Methods for high fidelity production of long nucleic acid molecules
First Claim
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1. A method for removing or controlling errors in a plurality or pool of nucleic acid molecules, the method comprising:
- a) synthesizing said plurality or pool of nucleic acid molecules by the steps of;
providing a first immobilized nucleic acid comprising a first 5′
region and a first 3′
region;
providing a second immobilized nucleic acid comprising a second 5′
region and a second 3′
region, wherein said second 3′
region and said first 5′
region comprise identical nucleic acid sequences;
hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′
region, extending the hybridized oligonucleotide, and producing a first extension product comprising a 3′
region that is complementary to said first 5′
region; and
hybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′
region of said first extension product to said second 3′
region, extending the 3′
region of said first extension product, and producing a second extension product comprising a 3′
region that is complementary to said second 5′
region, wherein said second extension product comprises a sequence complementary to said first 3′ and
5′
regions and said second 3′ and
5′
regions, and the individual nucleic acid molecules of said second extension product comprise said plurality or pool of nucleic acid molecules;
b) distinguishing between error-free and error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules; and
c) selectively amplifying only the error-free nucleic acid molecules from said plurality or pool of nucleic acid molecules, thereby decreasing the relative amount of the error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules.
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Abstract
This invention generally relates to nucleic acid synthesis, in particular DNA synthesis. More particularly, the invention relates to the production of long nucleic acid molecules with precise user control over sequence content. This invention also relates to the prevention and/or removal of errors within nucleic acid molecules.
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Citations
13 Claims
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1. A method for removing or controlling errors in a plurality or pool of nucleic acid molecules, the method comprising:
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a) synthesizing said plurality or pool of nucleic acid molecules by the steps of; providing a first immobilized nucleic acid comprising a first 5′
region and a first 3′
region;providing a second immobilized nucleic acid comprising a second 5′
region and a second 3′
region, wherein said second 3′
region and said first 5′
region comprise identical nucleic acid sequences;hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′
region, extending the hybridized oligonucleotide, and producing a first extension product comprising a 3′
region that is complementary to said first 5′
region; andhybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′
region of said first extension product to said second 3′
region, extending the 3′
region of said first extension product, and producing a second extension product comprising a 3′
region that is complementary to said second 5′
region, wherein said second extension product comprises a sequence complementary to said first 3′ and
5′
regions and said second 3′ and
5′
regions, and the individual nucleic acid molecules of said second extension product comprise said plurality or pool of nucleic acid molecules;b) distinguishing between error-free and error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules; and c) selectively amplifying only the error-free nucleic acid molecules from said plurality or pool of nucleic acid molecules, thereby decreasing the relative amount of the error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules. - View Dependent Claims (2, 3, 4)
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5. A method for removing or controlling errors in a plurality or pool of nucleic acid molecules, the method comprising:
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a) synthesizing said plurality or pool of nucleic acid molecules by the steps of; providing a first immobilized nucleic acid comprising a first 5′
region and a first 3′
region;providing a second immobilized nucleic acid comprising a second 5′
region and a second 3′
region, wherein said second 3′
region and said first 5′
region comprise identical nucleic acid sequences;hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′
region, extending the hybridized oligonucleotide, and producing a first extension product comprising a 3′
region that is complementary to said first 5′
region; andhybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′
region of said first extension product to said second 3′
region, extending the 3′
region of said first extension product, and producing a second extension product comprising a 3′
region that is complementary to said second 5′
region, wherein said second extension product comprises a sequence complementary to said first 3′ and
5′
regions and said second 3′ and
5′
regions, and the individual nucleic acid molecules of said second extension product comprise said plurality or pool of nucleic acid molecules;b) distinguishing between error-free and error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules; and c) correcting errors in said plurality or pool of nucleic acid molecules by using the error-free nucleic acid molecules in said plurality or pool of nucleic acid molecules as templates for repairing said error-containing nucleic acid molecules. - View Dependent Claims (6, 7, 8, 9)
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10. A method for removing or controlling errors in a plurality or pool of nucleic acid molecules, the method comprising:
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a) synthesizing said plurality or pool of nucleic acid molecules by the steps of; providing a first immobilized nucleic acid comprising a first 5′
region and a first 3′
region;providing a second immobilized nucleic acid comprising a second 5′
region and a second 3′
region, wherein said second 3′
region and said first 5′
region comprise identical nucleic acid sequences;hybridizing said first immobilized nucleic acid with an oligonucleotide under conditions promoting hybridization of said oligonucleotide to said first 3′
region, extending the hybridized oligonucleotide, and producing a first extension product comprising a 3′
region that is complementary to said first 5′
region; andhybridizing said second immobilized nucleic acid with said first extension product under conditions promoting hybridization of said 3′
region of said first extension product to said second 3′
region, extending the 3′
region of said first extension product, and producing a second extension product comprising a 3′
region that is complementary to said second 5′
region, wherein said second extension product comprises a sequence complementary to said first 3′ and
5′
regions and said second 3′ and
5′
regions, and the individual nucleic acid molecules of said second extension product comprise said plurality or pool of nucleic acid molecules;b) identifying error-containing nucleic acid molecules within said plurality or pool of nucleic acid molecules; and c) removing the error-containing portions of said error-containing nucleic acid molecules, thereby producing error-free nucleic acid molecules and removing or controlling errors in said plurality or pool of nucleic acid molecules. - View Dependent Claims (11, 12, 13)
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Specification