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 oligonucleotide 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.
84 Citations
83 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 oligonucleotide 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, 61, 62, 63, 64, 65, 66, 67, 68, 69)
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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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70. 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 serves 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 (71)
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72. 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 serves 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 (73)
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74. 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 comprise complementary sequences with a terminal region of said first plurality of oligonucleotides; and b) contacting said first and second pluralities of oligonucleotides under conditions that allow one or more of;
annealing, chain extension, and denaturing. - View Dependent Claims (75, 76, 77)
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78. 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 support 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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79. A method for synthesizing a plurality of oligonucleotide 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 oligonucleotide; 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 (80, 81, 82, 83)
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Specification