Methods and Devices for High Fidelity Polynucleotide Synthesis
First Claim
1. A method for assembling a polynucleotide having a predefined sequence from a plurality of different oligonucleotides, the method comprising:
- a) providing a plurality of single-stranded template oligonucleotides on a support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site;
b) generating a complementary oligonucleotide for each of the plurality of template oligonucleotides by enzyme-catalyzed synthesis within a primary droplet, thereby producing a plurality of double-stranded oligonucleotides;
c) releasing the complementary oligonucleotides from the double-stranded oligonucleotides into the primary droplet;
d) combining at least a first and second primary droplets, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a portion that is complementary to a portion of a released or template oligonucleotide from the second primary droplet; and
e) exposing the secondary droplet to conditions suitable for hybridization and ligation, polymerase extension, or polymerase extension and ligation to assemble a double-stranded polynucleotide having a predefined sequence.
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Abstract
Disclosed are methods for synthesizing and/or assembling at least one polynucleotide product having a predefined sequence from a plurality of different oligonucleotides. In exemplary embodiments, the methods involve synthesis and/or amplification of different oligonucleotides immobilized on a solid support, release of synthesized/amplified oligonucleotides in solution to form droplets, recognition and removal of error-containing oligonucleotides, moving or combining two droplets to allow hybridization and/or ligation between two different oligonucleotides, and further chain extension reaction following hybridization and/or ligation to hierarchically generate desired length of polynucleotide products.
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Citations
84 Claims
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1. A method for assembling a polynucleotide having a predefined sequence from a plurality of different oligonucleotides, the method comprising:
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a) providing a plurality of single-stranded template oligonucleotides on a support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site; b) generating a complementary oligonucleotide for each of the plurality of template oligonucleotides by enzyme-catalyzed synthesis within a primary droplet, thereby producing a plurality of double-stranded oligonucleotides; c) releasing the complementary oligonucleotides from the double-stranded oligonucleotides into the primary droplet; d) combining at least a first and second primary droplets, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a portion that is complementary to a portion of a released or template oligonucleotide from the second primary droplet; and e) exposing the secondary droplet to conditions suitable for hybridization and ligation, polymerase extension, or polymerase extension and ligation to assemble a double-stranded polynucleotide having a predefined sequence. - 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, 60)
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25. A method for assembling at least one polynucleotide having a predefined sequence, the method comprising:
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a) providing a plurality of different oligonucleotides segregated in separate primary droplets;
wherein the plurality of oligonucleotides together comprise the polynucleotide sequence; and
wherein each of said primary droplets comprises multiple copies of at least one of said plurality of different oligonucleotides;b) combining at least two primary droplets, thereby forming a secondary droplet; c) exposing the secondary droplet to hybridization conditions and ligation, chain extension, or chain extension and ligation to generate a polynucleotide having a predefined sequence. - View Dependent Claims (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)
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- 53. A polynucleotide assembly system comprising an acoustic liquid handling device operably connected to a microfluidic device, wherein the microfluidic device comprises an assembly station, a sequencing station and a polynucleotide isolation station.
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62. The method of claim 61, wherein the solid support is a microarray.
- 63. The method of claim 61, wherein each of the first plurality of oligonucleotides comprises a detectable tag at one terminus.
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64. The method of claim 61, wherein each of the second plurality of oligonucleotides comprises a detectable tag at one terminus.
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66. The method of claim 61, further comprising prior to step a):
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synthesizing the first plurality of oligonucleotides in a chain extension reaction, wherein the second plurality of oligonucleotides serve as templates in the chain extension reaction; and denaturing products of the chain extension reaction. - View Dependent Claims (67)
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68. The method of claim 61, further comprising reusing the solid support in synthesizing oligonucleotides, hybridizing oligonucleotides, or both.
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69. The method of claim 61, further comprising using a digital mirror device to selectively heat different spots on the solid support.
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70. The method of claim 61, further comprising repeating a)-c) at least one time prior to forming the purified plurality of oligonucleotides.
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71. A method for removing error-containing oligonucleotides synthesized on a solid support, the method comprising:
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a) synthesizing a first plurality of oligonucleotides in a chain extension reaction, wherein a second plurality of oligonucleotides immobilized on said solid support serve as templates in the chain extension reaction; b) denaturing products of the chain extension reaction; c) contacting the first plurality of oligonucleotides with the second plurality of oligonucleotides under hybridization conditions to form duplexes; and d) separating error-containing oligonucleotides from oligonucleotides with error-free sequences using a component which actively selects for a sequence error. - View Dependent Claims (72)
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73. A method for removing error-containing oligonucleotides synthesized on a solid support, the method comprising:
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a) synthesizing a first plurality of oligonucleotides in a chain extension reaction on a first spot on said solid support, wherein a second plurality of oligonucleotides immobilized on said first spot on said solid support serve as templates in the chain extension reaction; b) denaturing products of the chain extension reaction; c) contacting the first plurality of oligonucleotides with a third plurality of oligonucleotides under hybridization conditions to form duplexes, wherein said third plurality of oligonucleotides are synthesized on a second spot on said solid support substantially in parallel to step a), and wherein said first and third plurality of oligonucleotides comprise sequences that are complementary; and d) separating error-containing oligonucleotides from oligonucleotides with error-free sequences using a component which actively selects for a sequence error. - View Dependent Claims (74)
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75. A method of assembling a polynucleotide product on a solid support comprising:
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a) moving a first droplet comprising a first plurality of oligonucleotides from a first spot on said solid support to a second spot on said solid support, wherein said second spot comprises a second plurality of oligonucleotides, wherein a terminal region of said second plurality of oligonucleotides comprises complementary sequences with a terminal region of said first plurality of oligonucleotides; and b) contacting said first and second plurality of oligonucleotides under conditions that allow one or more of;
annealing, chain extension, and denaturing. - View Dependent Claims (76, 77, 78)
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79. A method for qualitatively confirming a sequence of a polynucleotide product on a solid support comprising:
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a) synthesizing said polynucleotide product on said solid support, wherein said solid support comprises one of more spots comprising immobilized oligonucleotides, and wherein said polynucleotide product comprises a detectable tag; b) recycling said solid support; c) contacting said polynucleotide product with said recycled solid supports under hybridization conditions to form duplexes between said polynucleotide product and said immobilized oligonucleotides; and d) detecting a presence of said detectable tag at said one or more spots, thereby confirming the sequence of said polynucleotide product.
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80. A method for synthesizing a plurality of oligonucleotides having a predefined sequence, the method comprising:
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a) providing a plurality of support-bound template oligonucleotides in a solution comprising a primer, a polymerase and nucleotides, wherein each of the plurality of template oligonucleotides comprises a predefined sequence and includes a primer binding site, and wherein the primer comprises at least one nuclease recognition site; b) exposing the plurality of template oligonucleotides to conditions suitable for primer hybridization and polymerase extension, thereby extending the primers to produce a complementary oligonucleotide for each of the plurality of template oligonucleotides; c) releasing the complementary oligonucleotides; d) exposing the complementary oligonucleotides to a nuclease under conditions suitable for the nuclease to bind to the nuclease recognition site on the primer and cleave the primer from complementary oligonucleotides; and e) exposing the complementary and template oligonucleotides to conditions suitable for hybridization;
thereby to produce a plurality of partially double-stranded oligonucleotides. - View Dependent Claims (81, 82, 83, 84)
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Specification