阅读说明：本技术 用于分离靶核酸的组合物和方法 (Compositions and methods for isolating target nucleic acids ) 是由 A·沙阿 于 2018-05-10 设计创作，主要内容包括：提供了能够用于核酸分离和纯化的靶捕获探针的群体。所述群体的探针包括：第一区域和第二区域,所述第一区域的长度为至少约12个残基并且包括聚(r)序列,所述聚(r)序列包括(i)包括G和A核苷酸的随机序列或(ii)非随机重复(A和G)序列；所述第二区域包括第一特异性结合配偶体(SBP),其中所述SBP能够特异性结合第二特异性结合配偶体(SBP2)。还提供了相关的组合、方法、用途、试剂盒和反应混合物。(Populations of target capture probes are provided that can be used for nucleic acid isolation and purification. The probes of the population include: a first region and a second region, the first region being at least about 12 residues in length and comprising a poly (r) sequence comprising (i) a random sequence comprising G and a nucleotides or (ii) a non-random repeating (a and G) sequence; the second region comprises a first Specific Binding Partner (SBP), wherein the SBP is capable of specifically binding to a second specific binding partner (SBP 2). Related combinations, methods, uses, kits and reaction mixtures are also provided.)

1. A population of capture probes for isolating a target nucleic acid from a sample, comprising: a first region and a second region, the first region being at least about 12 residues in length and comprising at least one poly (r) sequence comprising (i) a random sequence comprising G and a nucleotides or (ii) a non-random repeating (a and G) sequence; the second region comprises a first Specific Binding Partner (SBP), wherein the SBP is capable of specifically binding to a second specific binding partner (SBP 2).

2. The population of capture probes of claim 1, wherein the poly (r) sequence comprises a random sequence comprising G and A nucleotides.

3. The population of capture probes of claim 2, wherein the first region comprises at least about 2, 3,4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 random poly (r) sequence nucleotides.

4. The population of capture probes of any one of the preceding claims, wherein the poly (r) sequence comprises at least about 4, 5,6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 non-randomly repeating (a and G) sequence nucleotides.

5. The population of capture probes of any one of the preceding claims, wherein the first region is at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

6. The population of capture probes of any one of the preceding claims, wherein the first region consists of random G and a nucleotides, non-random repeating (a and G) sequences, or a combination thereof.

7. The population of capture probes of any one of claims 1-5, wherein the first region further comprises a linker sequence between the poly (r) sequence and a second poly (r) sequence, and the second poly (r) sequence comprises (i) a random sequence comprising G and A nucleotides or (ii) a non-random repeating (A and G) sequence.

8. The population of capture probes of claim 7, wherein the poly (r) sequence is at least about 6 residues in length and the second poly (r) sequence is at least about 6 residues in length.

9. The population of capture probes of any of the preceding claims, wherein the first region comprises 2' -O-methyl modified RNA residues.

10. The population of capture probes of any of the preceding claims, wherein the first region comprises poly (r)₁₈Poly (r)₂₄Or poly (r)₂₅And (4) sequencing.

11. The population of capture probes of any one of the preceding claims, wherein the SBPs are non-nucleic acid moieties.

12. The population of capture probes of any one of claims 1-10, wherein the SBPs comprise homopolymeric sequences.

13. The population of capture probes of claim 12, whereinThe SBP comprises dT₃dA₃₀(SEQ ID NO:10) or dA₃₀(SEQ ID NO: 11).

14. The population of capture probes of any one of the preceding claims, wherein the SBPs are located 3' to the first region.

15. A combination comprising the population of capture probes of any of the preceding claims and a second population of capture probes comprising a first region and a second region, the first region being at least about 12 residues in length and comprising a poly (k) sequence comprising (i) a random sequence comprising G and U/T nucleotides or (ii) a non-randomly repeating (G and U/T) sequence; the second region comprises a third specific binding partner (SBP3), wherein the SBP3 is capable of specifically binding a fourth specific binding partner (SBP 4).

16. The combination of claim 15, wherein the SBP and the SBP3 are capable of binding the same SBP2/SBP 4.

17. The combination of claim 16, wherein the SBP and the SBP3 are the same as each other.

18. The combination of any one of claims 15-17, wherein the first region of the second population is at least 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

19. The combination of any one of claims 15-18, wherein the first region of the second population comprises poly (k)₁₈Poly (k)₂₄Or poly (k)₂₅And (4) sequencing.

20. The combination of any one of claims 15-19, wherein the first region of the second population consists of random G and U/T nucleotides or non-random repeats (G and U/T).

21. A kit or reaction mixture for isolating a target nucleic acid from a sample, the reaction mixture comprising:

a. the population of capture probes of any one of claims 1 to 14 or the combination of any one of claims 15 to 20; and

b. SBP2 immobilized on a carrier.

22. The kit or reaction mixture of claim 21, wherein the SBP and SBP2 are substantially complementary nucleic acid sequences.

23. The kit or reaction mixture of claim 21, wherein the SBP and SBP2 are non-nucleic acid moieties.

24. The kit or reaction mixture of any one of claims 21-23, further comprising a detergent.

25. A kit or reaction mixture according to any one of claims 21 to 24, further comprising lithium dodecyl sulphate or sodium dodecyl sulphate and/or lithium hydroxide.

26. The kit or reaction mixture of any one of claims 21 to 25, comprising a combination of capture probes according to any one of claims 15 to 20.

27. The kit or reaction mixture of claim 26, wherein the SBP and the SBP3 are capable of binding to the SBP 2.

28. The kit or reaction mixture of claim 26, further comprising SBP4 immobilized on a support.

29. The kit or reaction mixture of any one of claims 21-28, further comprising a solution phase.

30. The reaction mixture of claim 29, wherein the reaction mixture comprises a target nucleic acid in the solution phase and/or associated with the capture probe.

31. The reaction mixture of claim 30, wherein the target nucleic acid is derived from a cell treated to release intracellular components into the solution phase.

32. The reaction mixture of any one of claims 29-31, wherein the solution phase comprises a sample from an animal, environmental, food, or industrial source.

33. The reaction mixture of any one of claims 29-32, wherein the solution phase comprises a sample comprising peripheral blood, serum, plasma, cerebrospinal fluid, sputum, or swab specimen.

34. A method for isolating a target nucleic acid from a sample, the method comprising:

a. contacting the population of capture probes of any one of claims 1-14 or the combination of any one of claims 15-20 with a solution containing nucleic acids to form a reaction mixture, wherein the reaction mixture further comprises a support comprising the SBP 2;

b. incubating the reaction mixture under conditions that allow hybridization of the first region to the target nucleic acid and association of the SBP with the SBP2 immobilized on the support, thereby forming a hybridization complex in contact with a solution; and

c. separating the support from the solution phase, thereby separating the target nucleic acid from other components in the sample.

35. A method for isolating a target nucleic acid from a sample, the method comprising:

a. incubating the reaction mixture of any one of claims 21 to 33 with the sample under conditions that allow hybridization of the first region to the target nucleic acid and association of the SBP with SBP2 immobilized on a support, thereby forming a hybridization complex in contact with the solution; and

b. separating the support from the solution phase, thereby separating the target nucleic acid from other components in the sample.

36. The method of claim 34 or 35, wherein the sample contains cells and is treated to release intracellular components into the solution prior to the contacting step.

37. The method of claim 36, wherein the treating comprises treating the sample with a solution comprising a detergent.

38. The method of claims 34-37, wherein the sample is from an animal, environmental, food, or industrial source.

39. The method of any one of claims 34 to 38, wherein the sample comprises peripheral blood, serum, plasma, cerebrospinal fluid, sputum, or a swab specimen.

40. The method of any one of claims 34-39, wherein the sample comprises a cell lysate.

41. The method of any one of claims 34-40, wherein said SBP and said SBP2 are non-nucleic acid moieties.

42. The method of any one of claims 34-40, wherein said SBP and SBP2 are substantially complementary nucleic acid sequences.

43. The method of any one of claims 34-42, wherein the combination of any one of claims 15-20 is contacted with the solution containing nucleic acids.

44. The method of claim 43, wherein the SBP and the SBP3 are capable of binding to the SBP 2.

45. The method of claim 43, wherein the reaction mixture further comprises a support comprising SBP 4.

46. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises DNA.

47. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises RNA.

48. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises a viral nucleic acid.

49. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises a prokaryotic nucleic acid.

50. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises a eukaryotic nucleic acid.

51. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises a synthetic nucleic acid.

52. The population, combination, reaction mixture or method of any one of the preceding claims, wherein the target nucleic acid comprises a combination of DNA, RNA, viral nucleic acid, bacterial nucleic acid, eukaryotic nucleic acid and/or synthetic nucleic acid.

Background and summary of the invention

Many molecular biological procedures, such as in vitro amplification and in vitro hybridization of nucleic acids, involve the preparation of some nucleic acids to facilitate subsequent procedures. Methods of nucleic acid purification can separate all nucleic acids present in a sample, separate different types of nucleic acids based on physical characteristics, or separate specific nucleic acids from a sample. Many methods involve complicated procedures, use harsh chemicals or conditions, or take a long time to complete the nucleic acid isolation. Some methods involve the use of specialized oligonucleotides, each specific for the intended target nucleic acid, which adds complexity to the design, optimization, and performance of the method, particularly if more than one target nucleic acid needs to be isolated or the sequence of the desired target nucleic acid is unknown. Some methods do not require specific target sequences to isolate the target nucleic acid, but do not efficiently isolate all sequences. Thus, there remains a need for a simple, efficient and rapid method for separating nucleic acids of interest from other sample components.

Accordingly, the following examples are among the examples provided by this disclosure.

Drawings

FIG. 1 shows that (r) is to be used₁₈/(k)₁₈Extraction and use of Capture Probe mixtures Only (k)₁₈Extraction of capture probes Δ CT of 49 clinical specimens to be compared was performed as described in example 2. Each histogram represents results from a single specimen.

Example 1 is a population of capture probes for isolating a target nucleic acid from a sample, comprising: a first region and a second region, the first region being at least about 12 residues in length and comprising at least one poly (r) sequence comprising (i) a random sequence comprising G and a nucleotides or (ii) a non-random repeating (a and G) sequence; the second region comprises a first Specific Binding Partner (SBP), wherein the SBP is capable of specifically binding to a second specific binding partner (SBP 2).