Nucleic acid hybridization methods
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.
1. A method of nucleic acid strand invasion, said method comprising:
obtaining a sample from an individual, said sample comprising a target sequence;
copying said target sequence to generate a double-stranded DNA comprising a first strand hybridized to a second strand, wherein said first strand comprises a copy of said target sequence, said copy of said target sequence comprising at least one non-natural DNA base;
incubating said double-stranded DNA with an enzyme that removes said non-natural DNA base so as to destabilize hybridization between said first strand and said second strand, thereby generating an exposed region of said second strand; and
contacting said exposed region of said second strand with a capture probe attached to a surface, which is complementary to at least a portion of the exposed region of said second strand, under conditions which permit hybridization of said capture probe to said second strand.
2. The method of claim 1 , wherein said enzyme comprises a DNA N-glycosylase activity.
3. The method of claim 2 , wherein said enzyme is uracil-DNA glycosylase.
4. The method of claim 1 , wherein said enzyme comprises both a DNA N-glycosylase activity and an AP-lyase activity.
5. The method of claim 1 , wherein said enzyme is selected from the group consisting of Fpg, hOGG1, endonuclease VIII and endonuclease III.
6. The method of claim 1 further comprising incubating said double-stranded DNA with an AP-endonuclease thereby partially degrading said first strand.
7. The method of claim 1 , wherein said non-natural DNA base is selected from the group consisting of uracil, 7,8-dihydro-8-oxoguanine (8-oxoguanine), 8-oxoadenine, foramidopyrimidine (fapy)-guanine, methyl-fapy-guanine, fapy-adenine, aflatoxin B 1 -fapy-guanine, 5-hydroxy-cytosine, 5-hydroxy-uracil, urea, 5,6-dihydroxythymine, thymine glycol, 5-hydroxy-5-methylhydanton, uracil glycol, 6-hydroxy-5,6-dihydrothymine and methyltartronylurea.
8. The method of claim 1 , wherein said non-natural DNA base is uracil.
9. A method of nucleic acid strand invasion, said method comprising:
obtaining a sample from an individual, said sample comprising a target sequence;
copying said target sequence to generate a double-stranded DNA comprising a first strand hybridized to a second strand, wherein said first strand comprises a copy of said target sequence, and wherein uracil replaces at least one thymine in said copy of said target sequence;
incubating said double-stranded DNA with uracil-DNA glycosylase so as to destabilize hybridization between said first strand and said second strand, thereby generating an exposed region of said second strand; and
contacting said exposed region of said second strand with a capture probe attached to a surface, which is complementary to at least a portion of the exposed region of said second strand, under conditions which permit hybridization of said capture probe to said second strand.
10. The method of claim 9 further comprising incubating said double-stranded DNA with AP-endonuclease thereby partially degrading said first strand.
11. The method of claim 9 further comprising hydrolyzing said first strand by heating said double-stranded DNA.
12. The method of claim 9 , wherein said double stranded DNA is circular.
13. The method of claim 12 , wherein uracil replaces every thymine in said first strand.
14. The method of claim 13 , wherein said first strand is hydrolyzed by heating said double-stranded DNA.
15. The method of claim 9 , wherein said double-stranded DNA is linear.
16. The method of claim 15 , wherein the first strand of said double-stranded DNA comprises a first 5′ flap sequence.
17. The method of claim 16 , wherein uracil replaces thymine only in said first 5′ flap sequence.
18. The method of claim 16 , wherein the exposed region of said second strand, which is complementary to said first 5′ flap sequence, comprises a tag sequence.
19. The method of claim 18 , wherein said first 5′ flap sequence comprises a sequence complementary to said tag sequence.
20. The method of claim 19 , wherein said tag sequence hybridizes with a capture probe.
21. The method of claim 15 , further comprising contacting the exposed region of said second strand with a primer for head-to-tail hybridization.
22. The method of claim 15 , further comprising contacting the exposed region of said second strand with a circular RCA template.
23. The method of claim 15 , further comprising contacting the exposed region of said second strand with a polynucleotide conjugated to a detector molecule.
24. The method of claim 23 , wherein the detector molecule is a redox enzyme.
25. The method of claim 23 , wherein said detector molecule is an antibody-horse radish peroxidase complex.
26. The method of claim 15 , wherein the first strand of said double-stranded DNA comprises a first 5′ flap sequence and wherein, the second strand of said double-stranded DNA comprises a second 5′ flap sequence.
27. The method of claim 26 , wherein uracil replaces thymine in said first 5′ flap sequence and in said second 5′ flap sequence.
28. The method of claim 27 , wherein the region of said first strand that is complementary to said second 5′ flap sequence becomes exposed after incubation of said double-stranded DNA with uracil-DNA glycosylase.
29. The method of claim 28 , further comprising:
contacting the exposed region on said second strand with a capture probe; and
contacting the exposed region on said first strand with a polynucleotide selected from the group consisting of a primer for head-to-tail hybridization, a circular RCA template and a polynucleotide conjugated to a detector molecule.
30. The method of claim 29 , wherein said detector molecule is a redox enzyme.
31. The method of claim 29 , wherein said detector molecule is an antibody-horse radish peroxidase complex.
32. A method of detecting a target DNA, said method comprising:
obtaining a sample from an individual, said sample comprising a target sequence;
copying said target sequence to generate a double-stranded target DNA comprising a first strand hybridized to a second strand, wherein said first strand comprises a copy of said target sequence, and wherein uracil replaces at least one thymine in said copy of said target sequence;
incubating said double-stranded target DNA with uracil-DNA glycosylase so as to destabilize hybridization between said first strand and said second strand, thereby generating an exposed region of said second strand;
contacting said second strand with a capture probe attached to a surface, which is complementary to at least a portion of the exposed region of said second strand, under conditions which permit hybridization of said capture probe to said second strand; and
detecting said target DNA by determining whether a signal indicative of the presence of said target DNA has been generated.
33. The method of claim 32 further comprising incubating said double-stranded target DNA with AP-endonuclease thereby partially degrading said first strand.
34. The method of claim 32 further comprising hydrolyzing said first strand by heating said double-stranded target DNA.
35. The method of claim 32 , wherein said double stranded DNA is circular.
36. The method of claim 32 , wherein said capture probe is coupled to a detection zone.
37. The method of claim 36 , wherein said detection zone is an electrode.
38. The method of claim 32 , wherein said double-stranded target DNA is linear.
39. The method of claim 38 , wherein the first strand of said double-stranded target DNA comprises a first 5′ flap sequence.
40. The method of claim 39 , wherein uracil replaces thymine only in said first 5′ flap sequence.
41. The method of claim 39 , wherein the exposed region of said second strand, which is complementary to said first 5′ flap sequence, comprises a tag sequence.
42. The method of claim 41 , wherein said first 5′ flap sequence comprises a sequence complementary to said tag sequence.
43. The method of claim 41 , wherein said tag sequence hybridizes with said capture probe.
44. The method of claim 38 , wherein the first strand of said double-stranded DNA comprises a first 5′ flap sequence and wherein, the second strand of said double-stranded DNA comprises a second 5′ flap sequence.
45. The method of claim 44 , wherein uracil replaces thymine in said first 5′ flap sequence and in said second 5′ flap sequence.
46. The method of claim 45 , wherein the region of said first strand that is complementary to said second 5′ flap sequence becomes exposed after incubation of said double-stranded DNA with uracil-DNA glycosylase.
47. The method of claim 46 , further comprising contacting the exposed region of said first strand with a primer for head-to-tail hybridization.
48. The method of claim 46 , further comprising contacting the exposed region of said first strand with a circular RCA template.
49. The method of claim 46 , further comprising contacting the exposed region of said first strand with a polynucleotide conjugated to a detector molecule.
50. The method of claim 49 , wherein the detector molecule is a redox enzyme.
51. The method of claim 49 , wherein said detector molecule is an antibody-horse radish peroxidase complex.