Microarray synthesis and assembly of gene-length polynucleotides
First Claim
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1. A process for creating a mixture of double-stranded fragments 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;
(b) amplifying each oligonucleotide sequence to form a plurality of double-stranded oligonucleotide sequences using a primer complementary to a primer region of at least one flanking sequence located at the 3′
end, the 5′
end, or the 3′
end and the 5′
end of each fragment, wherein the at least one flanking sequence is from about 7 to about 50 bases and comprises the primer region and a sequence segment having a restriction enzyme cleavable site; 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 double-stranded fragments of the target polynucleotide sequence,wherein the plurality of double-stranded 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.
228 Citations
28 Claims
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1. A process for creating a mixture of double-stranded fragments 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; (b) amplifying each oligonucleotide sequence to form a plurality of double-stranded oligonucleotide sequences using a primer complementary to a primer region of at least one flanking sequence located at the 3′
end, the 5′
end, or the 3′
end and the 5′
end of each fragment, wherein the at least one flanking sequence is from about 7 to about 50 bases and comprises the primer region and a sequence segment having a restriction enzyme cleavable site; 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 double-stranded fragments of the target polynucleotide sequence, wherein the plurality of double-stranded 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 double-stranded fragments in solution comprising:
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(a) providing a plurality of oligonucleotide sequences bound on a solid surface, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence; (b) amplifying each oligonucleotide sequence to form a plurality of double-stranded oligonucleotide sequences using a primer complementary to a primer region of at least one flanking sequence located at the 3′
end, the 5′
end, or the 3′
end and the 5′
end of each fragment, wherein the at least one flanking sequence comprises the primer region and a sequence segment having a restriction enzyme cleavable site, and wherein the plurality of oligonucleotides has two or more different flanking sequences; and(c) cleaving the primer region from the plurality of double-stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of double-stranded fragments, wherein the plurality of double-stranded 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 double-stranded fragments in solution comprising:
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providing a plurality of oligonucleotides sequences, wherein each oligonucleotide sequence comprises a fragment of a target polynucleotide sequence and further comprises at least one flanking sequence, at the 3′
end, the 5′
end, or the 3′
end and the 5′
end of each fragment, wherein each flanking sequence comprises a primer region and a sequence segment having a restriction enzyme cleavable site;amplifying each oligonucleotide sequence using a primer complementary to the primer region of the at least one flanking sequence to form a plurality of double-stranded oligonucleotide sequences; and cleaving the primer region from the plurality of double-stranded oligonucleotide sequences at the restriction enzyme cleavable sites, thereby to produce a plurality of double-stranded fragments, wherein the plurality of double-stranded fragments together comprise the target polynucleotide sequence. - View Dependent Claims (21)
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22. A process for creating a mixture of double-stranded oligonucleotide sequences in solution, the process comprising
providing a plurality of oligonucleotides sequences, wherein each oligonucleotide sequence comprises a fragment of a 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 the at least one flanking sequence comprises a primer binding site, each primer binding region being complementary to a primer permitting amplification of the plurality of oligonucleotides; andsubjecting the plurality of oligonucleotides sequences to amplification to form double-stranded oligonucleotides comprising double-stranded fragments, the double-stranded fragments together comprising the target polynucleotide sequence. - View Dependent Claims (23, 24, 25, 26, 27, 28)
- end, the 5′
Specification