Selective enrichment of nucleic acids

• US 9,365,897 B2
• Filed: 02/08/2011
• Issued: 06/14/2016
• Est. Priority Date: 02/08/2011
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Steemers, Frank J., Gunderson, Kevin, York, Kerri, Shults, Melissa D.
• Primary Examiner(s): Martinell, James
• Application Number:
  US13/984,005
• Publication Number:
  US 20130316917A1
• Time in Patent Office: 1,953 Days
• Field of Search: None
• US Class Current: 1/1
• 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 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/319 Exonuclease
• CPC Class Codes: C12Q 2537/159 Reduction of complexity, e.g. amplification of subsets, removing duplicated genomic regions
• CPC Class Codes: C12Q 2537/162 Helper probe

Abstract

Methods for the selective enrichment of nucleic acids.

Claims

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.