Methods for preparative in vitro cloning

• US 9,752,176 B2
• Filed: 06/15/2012
• Issued: 09/05/2017
• Est. Priority Date: 06/15/2011
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Ginkgo Bioworks, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Kung, Li-yun A., Schindler, Daniel, Jacobson, Joseph
• Primary Examiner(s): Priest, Aaron
• Application Number:
  US13/524,164
• Publication Number:
  US 20120322681A1
• Time in Patent Office: 1,908 Days
• Field of Search: None
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1065 Preparation or screening of tagged libraries, e.g. tagged microorganisms by STM-mutagenesis, tagged polynucleotides, gene tags
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6855 Ligating adaptors
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2533/107 Probe or oligonucleotide ligation
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid

Abstract

Methods and devices relate to the isolation of nucleic acids of interest from within a population of nucleic acids such as libraries of nucleic acid sequences.

Claims

1. A method of isolating a target nucleic acid molecule, the method comprising:

(a) providing a plurality of nucleic acid molecules;

(b) providing a library of oligonucleotide tags, each oligonucleotide tag comprising a region having a different sequence than the other tags in the library and comprising a primer binding site;

(c) ligating at least a subset of the plurality of nucleic acid molecules with the library of oligonucleotide tags, thereby generating a population of conjugates, wherein each conjugate has a unique oligonucleotide tag that is different in sequence than that of the other conjugates;

(d) amplifying in a pool at least a subset of the population of conjugates using one or more primers each having sequence complementarity to the primer binding site thereon;

(e) isolating at least one conjugate of the amplified conjugates from the pool, wherein the isolated conjugate comprises a target nucleic acid molecule having a predetermined sequence; and

(f) removing the oligonucleotide tag attached thereto to recover the target nucleic acid molecule.

2. The method of claim 1 further comprising sequencing the amplified conjugates.

3. The method of claim 2 wherein the sequencing step comprises high throughput sequencing.

4. The method of claim 2 wherein the step of isolating comprises clonally separating the target nucleic acid molecule from non-target by selectively amplifying, after the sequencing step, at least one conjugates.

5. The method of claim 1 wherein the step of isolating comprises limiting dilution.

6. The method of claim 5 further comprising amplifying the isolated conjugate.

13. The method of claim 1 wherein the step of amplifying comprises providing a plurality of primers in stoichiometry wherein the absolute number of primers is less than the absolute number of oligonucleotide tags.

14. The method of claim 1 wherein in the step of providing a library of oligonucleotide tags, each oligonucleotide tag in the library differs from one another by at least 2 nucleotides.

15. The method of claim 1 wherein step (d) is performed in solution.

16. The method of claim 1 wherein in step (b), the library of oligonucleotide tags is provided in stoichiometry wherein the absolute number of oligonucleotide tags is less than the absolute number of nucleic acid molecules.

17. The method of claim 1 further comprising isolating a plurality of target nucleic acid molecules having different predetermined sequences and assembling the plurality of target nucleic acid molecules.

18. The method of claim 1 wherein in step (a) the nucleic acid molecules are synthesized on a solid support.

20. The method of claim 1, wherein the removing step (f) comprises contacting the isolated conjugate with an endonuclease to produce a double-stranded break.

21. The method of claim 20 further comprising isolating a plurality of target nucleic acid molecules having different predetermined sequences that together comprise and can be assembled into a target polynucleotide.

22. The method of claim 1, wherein the removing step (f) comprises selectively amplifying the target nucleic acid molecule.

7. A method of isolating a target nucleic acid molecule, the method comprising:

(a) providing a population of nucleic acid molecules having different predetermined sequences;

(b) providing a plurality of oligonucleotide sequences that are different from one another in sequence and are immobilized on microparticles, each microparticle having a single copy of oligonucleotide sequence immobilized on its surface, wherein each single copy of oligonucleotide sequence is uniquely complementary to a corresponding nucleic acid molecule of the population;

(c) hybridizing the population of nucleic acid molecules to the plurality of oligonucleotide sequences, thereby forming a population of hybridized microparticles, each hybridized microparticle having a single nucleic acid molecule attached thereon via hybridization with its corresponding uniquely complementary oligonucleotide sequence;

(d) separating the population of hybridized microparticles into samples, each sample comprising a single hybridized microparticle; and

(e) isolating at least one hybridized microparticle comprising a target nucleic acid molecule having a predetermined sequence.

8. The method of claim 7 wherein each nucleic acid molecule comprises an oligonucleotide tag at one terminus.

9. The method of claim 8 wherein the plurality of nucleic acid molecules have the same oligonucleotide tag.

10. The method of claim 8 wherein the plurality of nucleic acid molecules have different oligonucleotide tags.

11. The method of claim 7 wherein the step of isolating comprises limiting dilution.

12. The method of claim 7 further comprising amplifying the target nucleic acid molecule.

19. A method of isolating a target nucleic acid molecule, the method comprising:

(a) providing a population of nucleic acid conjugates, each having a distinct predetermined sequence and a oligonucleotide tag that is different in sequence than that of the other conjugates, wherein each predetermined sequence and each oligonucleotide tag are conjugated via ligation;

(b) amplifying in a pool at least a subset of the population of nucleic acid conjugates using primers having sequence complementarity to the oligonucleotide tags conjugated thereto;

(c) isolating a target nucleic acid molecule having a predetermined sequence from the amplified nucleic acid molecules in the pool; and

(d) removing the oligonucleotide tag conjugated thereto to recover the target nucleic acid molecule.

23. The method of claim 19, wherein the removing step (d) comprises contacting the isolated target nucleic acid molecule with an endonuclease to produce a double-stranded break.

24. The method of claim 23 further comprising isolating a plurality of target nucleic acid molecules having different predetermined sequences that together comprise and can be assembled into a target polynucleotide.

25. The method of claim 19, wherein the removing step (d) comprises selectively amplifying the target nucleic acid molecule.