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 at different features on a solid support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence, a primer binding site and a sequence region that is the same or complementary to a sequence region of a single-stranded oligonucleotide present at a different feature of the solid support;
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 on the support, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a sequence region that is complementary to a sequence region 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
64 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 at different features on a solid support, wherein each of the plurality of template oligonucleotides comprises a predefined sequence, a primer binding site and a sequence region that is the same or complementary to a sequence region of a single-stranded oligonucleotide present at a different feature of the solid support; 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 on the support, thereby forming a secondary droplet, wherein the first primary droplet includes a released oligonucleotide that comprises a sequence region that is complementary to a sequence region 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 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 each of the plurality of oligonucleotides is provided on a different feature of a solid support, 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; d) combining at least two secondary droplets, each droplet comprising a different polynucleotide thereby generating a third stage droplet; and e) exposing the third stage droplet to hybridization conditions and ligation, chain extension, or chain extension and ligation to generate a longer 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)
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61. 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 (62, 63, 64)
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Specification