IP Library › Granted Patent US 12,668,791
Granted Patent B2
US 12,668,791 · App. 17/412,100 · Granted Jun 30, 2026

Compositions and methods for multiplex nucleic acids synthesis

Inventors: Joseph Jacobson (Newton, MA); Daniel Schindler (Newton Upper Falls, MA); Ishtiaq Saaem (Chelsea, MA); Scott S. Lawton (Bedford, MA); Martin J. Goldberg (Saratoga, CA); Michael E. Hudson (Framingham, MA); Li-Yun A. Kung (Arlington, MA)
Assignee: Twist Bioscience Corporation
C12N15/1031C12P19/34B01J2219/005B01J2219/00722
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Quick Facts
Patent No.
US 12,668,791
App. No.
17/412,100
Filed
Aug 25, 2021
Granted
Jun 30, 2026
Kind
B2
Art Unit
1683
USPC
435/6.11
Abstract

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.

Claims (20)

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.

Assignments (3)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Aug 22, 2025
From: GINKGO BIOWORKS, INC.
To: TWIST BIOSCIENCE CORPORATION
Reel/Frame 072092/0880 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jul 15, 2025
From: GEN9, INC.
To: GINKGO BIOWORKS, INC.
Reel/Frame 071945/0972 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Oct 4, 2021
From: JACOBSON, JOSEPH; SCHINDLER, DANIEL; SAAEM, ISHTIAQ; LAWTON, SCOTT; GOLDBERG, MARTIN J.; HUDSON, MICHAEL E.; KUNG, LI-YUN A.
To: GEN9, INC.
Reel/Frame 057692/0253 →
Continuity (4)
Continuation 16351488 · Mar 12, 2019
Division 14766195
Provisional Application 61792245 · Mar 15, 2013
Related Publication 20210380968A1 · Dec 9, 2021
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