Compositions and methods for multiplex nucleic acids synthesis

1 . A method of producing at least one target nucleic acid having a predefined sequence, the method comprising:

(a) providing a first plurality of double-stranded anchor oligonucleotides having a first plurality of overhangs, wherein the anchor oligonucleotides are attached to a support, and wherein the first plurality of overhangs are designed to be relatively mutually orthogonal to each other;

(b) providing a first plurality of double-stranded construction oligonucleotides having a second plurality of overhangs and a third plurality of overhangs, wherein the second plurality of overhangs are designed to be complementary to the first plurality of overhangs, wherein the second plurality of overhangs are designed to be relatively mutually orthogonal to each other, and wherein the third plurality of overhangs are designed to be relatively mutually orthogonal to each other;

(c) hybridizing the first plurality of overhangs with the second plurality of overhangs and ligating the double-stranded anchor oligonucleotides with the double-stranded construction oligonucleotides to form a second plurality of double-stranded anchor oligonucleotides with the third plurality of overhangs; and

(d) repeating steps (a) through (c), using the second plurality of double-stranded anchor oligonucleotides and a second plurality of double-stranded construction oligonucleotides having a fourth and fifth plurality of overhangs, wherein the fourth plurality of overhangs are designed to be complementary to the third plurality of overhangs, wherein the fourth plurality of overhangs are designed to be relatively mutually orthogonal to each other, and wherein the fifth plurality of overhangs are designed to be relatively mutually orthogonal to each other thereby generating the at least one target nucleic acid.

2 . The method of claim 1 , wherein the support is solid.

3 . The method of claim 1 , wherein the support is a bead.

4 . The method of claim 1 , wherein the support is a stem loop polynucleotide.

5 . The method of claim 2 , wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to a different support.

6 . The method of claim 2 , wherein each of the plurality of the double-stranded anchor oligonucleotides is immobilized to the same support.

7 . The method of claim 1 , wherein steps (a) through (d) are performed in a single reaction volume.

8 . The method of claim 1 , wherein each overhang of each plurality of overhangs has a length of between 8-14, 14-20, 20-25, 25-50, 50-100, or 100-500 nucleotides.

9 . The method of claim 1 , wherein each overhang of each plurality of overhangs has a length of 4 nucleotides.

10 . The method of claim 1 , wherein each pair of pluralities of complementary overhangs differ from any other pair of pluralities of complementary overhangs by at least one nucleotide.

11 . The method of claim 1 , wherein each plurality of double-stranded oligonucleotides having overhangs is generated by hybridizing partially complementary oligonucleotides.

12 . The method of claim 4 , wherein the stem loop polynucleotide is at least 100 bases long and wherein each of the first plurality of overhangs is 5-20 bases long.

13 . 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 substantially mutually orthogonal to each other.

14 . The method of claim 13 , wherein the first and second pluralities of blunt-ended double-stranded oligonucleotides are error corrected or error reduced prior to melting.