Compositions and methods for multiplex nucleic acids synthesis
Aspects of the invention relate to methods, compositions for designing and producing a target nucleic acid. In particular, aspects of the invention relate to the multiplex synthesis of target polynucleotides.
1. A method of producing a plurality of double-stranded oligonucleotides having a plurality of overhangs, comprising:
a) melting a first plurality of blunt-ended double-stranded oligonucleotides and a second plurality of blunt-ended double-stranded oligonucleotides to form a plurality of single stranded oligonucleotides; and
b) re-annealing the plurality of single-stranded oligonucleotides to form a plurality of double-stranded oligonucleotides having a plurality of overhangs, wherein the plurality of overhangs are designed to be relatively mutually orthogonal to each other.
2. The method of claim 1 , wherein the first and second pluralities of blunt ended double-stranded oligonucleotides are error-corrected or error-reduced prior to melting.
3. The method of claim 1 , wherein the first plurality of blunt-ended double-stranded oligonucleotides is attached to a support.
4. The method of claim 3 , wherein the support is solid.
5. The method of claim 3 , wherein the support is a bead.
6. The method of claim 3 , wherein the support is a stem-loop polynucleotide.
7. The method of claim 3 , wherein each oligonucleotide of the plurality of blunt-ended double-stranded oligonucleotides is immobilized to the same support.
8. The method of claim 3 , wherein each oligonucleotide of the plurality of blunt-ended double-stranded oligonucleotides is immobilized to a different support.
9. The method of claim 1 , wherein the second plurality of blunt-ended double-stranded oligonucleotides is attached to a support.
10. The method of claim 9 , wherein the support is solid.
11. The method of claim 9 , wherein the support is a bead.
12. The method of claim 9 , wherein the support is a stem-loop polynucleotide.
13. The method of claim 9 , wherein each oligonucleotide of the plurality of blunt-ended double-stranded oligonucleotides is immobilized to the same support.
14. The method of claim 9 , wherein each oligonucleotide of the plurality of blunt-ended double-stranded oligonucleotides is immobilized to a different support.
15. The method of claim 2 , wherein the first and second pluralities of blunt ended double-stranded oligonucleotides are error-corrected or error-reduced using a mismatch binding protein.
16. The method of claim 1 , wherein the first and second pluralities of blunt-ended double-stranded oligonucleotides are in the same reaction volume.
17. The method of claim 1 , wherein the first plurality of blunt-ended double-stranded oligonucleotides is in a first reaction volume and second plurality of blunt-ended double-stranded oligonucleotides is in a second reaction volume.
18. The method of claim 17 , further comprising combining the first and second reaction volumes.