Microarray synthesis and assembly of gene-length polynucleotides
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.
1. A process for creating a mixture of double-stranded fragments in solution, the process comprising:
a) predetermining a target polynucleotide sequence;
b) providing a plurality of single-stranded oligonucleotides bound on a solid or porous surface, wherein each single-stranded oligonucleotide comprise a fragment of the predetermined target polynucleotide sequence, and a flanking sequence, wherein the flanking sequence comprises a primer binding site and a restriction enzyme site permitting removal of the primer binding site, and
wherein each single-stranded oligonucleotide is bound to the surface via a cleavable linker moiety;
c) providing a plurality of complementary oligonucleotides, wherein each oligonucleotide of the plurality of complementary oligonucleotide is complementary to the flanking sequence;
d) hybridizing the plurality of said single-stranded oligonucleotides to the plurality of complementary oligonucleotides;
e) extending the hybridized plurality of complementary oligonucleotides to form double-stranded fragments; and
f) cleaving each oligonucleotides at the cleavable linker moiety to form a mixture of double-stranded fragments,
wherein the mixture of double-stranded fragments together comprise the predetermined target polynucleotide sequence.
2. The process of claim 1 wherein the restriction enzyme site is a class II endonuclease restriction site sequence capable of being cleaved by its corresponding class II restriction endonuclease enzyme.
3. The process of claim 1 , wherein the flanking sequence is located at the 3′ end, or the 5′ end, or the 3′ end and the 5′ end of each oligonucleotide of the plurality of single-stranded oligonucleotides.
4. The process of claim 3 wherein each oligonucleotide of the plurality of single-stranded oligonucleotides has a sequence region that is the same as or complementary to another sequence region of a next oligonucleotide.
5. The process of claim 1 , wherein the cleavable linker moiety is a chemical composition having a succinate moiety bound to a nucleotide moiety such that cleavage produces a 3′ hydroxy nucleotide.
6. The process of claim 1 , wherein the cleavable linker moiety is selected from the group consisting of 5′-dimethoxytrityl-thymidine-3′succinate, 4-N-benzoyl-5′-dimethoxytrityl-deoxycytidine-3′-succinate, 1-N-benzoyl-5′-dimethoxytrityl-deoxyadenosine-3′-succinate, 2-N-isobutyryl-5′-dimethoxytrityl-deoxyguanosine-3′-succinate, and combinations thereof.