Methods and devices for nucleic acid synthesis

Patent metadata

Field	Value
Publication number	US-9295965-B2
Application number	US-201113884463-A
Country	US
Kind code	B2
Filing date	Nov 10, 2011
Priority date	Nov 12, 2010
Publication date	Mar 29, 2016
Grant date	Mar 29, 2016

Abstract

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.

First claim

What is claimed is:

1. A method for producing at least one target polynucleotide having a predefined sequence, the method comprising:
a. providing at least a first and a second pluralities of support-bound, single-stranded, template oligonucleotides, wherein each of the first and second pluralities of template oligonucleotides has a predefined sequence and is bound to a first and second feature of one or more planar support, respectively, each of the first plurality of template oligonucleotides comprising a sequence region at its 5′ end complementary to a sequence region of a 5′ end of the second plurality of template oligonucleotides;
b. generating at least a first and a second pluralities of construction oligonucleotides complementary to the first and second pluralities of template oligonucleotides in a chain extension reaction, each of the first plurality of construction oligonucleotides comprising a sequence region at its 3′ end that is complementary to a sequence region of a 3′ end of the second plurality of construction oligonucleotides;
c. providing a plurality of support-bound, single-stranded, anchor oligonucleotides at a selected feature on a planar anchor support, wherein the 5′ end of each anchor oligonucleotide is complementary to the 5′ end of the first plurality of construction oligonucleotides; and
d. transferring the first plurality of construction oligonucleotides from the first feature to the selected feature on the planar anchor support, transferring the second plurality of construction oligonucleotides from the second feature to the selected feature on the planar anchor support, and hybridizing the at least first and second pluralities of construction oligonucleotides to the plurality of anchor oligonucleotides on the planar anchor support.

2. The method of claim 1 wherein the at least first and second pluralities of construction oligonucleotides are dissociated from the at least first and second plurality of template oligonucleotides.

3. The method of claim 1 wherein the selected feature is on the same planar support than the first and the second features.

4. The method of claim 1 wherein the selected feature is on a different planar support than the first and second features.

5. The method of claim 1 further comprising:
(i) providing a third plurality of support-bound, single-stranded, template oligonucleotides wherein each of the third plurality of template oligonucleotides has a predefined sequence and is bound to a third feature of the one or more planar support, each of the third plurality of template oligonucleotides comprising a sequence region at its 3′ end that is complementary to a sequence region of a 3′ end of the second plurality of template oligonucleotides;
(ii) generating a third plurality of construction oligonucleotides complementary to the third plurality of template oligonucleotides in a chain extension reaction;
(iii) hybridizing the first, second and third pluralities of construction oligonucleotides to the plurality of anchor oligonucleotides at the selected feature; and
(iv) ligating the first and third pluralities of construction oligonucleotides thereby producing the at least one target polynucleotide.

6. The method of claim 5 further comprising sequentially repeating steps (i) through (iii) till a Nth plurality of construction oligonucleotides.

7. The method of claim 1 wherein each plurality of template oligonucleotides has a primer binding site at its 3′ end.

8. The method of claim 7 wherein the primer binding site is a universal primer binding site.

9. The method of claim 7 further comprising annealing a primer to the at least first and second pluralities of template oligonucleotides and synthesizing the first and second plurality of construction oligonucleotides by chain extension from the primer, thereby forming extension product duplexes.

10. The method of claim 9 wherein the primer comprises at least one Uracil.

11. The method of claim 1 wherein the at least first and second pluralities of template oligonucleotides are synthesized or spotted on the one or more planar support.

12. The method of claim 1 wherein the at least first and second pluralities of template oligonucleotides are immobilized on the one or more planar support at their 3′ end.

13. The method of claim 1 wherein the one or more planar support is a microarray device.

14. The method of claim 9 wherein at least one of the first and second feature is subjected to chain extension conditions.

15. The method of claim 1 comprising:
providing N pluralities of support-bound, single-stranded, template oligonucleotides thereby generating the first plurality of construction oligonucleotides comprising at its 3′ end sequence complementarity to the 3′ end of the second plurality of construction oligonucleotides, and a Nth plurality of construction oligonucleotides comprising at its 3′ end sequence complementarity to the 3′ end of a (N−1)th plurality of construction oligonucleotides.

16. The method of claim 15 wherein the N pluralities of construction oligonucleotides span the entire sequence of the at least one target polynucleotide without gaps.

17. The method of claim 9 further comprising dissociating the extension product duplexes thereby releasing the first and second pluralities of construction oligonucleotides.

18. The method of claim 10 further comprising removing the primer using a mixture of Uracil DNA glycosylase (UDG) and a DNA glycosylase-lyase Endonuclease VIII.

19. A method for producing a plurality of target polynucleotides each having a predefined sequence, the method comprising:
(a). providing a first planar support having a plurality of first features and a second planar support having a plurality of second features, wherein each first or second feature comprises a plurality of different support-bound, template oligonucleotides;
(b). generating a first and second pluralities of construction oligonucleotides using the template oligonucleotides as templates in a chain extension reaction, the first and second pluralities of construction oligonucleotides having sequence complementarity to each other at their 3′ end;
(c). providing a planar anchor support comprising a plurality of features, wherein each feature comprises a plurality of support-bound, single-stranded, anchor oligonucleotides, wherein the 5′ end of each of the plurality of the anchor oligonucleotides is complementary to the 5′ end of the first plurality of construction oligonucleotides;
(d). transferring the first plurality of construction oligonucleotides to preselected features on the planar anchor support, and hybridizing the first plurality of construction oligonucleotides to the anchor oligonucleotides, wherein the preselected features correspond to the first features on the first planar support; and
(e). transferring the second plurality of construction oligonucleotides to the preselected features on the planar anchor support, and hybridizing the second plurality of construction oligonucleotides to the first plurality of construction oligonucleotides, wherein the preselected features correspond to the second features on the second planar support.

20. The method of claim 19 wherein each plurality of construction oligonucleotides is synthesized on a different planar support.

21. The method of claim 19 wherein each of the plurality of target polynucleotides is assembled at different preselected features of the planar anchor support.

22. The method of claim 19 wherein

Assignees

• Jacobson Joseph
• Kung Li-Yun A
• Wilson Andrew Kirk
• Ramu Senthil
• Schindler Daniel
• Hudson Mike
• Gen9 Inc

Inventors

• Jacobson Joseph
• Kung Li-Yun A
• Wilson Andrew Kirk
• Ramu Senthil
• Schindler Daniel
• Hudson Mike

Classifications

• B01J2219/00585

  Parallel processes · CPC title

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/10

  Processes for the isolation, preparation or purification of DNA or RNA (chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34) · CPC title

• B01J2219/00722

  Nucleotides · CPC title

• C12N15/1031

  mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR · CPC title