Methods for sorting nucleic acids and preparative in vitro cloning

• US 20130281308A1
• Filed: 04/24/2013
• Published: 10/24/2013
• Est. Priority Date: 04/24/2012
• Status: Abandoned Application

• Current Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Original Assignee: Gen9, Inc. (Ginkgo Bioworks Holdings, Inc.)
• Inventors: Kung, Li-yun A., Jacobson, Joseph
• Application Number:
  US13/986,368
• Publication Number:
  US 20130281308A1
• Time in Patent Office: Days
• US Class Current: 506/4
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups

Abstract

Methods and compositions relate to the sorting and cloning of high fidelity nucleic acids using high throughput sequencing. Specifically, nucleic acid molecules having the desired predetermined sequence can be sorted from a pool comprising a plurality of nucleic acids having correct and incorrect sequences.

Claims

1. A method of sorting nucleic acid molecules having a predetermined sequence, the method comprising:

(a) contacting a pool of nucleic acid molecules comprising at least two populations of nucleic acid molecules, each population of nucleic acid molecule having a unique nucleic acid sequence;

(b) tagging the 5′

end and the 3′

end of the nucleic acid molecules with an oligonucleotide tag sequence, wherein the oligonucleotide tag sequence comprises a unique nucleotide tag and a primer region;

(c) subjecting the nucleic acid molecules to a sequencing reaction from both ends to obtain a paired end read; and

(d) sorting the nucleic acid molecules having the predetermined sequence according to the identity of their corresponding unique nucleotide tags.

2. The method of claim 1 further comprising amplifying the nucleic acid molecules having the predetermined sequence.

3. The method of claim 2 further comprising amplifying the constructs having the predetermined sequence using primers complementary to the primer region.

4. The method of claim 1 further comprising pooling a plurality of nucleic acid molecules to form the pool of nucleic acid molecules, wherein each plurality of nucleic acid molecules comprises a population of nucleic acid sequences having the predetermined sequence and a population of nucleic acid sequences having a sequence different than the predetermined sequence.

5. The method of claim 4 further comprising assembling the plurality of nucleic acid molecules onto a solid support prior to pooling the plurality of nucleic acid molecules.

6. The method of claim 4 further comprising diluting the plurality of nucleic acid molecules prior to the step of pooling or after the step of pooling.

7. The method of claim 1 wherein in the step of contacting, the pool of nucleic acid molecules is normalized.

8. The method of claim 1 wherein in the step of tagging, the oligonucleotide tag sequences are ligated to the 5′

and 3′

end of the nucleic acid molecules.

9. The method of claim 1 wherein in the step of tagging, the oligonucleotide tag sequences are joined to the 5′

and 3′

end of the nucleic acid molecules by polymerase chain reaction.

10. The method of claim 1 wherein in the step of contacting, each nucleic acid molecule comprises a 5′

end common adaptor sequence and 3′

end common adaptor sequence and wherein the oligonucleotide tag sequence further comprises a common adaptor sequence.

11. The method of claim 1 wherein in the step of contacting, each population of nucleic acid molecules has a different desired nucleic acid sequence.

12. The method of claim 1 wherein in the step of tagging the unique oligonucleotide tag is a degenerate nucleotide sequence.