Selective enrichment of nucleic acids

• US 9,879,312 B2
• Filed: 05/10/2016
• Issued: 01/30/2018
• 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, Won, Melissa Shults
• Primary Examiner(s): Martinell, James
• Application Number:
  US15/150,716
• Publication Number:
  US 20160289742A1
• Time in Patent Office: 630 Days
• Field of Search: None
• 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) hybridizing said target with a capture probe;

(c) locking said hybridized capture probe to said target; and

(d) separating a nucleic acid locked to the capture probe from a nucleic acid that is not locked to a capture probe.

2. The method of claim 1, wherein said locking comprises contacting psoralen to said capture probe hybridized to said target.

3. The method of claim 1, wherein said target comprises a 5′

portion and a 3′

portion, and wherein said capture probe comprises a first end complementary to at least a portion of said 5′

portion of said target, an intermediary region, and a second end complementary to at least a portion of said 3′

portion of said target; and

wherein said locking comprises joining together the first end to the second end of said capture probe, thereby locking said capture probe to said target.

4. The method of claim 3, wherein said joining comprises extending and ligating the first end to the second end of said capture probe.

5. The method of claim 4, wherein said extending incorporates a capture moiety into said capture probe.

6. The method of claim 1, wherein said population comprises circularized nucleic acids.

7. The method of claim 1, further comprising repeating steps (a)-(d).

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

9. 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, said target comprising a portion of the 5′

end of a nucleic acid and a portion of the 3′

end of the nucleic acid;

(b) obtaining a selector probe comprising a first and second oligonucleotide annealed together, wherein said first oligonucleotide is complementary to at least a portion of said 5′

end of the nucleic acid and complementary to at least a portion of said second oligonucleotide, and said second oligonucleotide is complementary to at least a portion of said 3′

end of the nucleic acid;

(c) contacting said selector probe to said target, wherein said selector probe hybridizes to said target;

(d) joining said selector probe hybridized to said target; and

(e) separating a nucleic acid joined to said selector probe from a nucleic acid not joined to a selector probe.

10. The method of claim 9, further comprising sequencing at least a portion of said target.

11. The method of claim 9, wherein said contacting comprises contacting said selector probe and target with Rec A.

12. The method of claim 9, wherein said separating further comprises contacting a binding moiety to said selector probe joined to said target.

13. The method of claim 9, further comprising repeating steps (a)-(e).

14. The method of claim 9, wherein said target comprises a first capture moiety, and said selector probe comprises a second capture moiety, the method 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 selector probe.

15. A method for selective enrichment of a nucleic acid, said method comprising the steps of:

(a) obtaining a population of single-stranded nucleic acids, wherein at least some of the nucleic acids in said population comprise a target, said target comprising the 5′

end of a nucleic acid and the 3′

end of the nucleic acid;

(b) obtaining a selector probe comprising a first and second oligonucleotide annealed together, wherein said first oligonucleotide comprises a 5′

portion complementary to said 3′

end of the nucleic acid, a spacer portion, and a 3′

portion complementary to said 5′

end of the nucleic acid, said second oligonucleotide being complementary to said spacer portion;

(c) contacting said selector probe to said target, wherein said selector probe hybridizes to said target;

(d) joining said selector probe hybridized to said target; and

(e) separating a nucleic acid joined to said selector probe from a nucleic acid not joined to a selector probe;

wherein said target comprises a first capture moiety, and said selector probe comprises a second capture moiety, the method 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 selector probe.

16. The method of claim 15, wherein a 5′

end and a 3′

end of the selector probe have different annealing temperatures from one another.

17. The method of claim 15, further comprising sequencing at least a portion of said target.

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

19. A method for normalizing amplified nucleic acids comprising:

(a) selecting a first population of oligonucleotides capable of hybridizing to a first target, wherein the first population of oligonucleotides comprises oligonucleotides comprising capture moieties and oligonucleotides lacking capture moieties;

(b) obtaining a second population of oligonucleotides capable of hybridizing to a second target, wherein the second population of oligonucleotides comprises oligonucleotides comprising capture moieties;

(c) amplifying first and second target nucleic acids with said first and second populations of oligonucleotides; and

(d) separating amplified targets having incorporated oligonucleotide comprising capture moieties from amplified targets lacking incorporated oligonucleotide capture moieties, thereby obtaining a population of normalized amplified nucleic acids comprising capture moieties.