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1. WO2006044956 - METHODS FOR ASSEMBLY OF HIGH FIDELITY SYNTHETIC POLYNUCLEOTIDES

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[ EN ]
What is claimed is:
1. A method for assembling a long polynucleotide construct having a predefined sequence, comprising:
a) providing a pool of construction oligonucleotides;
b) conducting i), ii), or i) and ii) in either order or simultaneously:
i) amplifying said construction oligonucleotides; and
ii) subjecting said construction oligonucleotides to an error reduction process; and
c) exposing said pool of construction oligonucleotides to hybridization
conditions and one or more of the following conditions: (i) ligation, (ii) chain extension, or (iii) chain extension and ligation conditions, thereby forming a
plurality of copies of at least one double stranded subassembly construct that is longer than said construction oligonucleotides;
d) conducting i), ii), or i) and ii) in either order or simultaneously:
i) amplifying said subassembly construct; and
ii) subjecting said subassembly construct to an error reduction process; and e) incubating two or more subassembly constructs under hybridization conditions and one or more of the following conditions: (i) ligation, (ii) chain extension, or (iii) chain extension and ligation conditions, thereby forming a plurality of copies of a long polynucleotide construct.
2. The method of claim 1, wherein the construction oligonucleotides are subjected to an error reduction process comprising an error filtration process.
3. The method of claim 2, wherein the error filtration process compxises:
a) contacting the pool of construction oligonucleotides with a pool of
selection oligonucleotides under hybridization conditions to form
duplexes, wherein the selection oligonucleotides comprise sequences that are complementary to at least portions of the construction
oligonucleotides, and wherein at least a portion of the duplexes are stable duplexes comprising a copy of a construction oligonucleotide and a copy
of a selection oligonucleotide that do not contain a mismatch in the
complementary region and a portion of the duplexes are unstable duplexes comprising a copy of a construction oligonucleotide and a. copy of a

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9840615 3 selection oligonucleotide that contain one or more mismatches in the
complementary region;
b) removing copies of the construction oligonucleotides that have formed
unstable duplexes; and
c) denaturing the remaining duplexes thereby forming a purified pool of
construction oligonucleotides.
4. The method of claim 3, wherein the selection oligonucleotides are immobilized on a solid support.
5. The method of claim 3 or 4, wherein the selection oligonucleotides comprise a biotin group at one terminus.
6. The method of claim 3, wherein copies of the construction oligonucleotides that have formed unstable duplexes are removed from the pool by controlling the stringency of hybridization or wash conditions, or both.
7. The method of claim 4, wherein the solid support is a column or beads.
8. The method of claim 3, further comprising amplifying the construction
oligonucleotides prior to forming the subassembly construct.
9. The method of claim 3, further comprising repeating a)-c) at least one time prior to forming the subassembly construct.
10. The method of claim 1, wherein the construction oligonucleotides or the
subassembly constructs are subjected to an error filtration process, comprising:
a) incubating the construction oligonucleotides or the subassembly construct with at least one agent that binds to a DNA mismatch; and
b) removing copies of the construction oligonucleotides or the subassembly construct that bound to the agent.
11. The method of claim 10, wherein the agent is a mismatch binding protein.
12. The method of claim 11 , wherein the mismatch binding protein is one or more of the following: Fok I, T7 endonuclease, mutH, mutL, mutM, mutS, mutY, dam, thymidine DNA glycosylase (TDG), uracil DNA glycosylase, AIkA, MLHl,
MSH2, MSH3, MSH6, Exonuclease I, T4 endonuclease V, Exonuclease V, RecJ exonuclease, FENl (RAD27), dnaQ (mutD), or polC (dnaE).
13. The method of claim 12, wherein the mismatch binding protein is mutS .
14. The method of claim 13, wherein the construction oligonucleotides or the
subassembly construct are incubated with mutS in the presence of ATP.

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15. The method of claim 14, wherein the ATP is present in an amount sufficient to increase the affinity of mutS for a duplex containing a mismatch to> less than about 10 nanomolar.
16. The method of claim 12, wherein the mismatch binding protein is a DNA
glycosylase.
17. The method of claim 12, wherein the mismatch binding protein is XDG.
18. The method of claim 10, further comprising amplification of the copies of the construction oligonucleotides or the subassembly construct that did. not bind to the agent.
19. The method of claim 18, further comprising repeating a) and b) at least one time.

20. The method of claim 18, wherein copies of the construction oligonucleotides or the subassembly construct that bound to the agent are removed by gel filtration.

21. The method of claim 18, wherein the agent is immobilized on a substrate.
22. The method of claim 18, wherein the substrate is a column or beads.
23. The method of claim 18, further comprising cross-linking the agent to the
construction oligonucleotides or the subassembly construct.
24. The method of claim 2, wherein the error filtration process comprises:
a) exposing the construction oligonucleotides or the subassenfbly construct to a single stranded nuclease under conditions that permit clearvage of the
oligonucleotides or subassembly having at least one mismatch by the
nuclease; and
b) separating full length oligonucleotides or subassemblies from cleaved
oligonucleotides or subassemblies.
25. The method of claim 24, further comprising a round of denaturation and
renaturation prior to exposing the oligonucleotides or the subassem.~bly to the
single stranded nuclease.
26. The method of claim 24, further comprising amplification of the full length copies of the oligonucleotides or the subassembly remaining after digestion with the
single stranded nuclease.
27. The method of claim 24, wherein full length oligonucleotides or subassemblies are separated from cleaved oligonucleotides or subassemblies by size separation.

28. The method of claim 27, wherein the size separation is gel electrophoresis.

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29. The method of claim 27, wherein the size separation is column chromatography.

30. The method of claim 1, wherein the construction oligonucleotides or the
subassembly construct are subjected to an error reduction process comprising an error neutralization process, the error neutralization process comprising:
a) incubating the construction oligonucleotides or the subassembly construct with an agent that binds to a mismatch; and
b) crosslinking the agent to the copies of the construction oligonucleotides or the subassembly construct that contain a mismatch; and
c) amplifying the construction oligonucleotides or the subassembly construct, wherein copies of the construction oligonucleotides or the subassembly
construct that are crosslinked to the agent are not amplified exponentially and are diluted out.
31. The method of claim 1 , wherein the construction oligonucleotides or the
subassembly construct are subjected to an error reduction process comprising an error neutralization process, the error neutralization process comprising:
a) exposing the construction oligonucleotides or the subassembly construct to a single stranded nuclease under conditions that permit cleavage of the
construction oligonucleotides or the subassembly construct having at least one mismatch by the nuclease;
b) subjecting the digested construction oligonucleotides or subassembly
construct to a round of denaturation and renaturation; and
c) incubating the digested construction oligonucleotides or subassembly
construct under chain extension, or chain extension and ligation,
conditions thereby reforming the full length construction oligonucleotides or the full length subassembly construct.
32. The method of claim 31, further comprising repeating b) and c) at least two times.

33. The method of claim 31 or 32, further comprising adding primers to the pool of digested construction oligonucleotides or subassembly construct under chain
extension, or chain extension and ligation, conditions.
34. The method of claim 1, wherein the subassembly construct is subjected to an error reduction process comprising an error correction process.
35. The method of claim 34, wherein the error correction process comprises:

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9840615 3 a) incubating a plurality of copies of the subassembly construct with an agent that cleaves the subassembly construct to create a double stranded break
and remove the mismatch;
b) melting and reannealing the plurality of copies of the subassembly
construct; and
c) incubating the plurality of copies of the subassembly construct under
hybridization and chain extension conditions, wherein strands of the
subassembly construct that were cleaved by the agent can hybridize to
overlapping, complementary strands and serve as primers for chain
extension.
36. The method of claim 35, wherein the agent is a fusion protein comprising a
mismatch binding protein, or a functional fragment thereof, and a nuclease, or a functional fragment thereof.
37. The method of claim 35, wherein the agent is Fok I, T7 endonuclease I or T4 endonuclease.
38. The method of claim 34, wherein the error correction process comprises:
a) contacting a plurality of copies of the subassembly construct with a
methylation agent;
b) contacting the plurality of copies of the subassembly construct with a site- specific demethylation agent;
c) denaturing the plurality of copies of the subassembly construct;
d) renaturing the plurality of copies of the subassembly construct thereby
forming a plurality of copies of the double stranded subassembly construct at least a portion of which comprise at least one hemimethylated region;
and
e) contacting the plurality of copies of the subassembly construct with a
mismatch repair system.
39. The method of claim 38, wherein the site-specific demethylation agent is a fusion protein comprising a mismatch binding protein, or a functional fragment thereof, and a demethylase, or a functional fragment thereof.
40. The method of claim 34, wherein the error correction process comprises:
a) incubating a plurality of copies of the subassembly construct with an agent that cleaves the subassembly construct to create a double stranded break
and remove the mismatch;
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9840615 3 b) contacting the plurality of copies of the subassembly construct with an
agent that promotes formation of Holliday junctions;
c) incubating the plurality of copies of the subassembly construct under
conditions that promote chain extension; and
d) contacting the plurality of copies of the subassembly construct with an
agent that promotes resolution of the Holliday junctions.
41. The method of claim 1, further comprising subjecting the long polynucleotide construct to an error reduction process.
42. The method of claim 41 , wherein the error reduction process conducted on the long polynucleotide construct comprises:
a) contacting the plurality of copies of the long polynucleotide construct with an agent that causes site-specific double stranded breaks and cohesive ends thereby producing fragments;
b) contacting the fragments with an agent that binds to a mismatch;
c) removing fragments that bound to the agent; and
d) incubating the fragments under conditions that promote hybridization and ligation of the cohesive ends, thereby reforming the long polynucleotide
construct.
43. The method of claim 42, wherein the agent that causes site-specific double
stranded breaks is Fokl, T7 endonuclease I, or T4 endonuclease.
44. The method of claim 41, wherein the error reduction process comprises:
a) contacting the plurality of copies of the long polynucleotide construct with an agent that causes non-specific single-stranded breaks;
b) contacting the plurality of copies of the long polynucleotide construct with an agent that promotes formation of Holliday junctions;
c) incubating the plurality of copies of the long polynucleotide construct
under conditions that promote chain extension; and
d) contacting the plurality of copies of the long polynucleotide construct with an agent that promotes resolution of the Holliday junctions
45. The method of claim 41 , wherein the error reduction process comprises :
a) contacting the plurality of copies of the long polynucleotide construct with an agent that cause non-specific double stranded breaks thereby forming
double stranded fragments;
b) denaturing the fragments thereby forming single stranded fragments;
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9840615 3 c) incubating the fragments under conditions which promote hybridization
between complementary overlapping single stranded fragments;
d) contacting the fragments with an agent that binds to a mismatch;
e) removing fragments that bound to the agent; and
f) incubating fragments under hybridization conditions and at least one of the following conditions: (i) ligation, (ii) chain extension, or (iii) chain
extension and ligation conditions, to reassemble the long polynucleotide
construct.
46. The method of claim 1, wherein said pool forms a plurality of double stranded subassembly constructs.
47. The method of claim 1, wherein the two or more subassembly constructs are assembled in separate reactions.
48. The method of claim 1, wherein the two or more subassembly constructs are assembled in the same reaction.
49. The method of claim 1 , wherein a plurality of subassembly constructs are
incubated under hybridization conditions and at least one of the following
conditions: (i) ligation conditions, (ii) chain extension conditions, (iii) chain
extension and ligation conditions, thereby forming a plurality of long
polynucleotide constructs.
50. The method of any of claims 1 -49, further comprising a round of denaturation and renaturation prior to conducting an error reduction process.
51. The method of claim 1 , wherein the construction oligonucleotides are from about 20 to about 150 nucleotides in length.
52. The method of claim 1, wherein the subassembly construct is at least about 200 to about 750 nucleotides in length.
53. The method of claim 1 , wherein the subassembly construct is at least about 5 times as long as the construction oligonucleotides.
54. The method of claim 1, wherein the polynucleotide construct is at least about 5 times as long as the subassembly construct.
55. The method of claim 1, wherein the polynucleotide construct is at least about 1 kilobase in length.
56. The method of claim 55, wherein the polynucleotide construct is at least about 10 kilobases in length.

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57. The method of claim 56, wherein the polynucleotide construct is at least abcmt 100 kilobases in length.
58. The method of claim 1, wherein less than about 99% of the copies of the
subassembly or polynucleotide constructs have a sequence error.
59. The method of claim 58, wherein less than about 95% of the copies of the
subassembly or polynucleotide constructs have a sequence error.
60. The method of claim 59, wherein less than about 90% of the copies of the
subassembly or polynucleotide constructs have a sequence error.
61. The method of claim 60, wherein less than about 50% of the copies of the
subassembly or polynucleotide constructs have a sequence error.
62. The method of claim 1 or 3, wherein said oligonucleotides are synthesized on a solid support.
63. The method of claim 62, wherein the oligonucleotides are attached to the solid support by a cleavable linker.
64. The method of claim 63, wherein the linker is a chemically cleavable linker, a thermally cleavable linker, an enzymatically cleavable linker, or a photoclearvable linker.
65. The method of claim 62, wherein the synthesis uses light triggered reactions at discrete location on said support.
66. The method of claim 65, wherein the light is directed to discrete locations using masks.
67. The method of claim 66, wherein the light is directed to discrete locations using light directing maskless optics.
68. The method of claim 62, wherein the oligonucleotides are severed from the solid support prior to amplification.
69. The method of claim 1 or 3, wherein said oligonucleotides comprise at least one pair of primer hybridization sites flanking at least a portion of said
oligonucleotides and common to at least a subset of said oligonucleotides.
70. The method of claim 69, wherein all of the oligonucleotides comprise at least one pair of primer hybridization sites in common.
71. The method of claim 69, wherein said oligonucleotides comprise cleavage sites between at least a portion of the primer hybridization sites and the
oligonucleotides.

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72. The method of claim 71, wherein the cleavage site is a restriction endonuclease site.
73. The method of claim 72, wherein the restriction endonuclease is a type IIS
endonuclease.
74. The method of claim 1 or 46, wherein said subassembly constructs comprise at least one pair of primer hybridization sites flanking at least a portion of said
subassembly constructs and common to at least a subset of said subassembly constructs.
75. The method of claim 74, wherein the subassembly constructs comprise a cleavage site between at least one of the primer hybridization sites and the subassembly constructs.
76. The method of claim 1, wherein the amplification of the construction
oligonucleotides or the subassembly constructs uses at least one primer containing at least one uracil residue.
77. The method of claim 76, wherein the uracil residue is located at the junction
between the primer hybridization site and the construction oligonucleotides or the subassembly constructs.
78. The method of claim 76, wherein the primer hybridization site is removed using uracil DNA glycosylase and an AP endonuclease.
79. The method of claim 1, wherein at least two subassembly constructs are formed in the same reaction mixture.
80. The method of claim 79, wherein at least four subassembly constructs are formed in the same reaction mixture.
81. The method of claim 80, wherein at least ten subassembly constructs are formed in the same reaction mixture.
82. The method of claim 1, wherein at least two polynucleotide constructs are formed in the same reaction mixture.
83. The method of claim 82, wherein at least four polynucleotide constructs are
formed in the same reaction mixture.
84. The method of claim 83, wherein at least ten polynucleotide constructs are formed in the same reaction mixture.
85. A method for preparing a long polynucleotide construct having a predefined
sequence, comprising:

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9840615 3 a) providing a pool of construction oligonucleotides under hybridization
conditions and at least one of the following conditions: (i) ligation
conditions, (ii) chain extension conditions, or (iii) chain extension and
ligation conditions, wherein said pool comprises a plurality of overlapping sequences, and wherein said pool forms at least one polynucleotide
construct that is longer than said construction oligonucleotides;
b) conducting i) and ii) in either order or simultaneously:
i) amplifying said polynucleotide constructs; and
ii) subjecting said polynucleotide constructs to an error reduction process;
and
c) repeating steps a) and b) at least two times, wherein said polynucleotide
constructs constitute the construction oligonucleotides in the next cycle.

86. The method of claim 85, wherein the pool of construction oligonucleotides
comprises positive and negative strands that are complementary in the overlapping regions.
87. The method of claim 85, wherein the pool of construction oligonucleotides is amplified prior to forming a polynucleotide construct.
88. The method of claim 85 or 87, wherein the pool of construction oligonucleotides is subjected to an error reduction process prior to forming a polynucleotide
construct.
89. The method of claim 88, wherein the error reduction process is error filtration using a pool of selection oligonucleotides.
90. A method for preparing, in a single pool, a plurality of polynucleotide constructs having different predefined sequences and at least one region of internal
homology, comprising:
a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequence of each of said plurality of
polynucleotide constructs;
b) incubating said pool of construction oligonucleotides under hybridization
conditions and at least one of the following conditions: (i) ligation conditions, (2) chain extension conditions, or (iii) chain extension and ligation conditions; and

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9840615 3 c) separating constructs having said predefined sequences from constructs not
having said predefined sequences, thereby forming a plurality of
polynucleotide constructs having predefined sequences.
91. The method of claim 90, wherein the polynucleotide constructs encode a plurality of polypeptides having at least one region of internal homology.
92. The method of claim 91 , wherein at least a portion of the sequence of one or more polynucleotide constructs has been codon remapped to reduce the homology with at least one other polynucleotide construct.
93. The method of claim 91 , wherein at least 10 polynucleotide constructs are
prepared in a single pool.
94. The method of claim 93, wherein at least 100 polynucleotide constructs are
prepared in a single pool.
95. The method of claim 94, wherein at least 1,000 polynucleotide constructs are prepared in a single pool.
96. The method of claim 90, wherein the polynucleotide constructs encode a plurality of RNAi molecules.
97. The method of claim 90, further comprising subjecting the polynucleotide
constructs to further assembly thereby forming at least one longer polynucleotide construct.
98. The method of claim 90 or 97, further comprising subjecting said construction oligonucleotides or polynucleotide constructs, or both, to at least one round of (i) amplification, (ii) error reduction, or (iii) amplification and error reduction in either order.
99. The method of claim 90, further comprising introducing the polynucleotide
constructs into a vector.
100. The method of claim 90 or 99, further comprising introducing the polynucleotide constructs into a host cell.
101. The method of claim 90, 99 or 100, further comprising expressing a polypeptide or ribonucleic acid from the polynucleotide construct.
102. The method of claim 101, further comprising assaying the polypeptide or
ribonucleic acid for a physical or functional characteristic.
103. A method for assembling at least two polynucleotide constructs having at least one region of internal homology in a single reaction mixture, comprising:
a) providing a pool of construction oligonucleotides comprising:
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9840615 3 i) partially overlapping sequences that define the sequences of said
polynucleotide constructs; and
ii) sequence tags on one or more of the construction oligonucleotides,
wherein a set of construction oligonucleotides that defines a
polynucleotide construct having a desired sequence has a distinguishable complement of sequence tags as compared to a set of construction
oligonucleotides that defines an incorrect crossover product;
b) exposing said pool of construction oligonucleotides to hybridization
conditions and at least one of the following conditions: (i) ligation conditions, (ii) chain extension conditions, or (iii) chain extension and ligation conditions - and
c) separating polynucleotide constructs having a desired sequence from incorrect crossover products based on size or electrophoretic mobility, thereby forming polynucleotide constructs having a region of internal homology.
104. The method of claim 103, wherein the size separation is conducted by column chromatography.
105. The method of claim 103, wherein the polynucleotide constructs have at least two> internal regions of homology.
106. A method for assembling at least two polynucleotide constructs having at least one region of internal homology in a single reaction mixture, comprising:
a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequences of said polynucleotide
constructs, wherein the construction oligonucleotides that define the termini o f a first polynucleotide construct have complementary regions, and wherein the construction oligonucleotides that define the termini of a second
polynucleotide construct have different complementary regions; and
b) exposing said pool of construction oligonucleotides to hybridization
conditions and at least one of the following conditions: (i) ligation conditions^ (ii) chain extension conditions, or (iii) chain extension and ligation conditions ; and
c) separating circularized products from linear products, thereby forming
polynucleotide constructs having a region of internal homology.
107. The method of claim 106, wherein the circularized products are separated from tbre linear products by digesting the linear products with an exonuclease.
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108. The method of claim of claim 106, wherein the circularized products are separated from the linear products using gel electrophoresis.
109. A method for assembling at least two polynucleotide constructs having at least one region of internal homology in a single reaction mixture, comprising:
a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequences of said polynucleotide
constructs; and
b) providing bridging oligonucleotides to the pool of construction
oligonucleotides, wherein the bridging oligonucleotides are complementary to the termini of the correct polynucleotide constructs but which are not
complementary to both termini of an incorrect crossover product;
c) exposing said pool of construction oligonucleotides to hybridization and
ligation conditions thereby by forming a mixture of linear and circularized
products; and
d) denaturing the mixture to form a mixture of single stranded circular and linear products;
e) contacting the mixture with a single stranded exonuclease to remove the linear fragments;
f) contacting the single stranded circular products with a primer pair under chain extension conditions, wherein the primer pair flanks the sequence of the
desired polynucleotide construct, thereby forming a plurality of copies of said polynucleotide constructs having regions of internal homology.
110. The method of any one of claims 103-109, wherein at least three polynucleotide constructs having at least one region of internal homology are assembled in a single reaction mixture.
111. The method of claim 110, wherein at least five polynucleotide constructs having at least one region of internal homology are assembled in a single reaction
mixture.
112. The method of claim 110, wherein at least ten polynucleotide constructs having at least one region of internal homology are assembled in a single reaction mixture.

113. The method of claim 110, wherein at least 100 polynucleotide constructs having at least one region of internal homology are assembled in a single reaction mixture.

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114. The method of any one of claims 103-113, wherein the pool of construction
oligonucleotides comprises positive and negative strands that are complementary in the overlapping regions.
115. The method of any one of claims 103-114, wherein the pool of construction
oligonucleotides is amplified prior to forming a polynucleotide construct.
116. The method of any one of claims 103-115, wherein the polynucleotide constructs are further assembled into longer polynucleotide constructs.
117. The method of claim 116, wherein the polynucleotide constructs are subjected to (i) amplification, (ii) an error reduction process, or (i) and (ii) in either order prior to further assembly.
118. The method of claim 116, wherein further assembly comprises:
a) melting the polynucleotide constructs; and
b) exposing the polynucleotide constructs to hybridization conditions and art least one of the following conditions: (i) ligation conditions, (ii) chain extension
conditions, or (iii) chain extension and ligation conditions, thereby forming
longer polynucleotide constructs.
119. The method of claim 116, wherein further assembly comprises:
a) contacting the polynucleotide constructs with a restriction enzyme; and
b) exposing the polynucleotide constructs to hybridization conditions and art least one of the following conditions: (i) ligation conditions, (ii) chain extension
conditions, or (iii) chain extension and ligation conditions, thereby forming
longer polynucleotide constructs.
120. A method for assembling at least two polynucleotide constructs having at least one region of internal homology in a single reaction mixture, comprising:
d) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequences of said polynucleotide
constructs, wherein the sequences of said construction oligonucleotides <do not terminate within the region of internal homology; and
e) exposing said pool of construction oligonucleotides to hybridization
conditions and at least one of the following conditions: (i) ligation conditions, (ii) chain extension conditions, or (iii) chain extension and ligation conditions,

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9840615 3 thereby forming polynucleotide constructs having a region of internal
homology.
121. The method of claim 120, wherein at least two construction oligonucleotides for assembly of each polynucleotide construct comprise a sequence spanning said region of internal homology and terminating in sequences flanking said region of internal homology.
122. The method of claim 121 , wherein one end of said oligonucleotides terminates about 5 basepairs into the flanking sequences.
123. The method of claim 120, wherein at least one polynucleotide construct further comprises self-complementary regions, wherein said construction
oligonucleotides are designed so that the melting temperature of a duplex between self-complementary regions within a single construction oligonucleotide is lower than the melting temperature of a duplex between complementary strands of two construction oligonucleotides having overlapping sequences.
124. The method of claim 123, further comprising exposing said pool of construction oligonucleotides to hybridization conditions that favor duplex formation between the complementary strands of two construction oligonucleotides having
overlapping sequences over duplex formation between self-complementary
regions within a single oligonucleotide.
125. A method for assembling a polynucleotide construct having self-complementary regions, comprising:
a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequence of said polynucleotide
construct, wherein the melting temperature of a duplex between self- complementary regions within a single construction oligonucleotide is lower than the melting temperature of a duplex between complementary strands of two construction oligonucleotides having overlapping sequences;
b) exposing said pool of construction oligonucleotides to hybridization
conditions that favor duplex formation between the complementary strands of two construction oligonucleotides having overlapping sequences over duplex formation between self-complementary regions within a single
oligonucleotide; and
c) exposing said pool of construction oligonucleotides to at least one of the
following conditions: (i) ligation conditions, (ii) chain extension conditions, or
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9840615 3 (iii) chain extension and ligation conditions, thereby forming polynucleotide
constructs having at least two self-complementary regions.
126. A method for assembling a plurality of polynucleotide constructs encoding a
plurality of interfering RNA (RNAi) molecules comprising a hairpin structure having a sense region and an antisense region joined by a loop region, comprising: a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequences of said polynucleotide
constructs, wlierein the sequences of said construction oligonucleotides do not terminate within the loop region, and wherein the melting temperature of a
duplex between the sense and antisense regions within a single construction
oligonucleotide is lower than the melting temperature of a duplex between
complementary strands of two construction oligonucleotides having
overlapping sequences;
b) exposing said pool of construction oligonucleotides to hybridization
conditions that favor duplex formation between the complementary strands of two construction oligonucleotides having overlapping sequences over duplex formation between the sense and antisense regions within a single
oligonucleotide; and
c) exposing said, pool of construction oligonucleotides to at least one of the
following conditions: (i) ligation conditions, (ii) chain extension conditions, or (iii) chain extension and ligation conditions, thereby forming a plurality of
polynucleotide constructs encoding RNAi molecules.
127. The method of any one of claims 123, 125 or 126, wherein said oligonucleotides are designed to have said melting temperatures by selection of nucleotide length and/or GC content.
128. The method of claim 126, wherein at least two construction oligonucleotides for assembly of each, polynucleotide construct comprise a sequence spanning said region of internal homology and terminating in sequences flanking said region of internal homology.
129. The method of claim 128, wherein a first construction oligonucleotide spans the sense region, the loop region, and a portion of the antisense region and a second, overlapping, construction oligonucleotide spans the antisense region, the loop region, and a portion of the sense region.

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130. The method of claim 129, wherein said portions of the sense and antisense regions spanned by the construction oligonucleotides are about 5 basepairs in length.

131. The method of claim 126, wherein said polynucleotide constructs further comprise a plurality of unique bar code sequences that permit identification of individual polynucleotide constructs.
132. The method of claim 131, wherein said polynucleotide constructs further comprise a primer hybridization site upstream of the bar code sequences.
133. The method of claim 132, wherein said primer hybridization sites are common to a plurality of polynucleotide constructs.
134. The method of claim 126, wherein said polynucleotide constructs further comprise at least one pair of primer hybridization sites near the termini of said
polynucleotide constructs.
135. The method of claim 134, wherein said primer hybridization sites are common to a plurality of polynucleotide constructs.
136. The method of claim 134, wherein said primer hybridization sites are removable upon chemical or enzymatic treatment.
137. The method of claim 136, wherein said primer hybridization sites are removable with a restriction enzyme.
138. The method of claim 137, wherein said restriction enzyme is a type IIS
endonuclease.
139. The method of claim 136, wherein said polynucleotide constructs comprise at least one uracil residue at the junction between the primer hybridization site and the polynucleotide construct.
140. The method of claim 139, wherein said primer hybridization sites are removable with uracil DNA glycosylase and an AP endonuclease.
141. The method of claim 126, wherein said plurality of RNAi molecules comprise sequences for attenuation of a plurality of mammalian genes.
142. The method of any one of claims 90, 103, 1O6, 109, 120, 125, or 126, wherein the assembled polynucleotide constructs are free, or substantially free, of unwanted cross-over products.

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143. The methods of any one of claims 120, 125, 126, or 142, wherein the assembled polynucleotide constructs are free, or substantially free, of products ha-ving
unwanted sequence repeats.
144. The method of any one of claims 90, 103, 106, 109, 120, 125, or 126, xvherein said construction oligonucleotides comprise at least one pair of primer
hybridization sites flanking at least a portion of said construction oligonucleotides and common to at least a subset of said construction oligonucleotides.
145. The method of claim 144, wherein all of said construction oligonucleotides
comprise at least one pair of primer hybridization sites in common.
146. The method of claim 144, wherein said primer hybridization sites are removable upon chemical or enzymatic treatment.
147. The method of claim 146, wherein said primer hybridization sites are removable with a restriction enzyme.
148. The method of claim 147, wherein said restriction enzyme is a type IIS
endonuclease.
149. The method of claim 146, wherein said polynucleotide constructs comprise at least one uracil residue at the junction between the primer hybridization site and the construction oligonucleotides.
150. The method of claim 149, wherein said primer hybridization sites are removable with uracil DNA glycosylase and an AP endonuclease.
151. The method any one of claims 90, 103, 106, 109, 120, 125, or 126, further
comprising subjecting said construction oligonucleotides or polynucleotide
constructs, or both, to at least one round of (i) amplification, (ii) error reduction, or (iii) amplification and error reduction in either order.
152. The method of claim 151, wherein said pool of construction oligonucleotides is amplified prior to assembling a polynucleotide construct.
153. The method of claim 151, wherein the pool of input oligonucleotides is subjected to an error reduction process prior to assembling a polynucleotide construct.

154. The method of claim 153, wherein the error reduction process is error -filtration using a pool of selection oligonucleotides.
155. The method of claim 151, further comprising a round of denaturation and
renaturation prior to conducting an error reduction process.

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156. The method of any one of claims 90, 1O3, 106, 109, 120, 125, or 126, wherein the pool of construction oligonucleotides comprises positive and negative strands that are complementary in the overlapping regions.
157. The method of claim 156, wherein complementary overlapping regions comprise from about 10 to about 30 bases.
158. The method of claim 157, wherein complementary overlapping regions comprise from about 14 to about 20 bases.
159. The method of any one of claims 90, 1O3, 106, 109, 120, 125, or 126, further comprising introducing the polynucleotide constructs into a vector.
160. The method of any one of claims 90, 1O3, 106, 109, 120, 125, 126, or 159, further comprising introducing the polynucleotide constructs into a host cell.
161. The method of any one of claims 90, 1 O3, 106, 109, 120, 125, 126, 159, or 160, further comprising expressing a polypeptide or ribonucleic acid from the
polynucleotide construct.
162. The method of claim 161, further comprising assaying the polypeptide or
ribonucleic acid for a physical or functional characteristic.
163. The method of claim 120 or 126, wherein at least 10 polynucleotide constructs are assembled in a single pool.
164. The method of claim 163, wherein at least 100 polynucleotide constructs are assembled in a single pool.
165. The method of claim 164, wherein at least 1 ,000 polynucleotide constructs are assembled in a single pool.
166. A composition comprising a plurality of oligonucleotides, wherein said
oligonucleotides comprise overlapping sequences that define the sequences of a plurality of polynucleotide constructs comprising at least one region of internal homology, and wherein the sequences of said oligonucleotides do not terminate within the region of internal homology.
167. The composition of claim 166, wherein at least two oligonucleotides that define the sequence of each polynucleotide construct comprise a sequence spanning said region of internal homology and terminating in sequences flanking said region of internal homology.
168. The composition of claim 167, wherein one end of said oligonucleotides
terminates about 5 basepairs into the flanking sequences.

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169. The composition of claim 166, wherein at least one polynucleotide construct further comprises self-complementary regions, wherein said oligonucleotides are designed so that the melting temperature of a duplex between self-complementary regions within a single oligonucleotide is lower than the melting temperature of a duplex between complementary strands of two oligonucleotides having
overlapping sequences.
170. The composition of claim 166 or 169, wherein said oligonucleotides comprise at least one pair of primer hybridization sites flanking at least a portion of said
oligonucleotides and common to at least a subset of said oligonucleotides.
171. The composition of claim 170, wherein all of said oligonucleotides comprise at least one pair of primer hybridization sites in common.
172. The composition of claim 170, wherein said primer hybridization sites are
removable upon chemical or enzymatic treatment.
173. The composition of claim 172, wherein said primer hybridization sites are
removable with a restriction enzyme.
174. The composition of claim 173, wherein said restriction enzyme is a type IIS
endonuclease.
175. The composition of claim 172, wherein said polynucleotide constructs comprise at least one uracil residue at the junction between the primer hybridization site and the construction oligonucleotides.
176. The composition of claim 175, wherein said primer hybridization sites are
removable with uracil DNA glycosylase and an AP endonuclease.
177. The composition of claim 166, wherein said oligonucleotides are double stranded.

178. The composition of claim 166, wherein said composition comprises
oligonucleotides having positive and negative strands that are complementary in the overlapping regions.
179. The composition of claim 178, wherein complementa.τy overlapping regions
comprise from about 10 to about 30 bases.
180. The composition of claim 179, wherein complementary overlapping regions
comprise from about 14 to about 20 bases.

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181. The composition of claim 166 or 169, wherein said polynucleotide constructs encode a plurality of interfering RNA (RNAi) molecules comprising a hairpin structure having a sense region and an antisense region joined by a loop region.

182. The composition of claim 181, wherein at least two oligonucleotides defining the sequence of each polynucleotide construct comprise a sequence spanning said region of internal homology and terminating in sequences flanking said region of internal homology.
183. The composition of claim 182, wherein a first oligonucleotide spans the sense region, the loop region, and a portion of the antisense region and a second,
overlapping, oligonucleotide spans the antisense region, the loop region, and a portion of the sense region.
184. The composition of claim 183, wherein said portions of the sense and antisense regions spanned by the oligonucleotides are about 5 basepairs in length.
185. The composition of claim 181, wherein said polynucleotide constructs further comprise a plurality of unique bar code sequences that permit identification of individual polynucleotide constructs.
186. The composition of claim 185, wherein said polynucleotide constructs further comprise a primer hybridization site upstream of the bar code sequences.
187. The composition of claim 166 or 169, wherein said oligonucleotides are
immobilized on a substrate.
188. A method for assembling a polynucleotide construct having regions of
self-homology, comprising:
a) providing a pool of construction oligonucleotides comprising partially
overlapping sequences that define the sequence of said polynucleotide
construct, wherein the sequences of said construction oligonucleotides do not terminate within the regions of self-homology; and
b) exposing said pool of construction oligonucleotides to hybridization
conditions and at least one of the following conditions: (i) ligation conditions, (ii) chain extension conditions, or (iii) chain extension and ligation conditions, thereby forming a polynucleotide construct having regions of self-homology.

189. The method of claim 188, wherein the regions of self-homology are direct repeats.

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190. The method of claim 188, wherein the regions of self-hology are regions of
self-complementarity.
191. The method of claim 188, wherein at least two construction oligonucleotides for assembly of the polynucleotide construct comprise a sequence spanning said region of self-homology and terminating in sequences flanking said region of self-homology.
192. The method of claim 191, wherein one end of said oligonucleotides terminates about 5 basepairs into the flanking sequences.
193. The method of claim 188, wherein two or more polynucleotide constructs
comprising regions of self-homology are assembled in a single reaction mixture.

194. The method of claim 188, wherein a region of self-homology encompasses at least one end of the polynucleotide construct and at least one construction
oligonucleotide further comprises a flanking sequence that is non-homologous to the regions of self-homology so that none of the construction oligonuceotides terminate within a region of self-homology.
195. The method of claim 194, further comprising removing the flanking sequence after assembly of the polynucleotide construct.
196. A composition comprising a plurality of construction oligonucleotides for
assembly into a larger double stranded polynucleotide construct wherein at least a portion of said construction oligonucleotides comprise a mutH cut site flanking the construction oligonucleotide at the 5' end, 3' end, or both ends.
197. The composition of claim 196, wherein at least a portion of said construction
oligonucleotides further comprise at least one pair of primer hybridization sites flanking the construction oligonucleotides and common to at least a subset of said construction oligonucleotides.
198. The composition of claim 196, wherein at least a portion of said construction
oligonucleotides further comprise a cleavage site between the construction
oligonucleotide and any flanking sequences and common to at least a subset of said construction oligonucleotides.
199. The composition of claim 198, wherein the cleavage site is a restriction
endonuclease cut site.
200. The composition of claim 199, wherein the cleavage site is a type IIS
endonuclease cut site.
201. The composition of claim 198, wherein the cleavage site is a uracil residue.
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202. The composition of claim 198, wherein the cleavage site between the co-nstruction oligonucleotide and any 5' flanking sequences is the same as the cleavage site between the construction oligonucleotide and any 3' flanking sequences.
203. The composition of claim 196, wherein at least a portion of said construction oligonucleotides further comprise an agent for detection, isolation or
immobilization that is common to at least a subset of said construction
oligonucleotides.
204. The composition of claim 203, wherein the agent is biotin, fluorescein, or an
aptamer.
205. A composition comprising a plurality of construction oligonucleotides for
assembly into a larger double stranded polynucleotide construct wherein, at least a portion of said construction oligonucleotides comprise (i) a mutH cut site flanking at least one end of the construction oligonucleotides, (ii) at least one pair of primer hybridization sites flanking the construction oligonucleotides and common to at least a subset of said construction oligonucleotides, and (iii) a cleavage site
between the construction oligonucleotide and any flanking sequences and
common to at least a subset of said construction oligonucleotides.
206. A method of preparing a purified pool of construction oligonucleotides
comprising:
a) providing a pool of double stranded construction oligonucleotides, wherein at least a portion of said construction oligonucleotides comprise an agent for
detection, isolation or immobilization and a mutH cut site flanking ttie
construction oligonucleotide at the 5 ' end or the 3 ' end;
b) contacting the pool of construction oligonucleotides with mutHLS, wherein
the mutHLS binds to copies of the construction oligonucleotides that contain
one or more mismatches and cleaves the copies that contain the one or more
mismatches at the mutH cut site; and
c) removing copies of the construction oligonucleotides that were cleaved at the mutH cut site, thereby forming a pool of purified construction
oligonucleotides.
207. The method of claim 206, wherein the agent is biotin.
208. The method of claim 206, further comprising amplifying the construction
oligonucleotides prior to addition of the mutHLS.

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209. The method of claim 206 or 208, further comprising denaturing and reannealing the pool of construction oligonucleotides prior to addition of the mutHLS.
210. An automated process for a manufacturer to satisfy customer orders for requested polynucleotide constructs having a specified sequence, the process comprising: a) obtaining a desired sequence from the customer;
b) computationally designing a set of construction oligonucleotides that define the desired sequence; and
c) synthesizing the set of construction oligonucleotides.
211. The process of claim 210, wherein the desired sequence encodes a polypeptide sequence.
212. The process of claim 211, further comprising computationally determining one or more polynucleotide constructs that encode the polypeptide sequence.
213. The process of claim 210, further comprising computationally designing a set of selection oligonucleotides.
214. The process of claim 213, further comprising synthesizing the set of selection oligonucleotides.
215. The process of claim 210, further comprising designing an assembly strategy for preparation of the polynucleotide construct.
216. The process of claim 215, further comprising assembling the polynucleotide construct using the assembly strategy.
217. The process of claim 215, wherein the assembly strategy is designed
computationally.
218. The process of claim 210, 214, or 215, further comprising shipping to the
customer one or more of the following: the set of construction oligonucleotides, the set of selection oligonucleotides, instructions for assembling the
polynucleotide construct.
219. The process of claim 218, further comprising shipping to the customer one or more reagents for assembling the polynucleotide construct.
220. The process of claim 219, wherein the reagents are buffers or enzymes.
221. The process of claim 216, further comprising shipping to the customer the
polynucleotide construct.
222. The process of claim 210, further comprising computationally adding one or more universal tags to the 5' flanking region, 3' flanking region, or both, of at least a subset of the construction oligonucleotides.

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223. The process of claim 210, wherein a plurality of desired sequences are obtained from the customer.
224. The process of claim 223, further comprising designing an assembly strategy for preparation of the polynucleotide constructs in a single pool.
225. The process of claim 223, further comprising designing a hierarchical assembly strategy for preparation of the polynucleotide constructs.
226. The process of claim 215 or 223, wherein the assembly strategy comprises one or more error reduction processes.
227. The process of claim 215 or 223, wherein the assembly strategy comprises one or more amplification steps.
228. The process of claim 210, 213, 215, or 223, further comprising one or more of the following: optimizing codon usage for expression in a particular host cell,
normalizing liybridization conditions for a set of construction oligonucleotides, normalizing liybridization conditions for a set of construction and selection
oligonucleotides, reducing homology between two or more desired sequences by codon remapping.
229. The process of claim 210, wherein the desired sequence is obtained from a
database.
230. The process of claim 210, wherein the communication from the customer is retained within a storage device of the manufacturer.
231. The process of claim 210, 213, or 215, wherein one or more of the following: the sequences of the construction oligonucleotides, the sequences of the selection oligonucleotides, or the assembly strategy, is retained within a storage device of the manufacturer.
232. A system for a manufacturer to obtain customer orders for custom designed
polynucleotide constructs, comprising:
a) a network-based receiving station for a manufacturer to receive desired
sequences from the customer;
b) a software means for designing a set of construction oligonucleotides, a set of selection oligonucleotides, or an assembly strategy; and
c) a manufacturing system for synthesizing the construction oligonucleotides,
selection oligonucleotides, or both.
233. The system of claim 232, wherein the system further comprises a manufacturing system for automated assembly of a polynucleotide construct.

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234. A composition comprising a plurality of copies of a synthetic nucleic acid having a predefined sequence wherein said nucleic acid has a length of at least about 5 kilobases and wherein at least about 1<M> of said copies do not contain an error in said predefined sequence.
235. The composition of claim 234, wherein said nucleic acid has a length of at least about 10 kilobases.
236. The composition of claim 235, wherein said nucleic acid has a length of at least about 100 kilobases.
237. The composition of claim 234, wherein at least about 5% of said copies do not contain an error is said predefined sequence.
238. The composition of claim 237, wherein at least about 10% of said copies do not contain an error is said predefined sequence.
239. The composition of claim 238, wherein at least about 20% of said copies do not contain an error is said predefined sequence.
240. The composition of claim 238, wherein at least about 50% of said copies do not contain an error is said predefined sequence.
241. The composition of claim 234, wherein the composition comprises at least about 1 mg of said synthetic nucleic acid.
242. The composition of claim 241, wherein the composition comprises at least about 1 g of said synthetic nucleic acid.
243. The composition of claim 242, wherein the composition comprises at least about 1 kg of said synthetic nucleic acid.
244. The composition of claim 234, wherein the composition is essentially free of at least one cellular contaminant without "using a purification step to remove said contaminant.
245. The composition of claim 244, wherein the composition is essentially free of at least one of the following cellular contaminants: lipids; lipopolysaccharides
(LPS); carbohydrates; pyrogens; a protein other than one or more of the following: polymerase, ligase, a mismatch binding protein, a mismatch repair protein, a
methylase, a demethylase, a restriction endonuclease, or an exonuclease; or a small molecule other than one or more of the following: dNTPs, biotin, or a
chemical cross-linker.
246. The composition of claim 234, wherein the composition is essentially free of at least one type of nucleic acid modification.
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247. The composition of claim 245, wherein the modification is methylation.
248. A method for reducing the error rate in a pool of nucleic acids, comprising:
a) exposing the pool of nucleic acids to a single stranded nuclease under
conditions that permit cleavage of nucleic acids having at least one
mismatch by the nuclease thereby forming a pool of digested nucleic
acids; and
b) separating full length nucleic acids from cleaved nucleic acids in the
digested pool of nucleic acids, thereby removing copies of nucleic acids containing errors from the pool and resulting in a pool having a reduced
error rate.
249. The method of claim 248, further comprising a round of denaturation and
renaturation prior to exposing the pool of nucleic acids to the single stranded nuclease.
250. The method of claim 248, further comprising amplification of the full length nucleic acids remaining in the digested pool of nucleic acids.
251. The method of claim 248, wherein full length nucleic a.cids are separated from cleaved nucleic acids by size separation.
252. The method of claim 251 , wherein the size separation is gel electrophoresis.

253. The method of claim 251, wherein the size separation i s column chromatography.

254. The method of claim 248, further comprising repeating a) and b) at least two times.
255. A method for reducing the error rate in a pool of nucleic acids, comprising:
a) exposing the pool of nucleic acids to a single stranded nuclease under
conditions that permit cleavage of nucleic acids having at least one
mismatch by the nuclease thereby forming a pool of digested nucleic
acids;
b) subjecting the pool of digested nucleic acids to a round of denaturation and renaturation; and

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chain extension and ligation, conditions thereby reforming a pool of full
length nucleic acids having a reduced error rate.
256. The method of claim 255, further comprising repeating b) and c) at least two times.
257. The method of claim 255, further comprising repeating a), b) and c) at least two times.
258. The method of claim 255 or 256, further comprising adding primers to the pool of digested nucleic acids under chain extension, or chain extension and ligation,
conditions.
259. The method of claim 248 or 255, wherein the single stranded nuclease is mung bean nuclease.
260. The method of claim 248 or 255, wherein the single stranded nuclease is Sl
nuclease.
261. The method of claim 248 or 255, wherein the error rate is reduced by at least 5O%.

262. The method of claim 261, wherein the error rate is reduced by at least 75%.
263. The method of claim 262, wherein the error rate is reduced by at least 90%.
264. The method of claim 248 or 255, further comprising introducing the nucleic acids into a vector.
265. The method of any of claims 248, 255 or 264, further comprising introducing thie nucleic acids into a host cell.
266. The method of any of claims 248, 255, 264 or 265, further comprising expressing a polypeptide or ribonucleic acid from the nucleic acids.
267. The method of claim 266, further comprising assaying the polypeptide or
ribonucleic acid for a physical or functional characteristic.
268. The method of claim 248 or 255, wherein at least a portion of the nucleic acids are circular.
269. The method of claim 248 or 255, wherein at least a portion of the nucleic acids are linear.
270. The method of claim 269, wherein the linear nucleic acids are blocked at one ox both ends with an agent that prevents cleavage of the ends by a single stranded

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271. The method of claim 270, wherein the linear nucleic acids are blocked at one or both ends with biotin.
272. A nucleic acid array, comprising: a solid support; and a plurality of discrete features associated with said solid support, wherein each feature independently comprises a population of nucleic acids collectively having a defined consensus sequence but in which no more than 10 percent of said nucleic acids of said
feature have the identical sequence.
273. The array of claim 272, wherein no more than 5 percent of said nucleic acids of a feature have the identical sequence.
274. The array of claim 272, wherein no more than 2 percent of said nucleic acids of a feature have the identical sequence.
275. The array of claim 272, wherein said nucleic acids are at least 50 nucleotides in length.
276. The array of claim 272, wherein said nucleic acids are at least 100 nucleotides in length.
277. The array of claim 272, wherein said nucleic acids are at least 200 nucleotides in length.
278. The array of claim 272, wherein said nucleic acids are releasable from said solid support.
279. The array of claim 278, wherein said feature includes means for selectively
releasing nucleic acids from said solid support.
280. The array of claim 279, wherein said means for selectively releasing nucleic acids from said solid support includes means for releasing said nucleic acids by
electrostatic or controlled field means.
281. The array of claim 279, wherein said means for selectively releasing nucleic acids from said solid support includes a photolabile linker.
282. The array of claim 278, wherein said features include a chemical agent for
forming a reversible non-covalent interaction with said nucleic acids, which
interaction can be selectively dissociated to release the nucleic acids from
predetermined subsets of said features.

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283. The array of claim 278, wherein said features include a chemical agent for
forming a covalent bond with said nucleic acids, which bond can be selectively cleaved to release the nucleic acids from predetermined subsets of said features.

284. The array of claim 272, comprising at least 100 different features per square centimeter.
285. The array of claim 272, comprising at least 500 different features per square centimeter.
286. The array of claim 272, comprising at least 1000 different features per square centimeter.
287. The array of claim 272, wherein said features have a feature size of less than 500 microns.
288. The array of claim 272, wherein said features have a feature size of less than 100 microns.
289. The array of claim 272, wherein said solid support is selected from the group consisting of glass, silicon, ceramic and nylon.
290. The array of claim 272, wherein said features are provided on a surface of said solid support composed of a polymer selected from the group consisting of
polytetrafluoroethylene, polyvinylidene difluoride, polystyrene, polycarbonate, and combinations thereof.
291. The array of claim 272, wherein said features are in fluid connection with one and other.
292. A method for assembling at least one polynucleotide construct having a
predefined sequence, comprising:
a) providing one or more nucleic acid arrays comprising a solid support and a plurality of discrete features associated with said solid support, wherein
each feature independently comprises a population of construction
oligonucleotides collectively having a defined consensus sequence but in which no more than 10 percent of said construction oligonucleotides of
said feature have the identical sequence;
b) simultaneously or sequentially releasing construction oligonucleotides
from one or more of said features to form a pool of construction
oligonucleotides comprising partially overlapping sequences that define
the sequence of said polynucleotide construct; and
c) providing conditions promoting:
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construction oligonucleotides;
ii) ligation, chain extension, or chain extension and ligation, of
hybridized construction oligonucleotides to form a polynucleotide
construct; and
iii) error reduction to provide a polynucleotide construct; having said
predetermined sequence.
293. The method of claim 292, wherein said polynucleotide construct is at least 1000 bases in length.
294. The method of claim 292, wherein said polynucleotide construct is at least 5000 bases in length.
295. The method of claim 292, wherein said pool of construction oligonucleotides comprises positive and negative strands that are complementary in the overlapping regions.
296. The method of claim 292, further comprising amplifying said pool of construction oligonucleotides prior to forming the polynucleotide construct.
297. The method of claim 292, further comprising subjecting said pool of construction oligonucleotides to an error reduction process prior to forming the polynucleotide construct.
298. The method of claim 292, wherein said error reduction is an error filtration
process.
299. The method of claim 292, wherein said error reduction is an error neutralization process.
300. The method of claim 292, wherein said error reduction is an error c orrection
process.
301. The method of claim 292, wherein construction oligonucleotides comprise at least one pair of primer hybridization sites flanking at least a portion of said
oligonucleotides and common to at least a subset of said construction
oligonucleotides.
302. The method of claim 301, wherein said primer hybridization sites are removable.

303. The method of claim 292, wherein at least two polynucleotide constructs are
formed in the same reaction mixture.
304. The method of claim 292, further comprising amplifying the polynucleotide
construct.

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