COMPOSITIONS AND METHODS FOR SYNTHETIC GENE ASSEMBLY
First Claim
Patent Images
1. A method for nucleic acid assembly, the method comprising:
- a) providing a predetermined nucleic acid sequence;
b) providing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or 5′
-G (Nx) C-3′
(SEQ ID NO.;
16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence;
c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′
-A (Nx) U-3′
(SEQ ID NO.;
80) corresponding to each of the different sticky end sequences of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or (ii) 5′
-G (Nx) U-3′
(SEQ ID NO.;
81) corresponding to each of the different sticky end sequences of 5′
-G (N′
) C-3′
(SEQ ID NO.;
16);
d) performing a polynucleotide extension reaction to form double-stranded nucleic acid fragments;
e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′
overhangs; and
f) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site.
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Abstract
Methods and compositions are provided for assembly of large nucleic acids where the assembled large nucleic acids lack internal sequence modifications made during the assembly process.
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Citations
79 Claims
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1. A method for nucleic acid assembly, the method comprising:
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a) providing a predetermined nucleic acid sequence; b) providing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or 5′
-G (Nx) C-3′
(SEQ ID NO.;
16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence;c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′
-A (Nx) U-3′
(SEQ ID NO.;
80) corresponding to each of the different sticky end sequences of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or (ii) 5′
-G (Nx) U-3′
(SEQ ID NO.;
81) corresponding to each of the different sticky end sequences of 5′
-G (N′
) C-3′
(SEQ ID NO.;
16);d) performing a polynucleotide extension reaction to form double-stranded nucleic acid fragments; e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′
overhangs; andf) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site. - View Dependent Claims (2, 3, 7, 11, 13, 14, 15, 16, 17, 18, 19, 20, 21, 23, 24)
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4-6. -6. (canceled)
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8-10. -10. (canceled)
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12. (canceled)
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22. (canceled)
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25. A method for nucleic acid assembly, the method comprising:
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a) providing a predetermined nucleic acid sequence; b) synthesizing a plurality of precursor double-stranded nucleic acid fragments, each precursor double-stranded nucleic acid fragment having two strands, wherein each of the two strands comprises a sticky end sequence of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or 5′
-G (Nx) C-3′
(SEQ ID NO.;
16), wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two precursor double-stranded nucleic acid fragments comprise the same sticky end sequence;c) providing primers comprising a nicking endonuclease recognition site and a sequence comprising (i) 5′
-A (Nx) M-3′
(SEQ ID NO.;
82) corresponding to each of the different sticky end sequences of 5′
-A (Nx) T-3′
(SEQ ID NO.;
1) or (ii) 5′
-G (Nx) M-3′
(SEQ ID NO.;
83) corresponding to each of the different sticky end sequences of 5′
-G (Nx) C-3′
(SEQ ID NO.;
16), wherein M is a non-canonical base, wherein the primers are each 7 to 70 bases in length;d) performing a polynucleotide extension reaction to form double-stranded nucleic acid fragments; e) subjecting the polynucleotide extension reaction product to nicking and cleavage reactions to form double-stranded nucleic acid fragments with 3′
overhangs; andf) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site. - View Dependent Claims (26, 28, 30, 33, 34, 35, 36, 38)
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27. (canceled)
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29. (canceled)
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31. (canceled)
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32. (canceled)
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37. (canceled)
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39. A method for nucleic acid assembly, the method comprising:
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a) providing a predetermined nucleic acid sequence; b) synthesizing a plurality of single-stranded nucleic acid fragments, wherein each single-stranded nucleic acid fragment encodes for a portion of the predetermined nucleic acid sequence and comprises at least one sticky end motif, wherein the sticky end motif comprises a sequence of 5- A(Nx)T -3′
(SEQ ID NO.;
1) or 5′
- G(Nx)C -3′
(SEQ ID NO.;
16) in the predetermined nucleic acid sequence, wherein N is a nucleotide, wherein x is the number of nucleotides between nucleotides A and T or between G and C, and wherein x is 1 to 10, and wherein no more than two single-stranded nucleic acid fragments comprise the same sticky end sequence;c) amplifying the plurality of single-stranded nucleic acid fragments to generate a plurality of double-stranded nucleic acid fragments, wherein the plurality of double-stranded nucleic acid fragments are modified from the predetermined nucleic acid sequence to comprise (i) a non-canonical base located at a 3′
end of the sticky end motif on a first strand and (ii) a first adaptor region located 5′
of the non-canonical base on the first strand, wherein the first adaptor region comprises a nicking enzyme recognition site;d) creating sticky ends, wherein creating sticky ends comprises; i) treating the plurality of double-stranded fragments with a first nicking enzyme that nicks the non-canonical base on a first strand of each double-stranded fragment, and cleaving the nicked non-canonical base; and ii) treating the plurality of double-stranded fragments with a second nicking enzyme, wherein the second nicking enzyme binds to the first strand at the nicking enzyme recognition site and cleaves a second strand of each double-stranded fragment, wherein a cleavage site for the nicking enzyme is located at a junction between the sticky end motif a sequence reverse complementary to the first adaptor region of the first strand; and e) annealing the double-stranded nucleic acid fragments to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site. - View Dependent Claims (40, 41, 45, 49, 51, 52, 53, 54, 55, 56, 57, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 73, 74, 76)
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42-44. -44. (canceled)
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46-48. -48. (canceled)
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50. (canceled)
- 58. The method of 39, wherein the first nicking enzyme comprises a base excision activity.
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72. (canceled)
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75. (canceled)
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77. (canceled)
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78. A DNA library comprising:
n DNA fragments, each comprising a first strand and a second strand, each of the n DNA fragments comprising, in order 5′
to 3′
;
a first nicking endonuclease recognition site, a first sticky end motif, a template region, a second sticky end motif, and a second nicking endonuclease recognition site, wherein the first sticky end motif comprises a sequence of 5′
-A (Nx)i,1U-3′
(SEQ ID NO.;
13) in the first strand; and
wherein the second sticky end motif comprises a sequence of 5′
-A (Nx)i,2U-3′
(SEQ ID NO.;
14) in the second strand;
wherein Nx denotes x nucleosides;
wherein (Nx)i,2 is reverse complementary to (Nx)i, 1 and different from every other Nx found in any sticky end motif sequence within the fragment library, wherein the first nicking endonuclease recognition site in each of the DNA fragments are positioned such that there is a corresponding cleavage site immediately 3′
of the sticky end motif in the second strand, and wherein the second nicking endonuclease recognition sites are positioned such that there is a corresponding cleavage site immediately 3′
of the second sticky end motif in the first strand.
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79-89. -89. (canceled)
Specification