Methods for Preparative In Vitro Cloning

• US 20120322681A1
• Filed: 06/15/2012
• Published: 12/20/2012
• Est. Priority Date: 06/15/2011
• Status: Active Grant

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Jacobson, Joseph, Kung, Li-yun A., Schindler, Daniel
• Granted Patent: US 9,752,176 B2
• Time in Patent Office: Days
• US Class Current: 506/9
• 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, the method comprising:

(a) providing a population of nucleic acid molecules comprising a plurality of distinct nucleic acid molecules;

(b) providing a plurality of oligonucleotide tags;

(c) attaching the oligonucleotide tags to one terminus of the plurality nucleic acid molecules thereby generating a subpopulation of nucleic acid-oligonucleotide tag molecules;

(d) amplifying the plurality of nucleic acid-oligonucleotide tag molecules using primers complementary to the plurality of oligonucleotide tags; and

(e) isolating at least one target nucleic acid-oligonucleotide tag molecule of the subpopulation of nucleic acid-oligonucleotide tag molecules.

2. The method of claim 1 further sequencing the at least one isolated target nucleic acid-oligonucleotide tag molecule.

3. The method of claim 2 further identifying the target nucleic acid.

4. The method of claim 2 wherein the sequencing step is by high throughput sequencing.

5. The method of claim 2 further amplifying the at least one target nucleic acid-oligonucleotide tag molecule.

6. The method of claim 1 wherein the step of isolating is by limiting dilution.

7. The method of claim 6 further amplifying the target nucleic acid-oligonucleotide tag molecule.

8. The method of claim 1 wherein the target nucleic acid has a predefined sequence.

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

(a) providing a population comprising a plurality of different nucleic acid molecules;

(b) providing a plurality of microparticles, wherein each microparticle has an oligonucleotide sequence complementary to a portion of the nucleic acid molecules immobilized on its surface;

(c) forming a population of nucleic acid molecules hybridized to the complementary oligonucleotide sequence on the microparticles; and

(d) isolating at least one target nucleic acid molecule.

10. The method of claim 9 wherein each microparticle has a single complementary sequence on its surface.

11. The method of claim 10 wherein the complementary oligonucleotide sequences are identical.

12. The method of claim 10 wherein the complementary oligonucleotide sequences are different.

13. The method of claim 9 wherein the plurality of nucleic acid molecules comprises an oligonucleotide tag at one end terminus.

14. The method of claim 13 wherein the plurality of nucleic acid molecules has the same oligonucleotide tag.

15. The method of claim 13 wherein the plurality of nucleic acid molecules has different oligonucleotide tags.

16. The method of claim 9 wherein the step of isolating is by limiting dilution.

17. The method of claim 9 further amplifying the at least one target molecule.

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

(a) providing a population comprising a plurality of different nucleic acid molecules;

(b) providing a dilution of the population of nucleic acid molecules;

(c) separating the nucleic acid molecules into samples comprising a single nucleic acid molecule or a smaller number of nucleic acid molecules;

(d) amplifying the nucleic acid molecules from step c thereby forming amplified nucleic acid molecules;

(e) optionally repeating step c and d in the samples that do not comprise a nucleic acid molecule; and

(f) isolating at least one target nucleic acid molecule.

19. The method of claim 18 wherein the target nucleic acid has a predefined sequence.

20. The method of claim 18 further sequencing the at least one target molecule.