nature of single nucleic acid polymorphisms (SNPs) in a genetic target. In one method of this invention, the nature of the SNPs in the genetic target is determined by the steps of providing a plurality of hybridization complexes arrayed on a plurality of test sites on an electronically bioactive microchip, where the hybridization complex includes at least a nucleic acid target containing a SNP, a stabilizer probe having a sequence complementary to the target sequence and/or reporter probe, and a reporter probe having a selected sequence complementary to either the stabilizer or the same target sequence strand wherein a selected sequence of the reporter includes either a wild type nucleotide or a nucleotide corresponding to the SNP of the target. In accordance with the invention, the stabilizer, reporter and target amplicons are hybridized using electronic assistance of the microchip system such that base-stacking energies are utilized in discerning among other identifying indicators, the presence of wild type or polymorphism sequence. Applications include disease diagnostics, such as for the identification of polymorphisms in structural genes, regulatory regions, antibiotic or chemotherapeutic resistance conferring regions, or for SNPs associated with speciation or used for determination of genetic linkage.

Claims

1. A method for determining the presence of a specific sequence in at least one genetic locus of one or more target nucleic acids of interest in at least one sample of interest using an electronically addressable microchip comprising a plurality of addressable capture sites with associated electrodes, the method comprising, for each genetic locus:

(a) contacting a single stranded target nucleic acid of interest with at least one stabilizer oligonucleotide, wherein the stabilizer oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein at least one terminus of the stabilizer oligonucleotide hybridizes to the target nucleic acid of interest at or adjacent to a region of expected variance in the specific sequence;

(b) contacting the target nucleic acid of interest with at least one reporter oligonucleotide, wherein the reporter oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein a terminus of the reporter oligonucleotide further fully hybridizes to the target nucleic acid at a position contiguous with the terminus of the stabilizer oligonucleotide if the specific sequence is present, further wherein the contiguously hybridized termini of the reporter and stabilizer form a stabilizing base-stacking interaction if the specific sequence is present;

(c) electronically addressing the target nucleic acid to at least one capture site on the bioelectronic microchip, wherein the target nucleic acid is captured at the capture site by a capturing means;

(d) after (a), (b), and (c), subjecting the captured target nucleic acid and hybridized stabilizer and reporter oligonucleotides to destabilizing conditions, wherein the destabilizing conditions are sufficient to cause the reporter oligonucleotide to dissociate in the absence of the stabilizing base-stacking interaction; and

(e) detecting the hybridization of the reporter oligonucleotide to the target nucleic acid after (d), whereby the presence of the specific sequence in the target nucleic acid is determined.

2. The method of claim 1 wherein step (c) occurs before (a) and (b).

3. The method of claim 1 wherein step (a) occurs before (c).

4. The method of claim 1 wherein step (b) occurs before (c).

5. The method of claim 1 wherein steps (a) and (c) occur at the same time.

6. The method of claim 1 wherein steps (b) and (c) occur at the same time.

7. The method of claim 1 wherein steps (a) and (b) occur at the same time.

8. The method of claim 1 wherein steps (a), (b), and (c) occur at the same time.

9. The method of claim 1 wherein the capturing means is an attachment moiety on a terminal portion of the target nucleic acid.

10. The method of claim 9 wherein the attachment moiety on the target nucleic acid is biotin, and further wherein a biotin-binding moiety is present at the capture site.

11. The method of claim 1 wherein the capturing means is an attachment moiety on a terminal portion of the stabilizer, wherein step (a) occurs prior to or at the same time as step (c).

12. The method of claim 11 wherein the attachment moiety on the stabilizer is biotin, and further wherein a biotin-binding moiety is present at the capture site.

13. The method of claim 1 wherein the capturing means is by the hybridization of a portion of the target nucleic acid a capture probe previously attached to said capture site, wherein the capture probe comprises a sequence complementary to a portion of the target nucleic acid.

14. The method of claim 1 wherein the presence of at least two specific sequences in at least two genetic loci of a target nucleic acid of interest from a sample are determined.

15. The method of claim 1 wherein the presence of at least two specific sequences in at least two genetic loci of at least two target nucleic acids of interest from a sample are determined.

16. The method of claim 1 wherein the presence of at least one specific sequence in at least one genetic loci for of a target nucleic acid of interest is determined for at least two samples on the same bioelectronic microchip, wherein step (c) is performed individually for each sample on the bioelectronic microchip.

17. The method of claim 16 wherein step (e) is performed sequentially for each sample on the bioelectronic microchip.

18. The method of claim 16 wherein step (e) is performed at the same time for two or more samples on the bioelectronic microchip.

19. The method of claim 1 wherein the expected variance in the specific sequence is a substitution mutation, wherein one or more nucleotides are replaced with nucleotides other than those of the specific sequence.

20. The method of claim 1 wherein the expected variance in the specific sequence is a deletion mutation, wherein one or more nucleotides are missing from the specific sequence.

21. The method of claim 1 wherein the expected variance in the specific sequence is an insertion mutation, wherein one or more nucleotides are inserted into the specific sequence.

22. The method of claim 1 further comprising a preparatory step of amplifying one or more target nucleic acid sequences from the nucleic acids of a sample, wherein the amplicons become the target nucleic acids.

23. The method of claim 22 wherein at least one of the primers used in the amplification step is biotinylated.

24. The method of claim 22 wherein the amplification step utilizes a linear amplification protocol, wherein the amplification products are not utilized as amplification templates in a feed-back loop reaction.

25. The method of claim 22 wherein the amplification step utilizes an exponential amplification protocol.

26. The method of claim 22 wherein the amplification step produces single stranded amplicons, which are then utilized as the single stranded target nucleic acids.

27. The method of claim 22 wherein the amplification step produces double stranded amplicons, further comprising a step of subjecting the amplicons to denaturing conditions to form single stranded target nucleic acids.

28. The method of claim 22 wherein the amplification step utilizes at least one primer anchored to a capture site on the bioelectronic microchip, wherein anchored amplicons are formed for use as target nucleic acids.

29. The method of claim 22 wherein the amplification step is by an amplification method selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification, T7 mediated amplification, T3 mediated amplification, and SP6 mediated amplification.

30. The method of claim 1 further comprising a step of subjecting the target nucleic acids of the sample to denaturing conditions to form single stranded target nucleic acids.

31. The method of claim 1 wherein the detection of the hybridization of the reporter oligonucleotide is by the detection of a labeling moiety on the reporter oligonucleotide selected from the group consisting of fluorescent moieties, bioluminescent moieties, chemiluminescent moieties, and colorigenic moieties.

32. The method of claim 31 wherein the labeling moiety is a fluorescent moiety selected from the group consisting of fluorescein derivatives, Bodipy™

dyes, rhodamine derivatives, Lucifer Yellow derivatives, and cyanine (Cy) dyes.

33. The method of claim 1 wherein the destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, making adjustments in pH, and combinations thereof.

34. The method of claim 1 wherein the destabilizing conditions are created by performing electronic biasing.

35. The method of claim 1 wherein the destabilizing conditions are created by making temperature adjustments.

36. The method of claim 1 wherein the destabilizing conditions are created by making ionic strength adjustments.

37. The method of claim 1 wherein the destabilizing conditions are created by making adjustments in pH.

38. A method for determining the presence of at least a specific sequence in at least one genetic locus of one or more target nucleic acids of interest in at least one sample of interest using an electronically addressable microchip comprising a plurality of addressable capture sites with associated electrodes, the method comprising, for each genetic locus:

(a) contacting a single stranded target nucleic acid of interest with at least one stabilizer oligonucleotide, wherein the stabilizer oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein at least one terminus of the stabilizer oligonucleotide hybridizes to the target nucleic acid of interest at or adjacent to a first region of expected variance in the specific sequence;

(b) contacting the target nucleic acid of interest with

(i) a first reporter oligonucleotide, wherein the first reporter oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein a first terminus of the first reporter oligonucleotide further fully hybridizes to the target nucleic acid at a position contiguous with the terminus of the stabilizer oligonucleotide if the full specific sequence is present, further wherein the contiguously hybridized termini of the first reporter and stabilizer form a stabilizing base-stacking interaction if the full specific sequence is present, and further wherein a second terminus of the first reporter oligonucleotide hybridizes to the target nucleic acid of interest at or adjacent to a second region of expected variance in the specific sequence;

(ii) further contacting the target nucleic acid with at least a second reporter oligonucleotide, wherein the second reporter oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein a terminus of the second reporter oligonucleotide further fully hybridizes to the target nucleic acid at a position contiguous with the position on the target nucleic acid to which the second terminus of the first reporter oligonucleotide hybridizes if the full specific sequence is present, further wherein the contiguously hybridized termini of the first and second reporter oligonucleotides form a stabilizing base-stacking interaction if the full specific sequence is present;

(c) electronically addressing the target nucleic acid to at least one capture site on the bioelectronic microchip, wherein the target nucleic acid is captured at the capture site by a capturing means;

(d) after (a), (b), and (c), subjecting the captured target nucleic acid and hybridized stabilizer and reporter oligonucleotides to destabilizing conditions, wherein the destabilizing conditions are sufficient to cause the first and/or second reporter oligonucleotide to dissociate in the absence of the stabilizing base-stacking interaction; and

(e) detecting the hybridization of the first and second reporter oligonucleotide to the target nucleic acid after (d), whereby the presence of the full or partial specific sequence in the target nucleic acid is determined.

39. The method of claim 38 wherein the first and second reporter oligonucleotides are differently labeled with first and second labeling moieties.

40. The method of claim 38 wherein step (c) occurs before (a) and (b).

41. The method of claim 38 wherein step (a) occurs before (c).

42. The method of claim 38 wherein step (b) occurs before (c).

43. The method of claim 38 wherein steps (a) and (c) occur at the same time.

44. The method of claim 38 wherein steps (b) and (c) occur at the same time.

45. The method of claim 38 wherein steps (a) and (b) occur at the same time.

46. The method of claim 38 wherein steps (a), (b), and (c) occur at the same time.

47. The method of claim 38 wherein the capturing means is an attachment moiety on a terminal portion of the target nucleic acid.

48. The method of claim 47 wherein the attachment moiety on the target nucleic acid is biotin, and further wherein a biotin-binding moiety is present at the capture site.

49. The method of claim 38 wherein the capturing means is an attachment moiety on a terminal portion of the stabilizer, wherein step (a) occurs prior to or at the same time as step (c).

50. The method of claim 49 wherein the attachment moiety on the stabilizer is biotin, and further wherein a biotin-binding moiety is present at the capture site.

51. The method of claim 38 wherein the capturing means is by the hybridization of a portion of the target nucleic acid a capture probe previously attached to said capture site, wherein the capture probe comprises a sequence complementary to a portion of the target nucleic acid.

52. The method of claim 38 wherein the presence of at least two specific sequences in at least two genetic loci of a target nucleic acid of interest from a sample are determined.

53. The method of claim 38 wherein the presence of at least two specific sequences in at least two genetic loci of at least two target nucleic acids of interest from a sample are determined.

54. The method of claim 38 wherein the presence of at least one specific sequence in at least one genetic loci for of a target nucleic acid of interest is determined for at least two samples on the same bioelectronic microchip, wherein step (c) is performed individually for each sample on the bioelectronic microchip.

55. The method of claim 54 wherein step (e) is performed sequentially for each sample on the bioelectronic microchip.

56. The method of claim 54 wherein step (e) is performed at the same time for two or more samples on the bioelectronic microchip.

57. The method of claim 38 wherein one of the expected variances in the specific sequence is a substitution mutation, wherein one or more nucleotides are replaced with nucleotides other than those of the specific sequence.

58. The method of claim 38 wherein one of the expected variances in the specific sequence is a deletion mutation, wherein one or more nucleotides are missing from the specific sequence.

59. The method of claim 38 wherein one of the expected variances in the specific sequence is an insertion mutation, wherein one or more nucleotides are inserted into the specific sequence.

60. The method of claim 38 further comprising a preparatory step of amplifying one or more target nucleic acid sequences from the nucleic acids of a sample, wherein the amplicons become the target nucleic acids.

61. The method of claim 60 wherein at least one of the primers used in the amplification step is biotinylated.

62. The method of claim 60 wherein the amplification step utilizes a linear amplification protocol, wherein the amplification products are not utilized as amplification templates in a feed-back loop reaction.

63. The method of claim 60 wherein the amplification step utilizes an exponential amplification protocol.

64. The method of claim 60 wherein the amplification step produces single stranded amplicons, which are then utilized as the single stranded target nucleic acids.

65. The method of claim 60 wherein the amplification step produces double stranded amplicons, further comprising a step of subjecting the amplicons to denaturing conditions to form single stranded target nucleic acids.

66. The method of claim 60 wherein the amplification step utilizes at least one primer anchored to a capture site on the bioelectronic microchip, wherein anchored amplicons are formed for use as target nucleic acids.

67. The method of claim 60 wherein the amplification step is by an amplification method selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification, T7 mediated amplification, T3 mediated amplification, and SP6 mediated amplification.

68. The method of claim 38 further comprising a step of subjecting the target nucleic acids of the sample to denaturing conditions to form single stranded target nucleic acids.

69. The method of claim 38 wherein the detection of the hybridization of the reporter oligonucleotide is by the detection of a labeling moiety on the reporter oligonucleotide selected from the group consisting of fluorescent moieties, bioluminescent moieties, chemiluminescent moieties, and colorigenic moieties.

70. The method of claim 69 wherein the labeling moiety is a fluorescent moiety selected from the group consisting of fluorescein derivatives, Bodipy™

dyes, rhodamine derivatives, Lucifer Yellow derivatives, and cyanine (Cy) dyes.

71. The method of claim 38 wherein the destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, making adjustments in pH, and combinations thereof.

72. The method of claim 38 wherein the destabilizing conditions are created by performing electronic biasing.

73. The method of claim 38 wherein the destabilizing conditions are created by making temperature adjustments.

74. The method of claim 38 wherein the destabilizing conditions are created by making ionic strength adjustments.

75. The method of claim 38 wherein the destabilizing conditions are created by making adjustments in pH.

76. A method for determining the presence of a specific sequence in at least one genetic locus of one or more target nucleic acids of interest in at least one sample of interest using an electronically addressable microchip comprising a plurality of addressable capture sites with associated electrodes, the method comprising, for each genetic locus,:

(a) contacting a single stranded target nucleic acid of interest with first and second stabilizer oligonucleotides, wherein the first stabilizer oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein at least one terminus of the first stabilizer oligonucleotide hybridizes to the target nucleic acid of interest at or adjacent to a first region of expected variance in the first specific sequence, and further wherein the second stabilizer oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest, wherein at least one terminus of the second stabilizer oligonucleotide hybridizes to the target nucleic acid of interest at or adjacent to a second region of expected variance in the first specific sequence;

(b) contacting the target nucleic acid of interest with at least one reporter oligonucleotide, wherein the reporter oligonucleotide comprises a sequence complementary to at least a portion of the target nucleic acid of interest between the positions on the target nucleic acid at which the first and second stabilizer oligonucleotides hybridize, wherein a first terminus of at least one reporter oligonucleotide further fully hybridizes to the target nucleic acid at a position contiguous with the terminus of the first stabilizer oligonucleotide if the full specific sequence is present, further wherein the contiguously hybridized first termini of the at reporter and the termini of the first stabilizer form a stabilizing base-stacking interaction if the full specific sequence is present, and further wherein a second terminus of at least one reporter oligonucleotide hybridizes to the target nucleic acid of interest at a position contiguous with the terminus of the second stabilizer oligonucleotide if the full specific sequence is present, further wherein the contiguously hybridized second termini of the reporter and the termini of the second stabilizer form a stabilizing base-stacking interaction if the full specific sequence is present;

(c) electronically addressing the target nucleic acid to at least one capture site on the bioelectronic microchip, wherein the target nucleic acid is captured at the capture site by a capturing means;

(d) after (a), (b), and (c), subjecting the captured target nucleic acid and hybridized stabilizer and reporter oligonucleotides to destabilizing conditions, wherein the destabilizing conditions are sufficient to cause the reporter oligonucleotide(s) to dissociate in the absence of the stabilizing base-stacking interactions with the first and second stabilizers; and

(e) detecting the hybridization of the reporter oligonucleotide(s) to the target nucleic acid after (d), whereby the presence of the specific sequence in the target nucleic acid is determined.

77. The method of claim 76 wherein a single reporter oligonucleotide hybridizes to a position on the target nucleic acid which is contiguous with both a terminus of the first stabilizer oligonucleotide and a terminus of the second stabilizer oligonucleotide, if the specific sequence is present.

78. The method of claim 76 wherein at least two reporter oligonucleotides hybridize to a position on the target nucleic acid which is contiguous with both a terminus of the first stabilizer oligonucleotide and a terminus of the second stabilizer oligonucleotide, if the specific sequence is present, further wherein the termini of the reporter oligonucleotides hybridize to positions on the target nucleic acids which are contiguous with each other if the specific sequence is present, further forming stabilizing base-stacking interactions between the termini of the reporter oligonucleotides if the full specific sequence is present.

79. The method of claim 78 wherein at least a first and second reporter oligonucleotides are differently labeled with first and second labeling moieties.

80. The method of claim 76 wherein step (c) occurs before (a) and (b).

81. The method of claim 76 wherein step (a) occurs before (c).

82. The method of claim 76 wherein step (b) occurs before (c).

83. The method of claim 76 wherein steps (a) and (c) occur at the same time.

84. The method of claim 76 wherein steps (b) and (c) occur at the same time.

85. The method of claim 76 wherein steps (a) and (b) occur at the same time.

86. The method of claim 76 wherein steps (a), (b), and (c) occur at the same time.

87. The method of claim 76 wherein the capturing means is an attachment moiety on a terminal portion of the target nucleic acid.

88. The method of claim 87 wherein the attachment moiety on the target nucleic acid is biotin, and further wherein a biotin-binding moiety is present at the capture site.

89. The method of claim 76 wherein the capturing means is an attachment moiety on a terminal portion of the stabilizer, wherein step (a) occurs prior to or at the same time as step (c).

90. The method of claim 89 wherein the attachment moiety on the stabilizer is biotin, and further wherein a biotin-binding moiety is present at the capture site.

91. The method of claim 76 wherein the capturing means is by the hybridization of a portion of the target nucleic acid a capture probe previously attached to said capture site, wherein the capture probe comprises a sequence complementary to a portion of the target nucleic acid.

92. The method of claim 76 wherein the presence of at least two specific sequences in at least two genetic loci of a target nucleic acid of interest from a sample are determined.

93. The method of claim 76 wherein the presence of at least two specific sequences in at least two genetic loci of at least two target nucleic acids of interest from a sample are determined.

94. The method of claim 76 wherein the presence of at least one specific sequence in at least one genetic loci for of a target nucleic acid of interest is determined for at least two samples on the same bioelectronic microchip, wherein step (c) is performed individually for each sample on the bioelectronic microchip.

95. The method of claim 94 wherein step (e) is performed sequentially for each sample on the bioelectronic microchip.

96. The method of claim 94 wherein step (e) is performed at the same time for two or more samples on the bioelectronic microchip.

97. The method of claim 76 wherein one of the expected variances in the specific sequence is a substitution mutation, wherein one or more nucleotides are replaced with nucleotides other than those of the specific sequence.

98. The method of claim 76 wherein one of the expected variances in the specific sequence is a deletion mutation, wherein one or more nucleotides are missing from the specific sequence.

99. The method of claim 76 wherein one of the expected variances in the specific sequence is an insertion mutation, wherein one or more nucleotides are inserted into the specific sequence.

100. The method of claim 76 further comprising a preparatory step of amplifying one or more target nucleic acid sequences from the nucleic acids of a sample, wherein the amplicons become the target nucleic acids.

101. The method of claim 100 wherein at least one of the primers used in the amplification step is biotinylated.

102. The method of claim 100 wherein the amplification step utilizes a linear amplification protocol, wherein the amplification products are not utilized as amplification templates in a feed-back loop reaction.

103. The method of claim 100 wherein the amplification step utilizes an exponential amplification protocol.

104. The method of claim 100 wherein the amplification step produces single stranded amplicons, which are then utilized as the single stranded target nucleic acids.

105. The method of claim 100 wherein the amplification step produces double stranded amplicons, further comprising a step of subjecting the amplicons to denaturing conditions to form single stranded target nucleic acids.

106. The method of claim 100 wherein the amplification step utilizes at least one primer anchored to a capture site on the bioelectronic microchip, wherein anchored amplicons are formed for use as target nucleic acids.

107. The method of claim 100 wherein the amplification step is by an amplification method selected from the group consisting of polymerase chain reaction (PCR), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), rolling circle amplification, T7 mediated amplification, T3 mediated amplification, and SP6 mediated amplification.

108. The method of claim 76 further comprising a step of subjecting the target nucleic acids of the sample to denaturing conditions to form single stranded target nucleic acids.

109. The method of claim 76 wherein the detection of the hybridization of the reporter oligonucleotide is by the detection of a labeling moiety on the reporter oligonucleotide selected from the group consisting of fluorescent moieties, bioluminescent moieties, chemiluminescent moieties, and colorigenic moieties.

110. The method of claim 109 wherein the labeling moiety is a fluorescent moiety selected from the group consisting of fluorescein derivatives, Bodipy™

dyes, rhodamine derivatives, Lucifer Yellow derivatives, and cyanine (Cy) dyes.

111. The method of claim 76 wherein the destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, making adjustments in pH, and combinations thereof.

112. The method of claim 76 wherein the destabilizing conditions are created by performing electronic biasing.

113. The method of claim 76 wherein the destabilizing conditions are created by making temperature adjustments.

114. The method of claim 76 wherein the destabilizing conditions are created by making ionic strength adjustments.

115. The method of claim 76 wherein the destabilizing conditions are created by making adjustments in pH.

116. A method for determining the presence of at least two specific sequences in at least one pair of genetic loci of at least two target nucleic acids of interest in at least one sample of interest using an electronically addressable microchip comprising a plurality of addressable capture sites with associated electrodes, the method comprising, for each pair of genetic loci,:

(a) contacting a first and second single stranded target nucleic acids of interest with a stabilizer-bridge oligonucleotide, wherein the stabilizer-bridge oligonucleotide comprises a first sequence complementary to at least a portion of the first target nucleic acid of interest, wherein at least one terminus of the first target nucleic acid hybridizes to the stabilizer-bridge oligonucleotide at a region of expected variance in a first specific sequence, and wherein the stabilizer-bridge oligonucleotide comprises a second sequence complementary to at least a portion of the second target nucleic acid of interest, wherein at least one terminus of the second target nucleic acid hybridizes to the stabilizer-bridge oligonucleotide at a region of expected variance in a second specific sequence, whereby a bridged hybridized complex is formed;

(b) contacting the bridged hybridized complex with least one reporter oligonucleotide, wherein the reporter oligonucleotide comprises a sequence complementary to at least a portion of the stabilizer-bridge oligonucleotide between the positions on the stabilizer-bridge oligonucleotide at which the first and second target nucleic acids hybridize, wherein a first terminus of at least one reporter oligonucleotide further fully hybridizes to the stabilizer-bridge at a position contiguous with the terminus of first target nucleic acid if the first specific sequence is present, further wherein the contiguously hybridized first termini of at least one reporter and the termini of the first target nucleic acid form a stabilizing base-stacking interaction if the first specific sequence is present, and further wherein a second terminus of at least one reporter oligonucleotide hybridizes to the stabilizer-bridge at a position contiguous with the terminus of the second target nucleotide if the second specific sequence is present, further wherein the contiguously hybridized second termini of the reporter and the termini of the second target nucleic acid form a stabilizing base-stacking interaction if the second specific sequence is present;

(c) electronically addressing the first and second target nucleic acids to at least one capture site on the bioelectronic microchip, wherein the target nucleic acids are captured at the capture site by a capturing means;

(d) after (a), (b), and (c), subjecting the captured target nucleic acids and hybridized stabilizer-bridge and reporter oligonucleotides to destabilizing conditions, wherein the destabilizing conditions are sufficient to cause the reporter oligonucleotide(s) to dissociate in the absence of the stabilizing base-stacking interactions; and

(e) detecting the hybridization of the reporter oligonucleotide to the target nucleic acid after (d), whereby the presence of the first and second specific sequence in the target nucleic acids is determined.

117. The method of claim 116 wherein step (c) occurs before (a) and (b).

118. The method of claim 116 wherein step (b) occurs before (c).

119. The method of claim 116 wherein the capturing means is an attachment moiety on a terminal portion of the target nucleic acid.

120. The method of claim 116 wherein the capturing means is by the hybridization of a portion of the target nucleic acid a capture probe previously attached to said capture site, wherein the capture probe comprises a sequence complementary to a portion of the target nucleic acid.

121. The method of claim 116 wherein the presence of at least one specific sequence in at least one genetic loci for of a target nucleic acid of interest is determined for at least two samples on the same bioelectronic microchip, wherein step (c) is performed individually for each sample on the bioelectronic microchip.

122. The method of claim 116 further comprising a preparatory step of amplifying one or more target nucleic acid sequences from the nucleic acids of a sample, wherein the amplicons become the target nucleic acids.

123. The method of claim 122 wherein the amplification step utilizes at least one primer anchored to a capture site on the bioelectronic microchip, wherein anchored amplicons are formed for use as target nucleic acids.

124. The method of claim 116 wherein the detection of the hybridization of the reporter oligonucleotide is by the detection of a labeling moiety on the reporter oligonucleotide selected from the group consisting of fluorescent moieties, bioluminescent moieties, chemiluminescent moieties, and colorigenic moieties.

125. The method of claim 116 wherein the destabilizing conditions are created by methods selected from the group consisting of performing electronic biasing, making temperature adjustments, making ionic strength adjustments, making adjustments in pH, and combinations thereof.