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.
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Citations
40 Claims
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1-20. -20. (canceled)
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21. A composition for assembly of a target polynucleotide sequence, the composition comprising a plurality of different oligonucleotides, each oligonucleotide sequence comprising a fragment of a target polynucleotide sequence,
wherein the fragments together comprise the target polynucleotide sequence, and wherein each oligonucleotide further comprises at least one flanking region sequence, at the 3′ - end or the 5′
end or the 3′
end and the 5′
end of each fragment, each flanking sequence comprising a primer binding site. - View Dependent Claims (22, 23, 24, 25, 26, 27, 28, 29)
- end or the 5′
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30. A solid or porous surface comprising a plurality of cleavable oligonucleotides sequences immobilized thereon, wherein each oligonucleotide sequence has an overlapping sequence corresponding to a next oligonucleotide of the composition, wherein the plurality of overlapping sequences of the composition together comprise the polynucleotide sequence and wherein each oligonucleotide further comprises at least one flanking sequence at the 3′
- end, the 5′
end, or at the 3′
end and 5′
end of each overlapping sequence, wherein each flanking sequence comprises a primer binding site and a sequence segment permitting removal of the primer binding site. - View Dependent Claims (31, 32, 33, 34, 35)
- end, the 5′
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36. A process for creating a mixture of oligonucleotide sequences in solution, the process comprising:
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(a) providing a plurality of oligonucleotides sequences bound on a solid or porous surface, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and the fragments together comprising the target polynucleotide sequence and wherein each oligonucleotide further comprises at least one flanking sequence at the 3′
end, the 5′
end, or at the 3′
end and the 5′
end of each fragment, wherein each flanking sequence comprises a primer binding site and a sequence segment having a restriction enzyme cleavable site and wherein each oligonucleotide sequence has a cleavable site; and(b) cleaving the oligonucleotide sequences at the cleavable site to form a soluble mixture of oligonucleotides. - View Dependent Claims (37, 38, 39, 40)
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Specification