METHODS FOR RETRIEVAL OF SEQUENCE-VERIFIED DNA CONSTRUCTS

• US 20120283110A1
• Filed: 04/23/2012
• Published: 11/08/2012
• Est. Priority Date: 04/21/2011
• Status: Active Grant

• Current Assignee: University of Washington
• Original Assignee: University of Washington
• Inventors: Shendure, Jay, Kitzman, Jacob, Hiatt, Joseph, Schwartz, Jerrod, Patwardhan, Rupali
• Granted Patent: US 9,809,904 B2
• Time in Patent Office: Days
• US Class Current: 506/4
• 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 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/191 incorporating an adaptor
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2563/179 the label being a nucleic acid
• CPC Class Codes: C40B 20/04 Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes

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.

Claims

1. A method of recovering a sequence-verified target nucleic acid comprising

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.

2. The method of claim 1, wherein the set of adaptor sequences comprises a dial-out tag sequence.

3. The method of claim 2, wherein the dial-out tag sequence is a completely degenerate sequence.

4. The method of claim 2, wherein the dial-out tag sequence is a partially degenerate sequence.

5. The method of claim 4, further comprising tagging each member of a nucleic acid library with a second set of adaptor sequences.

6. The method of claim 1, 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.

7. 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.

8. The method of claim 1, wherein the sequencing is performed using a platform selected from cyclic-array methods, nanopore sequencing methods, real-time observation of DNA synthesis, sequencing by electron microscopy, dideoxy termination and electrophoresis, microelectrophoretic methods, sequencing by hybridization, and mass spectroscopy methods.

9. 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.

10. The method of claim 2, wherein the dial-out selection method comprises:

targeting the dial-out tag sequence of the sequence-verified target nucleic acid with a complementary PCR primer; and

selectively amplifying the sequence-verified target nucleic acid.

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

20. The method of claim 2, wherein the dial-out selection method comprises:

targeting the dial-out tag sequences of the two or more sequence-verified target nucleic acid with a set of complementary PCR primers; and

selectively amplifying the two or more sequence-verified target nucleic acids.

12. A method of recovering a sequence-verified target nucleic acid variant comprising:

tagging each member of a mutagenesis library with a set of adaptor sequences;

sequencing the tagged members of the mutagenesis library; and

recovering the sequence-verified target nucleic acid variant from the tagged and sequenced members of the mutagenesis library using a dial-out selection method.

13. The method of claim 12, wherein the mutagenesis library is generated using a method of directed mutagenesis, random mutagenesis, insertional mutagenesis, PCR mutagenesis, or a multiplex programmed mutagenesis.

14. A method of synthesizing a DNA construct comprising:

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 two or more sequence-verified target nucleic acids from the tagged and sequenced members of the nucleic acid library using a dial-out selection method; and

assembling the DNA construct using the two or more sequence-verified target nucleic acids.

15. The method of claim 14, wherein the set of adaptor sequences comprises a dial-out tag sequence.

16. The method of claim 14, further comprising tagging each member of a nucleic acid library with a second set of adaptor sequences.

17. The method of claim 14, 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.

18. The method of claim 14, 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.

19. The method of claim 14, wherein the sequencing is performed using a platform selected from cyclic-array methods, nanopore sequencing methods, real-time observation of DNA synthesis, sequencing by electron microscopy, dideoxy termination and electrophoresis, microelectrophoretic methods, sequencing by hybridization, and mass spectroscopy methods.