Methods for nucleic acid assembly and high throughput sequencing

Patent metadata

Field	Value
Publication number	US-11072789-B2
Application number	US-201916714208-A
Country	US
Kind code	B2
Filing date	Dec 13, 2019
Priority date	Jun 25, 2012
Publication date	Jul 27, 2021
Grant date	Jul 27, 2021

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.

First claim

What is claimed is:

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 3′ flanking sequences, each of the flanking sequences comprising a common primer recognition site and a Type IIS restriction endonuclease recognition site, the Type IIS restriction enzyme endonuclease recognition sites being orientated so that Type IIS restriction enzyme digestion will remove the flanking sequences and expose the internal sequence; and

b) exposing the first pool of double-stranded oligonucleotides to a ligase and a Type IIS restriction enzyme that recognizes the Type IIS restriction endonuclease recognition site 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 a 3′ flanking sequence, each of the flanking sequences comprising a restriction enzyme recognition site.

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 Type IIS restriction enzyme of (b).

4. 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.

5. The method of claim 4 , further comprising subjecting the amplified oligonucleotides to mismatch binding or error removal.

6. The method of claim 5 , wherein the amplified oligonucleotides are contacted with a mismatch binding agent.

7. The method of claim 6 , 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 a 3′ flanking sequence, each of the flanking sequences comprising a restriction enzyme recognition site; and

d) exposing the mixture to a ligase and a restriction enzyme that recognizes the restriction endonuclease 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 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 3′ flanking sequences, each of the flanking sequences comprising a common primer recognition site and a Type IIS restriction endonuclease recognition site, the Type IIS restriction enzyme endonuclease recognition sites being orientated so that Type IIS restriction enzyme digestion will remove the flanking sequences and expose the internal sequence; and

b) exposing the second pool of double-stranded oligonucleotides to a ligase and a Type IIS restriction enzyme that recognizes the Type IIS restriction endonuclease recognition sites 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.

16. The method of claim 14 , further comprising subjecting the amplified oligonucleotides to error removal.

17. The method of claim 16 , wherein the error removal comprises contacting the amplified oligonucleotides with a mismatch binding agent.

18. The method of claim 17 , wherein the mismatch binding agent is MutS.

19. 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.

Assignees

• Gen9 Inc

Inventors

• Hudson Michael E
• Kung Li-Yun A
• Schindler Daniel
• Archer Stephen
• Saaem Ishtiaq

Classifications

• C12Q2521/501

  Ligase · CPC title

• C12Q2521/301

  Endonuclease · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12N15/1031 Primary

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

• C12Q1/686

  Polymerase chain reaction [PCR] · CPC title