Methods for retrieval of sequence-verified DNA constructs

Patent metadata

Field	Value
Publication number	US-9809904-B2
Application number	US-201213453947-A
Country	US
Kind code	B2
Filing date	Apr 23, 2012
Priority date	Apr 21, 2011
Publication date	Nov 7, 2017
Grant date	Nov 7, 2017

Abstract

In some embodiments, methods of recovering a sequence-verified target nucleic acid are provided. In some embodiments, such methods may include tagging each member of a nucleic acid library with a set of adaptor sequences; sequencing the tagged members of the nucleic acid library; and recovering the sequence-verified target nucleic acid from the tagged and sequenced members of the nucleic acid library using a dial-out selection method. In certain embodiments, the members of the nucleic acid library may be tagged with a second set of adaptor sequences.

First claim

What is claimed is:

1. A method of recovering a sequence-verified target nucleic acid from a sequenced nucleic acid library comprising
tagging each member of a nucleic acid library with at least one identification tag having a degenerate sequence;
sequencing the tagged members of the nucleic acid library; and
selectively recovering only the sequence-verified target nucleic acid from the tagged and sequenced members of the nucleic acid library by targeting the at least one identification tag that is unique to the sequence-verified target nucleic acid using a clone-free dial-out selection method and amplifying the sequence-verified target nucleic acids.

2. The method of claim 1 , further comprising tagging each member of the nucleic acid library with a common adaptor sequence.

3. The method of claim 2 , wherein the set of adaptor sequences comprises one or more sequences selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48.

4. The method of claim 1 , wherein the nucleic acid library comprises a population of oligonucleotides, a population of single stranded or double stranded gene fragments, a set of synthetic nucleic acids assembled from oligonucleotides, or a mutagenesis library.

5. The method of claim 1 , wherein the sequencing is performed using a massive parallel sequencing platform.

6. The method of claim 1 , wherein the dial-out selection method comprises a hybridization-based capture method, a 2-primer based PCR method, a 1-primer PCR method, a linear amplification method, a multiple displacement amplification method, a rolling circle amplification method, or a ligation-based method.

7. The method of claim 1 , wherein the dial-out selection method comprises:
targeting the at least one identification tag that is unique to the sequence-verified target nucleic acid with a complementary PCR primer.

8. The method of claim 1 , further comprising a subassembly step prior to or during the sequencing of the tagged members of the nucleic acid library.

9. A method of recovering a sequence-verified target nucleic acid variant comprising:
tagging each member of a mutagenesis library with at least one identification tag having a degenerate sequence;
sequencing the tagged members of the mutagenesis library; and
selectively recovering only the sequence-verified target nucleic acid variant from the tagged and sequenced members of the mutagenesis library by targeting the at least one identification tag that is unique to the sequence-verified target nucleic acid variant using a clone-free dial-out selection method and amplifying the sequence-verified target nucleic acids.

10. The method of claim 9 , wherein the mutagenesis library is generated using a method of directed mutagenesis, random mutagenesis, insertional mutagenesis, PCR mutagenesis, or a multiplex programmed mutagenesis.

11. A method of synthesizing a DNA construct comprising:
tagging each member of a nucleic acid library with at least one identification tag having a degenerate sequence;
sequencing the tagged members of the nucleic acid library; and
selectively recovering only two or more sequence-verified target nucleic acids from the tagged and sequenced members of the nucleic acid library by targeting the at least one identification tag that is unique to the two or more sequence-verified target nucleic acids using a clone-free dial-out selection method and amplifying the two or more sequence-verified target nucleic acids; and
assembling the DNA construct using the two or more sequence-verified target nucleic acids.

12. The method of claim 11 , further comprising tagging each member of the nucleic acid library with a common adaptor sequence.

13. The method of claim 12 , wherein the set of adaptor sequences comprises one or more sequences selected from SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48.

14. The method of claim 11 , wherein the nucleic acid library comprises a population of oligonucleotides, a population of single stranded or double stranded gene fragments, a set of synthetic nucleic acids assembled from oligonucleotides, or a mutagenesis library.

15. The method of claim 11 , wherein the sequencing is performed using a massive parallel sequencing platform.

16. The method of claim 11 , wherein the dial-out selection method comprises:
targeting the identification tag sequences of the two or more sequence-verified target nucleic acid with a set of complementary PCR primers.

Assignees

• Shendure Jay
• Schwartz Jerrod
• Kitzman Jacob
• Patwardhan Rupali
• Hiatt Joseph
• Univ Washington Through Its Center For Commercialization

Inventors

• Shendure Jay
• Schwartz Jerrod
• Kitzman Jacob
• Patwardhan Rupali
• Hiatt Joseph

Classifications

• C12Q1/6853

  using modified primers or templates · CPC title

• C40B20/04 Primary

  Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes · CPC title

• C12Q1/6806

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12Q2563/179

  the label being a nucleic acid · CPC title

• C12Q2525/155

  incorporating/generating a new priming site · CPC title