Methods for assembly of high fidelity synthetic polynucleotides
First Claim
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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.
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Abstract
Disclosed are methods of manufacturing synthetic DNAs, that is, DNAs made at least in significant part by chemical synthesis of polynucleotide polymers. Also provided are methods for assembling plural DNAs in the same pool by multiplexed assembly of synthetic oligonucleotides. In exemplary embodiments, the methods involve pre-amplification of one or more oligonucleotides using “universal” primers, reduction of the error rate in oligonucleotide and/or polynucleotide products, and sequence optimization and oligonucleotides design.
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Citations
81 Claims
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1. A method for assembling a long polynucleotide construct having a predefined sequence, comprising:
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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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75)
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76. A method for preparing a long polynucleotide construct having a predefined sequence, comprising:
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a) providing a pool of input 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 product polynucleotide that is longer than said oligonucleotides;
b) conducting i) and ii) in either order or simultaneously;
i) amplifying said product polynucleotides; and
ii) subjecting said product polynucleotides to an error reduction process; and
c) repeating steps a) and b) at least two times, wherein said product polynucleotides constitute the input oligonucleotides in the next cycle. - View Dependent Claims (77, 78, 79, 80)
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81-162. -162. (canceled)
Specification