IP Library › Granted Patent US 12,241,057
Granted Patent B2
US 12,241,057 · App. 17/361,091 · Granted Mar 4, 2025

Methods for nucleic acid assembly and high throughput sequencing

Inventors: Michael E. Hudson (Boston, MA); Li-Yun A. Kung (Boston, MA); Daniel Schindler (Boston, MA); Stephen Archer (Boston, MA); Ishtiaq Saaem (Boston, MA)
Assignee: Gen9, Inc.
C12N15/1031C12N15/1093C12N15/66C12Q1/686
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Quick Facts
Patent No.
US 12,241,057
App. No.
17/361,091
Granted
Mar 4, 2025
Kind
B2
Abstract

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

Claims (42)

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

(a) providing a first pool of double-stranded oligonucleotides, wherein the double-stranded oligonucleotides comprise:

(i) internal sequences identical to different portions of a first target nucleic acid, wherein the internal sequences comprise an overlapping region with another double-stranded oligonucleotide in the first pool; and

(ii) 5′ flanking sequences and/or 3′ flanking sequences, each of the flanking sequences comprising a common primer recognition site and a first restriction enzyme recognition site, the first restriction enzyme recognition site being oriented so that digestion with a first restriction enzyme that recognizes the first restriction enzyme recognition site will remove the flanking sequences and expose the internal sequence; and

(b) exposing the first pool of double-stranded oligonucleotides to a ligase and the first restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation, thereby generating the first target nucleic acid;

wherein the first target nucleic acid comprises:

(i) an internal sequence identical to a portion of a final target nucleic acid; and

(ii) a 5′ flanking sequence and/or a 3′ flanking sequence, each of the flanking sequences comprising a second restriction enzyme recognition site, wherein the first restriction enzyme recognition site and the second restriction enzyme recognition site have different sequences.

2. The method of claim 1 , wherein the first target nucleic acid is both strands of a double-stranded molecule.

3. The method of claim 1 , wherein the first target nucleic acid does not comprise a substrate of the first restriction enzyme of (b).

4. The method of claim 1 , wherein the first restriction enzyme recognition site is a Type IIs restriction enzyme recognition site, and wherein the first restriction enzyme is a Type IIs restriction enzyme.

5. The method of claim 1 , wherein the double-stranded oligonucleotides in the first pool are produced by amplifying a plurality of single-stranded oligonucleotides, each single-stranded oligonucleotide corresponding to one strand of a double-stranded oligonucleotide in the first pool, wherein amplification is performed using the common primer recognition sites of the single-stranded oligonucleotides.

6. The method of claim 5 , further comprising subjecting the amplified oligonucleotides to mismatch binding or error removal.

7. The method of claim 6 , wherein the amplified oligonucleotides are contacted with a mismatch binding agent, and wherein the mismatch binding agent is MutS.

8. The method of claim 1 , further comprising, after step (b), amplifying the first target nucleic acid.

9. The method of claim 1 , further comprising confirming the sequence accuracy of and isolating the first target nucleic acid.

10. The method of claim 1 , further comprising:

(c) providing a mixture comprising the first target nucleic acid and a second target nucleic acid, wherein the second target nucleic acid comprises:

(i) an internal sequence that differs from the internal sequence of the first target nucleic acid and is identical to a portion of the final target nucleic acid;

(ii) a 5′ flanking sequence and/or a 3′ flanking sequence, each of the flanking sequences comprising the second restriction enzyme recognition site; and

(d) exposing the mixture to a ligase and a second restriction enzyme that recognizes the second restriction enzyme recognition site, thereby generating the final target nucleic acid comprising the internal sequence of the first target nucleic acid and the internal sequence of the second target nucleic acid.

11. The method of claim 10 , wherein the first and the second target nucleic acids are subject in (d) to conditions suitable to promote concurrent digestion and ligation.

12. The method of claim 10 , wherein the final target nucleic acid does not comprise a substrate of the second restriction enzyme of (d).

13. The method of claim 10 , further comprising, prior to step (d), amplifying the first target nucleic acid and the second target nucleic acid.

14. The method of claim 10 , wherein the second target nucleic acid is produced by a method comprising:

(a) providing a second pool of double-stranded oligonucleotides, wherein the double-stranded oligonucleotides comprise:

(i) internal sequences identical to different portions of a second target nucleic acid, wherein the internal sequences comprise an overlapping region with another oligonucleotide in the second pool; and

(ii) 5′ flanking sequences and/or 3′ flanking sequences, each of the flanking sequences comprising a common primer recognition site and a third restriction enzyme recognition site, the third restriction enzyme recognition site being oriented so that digestion with a third restriction enzyme that recognizes the third restriction enzyme recognition site will remove the flanking sequences and expose the internal sequence; and

(b) exposing the second pool of double-stranded oligonucleotides to a ligase and the third restriction enzyme under conditions suitable to promote concurrent restriction enzyme digestion and ligation, thereby generating the second target nucleic acid.

15. The method of claim 14 , wherein the double-stranded oligonucleotides in the second pool are produced by amplifying a plurality of single-stranded oligonucleotides, each single-stranded oligonucleotide corresponding to one strand of a double-stranded oligonucleotide in the second pool, wherein amplification is performed using the common primer recognition sites of the single-stranded oligonucleotides, and

wherein the method further comprises subjecting the amplified oligonucleotides to error removal, wherein the amplified oligonucleotides are contacted with a mismatch binding agent, and wherein the mismatch binding agent is MutS.

16. The method of claim 14 , wherein the 5′ flanking sequences and 3′ flanking sequences of the second pool of double-stranded oligonucleotides have the same restriction enzyme recognition site sequences.

17. The method of claim 14 , wherein the 5′ flanking sequences and 3′ flanking sequences of the second pool of double-stranded oligonucleotides have different restriction endonuclease recognition site sequences.

18. The method of claim 14 , wherein the third restriction enzyme recognition site and the second restriction enzyme recognition site have the same sequence.

19. The method of claim 14 , wherein the third restriction enzyme recognition site and the second restriction enzyme recognition site have different sequences.

20. The method of claim 14 , wherein the third restriction enzyme and the second restriction enzyme are the same.

21. The method of claim 14 , wherein the third restriction enzyme and the second restriction enzyme are different.

22. The method of claim 14 , wherein the third restriction enzyme recognition site is a type IIs restriction enzyme recognition site, and wherein the third restriction enzyme is a type IIs restriction enzyme.

23. The method of claim 10 , further comprising, after step (d), confirming the sequence accuracy of the final target nucleic acid by sequencing and/or isolating the final target nucleic acid.

24. The method of claim 10 , wherein the second restriction enzyme recognition site is a Type IIs restriction enzyme recognition site, and wherein the second restriction enzyme is a Type IIs restriction enzyme.

25. The method of claim 1 , wherein the 5′ flanking sequences and 3′ flanking sequences of the first pool of double-stranded oligonucleotides have the same restriction enzyme recognition site sequences.

26. The method of claim 1 , wherein the 5′ flanking sequences and 3′ flanking sequences of the first pool of double-stranded oligonucleotides have different restriction enzyme recognition site sequences.

Assignments (2)
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 5, 2021
From: HUDSON, MICHAEL EDWARD; KUNG, LI-YUN ARTHUR; SCHINDLER, DANIEL; ARCHER, STEPHEN; SAAEM, ISHTIAQ
To: GEN9, INC.
Reel/Frame 057716/0057 →
Continuity (5)
Continuation 16714208 · Dec 13, 2019
Continuation 14408103
Provisional Application 61731627 · Nov 30, 2012
Provisional Application 61664118 · Jun 25, 2012
Related Publication 20210395724A1 · Dec 23, 2021
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