Methods for Nucleic Acid Assembly and High Throughput Sequencing
First Claim
1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
- a) providing a first mixture comprising;
(i) a first pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′
end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′
end of the target nucleic acid, and a plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the first pool together comprising the target nucleic acid sequence;
(ii) a restriction enzyme, andb) exposing the first mixture to a ligase and subjecting the first mixture comprising the ligase to conditions suitable to promote restriction enzyme digestion and ligation, thereby generating the target nucleic acid.
2 Assignments
0 Petitions
Accused Products
Abstract
Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.
-
Citations
29 Claims
-
1. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
-
a) providing a first mixture comprising; (i) a first pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′
end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′
end of the target nucleic acid, and a plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the first pool together comprising the target nucleic acid sequence;(ii) a restriction enzyme, and b) exposing the first mixture to a ligase and subjecting the first mixture comprising the ligase to conditions suitable to promote restriction enzyme digestion and ligation, thereby generating the target nucleic acid. - View Dependent Claims (3, 4, 5, 6, 7, 9, 10, 11, 14, 16, 17, 19, 20, 21)
-
-
2. (canceled)
-
8. (canceled)
-
12. (canceled)
-
13. (canceled)
-
15. (canceled)
-
18. (canceled)
-
22. (canceled)
-
23. A method of producing a target nucleic acid having a predefined sequence, the method comprising:
-
a) providing a plurality of oligonucleotides, wherein each oligonucleotide comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) 5′
flanking sequence flanking the 5′
end of the internal sequence and 3′
flanking sequences flanking the 3′
end of the internal sequence, each of the flanking sequences comprising a primer recognition site for a primer pair and a restriction enzyme recognition site for a restriction endonuclease;b) amplifying at least a subset of the oligonucleotides using the primer pair thereby generating a plurality of amplified oligonucleotides; c) optionally subjecting the plurality of amplified oligonucleotides to error removal; d) providing a circular vector having a restriction enzyme recognition site for the restriction endonuclease; and c) exposing the plurality of amplified oligonucleotides and circular vector to the restriction enzyme and ligase in a single pool, wherein the restriction enzyme is capable of recognizing the restriction enzyme recognition sites, thereby assembling the target nucleic acid in the vector. - View Dependent Claims (24)
-
-
25. A composition for the assembly of a target nucleic acid having a predefined sequence comprising:
-
a) a pool of oligonucleotides comprising a first plurality of oligonucleotides comprising a sequence identical to the 5′
end of the target nucleic acid, a second plurality of oligonucleotides comprising a sequence identical to the 3′
end of the target nucleic acid, and one or more plurality of oligonucleotides comprising a sequence identical to a different portion of a sequence of a target nucleic acid, each of the oligonucleotides having an overlapping sequence region corresponding to a sequence region in a next oligonucleotide, the oligonucleotides in the pool together comprising the target nucleic acid;b) a plurality of common sequences comprising a primer recognition site for a primer pair and a restriction endonuclease recognition site; c) a restriction endonuclease; d) a ligase; and e) a linearized vector having a 5′
end compatible with the first plurality of oligonucleotides and a 3′
end compatible with the second plurality of oligonucleotides. - View Dependent Claims (26, 29)
-
-
27. (canceled)
-
28. (canceled)
Specification