Single molecule arrays for genetic and chemical analysis

• US 20090137404A1
• Filed: 10/31/2007
• Published: 05/28/2009
• Est. Priority Date: 06/15/2005
• Status: Active Grant

• Current Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Original Assignee: Complete Genomics Incorporated (BGI Genomics Co., Ltd.)
• Inventors: Drmanac, Radoje T., Yeung, George, Drmanac, Snezana, Callow, Matthew J., Hauser, Brian K.
• Granted Patent: US 8,445,196 B2
• Time in Patent Office: Days
• US Class Current: 506/3
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C07K 1/047 Simultaneous synthesis of different peptide species; Peptide libraries
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/682 Signal amplification
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2525/151 repeat or repeated sequences, e.g. VNTR, microsatellite, concatemer
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/313 Branched oligonucleotides
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2535/122 Massive parallel sequencing
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides
• CPC Class Codes: C12Q 2565/514 characterised by the use of the arrayed oligonucleotides as identifier tags, e.g. universal addressable array, anti-tag or tag complement array
• CPC Class Codes: G01N 15/1404 Handling flow, e.g. hydrodynamic focusing
• CPC Class Codes: G01N 15/1434 Optical arrangements
• CPC Class Codes: Y10S 977/778 within specified host or matrix material, e.g. nanocomposite films
• CPC Class Codes: Y10S 977/789 in array format
• CPC Class Codes: Y10S 977/792 : Nucleic acid array, e.g. human genome array
• CPC Class Codes: Y10S 977/88 : with arrangement, process, or apparatus for testing
• CPC Class Codes: Y10S 977/882 : Assembling of separate components, e.g. by attaching

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.

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.

75. The array of claim 74, wherein the polymeric unit comprises the structure (M-L)_n, wherein L is a linker moiety, M is a monomer of a polymeric chemical structure, and n is a number from 3-100.

76. The array of claim 74, wherein said polymeric units comprise a reactive functionality and a polymer moiety comprising a second functionality complementary to said reactive functionality.

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.

79. The array of claim 78, wherein each polynucleotide comprises multiple copies of a polymeric unit having an alternating target sequence and adaptor oligonucleotide.

80. The array of claim 78, wherein said discrete regions has diameter of from 0.1 to 10 μ

m.

81. The array of claim 78, wherein said polynucleotides are randomly disposed on said planar surface such that at least seventy percent of said polynucleotides are optically resolvable.

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.

84. The array of claim 83, wherein each concatemer comprises multiple alternating copies of a target sequence polynucleotide and an adaptor oligonucleotide.

85. The array of claim 83, wherein the support surface comprises reactive functionalities, the polynucleotides comprise functionalities complementary to the reactive functionalities, and wherein said polynucleotides are each attached to said surface by one or more linkages formed between the reactive functionalities and the functionalities complementary to the reactive functionalities.

86. The array of claim 83, wherein said discrete regions have a diameter of from 0.1 to 10 μ

m.

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.

88. The array of claim 87, wherein said target sequences each have a length in the range of from 50 to 500 nucleotides and wherein said adaptor oligonucleotide has a length in the range of from 6 to 60 nucleotides.

89. The array of claim 87, wherein said polynucleotides are disposed on said support at a density that allows seventy percent of the polynucleotides to be optically resolvable.

90. The array of claim 87, wherein each of the concatemers comprises at least one sequence for identifying its respective target sequence using one or more decoding probes.

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.

92. The array of claim 91, wherein said discrete regions each have a nearest neighbor distance in the range of from 0.1 to 20 μ

m.

93. The array of claim 91, wherein said discrete regions form a plurality of Regular subarrays, wherein said discrete regions within the same subarray have capture oligonucleotides having substantially the same nucleotide sequence and said discrete regions within different subarrays have capture oligonucleotides having substantially different nucleotide sequences.

94. The array of claim 91, wherein each of said discrete regions is surrounded by an inter-regional space that is substantially free of said polynucleotides.

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.

96. The array of claim 95, wherein the fragments of DNA disposed on the array substantially represent the nucleotide sequence of the source DNA.

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.

98. The method of claim 97, wherein said concatemers are randomly distributed on said array and wherein said nearest neighbor distance between concatemers is in the range of from 0.3 to 3 μ

m.

99. The method of claim 97, wherein said concatemers on said array have a nearest neighbor distance such that at least seventy percent of the concatemers are optically resolvable.

100. The method of claim 97, wherein said concatemers 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.

101. The method of claim 97, wherein said step of generating includes the steps of fragmenting said source nucleic acid, circularizing said fragments, and forming concatemers of said fragments.

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.

103. The method of claim 102, wherein said disposing forms a regular array of concatemers with a nearest neighbor distance in the range of from 0.1 to 20 μ

m.

104. The method of claim 102, wherein said concatemers 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.

105. The method of claim 102, wherein said concatemers are randomly disposed on said planar surface such that at least seventy percent of the concatemers are optically resolvable.

106. The method of claim 102, wherein said concatemers are randomly distributed on said discrete regions and wherein said nearest neighbor distance of the concatemers is in the range of from 0.3 to 3 μ

m.

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.

108. The method of claim 107, wherein said concatemers on said array have a nearest neighbor distance such that at least seventy percent of the concatemers are optically resolvable.

109. The method of claim 107, wherein said step of generating includes the steps of fragmenting said target polynucleotide, circularizing said fragments, and forming concatemers of said fragments.

110. The method of claim 107 wherein said discrete regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete regions.

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.

112. The method of claim 111, wherein the fragments of DNA disposed on the array substantially represent the nucleotide sequence of the source DNA.

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.

114. The method of claim 113, wherein said concatemers on said discrete regions have a nearest neighbor distance such that at least seventy percent of the concatemers are optically resolvable.

115. The method of claim 113, wherein said step of generating further includes the steps of fragmenting said source DNA to produce said DNA fragments, circularizing said DNA fragments, and forming concatemers of said DNA fragments.

116. The method of claim 113 wherein said discrete regions are wells in said support, the wells each having an opening with an area equal to or less than that of said discrete regions.

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.

118. The method of claim 117, wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern.

119. The method of claim 117, wherein the concatemers comprise multiple alternating copies of a fragment of the target polynucleotide and an adaptor oligonucleotide.

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.

121. The method of claim 120, wherein said discrete spaced apart regions are disposed on said planar surface in a rectilinear pattern.

122. The method of claim 121, wherein the concatemers comprise multiple alternating copies of a fragment of the target polynucleotide and an adaptor oligonucleotide.

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.

124. The method of claim 123, wherein each concatemer comprises multiple alternating copies of a fragment of the target polynucleotide and an adaptor oligonucleotide.

125. The method of claim 123, wherein the surface comprises reactive functionalities, the concatemers comprise functionalities complementary to the reactive functionalities, and wherein said concatemers are each attached to said surface by one or more linkages formed between the reactive functionalities and the complementary functionalities of said concatemers.

126. The method of claim 123, wherein said surface is a planar surface having an array of discrete regions, wherein each discrete region has an area of less than 1 μ

m².

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.

128. The method of claim 127, wherein said discrete regions are disposed on said planar surface in a rectilinear pattern, and wherein each discrete region has an area of from 0.1 to 10 μ

m.

129. The method of claim 127, wherein said concatemers are randomly disposed on said planar surface such that at least seventy percent of the concatemers are optically resolvable.

130. The method of claim 127, wherein said discrete regions are wells in said support having an opening with an area equal to or less than that of said discrete regions.

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.

132. The method of claim 131, wherein said step of amplifying includes cleaving said concatemers to form second fragments, circularizing the second fragments, and amplifying the second fragments within said discrete region of each of said concatemers.

133. The method of claim 131, wherein said step of generating further includes the steps of fragmenting the target polynucleotide to produce said fragments, circularizing said fragments, and forming concatemers of said fragments.

134. The method of claim 131, wherein the concatemers are randomly disposed on a planar surface such that at least seventy percent of the concatemers are optically resolvable.

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.