NUCLEIC ACID HYBRIDIZATION METHODS

• US 20090227473A1
• Filed: 05/11/2009
• Published: 09/10/2009
• Est. Priority Date: 11/12/2003
• Status: Active Grant

• Current Assignee: Donald M. Crothers
• Original Assignee: Donald M. Crothers
• Inventors: Crothers, Donald M.
• Granted Patent: US 8,318,919 B2
• Time in Patent Office: Days
• US Class Current: 506/17
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/531 Glycosylase
• CPC Class Codes: C12Q 2531/125 Rolling circle
• CPC Class Codes: C12Q 2565/543 characterised by the use of two or more capture oligonucleotide primers in concert, e.g. bridge amplification

Abstract

Disclosed herein are methods of destabilizing double-stranded nucleic acid hybridization using an enzyme comprising DNA N-glycosylase activity. Also disclosed herein is the detection of a double-stranded target DNA wherein the hybridization of duplex strands has been at least partially disrupted thereby permitting invasion of a probe strand. Also disclosed herein are methods of using an enzyme comprising DNA N-glycosylase activity to generate single-stranded circular nucleic acids.

Claims

1. An array comprising:

a solid support comprising a capture probe; and

a double stranded nucleic acid comprising a first strand hybridized to second strand and at least one region where the hybridization of the first strand to the second strand is destabilized such that the first strand comprises an unhybridized flap sequence and the second strand comprises a sequence complementary to the flap sequence that is hybridized to the capture probe.

2. The array of claim 1, wherein the solid support comprises a plurality of different capture probes.

3. The array of claim 1, wherein the solid support comprises a plurality of capture probes having the same sequence.

4. The array of claim 1, wherein said flap sequence is a 5′

flap sequence.

5. The array of claim 1, wherein said sequence hybridized to the capture probe comprises a tag sequence.

6. The array of claim 1, wherein said capture probe is immobilized to said solid support.

7. The array of claim 1, wherein said solid support comprises a bead or particle.

8. The array of claim 1, wherein said at least one region where the hybridization of the first strand to the second strand is destabilized comprises one or more abasic sites.

9. The array of claim 8, wherein at least one of said one or more abasic sites is an apurinic site.

10. The array of claim 8, wherein at least one of said one or more abasic sites is an apyrimidinic site.

11. The array of claim 10, wherein said apyrimidinic site results from the hydrolysis of the bond between the N1 nitrogen of uracil and the C1′

carbon of deoxyribose.

12. The array of claim 1, wherein rehybridization of said at least one region where the hybridization of the first strand to the second strand is destabilized is energetically unfavorable

13. The array of claim 1, wherein said double stranded nucleic acid further comprises a second region where the hybridization of the first strand to the second strand is destabilized.

14. The array of claim 13, wherein said second region is destabilized such that said second strand comprises an unhybridized flap sequence.

15. The array of claim 13, wherein said second region is destabilized such that said second strand comprises an unhybridized flap sequence and said first strand comprises a sequence complementary to the flap sequence that is hybridized to a primer for head-to-tail hybridization.

16. The array of claim 13, wherein said second region is destabilized such that said second strand comprises an unhybridized flap sequence and said first strand comprises a sequence complementary to the flap sequence that is hybridized to a circular nucleic acid template.

17. The array of claim 16, wherein said first strand further comprises an extended region comprising a plurality of repeated sequences complementary to said circular nucleic acid template.

18. The array of claim 17, further comprising one or more bridge nucleic acids hybridized to said extended region.

19. The array of claim 18, wherein said one or more bridge nucleic acids further comprise an extended bridge sequence comprising a plurality of repeated sequences complementary to a second circular DNA template.

20. The array of claim 13, wherein said second region is destabilized such that said second strand comprises an unhybridized flap sequence and said first strand comprises a sequence complementary to the flap sequence that is hybridized to a polynucleotide conjugated to a detector molecule.