MICROARRAY SYNTHESIS AND ASSEMBLY OF GENE-LENGTH POLYNUCLEOTIDES
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Abstract
There is disclosed a process for in vitro synthesis and assembly of long, gene-length polynucleotides based upon assembly of multiple shorter oligonucleotides synthesized in situ on a microarray platform. Specifically, there is disclosed a process for in situ synthesis of oligonucleotide fragments on a solid phase microarray platform and subsequent, “on device” assembly of larger polynucleotides composed of a plurality of shorter oligonucleotide fragments.
60 Citations
72 Claims
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1-45. -45. (canceled)
- 46. A composition for assembly of a target polynucleotide sequence, the composition comprising a plurality of different synthesized oligonucleotide sequences, wherein the plurality of different synthesized oligonucleotide sequences are immobilized on a solid or porous surface, each oligonucleotide sequence comprising a fragment of a target polynucleotide sequence, a primer binding site and a sequence segment designed to permit removal of the primer binding site such that all fragments together comprise the target polynucleotide sequence.
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53. A device comprising a solid or porous surface having a plurality of synthesized oligonucleotides immobilized thereon, wherein each oligonucleotide has an overlapping sequence, a primer binding site and a sequence segment permitting removal of the primer binding site, such that all overlapping sequences together can be ligated and/or primer extended to form the target polynucleotide sequence,
wherein each overlapping sequence corresponds to a sequence of a next oligonucleotide of the plurality of oligonucleotides.
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60. A process for creating a mixture of oligonucleotides in solution, the process comprising:
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(a) providing a plurality of oligonucleotides bound on a solid or porous surface, wherein each oligonucleotide comprises a fragment of a target polynucleotide sequence, a primer binding site and a restriction enzyme site permitting removal of the primer binding site such that all fragments together comprise the target polynucleotide sequence, and wherein each oligonucleotide has a cleavable site; and (b) cleaving each oligonucleotide at the cleavable site to form a mixture of oligonucleotides. - View Dependent Claims (61, 62, 63, 64, 65)
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- 66. A composition for assembly of a target polynucleotide sequence, the composition comprising a plurality of different synthesized oligonucleotides immobilized on a solid or porous surface, each oligonucleotide comprising a fragment of a target polynucleotide sequence and a cleavable linker moiety, wherein the cleavable linker moiety permits cleavage of each oligonucleotide to release the fragment, and wherein all fragments together comprise the target polynucleotide sequence.
Specification