Single molecule arrays for genetic and chemical analysis
3 Assignments
0 Petitions
Accused Products
Abstract
Random arrays of single molecules are provided for carrying out large scale analyses, particularly of biomolecules, such as genomic DNA, cDNAs, proteins, and the like. In one aspect, arrays of the invention comprise concatemers of DNA fragments that are randomly disposed on a regular array of discrete spaced apart regions, such that substantially all such regions contain no more than a single concatemer. Preferably, such regions have areas substantially less than 1 μm2 and have nearest neighbor distances that permit optical resolution of on the order of 109 single molecules per cm2. Many analytical chemistries can be applied to random arrays of the invention, including sequencing by hybridization chemistries, sequencing by synthesis chemistries, SNP detection chemistries, and the like, to greatly expand the scale and potential applications of such techniques.
-
Citations
136 Claims
-
1-73. -73. (canceled)
-
74. An array of polymers comprising:
-
(a) a support having a surface; and (b) a plurality of polymers attached to the surface, wherein each polymer; (i) comprises multiple copies of a polymeric unit and (ii) has a random coil state, wherein the polymers are attached to the support surface within a region substantially equivalent to a projection of the random coil on the surface and wherein the polymers are randomly disposed on the support surface at a density allowing separate detection of at least thirty percent of the polymers. - View Dependent Claims (75, 76)
-
-
77. An array of single molecules comprising:
-
(a) a support having a surface having a regular array of discrete regions, wherein each discrete region has an area of less than 1 μ
m2 and contains reactive functionalities attached thereto; and(b) a plurality of single molecules attached to the surface, wherein each single molecule comprises a macromolecular structure and at least one analyte having an attachment moiety, such that each macromolecular structure comprises a unique functionality and a plurality of attachment functionalities that are capable of forming linkages with the reactive functionalities of the discrete regions, wherein the analyte is attached to the macromolecular structure by a linkage between the unique functionality and the attachment moiety of the analyte; and wherein the plurality of single molecules are randomly disposed on the discrete regions such that at least a majority of the discrete regions contain only one single molecule.
-
-
78. An array of polymeric units comprising:
-
(a) a support having a planar surface with a regular array of discrete regions, wherein each discrete region is less than 1 μ
m2 in area and contains reactive functionalities attached thereto; and(b) a plurality of polymeric units attached to the surface, wherein each polymeric unit comprises; (i) a polynucleotide comprising multiple copies of a target sequence; and (ii) at least one attachment moiety capable of forming a linkage with the reactive functionalities on said support surface; wherein the attachment moiety of the polynucleotide is linked to the support via the reactive functionalities, and wherein the polynucleotides are s are disposed on said planar surface in a rectilinear pattern, each disposed in an area having a diameter of from 0.1 to 10 μ
m. - View Dependent Claims (79, 80, 81)
-
-
82. An array of polynucleotides comprising:
-
(a) a support having a surface; and (b) a plurality of polynucleotides attached to the surface, wherein each polynucleotide comprises a concatemer having multiple copies of a target sequence and forms a random coil under conventional conditions; wherein the polynucleotides are attached to the surface within a discrete region substantially equivalent to a projection of the polynucleotide random coil on the support surface, and wherein the polynucleotides are randomly disposed on the support surface in a rectilinear pattern.
-
-
83. An array of polynucleotides comprising:
-
(a) a support having a surface; and (b) a plurality of polynucleotides attached to the surface, wherein each polynucleotide; (i) comprises a concatemer having multiple copies of a target sequence; and (ii) forms a random coil under conventional conditions; wherein the polynucleotides are attached to the surface within a discrete region substantially equivalent to a projection of the polynucleotide random coil on the support surface, and wherein the polynucleotides are randomly disposed on the support surface so that at least thirty percent of the polynucleotides have a nearest neighbor distance of at least fifty nm. - View Dependent Claims (84, 85, 86)
-
-
87. An array of polynucleotides comprising:
-
(a) a support having a surface with capture oligonucleotides attached thereto; and (b) a plurality of polynucleotides attached to the surface, wherein each polynucleotide; (i) comprises a concatemer having multiple copies of a polynucleotide with an alternating target sequence and adaptor oligonucleotide; and (ii) forms a random coil under conventional conditions; wherein the polynucleotides are attached to the surface within a region substantially equivalent to a projection of the random coil on the surface and wherein the polynucleotides are randomly disposed on the support surface in a rectilinear pattern at a density allowing separate detection of at least thirty percent of the polynucleotides. - View Dependent Claims (88, 89, 90)
-
-
91. An array of polynucleotides comprising:
-
(a) a support having a surface with capture oligonucleotides attached thereto; and (b) a plurality of polynucleotides attached to the surface, wherein each polynucleotide comprises a concatemer of multiple copies of a polynucleotide with alternating copies of a target sequence and an adaptor oligonucleotide, wherein the polynucleotides are attached to the surface by a complex formed between the capture oligonucleotides and adaptor oligonucleotides within the concatemers, and further wherein the individual polynucleotides are randomly disposed on discrete regions of the substrate so that at least thirty percent of the polynucleotides have a nearest neighbor distance of at least fifty nm. - View Dependent Claims (92, 93, 94)
-
-
95. An array of fragments from a source DNA, comprising:
-
(a) a support having a surface with capture oligonucleotides attached thereto; and (b) a plurality of concatemers, each comprising multiple alternating copies of a DNA fragment from the source DNA and an adaptor oligonucleotide; wherein the concatemers are attached to the surface by a complex formed between the capture oligonucleotides and adaptor oligonucleotides within the concatemers, and further wherein the individual polynucleotides are randomly disposed on discrete regions of the substrate so that at least thirty percent of the polynucleotides have a nearest neighbor distance of at least fifty nm. - View Dependent Claims (96)
-
-
97. A method of making an array of polynucleotides from a source nucleic acid, the method comprising the steps of:
-
(a) generating a plurality of concatemers, each comprising. (i) a polynucleotide fragment from a source nucleic acid and (ii) a functionality complementary to a reactive functionality; (b) disposing the plurality of concatemers onto a support having a surface comprising reactive functionalities attached thereto; and (c) fixing the concatemers to the surface by linkages formed between the reactive functionalities on the support surface and complementary functionalities on the concatemers; wherein the concatemers are randomly disposed on the surface at a density such that a majority of the polynucleotide molecules are optically resolvable, thereby forming an array of polynucleotides. - View Dependent Claims (98, 99, 100, 101)
-
-
102. A method of making an array of fragments of a target polynucleotide, the method comprising the steps of:
-
(a) providing a plurality of concatemers, each comprising multiple alternating copies of a fragment from a target polynucleotide and an adaptor oligonucleotide; (b) disposing the plurality of concatemers onto a support, said support comprising a planar surface with capture oligonucleotides attached thereto; and (c) fixing the concatemers to the surface by complexes formed between the capture oligonucleotides and adaptor oligonucleotides; wherein the concatemers are randomly distributed on the surface at a density where the concatemers have a nearest neighbor distance of at least fifty nm, thereby forming an array of concatemers which substantially represent the nucleotide sequence of the target polynucleotide. - View Dependent Claims (103, 104, 105, 106)
-
-
107. A method of making an array of a plurality of fragments of a target polynucleotide, the method comprising the steps of:
-
(a) generating a plurality of concatemers, each comprising multiple alternating copies of a fragment from a target polynucleotide and an adaptor oligonucleotide; (b) disposing the plurality of concatemers onto a support, wherein said surface is a planar surface having an array of discrete regions with an area of less than 1 μ
m2, said discrete regions having capture oligonucleotides attached thereto; and(c) fixing the concatemers to the surface by complexes formed between the capture oligonucleotides and the adaptor oligonucleotides within the concatemers; wherein the concatemers are distributed on the surface in a manner to promote the fixing of one concatemer in each discrete region, thereby forming an array of concatemers which substantially represent the nucleotide sequence of the target polynucleotide. - View Dependent Claims (108, 109, 110)
-
-
111. A method of making an array of a plurality of fragments from a source DNA, the method comprising the steps of:
-
(a) providing a plurality of concatemers, each comprising multiple alternating copies of a DNA fragment from the source DNA and an adaptor oligonucleotide; (b) disposing the plurality of concatemers onto a support, said support comprising a surface with capture oligonucleotides attached thereto; and (c) fixing the concatemers to the surface by complexes formed between the capture oligonucleotides and adaptor oligonucleotides; wherein the concatemers are randomly distributed on the surface at a density where the concatemers have a nearest neighbor distance of at least fifty nm, thereby forming an array of concatemers. - View Dependent Claims (112)
-
-
113. A method of making an array of a plurality of fragments from a source DNA, the method comprising the steps of:
-
(a) generating a plurality of concatemers, each comprising multiple alternating copies of a DNA fragment from the source DNA and an adaptor oligonucleotide; (b) disposing the plurality of concatemers onto a support surface, wherein said surface is a planar surface having an array of discrete regions with an area of less than 1 μ
m2, said discrete regions having capture oligonucleotides attached thereto; and(c) fixing the concatemers to the surface by complexes formed between the capture oligonucleotides and the adaptor oligonucleotides within the concatemers; wherein the concatemers are distributed on the surface in a manner to promote the fixing of one concatemer in each discrete region, thereby forming an array of concatemers which substantially represent the nucleotide sequence of the source DNA. - View Dependent Claims (114, 115, 116)
-
-
117. A method of creating random polynucleotide arrays of selected density, comprising the steps of:
-
(a) providing a substrate surface comprising multiple discrete spaced apart regions that are (b) substantially the sole sites for attaching a plurality of concatemers; applying a plurality of concatemers onto the substrate surface support; and (c) fixing single concatemers to the multiple discrete spaced apart regions on the substrate surface to produce a random array; wherein the concatemers are distributed on the surface in a manner to promote the fixing of one concatemer in each discrete region. - View Dependent Claims (118, 119)
-
-
120. A method of creating random polynucleotide arrays of selected density, providing the steps of:
-
(a) generating a plurality of concatemers, each comprising a polynucleotide fragment from a source nucleic acid; (b) applying the plurality of concatemers comprising target polynucleotide sequences onto a substrate surface comprising multiple discrete spaced apart regions that are substantially the sole sites for attaching the plurality of concatemers; and (c) fixing the concatemers to the multiple discrete spaced apart regions of the substrate surface to produce a random array; wherein the concatemers are distributed on the surface in a manner to promote the fixing of one concatemer in each discrete region. - View Dependent Claims (121, 122)
-
-
123. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:
-
(a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide, wherein the plurality of target concatemers substantially covers the nucleotide sequence of the target polynucleotide; (b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; (c) hybridizing one or more probes from a first set of probes under conditions that permit the formation of perfectly matched duplexes between the one or more of the first set of probes and complementary sequences on target concatemers; (d) hybridizing one or more probes from a second set of probes under conditions that permit the formation of perfectly matched duplexes between the one or more of the second set of probes and complementary sequences on target concatemers; (e) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (f) identifying the sequences of the ligated first and second probes; and (g) repeating steps (c) through (f) until a sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. - View Dependent Claims (124, 125, 126)
-
-
127. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:
-
(a) providing a random array of target concatemers fixed to a planar surface having an array of optically resolvable discrete regions, each target concatemer comprising multiple copies of a fragment of the target polynucleotide, wherein each discrete region has an area of less than 1 μ
m2, and wherein the target concatemers on said array substantially cover the nucleotide sequence of the target polynucleotide;(b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the first set of probes and complementary sequences on target concatemers; (c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the second set of probes and complementary sequences on target concatemers; (d) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (e) identifying the sequences of the ligated first and second probes; and (f) repeating steps (b) through (e) until a sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes. - View Dependent Claims (128, 129, 130)
-
-
131. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:
-
(a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple copies of a fragment of the target polynucleotide, wherein the plurality of target concatemers substantially covers the nucleotide sequence of the target polynucleotide; (b) forming a random array of target concatemers by disposing said concatemers to a surface at a density at which a majority of the target concatemers are optically resolvable; (c) identifying a sequence of at least a portion of each fragment in each target concatemer; and (d) reconstructing a nucleotide sequence of the target polynucleotide from the identities of the sequences of the portions of fragments of the concatemers. - View Dependent Claims (132, 133, 134)
-
-
135. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:
-
(a) generating a plurality of target concatemers from the target polynucleotide, each target concatemer comprising multiple alternating copies of a fragment of the target polynucleotide and an adaptor oligonucleotide, wherein the plurality of target concatemers substantially cover the nucleotide sequence of the target polynucleotide; (b) forming a random array of target concatemers fixed to a surface at a density such that at least a majority of the target concatemers are optically resolvable; (c) identifying a sequence of at least a portion of each fragment in each target concatemer; and (d) reconstructing the nucleotide sequence of the target polynucleotide from the identities of the sequences of the portions of fragments of the concatemers.
-
-
136. A method of determining a nucleotide sequence of a target polynucleotide, the method comprising the steps of:
-
(a) providing a random array of target concatemers fixed to a planar surface having an array of optically resolvable discrete regions, wherein each discrete region has an area of less than 1 μ
m2 and wherein substantially all such regions have one target concatemer attached, each target concatemer comprising multiple alternating copies of a fragment of the target polynucleotide and an adaptor oligonucleotide, wherein the target concatemers on said array substantially covers the nucleotide sequence of the target polynucleotide;(b) hybridizing one or more probes from a first set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the first set of probes and complementary sequences on target concatemers; (c) hybridizing one or more probes from a second set of probes to the random array under conditions that permit the formation of perfectly matched duplexes between the second set of probes and complementary sequences on target concatemers; (d) ligating probes from the first and second sets hybridized to a target concatemer at contiguous sites; (e) identifying the sequences of the ligated first and second probes; and (f) repeating steps (b) through (e) until the sequence of the target polynucleotide can be determined from the identities of the sequences of the ligated probes.
-
Specification