Selective enrichment of nucleic acids

Patent metadata

Field	Value
Publication number	US-9365897-B2
Application number	US-201113984005-A
Country	US
Kind code	B2
Filing date	Feb 8, 2011
Priority date	Feb 8, 2011
Publication date	Jun 14, 2016
Grant date	Jun 14, 2016

Abstract

Methods for the selective enrichment of nucleic acids.

First claim

What is claimed is:

1. A method for selective enrichment of a nucleic acid, said method comprising the steps of:
(a) obtaining a population of nucleic acids, wherein at least some of the nucleic acids in said population comprise a target;
(b) contacting said population of nucleic acids with a nickase, thereby producing a population of nicked nucleic acids;
(c) contacting said population of nicked nucleic acids with an exonuclease, thereby generating a nucleic acid having a single-stranded portion, wherein said single-strand portion comprises at least a portion of said target;
(d) contacting a capture probe to said at least a portion of said target, wherein said probe hybridizes to said target; and
(e) separating a nucleic acid hybridized to said capture probe from a nucleic acid not bound to said capture probe.

2. The method of claim 1 further comprising the step of releasing said hybridized nucleic acid from said capture probe.

3. The method of claim 1 , further comprising amplifying said target.

4. The method of claim 1 , further comprising sequencing at least a portion of said target.

5. The method of claim 1 , wherein step (a) further comprises selecting for a population of nucleic acids having an average length greater than about 10 kb.

6. The method of claim 1 , wherein step (a) further comprises:
contacting the population of double stranded nucleic acids with a type II restriction endonuclease comprising an isoschizomer of said nickase; and
recircularizing said cut double stranded nucleic acids under conditions that favor intramolecular recircularization of individual nucleic acids.

7. The method of claim 6 , wherein said restriction endonuclease comprises BbvCI.

8. The method of claim 1 , wherein said nickase is selected from Nb.BbvCI and Nt.BbvCI.

9. The method of claim 1 , wherein said probe comprises a capture moiety.

10. The method of claim 9 , wherein said capture moiety comprises biotin.

11. The method of claim 10 , wherein said separating further comprises contacting said hybridized target and probe to a binding moiety.

12. The method of claim 11 , wherein said binding moiety comprises streptavidin.

13. The method of claim 12 , wherein said binding moiety further comprises a microsphere.

14. The method of claim 1 , wherein said probe comprises RNA.

15. The method of claim 1 , further comprising repeating steps (a)-(e).

16. The method of claim 1 , wherein said target comprises a first capture moiety, and said probe comprises a second capture moiety.

17. The method of claim 16 , further comprising
contacting said first capture moiety to a first binding moiety, thereby enriching for said target, and
contacting said second capture moiety to a second binding moiety, thereby enriching for said probe.

Assignees

• Steemers Frank J
• Gunderson Kevin
• York Kerri
• Shults Melissa D
• Illumina Inc

Inventors

• Steemers Frank J
• Gunderson Kevin
• York Kerri
• Shults Melissa D

Classifications

• C12Q1/6806 Primary

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

• C12Q1/6869 Primary

  Methods for sequencing · CPC title

• C12Q2537/162

  Helper probe · CPC title

• C12Q2521/301

  Endonuclease · CPC title

• C12Q2521/319

  Exonuclease · CPC title