Arrays and Methods of Use

Abstract

Methods are provided for producing a molecular array comprising a plurality of molecules immobilised to a solid substrate at a density which allows individual immobilised molecules to be individually resolved, wherein each individual molecule in the array is spatially addressable and the identity of each molecule is known or determined prior to immobilization. The use of spatially addressable low density molecular arrays in single molecule detection and analysis techniques is also provided. Novel assays and methods are also provided.

1 - 107 . (canceled) 
     
     
         108 . A method of producing a biosensor comprising:
 depositing oligonucleotides on a solid support; and   labeling at least a portion of the oligonucleotides with at least two different labels before or after the depositing,   whereby at least a portion of the oligonucleotides that are deposited and labeled are separated from other deposited labeled oligonucleotides on the solid support to produce a plurality of separated labeled oligonucleotides.   
     
     
         109 . The method according to  claim 108 , the biosensor is a molecular array of two or more discrete elements each of which comprises at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         110 . The method according to  claim 109 , wherein the two or more discrete elements are separated by a raised region or an etched trench. 
     
     
         111 . The method according to  claim 110 , wherein the solid support is a microtiter plate comprising the raised region separating the two or more discrete elements. 
     
     
         112 . The method according to  claim 109 , wherein said each of the two or more discrete elements comprises the at least two different labels. 
     
     
         113 . The method according to  claim 109 , wherein each of the two or more discrete elements is from 1 μm to 300 μm apart. 
     
     
         114 . The method according to  claim 108 , wherein at least a portion of the oligonucleotides is amplified. 
     
     
         115 . The method according to  claim 108 , further comprising ligating at least a portion of the oligonucleotides to target strands and forming oligonucleotide-target molecule complexes. 
     
     
         116 . The method according to  claim 115 , wherein the oligonucleotide-target molecule complexes comprise circularized DNA. 
     
     
         117 . The method according to  claim 115 , wherein at least a portion of the oligonucleotide-target molecule complexes is amplified. 
     
     
         118 . The method according to  claim 115 , wherein at least a portion of the oligonucleotide-target molecule complexes is amplified by rolling circle amplification. 
     
     
         119 . The method according to  claim 115 , wherein the labeling comprises labeling at least a portion of the oligonucleotide-target molecule complexes. 
     
     
         120 . The method according to  claim 115 , wherein at least a portion of the oligonucleotide-target molecule complexes comprises said plurality of separated labeled oligonucleotides. 
     
     
         121 . The method according to  claim 115 , wherein the labeling comprises ligating an oligonucleotide probe to at least a portion of the oligonucleotide-target molecule complexes, wherein the labeling is performed by labeling the oligonucleotide probe after the ligation. 
     
     
         122 . The method according to  claim 115 , further comprising fragmenting a nucleotide sample to obtain the target strands. 
     
     
         123 . The method according to  claim 115 , further comprising denaturing a double stranded nucleotide sample to obtain the target strands. 
     
     
         124 . The method according to  claim 115 , further comprising primer extension of at least a portion of the oligonucleotide-target molecule complexes with labeled primers. 
     
     
         125 . The method according to  claim 115 , wherein the target strands are unamplified nucleotide molecules. 
     
     
         126 . The method according to  claim 108 , wherein the at least two different labels are dye loaded nanoparticles. 
     
     
         127 . The method according to  claim 108 , wherein the labeling is performed before the depositing. 
     
     
         128 . The method according to  claim 108 , wherein the oligonucleotides are deposited on a solid support thereby forming two or more separate and discrete elements, at least two elements of said two or more separate and discrete elements being spatially addressable. 
     
     
         129 . The method according to  claim 108 , wherein the oligonucleotides are deposited on a solid support thereby forming two or more separate and discrete elements, at least two elements of said two or more separate and discrete elements being spatially addressable, each of said at least two elements comprising the separated deposited labeled oligonucleotides. 
     
     
         130 . The method according to  claim 108 , wherein the oligonucleotides are deposited on a solid support thereby forming two or more separate and discrete elements, at least two elements of said two or more separate and discrete elements being spatially addressable, each of said at least two elements comprising the plurality of separated labeled oligonucleotides that are labeled with said at least two different labels. 
     
     
         131 . The method according to  claim 108 , wherein the oligonucleotides are deposited on a solid support thereby forming two or more separate and discrete elements, at least two elements of said two or more separate and discrete elements being spatially addressable, each of said at least two elements comprising the plurality of separated labeled oligonucleotides that are labeled with an identical label. 
     
     
         132 . The method according to  claim 108 , wherein the depositing comprises immobilizing the oligonucleotides on the solid support by a means selected from the group consisting of Biotin-oligonucleotide complexed with Avidin, Strepatavidin or Neutravidin; SH-oligonucleotide covalently linked via a disulphide bond to a SH-surface; Amine-oligonucleotide covalently linked to an activated carboxylate or an aldehyde group; Phenylboronic acid (PBA)-oligonucleotide complexed with salicylhydroxamic acid (SHA); and Acrydite-oligonucleotide reacted with thiol or silane surface or co-polyemerized with acrylamide monomer to form polyacrylamide. 
     
     
         133 . The method according to  claim 108 , wherein the depositing comprises immobilizing the oligonucleotides on the solid support by a covalent or non-covalent bond. 
     
     
         134 . The method according to  claim 108 , further comprising coating the substrate with polylysine. 
     
     
         135 . The method according to  claim 108 , wherein the plurality of separated labeled oligonucleotides are visually separated from each other. 
     
     
         136 . The method according to  claim 108 , wherein the plurality of separated labeled oligonucleotides are separated by a distance from 250 nm to 800 nm apart. 
     
     
         137 . The method according to  claim 108 , further comprising preselecting oligonucleotides to be deposited prior to the depositing. 
     
     
         138 . A method of performing an assay comprising: producing the biosensor according to the method of  claim 108 , and performing the assay comprising counting a number of at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         139 . The method according to  claim 138 , wherein the number is counted by counting a number of signals from the at least two different labels of the at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         140 . The method according to  claim 138 , wherein the performing further comprises comparing a number of the at least two different labels of the at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         141 . The method according to  claim 138 , wherein the number is counted by counting a number of repeated motifs in the at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         142 . The method according to  claim 138 , wherein the performing further comprises image processing the at least a portion of the plurality of separated labeled oligonucleotides, and the at least a portion of the plurality of separated labeled oligonucleotides are optically resolvable. 
     
     
         143 . The method according to  claim 138 , wherein the performing further comprises digital image processing the at least a portion of the plurality of separated labeled oligonucleotides. 
     
     
         144 . The method according to  claim 138 , wherein the performing further comprises scanning the solid support by an array scanner. 
     
     
         145 . The method according to  claim 138 , further comprising ligating at least a portion of the oligonucleotides to target strands to form probe-target molecule complexes, wherein the method excludes amplification of the target strands. 
     
     
         146 . The method according to  claim 138 , further comprising ligating at least a portion of the oligonucleotides to target strands to form probe-target molecule complexes, wherein the target strands are from a maternal blood. 
     
     
         147 . The method according to  claim 138 , wherein the assay is prenatal diagnosis of a genetic disorder. 
     
     
         148 . The method according to  claim 138 , wherein at least a part of the method is automated. 
     
     
         149 . The method according to  claim 138 , wherein
 the biosensor is a molecular array of two or more discrete elements each of which comprises at least a portion of the plurality of separated labeled oligonucleotides;   the two or more discrete elements are separated by a raised region;   the solid support is a microtiter plate comprising the raised region; and   the performing comprises using an array scanner.   
     
     
         150 . A method of producing a molecular array comprising:
 immobilising directly or indirectly a plurality of oligonucleotides to a solid phase to form at two or more separate and discrete elements, at least two of said two or more separate and discrete elements being spatially addressable and comprising a plurality of immobilised oligonucleotides; and   labeling, with two or more labels, at least a portion of the plurality of immobilised oligonucleotides, wherein said at least two elements comprise a plurality of labeled immobilised oligonucleotides, wherein at least a portion of the plurality of labeled immoblised oligonucleotides are individually resolvable.   
     
     
         151 . A method of producing a molecular array comprising:
 labeling, with two or more labels, a plurality of oligonucleotides to form a plurality of labeled oligonucleotides; and   immobilising directly or indirectly to a solid phase at least a portion of the plurality of labeled oligonucleotides to form two or more separate and discrete elements, at least two of said two or more separate and discrete elements being spatially addressable, said at least two elements comprising a plurality of labeled immobilised oligonucleotides, wherein at least a portion of the plurality of labeled immoblised oligonucleotides are individually resolvable.   
     
     
         152 . The method according to  claim 150  or  151 , wherein the one or more labels can be detected by optical methods. 
     
     
         153 . The method according to  claim 150  or  151 , wherein the oligonucleotides are cDNA and/or genomic DNA. 
     
     
         154 . The method according to  claim 150  or  151 , wherein the immobilisation comprises applying the plurality of the oligonucleotides by a method selected from the group consisting of printing, electronic addressing, in situ light-directed synthesis, ink jet synthesis, physical masking, and spotting of a dilute solution. 
     
     
         155 . The method according to  claim 150  or  151 , wherein the at least two elements are separated by a raised region or an etched trench. 
     
     
         156 . The method according to  claim 155 , wherein the solid phase is microtiter plate comprising the raised region or an etched trench. 
     
     
         157 . The method according to  claim 150  or  151 , wherein the one or more labels are fluorescent labels. 
     
     
         158 . The method according to  claim 150  or  151 , further comprising counting a number of the at least a portion of the plurality of labeled immoblised oligonucleotides. 
     
     
         159 . The method according to  claim 158 , wherein the number of the at least a portion of the plurality of labeled immoblised oligonucleotides is counted for one or more diagnostic purposes. 
     
     
         160 . The method according to  claim 150  or  151 , wherein the at least two elements are spatially addressable without sequencing of the at least a portion of the plurality of labeled immoblised oligonucleotides. 
     
     
         161 . The method according to  claim 150  or  151 , wherein the labeling comprises ligating at least a portion of the plurality of oligonucleotides to target strands and forming oligonucleotide-target molecule complexes. 
     
     
         162 . The method according to  claim 161 , wherein said at least a portion of the oligonucleotide-target molecule complexes comprises circularized DNA. 
     
     
         163 . The method according to  claim 161 , wherein at least a portion of the oligonucleotide-target molecule complexes is amplified. 
     
     
         164 . The method according to  claim 161 , wherein at least a portion of the oligonucleotide-target molecule complexes is amplified by rolling circle amplification. 
     
     
         165 . The method according to  claim 150  or  151 , wherein the immobilising is performed by a means selected from the group consisting of Biotin-oligonucleotide complexed with Avidin, Strepatavidin or Neutravidin; SH-oligonucleotide covalently linked via a disulphide bond to a SH-surface; Amine-oligonucleotide covalently linked to an activated carboxylate or an aldehyde group; Phenylboronic acid (PBA)-oligonucleotide complexed with salicylhydroxamic acid (SHA); and Acrydite-oligonucleotide reacted with thiol or silane surface or co-polyemerized with acrylamide monomer to form polyacrylamide. 
     
     
         166 . A method of performing an assay comprising: producing the molecular array according to the method of  claim 150  or  151 , and performing the assay comprising counting a number of at least a portion of the plurality of labeled immoblised oligonucleotides. 
     
     
         167 . The method according to  claim 166 , wherein the number is counted by counting a number of signals from the two or more different labels. 
     
     
         168 . The method according to  claim 166 , wherein the performing further comprises comparing numbers of the two or more different labels. 
     
     
         169 . The method according to  claim 166 , wherein the number is counted by counting a number of repeated motifs in the at least a portion of the plurality of immobilised labeled oligonucleotides. 
     
     
         170 . The method according to  claim 166 , wherein the performing further comprises image processing the at least a portion of the plurality of immobilised labeled oligonucleotides. 
     
     
         171 . The method according to  claim 166 , wherein the performing further comprises digital image processing the at least a portion of the plurality of immobilised labeled oligonucleotides. 
     
     
         172 . The method according to  claim 166 , further comprising ligating at least a portion of the plurality of oligonucleotides to target strands to form probe-target molecule complexes, wherein the method excludes amplification of the target strands. 
     
     
         173 . The method according to  claim 166 , further comprising ligating at least a portion of the plurality of oligonucleotides to target strands to form probe-target molecule complexes, wherein the target strands are from a maternal blood. 
     
     
         174 . The method according to  claim 166 , wherein the assay is prenatal diagnosis of a genetic disorder. 
     
     
         175 . The method according to  claim 166 , wherein at least a part of the method is automated. 
     
     
         176 . A method of performing a genetic analysis comprising:
 depositing a plurality of labeled oligonucleotides to one or more wells in a microtitre plate to form a plurality of labeled deposited oligonucleotides, and   performing the genetic analysis comprising counting a number of at least a portion of the plurality of labeled deposited oligonucleotides,   wherein the plurality of labeled deposited oligonucleotides are deposited at a density which allows the at least a portion of the plurality of labeled deposited oligonucleotides to be individually resolved, and the at least a portion of the plurality of labeled deposited oligonucleotides are labeled with at least two different labels.   
     
     
         177 . The method according to  claim 176 , wherein the at least two different labels are fluorescent labels. 
     
     
         178 . The method according to  claim 176 , wherein the plurality of labeled deposited oligonucleotides comprise circularized DNA. 
     
     
         179 . The method according to  claim 176 , wherein at least a portion of the plurality of labeled oligonucleotides is amplified by rolling circle amplification. 
     
     
         180 . The method according to  claim 176 , wherein depositing comprises immobilising the plurality of labeled oligonucleotides by a means selected from the group consisting of Biotin-oligonucleotide complexed with Avidin, Strepatavidin or Neutravidin; SH-oligonucleotide covalently linked via a disulphide bond to a SH-surface; Amine-oligonucleotide covalently linked to an activated carboxylate or an aldehyde group; Phenylboronic acid (PBA)-oligonucleotide complexed with salicylhydroxamic acid (SHA); and Acrydite-oligonucleotide reacted with thiol or silane surface or co-polyemerized with acrylamide monomer to form polyacrylamide. 
     
     
         181 . A method of prenatal diagnosis, comprising
 providing a plurality of probes complementary to at least a portion of nucleic acids present in a sample from a maternal blood;   hybridizing said at least a portion of nucleic acids to the plurality of probes to produce hybridized molecules; and   counting at least a portion of said hybridized molecules to determine frequency of the nucleic acids.   
     
     
         182 . The method according to  claim 181 , wherein at least a portion of said hybridized molecules are labeled with two or more labels directly or indirectly. 
     
     
         183 . The method according to  claim 182 , wherein the two or more labels are fluorescent labels. 
     
     
         184 . The method according to  claim 181 , further comprising depositing the hybridized molecules on a solid support. 
     
     
         185 . The method according to  claim 184 , wherein the solid support is a microtiter plate. 
     
     
         186 . The method according to  claim 185 , wherein the hybridized molecules are deposited in two or more wells of the microtiter plate. 
     
     
         187 . The method according to  claim 181 , further comprising amplifying the at least a portion of nucleic acids. 
     
     
         188 . The method according to  claim 181 , wherein the at least a portion of said hybridized molecules is amplified by rolling circle amplification. 
     
     
         189 . The method according to  claim 181 , further comprising fragmenting nucleotide molecules from the sample to obtain said at least a portion of nucleic acids. 
     
     
         190 . The method according to  claim 181 , further comprising denaturing a double stranded nucleotide molecules to obtain said at least a portion of nucleic acids. 
     
     
         191 . A method of performing a single molecule counting, comprising
 contacting a plurality of probes with target molecules to form probe-target molecule complexes in a solution, wherein the probe-target molecule complexes are labeled directly or indirectly with at least two different labels,   applying the solution comprising the probe-target molecule complexes to a solid phase before or after the contacting, and   determining relative numbers of the target molecules by comparing numbers of signals from the at least two different labels.   
     
     
         192 . The method according to  claim 191 , wherein the solid phase is a microtiter plate. 
     
     
         193 . The method according to  claim 191 , wherein the at least two different labels are fluorescent labels. 
     
     
         194 . The method according to  claim 191 , wherein the probe-target molecule complexes comprise circularized DNA. 
     
     
         195 . The method according to  claim 191 , wherein at least a portion of the probe-target molecule complexes are amplified by rolling circle amplification.