Microarray synthesis and assembly of gene-length polynucleotides
First Claim
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1. A process for creating a mixture of oligonucleotide sequences in solution comprising:
- (a) synthesizing in situ or spotting a plurality of oligonucleotide sequences on a microarray device or bead device each having a solid or porous surface, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface and wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and further comprises two flanking sequences, one at the 3′
end and the other at the 5′
end of each fragment, wherein each flanking sequence is from about 7 to about 50 bases and comprising a primer region and a sequence segment having a restriction enzyme cleavable site;
(b) amplifying each oligonucleotide sequence using primers complementary to the primer regions of the flanking sequence to form a plurality of double stranded oligonucleotide sequences; and
(c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments of the target polynucleotide sequence,wherein the plurality of fragments together comprise the target polynucleotide sequence.
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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
22 Claims
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1. A process for creating a mixture of oligonucleotide sequences in solution comprising:
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(a) synthesizing in situ or spotting a plurality of oligonucleotide sequences on a microarray device or bead device each having a solid or porous surface, wherein the plurality of oligonucleotide sequences are attached to the solid or porous surface and wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and further comprises two flanking sequences, one at the 3′
end and the other at the 5′
end of each fragment, wherein each flanking sequence is from about 7 to about 50 bases and comprising a primer region and a sequence segment having a restriction enzyme cleavable site;(b) amplifying each oligonucleotide sequence using primers complementary to the primer regions of the flanking sequence to form a plurality of double stranded oligonucleotide sequences; and (c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments of the target polynucleotide sequence, wherein the plurality of fragments together comprise the target polynucleotide sequence. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A process for creating a mixture of oligonucleotide sequences in solution comprising:
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(a) providing a plurality of oligonucleotides sequences bound on a solid surface, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and further comprises two flanking sequences, one at the 3′
end and the other at the 5′
end of each fragment, wherein each flanking sequence comprises a primer region and a sequence segment having a restriction enzyme cleavable site, and wherein the plurality of oligonucleotides has two or more different flanking region sequences;(b) amplifying each oligonucleotide sequence using primers complementary to a pair of primer regions of the flanking sequences to form a plurality of double stranded oligonucleotide sequences; and (c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments, wherein the plurality of fragments together comprise the target polynucleotide sequence. - View Dependent Claims (15, 16, 17, 18, 19)
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20. A process for creating a mixture of oligonucleotide sequences in solution 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 further comprises two flanking sequences, one at the 3′
end and the other at the 5′
end of each fragment, wherein each flanking sequence comprises a primer region and a sequence segment having a restriction enzyme cleavable site;(b) amplifying each oligonucleotide sequence using primers complementary to a pair of primer regions of the flanking sequences to form a plurality of double stranded oligonucleotide sequences; and (c) cleaving the primer regions from the plurality of double stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of fragments, wherein the plurality of fragments together comprise the target polynucleotide sequence. - View Dependent Claims (21, 22)
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Specification