Iterative nucleic acid assembly using activation of vector-encoded traits
First Claim
1. A method for assembling nucleic acid segments, the method comprisingdigesting a first population of nucleic acids having at least first, second, third and fourth restriction sites, using a first set of restriction enzymes that cleave the nucleic acids at the first and third sites,digesting a second population of nucleic acids having at least first, second, third and fourth restriction sites, using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth sites, wherein the first and second populations of nucleic acids comprise a first activation sequence located between the first and second restriction sites and a second activation sequence located between the third and fourth restriction sites, and digestion of the first population results in a first population of nucleic acid segments that comprises the first activation sequence but lacks the second activation sequence, and digestion of the second population results in a second population of nucleic acid segments that lacks the first activation sequence and comprises the second activation sequence,combining the first and second populations of nucleic acid segments with a first nucleic acid vector that is digested with one or more restriction enzymes that produce restriction site overhangs that are complementary to the overhangs generated by the first and fourth restriction enzymes on the first and second populations of nucleic acid segments, wherein the first nucleic acid vector comprises a coding sequence of a first marker gene 5′
- of the first restriction site and a coding sequence of a second marker gene 3′
of the fourth restriction site, andisolating ligated first nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the first and second populations of nucleic acid segments.
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Abstract
Certain aspects of the present invention provide methods for assembling nucleic acid molecules using iterative activation of one or more vector-encoded traits to progressively assemble a longer nucleic acid insert. Aspects of the invention also provide kits, compositions, devices, and systems for assembling synthetic nucleic acids using iterative activation of one or more vector-encoded traits.
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Citations
31 Claims
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1. A method for assembling nucleic acid segments, the method comprising
digesting a first population of nucleic acids having at least first, second, third and fourth restriction sites, using a first set of restriction enzymes that cleave the nucleic acids at the first and third sites, digesting a second population of nucleic acids having at least first, second, third and fourth restriction sites, using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth sites, wherein the first and second populations of nucleic acids comprise a first activation sequence located between the first and second restriction sites and a second activation sequence located between the third and fourth restriction sites, and digestion of the first population results in a first population of nucleic acid segments that comprises the first activation sequence but lacks the second activation sequence, and digestion of the second population results in a second population of nucleic acid segments that lacks the first activation sequence and comprises the second activation sequence, combining the first and second populations of nucleic acid segments with a first nucleic acid vector that is digested with one or more restriction enzymes that produce restriction site overhangs that are complementary to the overhangs generated by the first and fourth restriction enzymes on the first and second populations of nucleic acid segments, wherein the first nucleic acid vector comprises a coding sequence of a first marker gene 5′ - of the first restriction site and a coding sequence of a second marker gene 3′
of the fourth restriction site, andisolating ligated first nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the first and second populations of nucleic acid segments. - View Dependent Claims (2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 15, 16, 17, 18, 19)
- of the first restriction site and a coding sequence of a second marker gene 3′
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3. A method for assembling nucleic acid segments, the method comprising
digesting a first population of nucleic acids having at least first, second, third and fourth restriction sites, using a first set of restriction enzymes that cleave the nucleic acids at the first and third sites, digesting a second population of nucleic acids having at least first, second, third and fourth restriction sites, using a second set of restriction enzymes that cleave the nucleic acids at the second and fourth sites, wherein the first and second populations of nucleic acids comprise a 5′ - promoter sequence located between the first and second restriction sites and a 3′
promoter sequence located between the third and fourth restriction sites, and digestion of the first population results in a first population of nucleic acid segments that comprises the 5′
promoter sequence but lacks the 3′
promoter sequence, and digestion of the second population results in a second population of nucleic acid segments that lacks the 5′
promoter sequence and comprises the 3′
promoter sequence,combining in the presence of a ligase the first and second populations of nucleic acid segments with a first nucleic acid vector that is digested with restriction enzymes that cleave at the first and fourth restriction sites, wherein the first nucleic acid vector comprises a coding sequence of a first marker gene 5′
of the first restriction site and a coding sequence of a second marker gene 3′
of the fourth restriction site, andselecting for ligated first nucleic acid vectors that express the first and the second marker genes, wherein expression of the first and the second marker genes is indicative of correct assembly of the first and second populations of nucleic acid segments. - View Dependent Claims (13)
- promoter sequence located between the first and second restriction sites and a 3′
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20-29. -29. (canceled)
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30. A method of assembling a nucleic acid, the method comprising a plurality of consecutive alternating assembly cycles, wherein each alternating assembly cycle comprises:
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a) combining a first nucleic acid insert with a second nucleic acid insert and a first vector, wherein the first nucleic acid insert comprises a first portion of a target sequence and a first activation sequence, the second nucleic acid insert comprises a second portion of the target sequence and a second activation sequence, and the first vector comprises first and second activatable markers; b) selecting for correct assembly of the first and second nucleic acid inserts in the first vector by selecting for activation of the first and second activatable markers, wherein correct assembly results in activation of the first activatable marker by the first activation sequence and activation of the second activatable marker by the second activation sequence; c) isolating from step b) an assembled nucleic acid comprising the first and second portions of the target sequence and the first activation sequence, but not the second activation sequence; d) combining the nucleic acid of step c) with a third nucleic acid insert and a second vector, wherein the third nucleic acid insert comprises a third portion of the target sequence and the second activation sequence, and the second vector comprises third and fourth activatable markers; e) selecting for correct assembly of the nucleic acid of step c) and the third nucleic acid insert in the second vector by selecting for activation of the third and fourth activatable markers, wherein correct assembly results in activation of the third activatable marker by the first activation sequence and activation of the fourth activatable marker by the second activation sequence; and f) isolating from step e) an assembled nucleic acid comprising the first, second, and third portions of the target sequence and the first activation sequence, but not the second activation sequence;
wherein the nucleic acid of step f) can be combined in a subsequent assembly cycle starting at step a) with a with a fourth nucleic acid insert and the first vector, wherein the fourth nucleic acid insert comprises a fourth portion of the target sequence and the second activation sequence.
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31-33. -33. (canceled)
Specification