Methods and Devices for Nucleic Acid Synthesis
Methods and apparatus relate to the synthesis of polynucleotides having a predefined sequence on a support. Assembly methods include primer extension to generate overlapping construction oligonucleotides and assembly of the polynucleotides of interest onto an anchor support-bound oligonucleotides. Methods and apparatus for selection of polynucleotides having the predefined sequence and/or length are disclosed.
1 - 41 . (canceled)
42 . A nucleic acid array system comprising:
a. one or more solid support;
b. a plurality of discrete features associated with the one or more solid support wherein each feature comprises a plurality of support-bound oligonucleotides having a predefined sequence wherein a first support-bound oligonucleotide comprises a terminal region that is complementary to a terminal region of a second support-bound oligonucleotide, wherein a Nth support-bound oligonucleotide comprises a terminal sequence complementary to a terminal sequence region of a (N-1)th support-bound oligonucleotide, wherein N is no less than 3; and
c. at least a first plurality of anchor oligonucleotides comprising a terminal sequence that is identical to a sequence region of the first support-bound oligonucleotide.
43 . The nucleic acid array system of claim 42 further comprising a second plurality of support-bound anchor oligonucleotide having a sequence region that is identical to the Nth support-bound oligonucleotide.
44 - 65 . (canceled)
66 . The nucleic acid array system of claim 42 wherein the first to the Nth support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence.
67 . The nucleic acid array system of claim 66 wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel.
68 . The nucleic acid array system of claim 42 further comprising an anchor support wherein the anchor oligonucleotides are immobilized on the anchor support.
69 . The nucleic acid array system of claim 68 further comprising a microvolume of reagents localized on each feature for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template.
70 . The nucleic acid array system of claim 69 further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support.
71 . A system for producing at least one target nucleic acid, comprising:
a. one or more solid supports having at least a first and a second features thereon, the first feature having a first plurality of support-bound oligonucleotides, the second feature having a second plurality of support-bound oligonucleotides, wherein each of the first plurality of support-bound oligonucleotides comprises a terminal sequence region complementary to that of the second plurality of support-bound oligonucleotides;
b. an anchor support comprising a plurality of anchor oligonucleotides, each having a terminal region identical to that of the first plurality of support-bound oligonucleotides; and
c. a solution of reagents for synthesizing construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template.
72 . The system of claim 71 further comprising a Nth plurality of support-bound oligonucleotides comprising a terminal sequence complementary to that of a (N-1)th plurality of support-bound oligonucleotides, wherein N>=3.
73 . The system of claim 72 wherein the first to the Nth pluralities of support-bound oligonucleotides together comprise a target nucleic acid having a predetermined sequence.
74 . The system of claim 73 wherein each solid support comprises a plurality of features for assembling two or more target nucleic acids in parallel.
75 . The system of claim 71 wherein the solution comprises a plurality of microvolumes each localized on a feature.
76 . The system of claim 71 further comprising a fluid medium for transferring the construction oligonucleotides to the anchor support.
77 . The system of claim 71 wherein the support-bound oligonucleotides are synthesized or spotted on the one or more solid supports.
78 . A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 42 ;
b. synthesizing a first to Nth construction oligonucleotides in a chain extension reaction using the support-bound oligonucleotides as template;
c. transferring the first to Nth construction oligonucleotides to the first plurality of anchor oligonucleotides; and
d. hybridizing the first to Nth construction oligonucleotides with the first plurality of anchor oligonucleotides, thereby producing at least one target nucleic acid.
79 . A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 71 ;
b. synthesizing a first and second construction oligonucleotides using the support-bound oligonucleotides as template;
c. transferring the first and second construction oligonucleotides to the anchor support; and
d. hybridizing the first and second construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.
80 . A method for producing at least one target nucleic acid, comprising:
a. providing the nucleic acid array system of claim 73 ;
b. synthesizing a first to a Nth construction oligonucleotides using the support-bound oligonucleotides as template;
c. transferring the first to the Nth construction oligonucleotides to the anchor support; and
d. hybridizing the first to the Nth construction oligonucleotides with the anchor oligonucleotides, thereby producing at least one target nucleic acid.