Methods for determining nature of repeat units in DNA
First Claim
1. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
- providing a plurality of hybridization complex assays arrayed on a plurality of test sites by;
providing a nucleic acid target containing repetitive DNA sequences, providing a capture probe having a first unique flanking sequence and n repeat units, where n≧
0 complementary to the target sequence, and providing a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including attributes selected from the group consisting of;
a) a second unique flanking sequence, b) a sequence complementary to a variant region of the target sequence, and c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence, wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero, the sequence of the capture probe differing at at least two test sites of the array, determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability without the use of an enzymatic reaction and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site.
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Accused Products
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.
41 Citations
244 Claims
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1. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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providing a plurality of hybridization complex assays arrayed on a plurality of test sites by;
providing a nucleic acid target containing repetitive DNA sequences, providing a capture probe having a first unique flanking sequence and n repeat units, where n≧
0 complementary to the target sequence, andproviding a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including attributes selected from the group consisting of;
a) a second unique flanking sequence, b) a sequence complementary to a variant region of the target sequence, and c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence, wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero, the sequence of the capture probe differing at at least two test sites of the array, determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability without the use of an enzymatic reaction and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185)
increasing denaturation stringency to remove said reporter probe at all sites, whether concordant or discordant, hybridizing a second reporter probe where the number of repeat units in said second reporter probe differs from the number of repeat units in said reporter probe, wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site, wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat number in the capture probe and the number of repeat units in the reporter probe, determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and comparing these results with the initial complex hybridization assay for confirmation of target repeat unit number. -
114. The method of claim 111 further including the step of repeating the redundant assay until a statistically significant result is obtained.
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115. The method of claim 111 wherein the redundant assay includes multiple arrays.
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116. The method of claim 115 further including the step of hybridizing target to at least two independent sets of assays,
hybridizing said reporter to a first set and a second reporter to a second set, where the number of repeat units in said reporter probe differs from the number of repeat units in the second reporter, determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site, and p1 comparing these results with the two complexes'"'"' hybridization assay for confirmation of target repeat unit number. -
117. The method of claim 116 including the step of simultaneous hybridization of differently labeled reporters.
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118. The method of claim 117 wherein two reporters differing in the number of repeat units in their nucleic acid sequence, and which are differentially labeled, includes the reporters then being simultaneously provided to the device.
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119. The method of claim 117 wherein the differently labeled reporters are distinguishable chromophores.
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120. The method of claim 119 wherein the chromophores are fluorescent.
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121. The method of claim 119 wherein the chromophores are luminescent.
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122. The method of claim 119 wherein the chromophores are electrochemiluminescent.
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123. The method of claim 119 wherein the chromophores include combinations of fluorescent, luminescent and electrochemiluminescent materials.
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124. The method of claim 117 wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site.
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125. The method of claim 117 wherein the detected presence of a first label at a first test site indicates the pre-determined number of repeat units associated with that reporter and capture sequence at the test site, which is confirmed by the detected presence of a second label at the second test site which indicates the pre-determined number of repeat units associated with that reporter and capture sequence at the test site.
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126. The method of claim 115 wherein the two sets of arrays are on the same device.
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127. The method of claim 115 wherein the two sets of arrays are on different devices.
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128. The method of claim 1 wherein the target DNA is purified.
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129. The method of claim 1 wherein the target is unamplified.
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130. The method of claim 1 wherein the target is amplified.
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131. The method of claim 130 wherein the amplification is an exponential methods of target DNA amplification.
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132. The method of claim 131 wherein the amplification includes PCR amplified DNA.
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133. The method of claim 131 wherein the amplification includes strand displacement amplification (SDA) amplified DNA.
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134. The method of claim 131 wherein the amplification includes linear methods of DNA amplification.
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135. The method of claim 134 wherein the amplification includes rolling circle amplification.
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136. The method of claim 134 wherein the amplification includes transcriptional run-off amplification.
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137. The method of claim 1 wherein the target DNA is unpurified.
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138. The method of claim 137 wherein the target is unamplified.
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139. The method of claim 137 wherein the target is amplified.
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140. The method of claim 139 wherein the amplification is an exponential method of target DNA amplification.
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141. The method of claim 140 wherein the amplification includes PCR amplified DNA.
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142. The method of claim 140 wherein the amplification includes SDA amplified DNA.
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143. The method of claim 139 wherein the amplification is a linear method of DNA amplification.
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144. The method of claim 143 wherein the amplification includes rolling circle amplification.
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145. The method of claim 143 wherein the amplification includes run-off amplification.
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146. The method of claim 1 wherein the plurality of hybridization complex assays arrayed at the test sites include at least one site for each allele of a locus.
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147. The method of claim 146 wherein the allele includes an integral number of repeat units.
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148. The method of claim 146 wherein the allele includes an integral number of repeat units plus at least one microvariant.
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149. The method of claim 1 wherein each concordant test site identifies the repeat unit number of the target.
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150. The method of claim 1 wherein the group of discordant test sites indicates the repeat unit number of the target.
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151. The method of claim 1 wherein the number of concordant test sites per locus array is one or two for a homogeneous sample in a nonredundant, multiple array redundant or serial redundant assay.
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152. The method of claim 1 wherein the number of concordant test sites is more than one for a mixed sample.
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153. The method of claim 117 wherein the number of concordant test sites is more than one for a simultaneous hybridization of differently labeled reporters redundant assay.
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154. The method of claim 1 wherein the nature of the hybridization complex may be resolved into match, mismatch/gap, and mismatch/overlap.
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155. The method of claim 154 wherein the resolution is determined from the signal intensity of the hybridization complex assay.
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156. The method of claim 155 wherein the resolution is determined from gradient application of a selection of electronic, chemical and thermal stringency conditions and resulting change in the signal intensity of the hybridization complex assay.
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157. The method of claim 154 wherein the resolution is determined from the knowledge of the pre-determined sequences of the probes present in the hybridization complex assay.
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158. The method of claim 1 wherein the number of test sites is fewer than required to provide a test site for each allele.
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159. 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.
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160. The method of claim 159 wherein the reduction increases the statistical significance of results.
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161. The method of claim 1 wherein the target material constitutes homozygous allele for a locus.
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162. The method of claim 1 wherein the target material constitutes heterozygous allele for a locus.
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163. The method of claim 1 wherein the target material constitutes more than one allele per locus for a mixed sample.
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164. The method of claim 163 wherein the mixed sample further includes sample from more than one individual.
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165. The method of claim 164 wherein the mixed sample further includes tumor tissue mixed with normal tissue.
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166. The method of claim 165 wherein the tumor tissue is normal tissue.
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167. The method of claim 165 wherein the tumor tissue is diseased tissue.
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168. The method of claim 1 wherein the target material includes tumor tissue.
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169. The method of claim 1 wherein the target material includes plant material.
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170. The method of claim 1 wherein the target material includes animal material.
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171. The method of claim 170 wherein the animal material is mammal material.
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172. The method of claim 170 wherein the animal material is human material.
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173. The method of claim 170 wherein the target material includes bird material.
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174. The method of claim 170 wherein the target material includes fish material.
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175. The method of claim 1 wherein the target material constitutes microbial material.
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176. The method of claim 175 wherein the microbial materials is viral.
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177. The method of claim 175 wherein the microbial material s is bacterial.
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178. The method of claim 175 wherein the microbial materials is protozoa.
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179. The method of claim 1 wherein the method is used for identification.
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180. The method of claim 1 wherein the method is used for paternity testing.
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181. The method of claim 1 wherein the method is used for forensics.
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182. The method of claim 1 wherein the method is used for disease diagnostics.
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183. The method of claim 1 wherein the method is used for breeding.
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184. The method of claim 164 wherein the mixed sample further including sample from one individual which includes monoclonal and polyclonal cell sources.
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185. The method of claim 106 wherein the label is amplified by enzymatic label amplification.
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186. A method for utilizing electronic techniques in determining the nature of repeat units in a genetic target, comprising the steps of:
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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 repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n≧
0. complementary to the target sequence, anda reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including attributes selected from the group consisting of;
a) a second unique flanking sequence and n repeat units, where n≧
0,b) a sequence complementary to a microvariant region of the target sequence, c) a second unique flanking sequence and n repeat units, where n≧
0 and a sequence complementary to a microvariant region of the target sequence,wherein the sum of repeat units in the capture plus the reporter is greater than zero, the sequence of the capture probe differing at at least two test sites on the array, determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site.
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187. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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provide a platform for the identification of the genetic target, including a set of probes having a first unique flanking sequence, a variable number of intervening repeat units and a second unique flanking sequence, hybridizing the genetic target with the probes, including electronic hybridization stringency conditions during the hybridization, whereby the unique flanking sequence is properly indexed with the target, and determining concordance and discordance at the test sites as determined at least in part by electronic hybridization stability, whereby a loop out is formed in discordant hybridizations, thereby providing energetically less favorable condition than in the concordant hybridization.
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188. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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provide a platform for the identification of the genetic target, including a set of probes having a first unique flanking sequence, a variable number of intervening repeat units and a second unique flanking sequence, hybridizing the genetic target with the probes, including electronic hybridization stringency conditions during the hybridization, whereby the unique flanking sequence is properly indexed with the target, and determining concordance and discordance at the test sites, whereby a loop out is formed in discordant hybridizations, thereby providing energetically less favorable condition than in the concordant hybridization.
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189. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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provide a platform for the identification of the genetic target, including a set of probes having a first unique flanking sequence, a variable number of intervening repeat units and a second unique flanking sequence, hybridizing the genetic target with the probes, and determining concordance and discordance at the test sites as determined at least in part by electronic hybridization stability, whereby a loop out is formed in discordant hybridizations, thereby providing energetically less favorable condition than in the concordant hybridization.
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190. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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providing a platform for the identification of the genetic target, including probes selected from the group consisting of;
1) a probe having a first unique flanking sequence, an intervening repeat region and a second unique flanking sequence, and 2) a sandwich assay comprising a capture probe having a first unique flanking sequence and n≧
0 repeat units and a reporter probe having n≧
0 repeat units in sequence with a second unique flanking sequence, wherein the sum of the repeat units is >
0,hybridizing the target with the probes under electronic stringent conditions so as to provide proper indexing, and determining concordance and discordance at the test sites as determined at least in part by hybridization stability, wherein hybridization stability includes electronic hybridization stability.
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191. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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providing a plurality of hybridization complex assays on a plurality of test sites, where the hybridization complex assays includes at least;
a nucleic acid target containing repetitive DNA sequence, a capture probe having a first unique flanking sequence and n repeat units, where n≧
0, complementary to the target sequence, anda reporter probe having a selected sequence complementary to the same target sequence strand, the reporter including a second unique flanking sequence, and n≧
0 repeat units,wherein the sum of repeat units in the capture plus the reporter is greater then zero, the sequence of the capture probe differing at at least two test sites on the array, determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site.
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192. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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providing a plurality of hybridization complex assays arrayed on a plurality of test sites by;
providing a nucleic acid target containing repetitive DNA sequences, providing a capture probe having a first unique flanking sequence and n repeat units, where n≧
0 complementary to the target sequence, the capture probe having a terminal nucleotide, andproviding a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter probe having a terminal nucleotide, the juxtaposed terminal nucleotides of the capture probe and reporter probe selected to provide increased base stacking properties, the reporter including attributes selected from the group consisting of;
a) a second unique flanking sequence, b) a sequence complementary to a variant region of the target sequence, and c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence, wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero, the sequence of the capture probe differing at at least two test sites of the array, determining concordance and discordance among the hybridization complex assays at the test sites as determined at least in part by hybridization stability and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site. - View Dependent Claims (193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218)
increasing denaturation stringency to remove said reporter probe at all sites, whether concordant or discordant, hybridizing a second reporter probe where the number of repeat units in said second reporter probe differs from the number of repeat units in said reporter probe, wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site, wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat number in the capture probe and the number of repeat units in the reporter probe, determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and comparing these results with the initial complex hybridization assay for confirmation of target repeat unit number. -
207. The method of claim 192 wherein the plurality of hybridization complex assays arrayed at the test sites include at least one site for each allele of a locus.
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208. The method of claim 192 wherein each concordant test site identifies the repeat unit number of the target.
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209. The method of claim 192 wherein the group of discordant test sites indicates the repeat unit number of the target.
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210. The method of claim 192 wherein the resolution is determined from gradient application of a selection of electronic, chemical and thermal stringency conditions and resulting change in the signal intensity of the hybridization complex assay.
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211. The method of claim 192 wherein the number of test sites is fewer than required to provide a test site for each allele.
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212. The method of claim 192 wherein the target material constitutes more than one allele per locus for a mixed sample.
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213. The method of claim 192 further including a mixed sample which includes tumor tissue mixed with normal tissue.
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214. The method of claim 192 wherein the method is used for identification.
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215. The method of claim 192 wherein the method is used for disease diagnostics.
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216. The method of claim 192 wherein the method is used for breeding.
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217. The method of claim 192 further including a mixed sample having monoclonal and polyclonal cell sources.
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218. The method of claim 201 wherein the label is amplified by enzymatic label amplification.
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219. A method for determining the nature of repeat units in a genetic target, comprising the steps of:
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providing in the presence of an electric field a plurality of hybridization complex assays arrayed on a plurality of test sites by;
providing a nucleic acid target containing repetitive DNA sequences, providing a capture probe having a first unique flanking sequence and n repeat units, where n≧
0, complementary to the target sequence, the capture probe having a terminal nucleotide, andproviding a reporter probe having a selected sequence complementary to the same target sequence strand, the reporter probe having a terminal nucleotide, the juxtaposed terminal nucleotides of the capture probe and reporter probe selected to provide increased base stacking properties, the reporter including attributes selected from the group consisting of;
a) a second unique flanking sequence, b) a sequence complementary to a variant region of the target sequence, and c) a second unique flanking sequence and a sequence complementary to a variant region of the target sequence, wherein the sum of the number of repeat units in the capture plus the reporter is greater than zero, the sequence of the capture probe differing at at least two test sites of the array, determining concordance and discordance among the hybridization complex assays, at the test sites as determined at least in part by hybridization stability including thermal stringency and determining the nature of the repeat units in the target sequences based upon determination of the concordant site and further based upon the knowledge of the pre-determined sequences of the probes located in the hybridization complex at that site. - View Dependent Claims (220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244)
increasing denaturation stringency to remove said reporter probe at all sites, whether concordant or discordant, hybridizing a second reporter probe where the number of repeat units in said second reporter probe differs from the number of repeat units in said reporter probe, wherein the location of the concordant test site indicates the number of repeat units present in the target, based upon the knowledge of the pre-determined sequences of the probes located at that test site, wherein at the concordant test site, the number of repeat units in the target equals the sum of the number of repeat number in the capture probe and the number of repeat units in the reporter probe, determining concordant and discordant test sites among the hybridization complex assays at the test sites as determined at least in part by hybridization stability, and comparing these results with the initial complex hybridization assay for confirmation of target repeat unit number. -
233. The method of claim 219 wherein the plurality of hybridization complex assays arrayed at the test sites include at least one site for each allele of a locus.
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234. The method of claim 219 wherein each concordant test site identifies the repeat unit number of the target.
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235. The method of claim 219 wherein the group of discordant test sites indicates the repeat unit number of the target.
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236. The method of claim 219 wherein the resolution is determined from gradient application of a selection of electronic, chemical and thermal stringency conditions and resulting change in the signal intensity of the hybridization complex assay.
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237. The method of claim 219 wherein the number of test sites is fewer than required to provide a test site for each allele.
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238. The method of claim 219 wherein the target material constitutes more than one allele per locus for a mixed sample.
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239. The method of claim 219 further including a mixed sample which includes tumor tissue mixed with normal tissue.
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240. The method of claim 219 wherein the method is used for identification.
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241. The method of claim 219 wherein the method is used for disease diagnostics.
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242. The method of claim 219 wherein the method is used for breeding.
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243. The method of claim 219 further including a mixed sample having monoclonal and polyclonal cell sources.
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244. The method of claim 227 wherein the label is amplified by enzymatic label amplification.
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Specification