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 (N′
)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 (Nx)C-3′
(SEQ ID NO.;
16);
d) performing a polynucleotide extension reaction to form a polynucleotide extension reaction product that is 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 with 3′
overhangs 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
25 Claims
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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 (N′
)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 (Nx)C-3′
(SEQ ID NO.;
16);d) performing a polynucleotide extension reaction to form a polynucleotide extension reaction product that is 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 with 3′
overhangs 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, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16)
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17. 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 a polynucleotide extension reaction product that is 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 with 3′
overhangs to form a nucleic acid encoding for the predetermined nucleic acid sequence that does not include the nicking endonuclease recognition site. - View Dependent Claims (18, 19, 20, 21, 22, 23, 24, 25)
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Specification