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 an electronic 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, said capture probe and reporter probe being selected such that the base-stacking between the juxtaposed termini is disrupted within a discordant complex, and wherein the electronic test device subjects one or more of the assay components to an electric field.
- 2. The method of claim 1 for determining the nature of a genetic variant
wherein the electronic test device includes a plurality of test sites.
- 3. The method of claim 2 for determining the nature of a genetic variant
wherein a test site include a microelectrode.
- 4. The method of claim 3 for determining the nature of a genetic variant
wherein a test site includes a permeation layer disposed adjacent the microelectrode.
- 5. The method of claim 1 for determining the nature of a genetic variant
wherein two different hybridization complexes are distinguished at a single site with distinguishable reporters.
- 6. 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.
- 7. A kit for performing the method of claim 1 for determining the nature of a genetic variant including said reporter probe and said capture probe.
- 8. The method of claim 1 for determining the nature of a genetic variant
wherein the variant constitutes the number of repetitive DNA sequences.
- 9. The method of claim 1 wherein the genetic variants include deletions.
- 10. The method of claim 1 wherein the genetic variants include insertions.
- 11. The method of claim 1 wherein the genetic variants include transitions.
- 12. The method of claim 1 wherein the genetic variants include transversions.
- 13. The method of claim 1 wherein the genetic variants include single nucleotide variants.
- 14. The method of claim 1 wherein the genetic variants include mutations.
- 15. The method of claim 1 wherein the genetic variants include point mutations.
- 16. The method of claim 1 wherein the genetic variant affects a single base.
- 17. The method of claim 5 wherein the genetic variant affects more than a single base.
- 18. 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.
- 19. 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.
- 20. The method of claim 1 wherein the plurality of hybridization complex assays form an array.
- 21. The method of claim 20 wherein the location of the test site in the array indicates the nature of the genetic variant.
- 22. 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.
- 23. 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.
- 24. The method of claim 1 wherein the stability is enhanced by juxtaposed terminal nucleotides of the reporter and capture being contiguous to permit base stacking.
- 25. 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.
- 26. The method of claim 1 wherein the base stacked pair is 5′GpC3′.
- 27. The method of claim 1 wherein the base stacked pair is 5′TpA3′.
- 28. The method of claim 1 further including modifications of terminal nucleotides which increase base stacking.
- 29. The method of claim 1 wherein stability is enhanced with ligation techniques.
- 30. The method of claim 1 wherein discordance includes a genetic variant.
- 31. The method of claim 30 wherein the genetic variant is in the target sequence.
- 32. The method of claim 31 wherein the genetic variant in the target sequence is selected from the group comprising: insertions, deletions, transitions and transversions.
- 33. The method of claim 1 wherein discordance includes genetic variations which are greater than a single nucleotide.
- 34. The method of claim 1 wherein the number of loci analyzed at one time is one.
- 35. The method of claim 1 wherein the number of loci analyzed at one time is three.
- 36. The method of claim 1 wherein the number of loci analyzed at one time is less than five.
- 37. The method of claim 1 wherein the number of loci analyzed at one time is less than ten.
- 38. The method of claim 1 wherein the number of loci analyzed at one time is greater than ten.
- 39. The method of claim 1 wherein the number of loci analyzed at one time is less than one hundred.
- 40. The method of claim 1 wherein the number of loci analyzed at one time is greater than one hundred.
- 41. The method of claim 1 wherein the disruption of the juxtaposed terminii results in destabilization of the hybridization complex.
- 42. The method of claim 1 wherein the hybridization stability is determined, at least in part, by electronic stringency (ESC) control.
- 43. The method of claim 1 wherein the hybridization stability is determined, at least in part, by thermal regulation of stringency.
- 44. The method of claim 1 wherein the hybridization stability is determined, at least in part, by chemical regulation of stringency.
- 45. The method of claim 1 wherein the hybridization stability is determined, at least in part, by electronic stringency (ESC) and thermal control.
- 46. The method of claim 1 wherein the hybridization stability is determined, at least in part, by electronic stringency (ESC) and chemical control.
- 47. The method of claim 1 wherein the hybridization stability is determined, at least in part, by electronic stringency (ESC), thermal and chemical control.
- 48. The method of claim 1 wherein the electric field control includes electronic addressing of the target.
- 49. The method of claim 1 wherein the electric field control includes electronic addressing of the capture.
- 50. The method of claim 1 wherein the electric field control includes electronic addressing of the reporter.
- 51. The method of claim 1 wherein the electric field control includes electronic control of hybridizing the target in the complex.
- 52. The method of claim 1 wherein the electric field control includes electronic control of hybridizing the capture in the complex.
- 53. The method of claim 1 wherein the electric field control includes electronic control of hybridizing the reporter in the complex.
- 54. The method of claim 1 wherein the electric field control includes electronic control of formation of the hybridization complex.
- 55. The method of claim 1 further including electronic stringency conditions during the hybridizations of capture probe with the nucleic acid target.
- 56. The method of claim 55 wherein initial hybridization step occurs in 10 minutes or less.
- 57. The method of claim 55 wherein initial hybridization step occurs in 5 minutes or less.
- 58. The method of claim 55 wherein initial hybridization step occurs in 1 minute or less.
- 59. The method of claim 1 further including electronic stringency conditions during the hybridization of the reporter probe with the capture probe nucleic acid target hybridization complex.
- 60. The method of claim 1 wherein the hybridization complex is labeled.
- 61. The method of claim 60 further including the step of detecting the amounts of labeled hybridization complex at the test sites.
- 62. The method of claim 61 wherein the detecting is imaging.
- 63. The method of claim 62 wherein the imaging is optical imaging.
- 64. The method of claim 62 wherein the imaging is electronic imaging.
- 65. The method of claim 62 wherein the imaging is CCD imaging.
- 66. The method of claim 62 wherein the imaging is integrated optical imaging.
- 67. The method of claim 62 wherein the imaging detection is quantified.
- 68. The method of claim 1 further including a statistical analysis step.
- 69. The method of claim 60 wherein the labeled portion of complex is the target.
- 70. The method of claim 60 wherein the labeled portion of complex is the capture.
- 71. The method of claim 60 wherein the labeled portion of complex is the reporter.
- 72. The method of claim 60 wherein the labeling is by fluorescent labeling.
- 73. The method of claim 72 wherein the fluorescent labeling is with Bodipy Texas Red.
- 74. The method of claim 72 wherein the fluorescent labeling is with Bodipy 630/650.
- 75. The method of claim 72 wherein the fluorescent labeling is with Lucifer Yellow.
- 76. The method of claim 60 wherein the labeling is by colormetric labeling.
- 77. The method of claim 60 wherein the labeling is by chemiluminescent labeling.
- 78. The method of claim 1 further including energy transfer between molecules in the hybridization complex.
- 79. The method of claim 1 further including fluorescent perturbation analysis.
- 80. The method of claim 78 wherein the energy transfer includes quenching.
- 81. The method of claim 1 further including a redundant assay.
- 82. The method of claim 81 further including the step of repeating the redundant assay until a statistically significant result is obtained.
- 83. The method of claim 81 wherein the redundant assay includes multiple arrays.
- 84. The method of claim 1 wherein the target DNA is purified.
- 85. The method of claim 1 wherein the target is unamplified.
- 86. The method of claim 1 wherein the target is amplified.
- 87. 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.
- 88. The method of claim 87 wherein the reduction increases the statistical significance of results.
- 89. The method of claim 1 wherein the target material constitutes homozygous allele for a locus.
- 90. The method of claim 1 wherein the target material constitutes heterozygous allele for a locus.
- 91. The method of claim 1 wherein the target material constitutes more than one allele per locus for a mixed sample.
- 92. The method of claim 91 wherein the mixed sample further includes sample from more than one individual.
- 93. The method of claim 92 wherein the mixed sample further includes tumor tissue mixed with normal tissue.
- 94. 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 by varying at least the position of the juxtaposed termini relative to the genetic variant, such that the base-stacking stabilization between the juxtaposed termini is determined.
- 95. The method of claim 94 such that the base-stacking stabilization between the juxtaposed termini is maximized within a concordant hybridization complex.
- 96. The method of claim 94 such that the base-stacking stabilization between the juxtaposed termini is minimized within a discordant hybridization complex.
- 97. The method of claim 94 such that the differential base-stacking stabilization between the juxtaposed termini is maximized for a discordant and concordant hybridization complex.
- 98. The method of claim 94 wherein the genetic variant is immediately adjacent the termini.
- 99. The method of claim 94 wherein the genetic variant is one base removed from the termini.
- 100. The method of claim 94 wherein the genetic variant is two or more bases removed from the termini.
- 101. A kit for performing the method of claim 94 including said reporter probe and said capture probe.
- 102. The method of claim 94 wherein the base-stacking is maximized by selection of the terminal bases.
- 103. The method of claim 94 wherein the base-stacking is maximized by selection of the position of the genetic variant relative to the juxtaposed termini.
- 104. The method of claim 94 wherein the base-stacking is maximized by selection of complementary sequences.
- 105. The method of claim 94 wherein the base-stacking is maximized by selection of the terminal bases and complementary sequences.
- 106. The method of claim 94 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.
- 107. The method of claim 94 for determining the nature of a genetic variant wherein the test device includes a plurality of test sites.
- 108. The method of claim 107 for determining the nature of a genetic variant wherein a test site includes a permeation layer disposed adjacent the microelectrode.
- 109. The method of claim 94 for determining the nature of a genetic variant wherein two different hybridization complexes are distinguished at a single site with distinguishable reporters.
- 110. The method of claim 94 for determining the nature of a genetic variant wherein two different hybridization complexes are distinguished at multiple sites with different reporters.
- 111. A kit for performing the method of claim 94 for determining the nature of a genetic variant including said reporter probe and said capture probe.
- 112. The method of claim 94 for determining the nature of a genetic variant wherein the variant constitutes the number of repetitive DNA sequences.
- 113. The method of claim 94 wherein the genetic variants include deletions.
- 114. The method of claim 94 wherein the genetic variants include insertions.
- 115. The method of claim 94 wherein the genetic variants include transitions.
- 116. The method of claim 94 wherein the genetic variants include transversions.
- 117. The method of claim 94 wherein the genetic variants include single nucleotide variants.
- 118. The method of claim 94 wherein the genetic variants include mutations.
- 119. The method of claim 94 wherein the genetic variants include point mutations.
- 120. The method of claim 94 wherein the genetic variant affects a single base.
- 121. The method of claim 119 wherein the genetic variant affects more than a single base.
- 122. The method of claim 94 wherein multiple test locations indicate the nature of the target sequence by the detection of genetic variants in the target sequence.
- 123. The method of claim 94 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.
- 124. The method of claim 94 wherein the plurality of hybridization complex assays form an array.
- 125. The method of claim 124 wherein the location of the test site in the array indicates the nature of the genetic variant.
- 126. The method of claim 94 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.
- 127. The method of claim 94 wherein the stability is enhanced by complementary match of bases in the hybridization complex including the capture, reporter and target.
- 128. The method of claim 94 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.
- 129. The method of claim 94 wherein the base stacked pair is 5′GpC3′.
- 130. The method of claim 94 wherein the base stacked pair is 5′TpA3′.
- 131. The method of claim 94 further including modifications of terminal nucleotides which increase base stacking.
- 132. The method of claim 94 wherein stability is enhanced with ligation techniques.
- 133. The method of claim 94 wherein discordance includes a genetic variant.
- 134. The method of claim 133 wherein the genetic variant is in the target sequence.
- 135. The method of claim 133 wherein the genetic variant in the target sequence is selected from the group comprising: insertions, deletions, transitions and transversions.
- 136. The method of claim 94 wherein discordance includes genetic variations which are greater than a single nucleotide.
- 137. The method of claim 94 wherein the number of loci analyzed at one time is one.
- 138. The method of claim 94 wherein the number of loci analyzed at one time is three.
- 139. The method of claim 94 wherein the number of loci analyzed at one time is less than five.
- 140. The method of claim 94 wherein the number of loci analyzed at one time is less than ten.
- 141. The method of claim 94 wherein the number of loci analyzed at one time is greater than ten.
- 142. The method of claim 94 wherein the number of loci analyzed at one time is less than one hundred.
- 143. The method of claim 94 wherein the number of loci analyzed at one time is greater than one hundred.
- 144. The method of claim 94 wherein the disruption of the juxtaposed termini results in destabilization of the hybridization complex.
- 145. The method of claim 94 wherein the disruption of complementary sequences results in destabilization of the hybridization complex.
- 146. The method of claim 94 wherein the disruption of the juxtaposed termini and complementary sequences results in destabilization of the hybridization complex.
- 147. The method of claim 94 wherein the hybridization stability is determined, at least in part, by thermal regulation of stringency.
- 148. The method of claim 94 wherein the hybridization stability is determined, at least in part, by chemical regulation of stringency.
- 149. The method of claim 94 wherein initial hybridization step occurs in 10 minutes or less.
- 150. The method of claim 94 wherein initial hybridization step occurs in 5 minutes or less.
- 151. The method of claim 94 wherein initial hybridization step occurs in 1 minute or less.
- 152. The method of claim 94 wherein the hybridization complex is labeled.
- 153. The method of claim 152 further including the step of detecting the amounts of labeled hybridization complex at the test sites.
- 154. The method of claim 153 wherein the detecting is imaging.
- 155. The method of claim 154 wherein the imaging is optical imaging.
- 156. The method of claim 154 wherein the imaging is electronic imaging.
- 157. The method of claim 154 wherein the imaging is CCD imaging.
- 158. The method of claim 154 wherein the imaging is integrated optical imaging.
- 159. The method of claim 154 wherein the imaging detection is quantified.
- 160. The method of claim 94 further including a statistical analysis step.
- 161. The method of claim 94 wherein the labeled portion of complex is the target.
- 162. The method of claim 94 wherein the labeled portion of complex is the capture.
- 163. The method of claim 94 wherein the labeled portion of complex is the reporter.
- 164. The method of claim 94 wherein the labeling is by fluorescent labeling.
- 165. The met hod of claim 164 wherein the fluorescent labeling is with Bodipy Texas Red.
- 166. The method of claim 164 wherein the fluorescent labeling is with Bodipy 630/650.
- 167. The method of claim 164 wherein the fluorescent labeling is with Lucifer Yellow.
- 168. The method of claim 152 wherein the labeling is by colormetric labeling.
- 169. The method of claim 152 wherein the labeling is by chemiluminescent labeling.
- 170. The method of claim 94 further including energy transfer between molecules in the hybridization complex.
- 171. The method of claim 170 wherein the energy transfer includes quenching.
- 172. The method of claim 94 further including a redundant assay.
- 173. The method of claim 172 further including the step of repeating the redundant assay until a statistically significant result is obtained.
- 174. The method of claim 172 wherein the redundant assay includes multiple arrays.
- 175. The method of claim 94 wherein the target DNA is purified.
- 176. The method of claim 94 wherein the target is unamplified.
- 177. The method of claim 94 wherein the target is amplified.
- 178. The method of claim 94 wherein the target is applied to a reduction of test sites necessary to identify the number of repeat units in the target DNA.
- 179. The method of claim 178 wherein the reduction increases the statistical significance of results.
- 180. The method of claim 94 wherein the target material constitutes homozygous allele for a locus.
- 181. The method of claim 94 wherein the target material constitutes heterozygous allele for a locus.
- 182. The method of claim 94 wherein the target material constitutes more than one allele per locus for a mixed sample.
- 183. The method of claim 182 wherein the mixed sample further includes sample from more than one individual.
- 184. The method of claim 183 wherein the mixed sample further includes tumor tissue mixed with normal tissue.
- 185. 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 at a plurality of locations on an electronic 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 at least two sites contain target from different sources, and 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.
- 186. The method of claim 185 wherein the capture probe and reporter probe are selected such that the base-stacking between the juxtaposed termini which is disrupted within a discordant complex.
- 187. The method of claim 185 wherein the genetic variant is immediately adjacent the termini.
- 188. The method of claim 185 wherein the genetic variant is one base removed from the termini.
- 189. The method of claim 184 wherein the genetic variant is two or more bases removed from the termini.
- 190. A method for uniquely identifying any of N alleles in a genetic sample comprising the steps of:
performing M tests on the genetic sample, where M is less than N, wherein each test detects concordance, discordance, and each of at least two degrees of discordance, and combining the data from the M tests to uniquely identify the alleles.
- 191. The method of claim 190 wherein the selection of the range of tests spans the set of N alleles.
RELATED APPLICATION INFORMATION
[0001] This application is a continuation of U.S. Ser. No. 09/645,757, filed Aug. 24, 2000, entitled “METHODS AND APPARATUS FOR DETERMINATION OF LENGTH POLYMORPHISMS IN DNA”, now allowed, which is a continuation of U.S. Ser. No. 09/030,156, filed Feb. 25, 1998, entitled “METHODS AND APPARATUS FOR DETERMINATION OF LENGTH POLYMORPHISMS IN DNA”, now issued as U.S. Pat. No. 6,207,363.
[0002] Further, this application is related to Application Ser. No. 08/986,065, filed Dec. 5, 1997, entitled “METHODS AND PARAMETERS FOR ELECTRONIC BIOLOGICAL DEVICES”, now issued as U.S. Pat. No. 6,051,380, which is a continuation-in-part of application Ser. No. 08/534,454, filed Sep. 27, 1995, entitled “APPARATUS AND METHODS FOR ACTIVE PROGRAMMABLE MATRIX DEVICES”, now issued as U.S. Pat. No. 5,849,486, which is a continuation-in-part of application Ser. No. 08/304,657, filed Sep. 9, 1994, entitled “AUTOMATED MOLECULAR BIOLOGICAL DIAGNOSTIC SYSTEM,” now issued as U.S. Pat. No. 5,632,957, which is a continuation-in-part of application Ser. No. 08/271,882, filed Jul. 7, 1994, entitled “METHODS FOR ELECTRONIC STRINGENCY CONTROL FOR MOLECULAR BIOLOGICAL ANALYSIS AND DIAGNOSTICS,” now aissued as U.S. Pat. No. 6,017,696, which is a continuation-in-part of Ser. No. 08/146,504, filed Nov. 1, 1993, entitled “ACTIVE PROGRAMMABLE ELECTRONIC DEVICES FOR MOLECULAR BIOLOGICAL ANALYSIS AND DIAGNOSTICS”, now issued as U.S. Pat. No. 5,605,662, all incorporated herein by reference as if fully set forth herein.
Continuations (2)
|
Number |
Date |
Country |
Parent |
09645757 |
Aug 2000 |
US |
Child |
10108280 |
Mar 2002 |
US |
Parent |
09030156 |
Feb 1998 |
US |
Child |
09645757 |
Aug 2000 |
US |