Methods of nonspecific target capture of nucleic acids
View Patent ↗Methods for capturing a target nucleic acid from a sample by using a capture probe that binds nonspecifically to the target nucleic acid and binds specifically to an immobilized probe via a specific binding pair that has one member on the capture probe and one member on the immobilized probe are disclosed. Compositions that include a capture probe that binds nonspecifically to a target nucleic acid and specifically to an immobilized probe via binding of members of a specific binding pair in a solution phase of a reaction mixture are disclosed.
1. A method for isolating nucleic acid from a sample, comprising the steps of:
a. mixing a sample containing nucleic acids with a non-specific capture probe comprising a first region that is a randomized poly(k) sequence at a length of 18 residues comprising G and T nucleotides or G and U nucleotides attached to a second region that is a first specific binding partner (SBP);
b. incubating a reaction mixture containing a support and the mixture of nucleic acids and non-specific capture probe in conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample and that allow for hybridization of the SBP with a second specific binding partner (SBP′) immobilized to the support, thereby forming a hybridization complex; and
c. separating the support from the solution phase of the reaction mixture, thereby isolating the nucleic acids away from other components in the sample.
2. The method of claim 1 , wherein both the SBP and the SBP′ are non-nucleic acid moieties.
3. The method of claim 1 , wherein both the SBP and the SBP′ are nucleic acid sequences.
4. The method of claim 1 , wherein the SBP and the SBP′ are selected from the group consisting of: (a) a receptor and ligand pair, (b) an enzyme and substrate pair, (c) an enzyme and cofactor pair, (d) an enzyme and coenzyme pair, (e) an antibody and antigen pair, (f) an antibody fragment and antigen pair, (g) a sugar and lectin pair, (h) a ligand and chelating agent pair, (i) biotin and avidin, (j) biotin and streptavidin, (k) nickel and histidine, (l) a first and second nucleic acid sequence that are substantially complementary one to the other, and (m) dT 0-3 dA30 and dT10-30.
5. The method of claim 1 , wherein the non-specific capture probe is 5′-poly(k)- dT 0-3 dA30.
6. The method of claim 1 , wherein the SBP′ is dT14.
7. The method of claim 1 , further comprising a washing step following the separating step wherein additional sample components are removed from the captured nucleic acids.
8. The method of claim 1 , wherein the incubating step is performed at about 25 degrees Celsius.
9. The method of claim 8 , wherein the incubating step is performed for about 5 to about 90 minutes.
10. The method of claim 1 , further comprising an in vitro amplification following the separating step.
11. The method of claim 10 , further comprising an elution step to elute the nucleic acids for amplification.
12. The method of claim 1 , further comprising a detecting step following the separating step, wherein the detecting step includes, nucleic acid amplification, sequencing, probe hybridization or a combination thereof.
13. The method of claim 1 , wherein one or more of the poly(k) residues are 2′-methoxy.
14. A method for isolating nucleic acid from a sample, comprising the steps of:
a. mixing a sample containing nucleic acids with a non-specific capture probe comprising a first region that is from 6 to 24 randomized poly(k) residues, and from 0 to 2 C9 linkers or from 0 to 1 5-nitroindole, and the first region is attached to a second region that is a specific binding partner (SBP);
b. incubating the mixture of nucleic acids and non-specific capture probe in a target capture mixture under conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample; and
c. separating out of the solution phase all or a portion of the nucleic acids hybridized with a non-specific capture probe, thereby isolating the nucleic acids away from other components in the sample.
15. The method of claim 14 , wherein the first region is selected from the group consisting of: poly(k)18, poly(k)24, poly(k)6-C9-poly(k)6, poly(k)6-C9-poly(k)6-C9-poly(k)6, poly(k)6-5-nitroindole-poly(k)6, poly(k)12; 6 to 24 poly(k) residues and 1 to 2 C9 linkers, and 6 to 24 poly(k) residues and 1 5-nitroindole.
16. The method of claim 14 , wherein the target capture mixture uses a magnetic solid support attached to an immobilized nucleotide that is dT 10-30 .
17. The method of claim 16 , wherein the SBP is dT 0-3 dA30.
18. The method of claim 14 , further comprising a washing step following the separating step wherein additional sample components are removed from the captured nucleic acids.
19. The method of claim 14 , wherein the incubating step is performed for about 5 to about 90 minutes.
20. The method of claim 19 , wherein the incubating step is performed at about 25 degrees Celsius.
21. The method of claim 14 , further comprising an in vitro amplification following the separating step.
22. The method of claim 21 , wherein the in vitro amplification amplifies an HIV target nucleic acid.
23. The method of claim 21 , wherein the in vitro amplification amplifies a ribosomal target nucleic acid.
24. The method of claim 21 , further comprising an elution step to elute the nucleic acids for amplification.
25. The method of claim 14 , further comprising a detecting step following the separating step, wherein the detecting step includes, nucleic acid amplification, sequencing, probe hybridization or a combination thereof.
26. The method of claim 14 , wherein one or more of the poly(k) residues are 2′-methoxy.
27. A method for isolating nucleic acid from a sample, comprising the steps of:
a. forming a target capture reaction mixture to include nucleic acids from a sample and a non-specific capture probe comprising a first region that is from 6 to 24 randomized poly(k) residues, and from 0 to 2 non-nucleotide linkers or from 0 to 1 nucleotide analogs, and the first region is attached to a second region that is a specific binding partner (SBP);
b. incubating the mixture at step a for at least 1 minute and under conditions that allow hybridization of the first region with all or a portion of the nucleic acids in the sample; and
c. separating out of the solution phase all or a portion of the nucleic acids hybridized with a non-specific capture probe, thereby isolating the nucleic acids away from other components in the sample.
28. The method of claim 27 , wherein the SBP is dT0 — 3dA30o and the first region is selected from the group consisting of: poly(k)18, poly(k)24, poly(k)6-C9-poly(k)6, poly(k)6-C9-poly(k)6-C9-poly(k)6, poly(k)6-5-nitroindole-poly(k)6, poly(k)12; 6 to 24 poly(k) residues and 1 to 2 C9 linkers, and 6 to 24 poly(k) residues and 1 5-nitroindole.
29. The method of claim 27 , wherein the target capture mixture uses a magnetic solid support attached to an immobilized nucleotide that is dT 10-30 .
30. The method of claim 27 , further comprising a washing step following the separating step wherein additional sample components are removed from the captured nucleic acids.
31. The method of claim 27 , further comprising an in vitro amplification following the separating step.
32. The method of claim 31 , wherein the in vitro amplification amplifies an HIV target nucleic acid or a ribosomal target nucleic acid.
33. The method of claim 31 , further comprising a detecting step following the separating step, wherein the detecting step includes, nucleic acid amplification, sequencing, probe hybridization or a combination thereof.
34. The method of claim 31 , wherein one or more of the poly(k) residues are 2′-methoxy.
35. A method for isolating a target DNA from a sample, said method comprising:
1) mixing a sample containing the target DNA in a solution phase with a non-specific capture probe, wherein said non-specific capture probe comprises
(i) a randomized poly-(k) sequence that is at least 12 residues in length and comprising G and T nucleotides or G and U nucleotides, or a nonrandom repeating (GU) sequence that is at least 12 residues in length, and
(ii) a first specific binding partner (SBP), wherein said SBP is capable of specifically binding a second specific binding partner (SBP′);
2) incubating a reaction mixture containing (a) the SBP′ immobilized on a support and (b) the mixture of the non-specific capture probe and the sample containing the target DNA, wherein said incubating is under conditions that allow hybridization of the capture probe's poly-(k) sequence with the target DNA and that allow specific binding of the SBP to the SBP′; and
3) separating the support from the solution phase of the reaction mixture, thereby isolating the target DNA from other sample components.
36. The method of claim 35 , wherein the sample contains cells and is treated before the mixing step to release intracellular components into the solution phase.
37. The method of claim 36 , wherein said treatment comprises treating the sample with a solution containing a detergent.
38. The method of claim 36 , wherein the sample comprises peripheral blood.
39. The method of claim 35 , wherein the poly-(k) sequence comprises G and U nucleotides.
40. The method of claim 39 , wherein the poly-(k) sequence comprises 2′-O-methyl substituted RNA bases, or wherein the nonrandom repeating (GU) sequence comprises 2′-O-methyl substituted RNA bases.
41. The method of claim 35 , wherein the poly-(k) sequence is a poly-(k)18, poly-(k)24, or poly-(k)25 sequence.
42. The method of claim 35 , wherein the SBP and SBP′ are non-nucleic acid moieties.
43. The method of claim 35 , wherein the SBP and SBP′ are substantially complementary nucleic acid sequences.
44. The method of claim 43 , wherein the SBP is situated 3′ to the poly-(k) sequence.
45. The method of claim 43 , wherein the SBP is a dT3dA30 or dA30 sequence.
46. The method of claim 35 , wherein the solution phase comprises a detergent.
47. The method of claim 35 , wherein the solution phase comprises lithium lauryl sulfate and lithium hydroxide.
48. The method of claim 35 , further comprising detecting the presence of the target DNA isolated from other sample components, amplifying in vitro a sequence contained in the target DNA isolated from other sample components, or determining a sequence contained the target DNA isolated from other sample components.
49. A reaction mixture for isolating a target DNA from a sample, said reaction mixture comprising:
a) a sample containing the target DNA in a solution phase;
b) a non-specific capture probe, wherein said non-specific capture probe comprises
(i) a randomized poly-(k) sequence that is at least 12 residues in length and comprising G and T nucleotides or G and U nucleotides, and
(ii) a first specific binding partner (SBP), wherein said SBP is capable of specifically binding a second specific binding partner (SBP′); and
c) the SBP′ immobilized on a support.
50. The reaction mixture of claim 49 , wherein the poly-(k) sequence comprises G and U nucleotides.
51. The reaction mixture of claim 49 , wherein the poly-(k) sequence comprises 2′-O-methyl substituted RNA bases.
52. The reaction mixture of claim 49 , wherein the poly-(k) sequence is a poly-(k)18, poly-(k)24, or poly-(k)25 sequence.
53. The reaction mixture of claim 49 , wherein the SBP and SBP′ are non-nucleic acid moieties.