Methods for targeting, enriching, detecting and/or isolating target nucleic acid sequence using RecA-like recombinase

• US 6,335,164 B1
• Filed: 05/18/1999
• Issued: 01/01/2002
• Est. Priority Date: 08/29/1996
• Status: Expired due to Fees

• Current Assignee: TAPESTRY PHARACEUTICALS, INC.
• Original Assignee: Daikin Industries Ltd
• Inventors: Kigawa, Koji, Yamanaka, Mikayo, Mukai, Eli, Obata, Kazuaki, Kusumi, Kayo
• Primary Examiner(s): Carlson, Karen Cochrane
• Assistant Examiner(s): Robinson, Hope A.
• Application Number:
  US09/147,751
• Time in Patent Office: 959 Days
• Field of Search: 435/6, 435/172.3, 435/240.2, 435/4, 435/172.1, 435/320.1, 435/69.1, 530/350, 514/44
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/6841 In situ hybridisation
• CPC Class Codes: C12Q 2521/507 Recombinase

Abstract

A method for targeting, enriching, detecting and isolating a double-stranded nucleic acid target sequence by adding heterologous probes to the reaction system along with the reduced amount of homologous probes in the process of homologous pairing and/or strand exchange between the homologous probe and the target sequence in the presence of a RecA-like recombinase.

Claims

1. A method for improving specificity of homologous pairing or strand exchange or a combination of homologous pairing and strand exchange between a double-stranded target nucleic acid sequence and a homologous nucleic acid probe by using a RecA-like recombinase, comprising the steps of:

(a) providing at least one recombinase, at least one homologous probe, and at least one heterologous probe, wherein the probes are bound to the recombinase, and

(b) mixing the recombinase-bound homologous probe and the recombinase-bound heterologous probe with the double-stranded nucleic acid target sequence in a sample,

wherein the weight ratio of the homologous probe to the heterologous probe is about 1;

5 to about 1;

500, wherein the combination of recombinase-bound heterologous probe and recombinase-bound homologous probe improves the specificity of the homologous pairing or strand exchange or combination of homologous pairing and strand exchange relative to a method wherein recombinase-bound homologous probe is used alone.

2. The method of claim 1, wherein the double-stranded nucleic acid target sequence is double-stranded target DNA.

3. The method of claim 1, wherein the step (a) provides at least two types of homologous probes.

4. The method of claim 3, wherein the homologous probes are two types of homologous probes which are sufficiently complementary to each other.

5. The method of claim 1, wherein the homologous probe is at least 15 nucleic acid residues in length.

6. The method of claim 5, wherein the homologous probe is about 15 to 2000 nucleic acid residues in length.

7. The method of claim 1, wherein the homologous probe comprises a label or a ligand.

8. The method of claim 1, wherein the homologous probe is a double-stranded DNA probe.

9. The method of claim 1, wherein the homologous probe is a single-stranded DNA probe.

10. The method of claim 1, wherein the heterologous probe is about 15 bases or longer.

11. The method of claim 1, wherein the heterologous probe is a single-stranded DNA probe.

12. The method of claim 1, wherein the heterologous probe is a RNA probe.

13. The method of claim 1, wherein the weight ratio of the homologous probe to the heterologous probe is about 1:

5 to about 1;

250.

14. The method of claim 1, wherein the RecA-like recombinase is derived from a prokaryote.

15. The method of claim 13, wherein the RecA-like recombinase is derived from Escherichia coli.

16. The method of claim 14, wherein the RecA-like recombinase is RecA.

36. The methods of claims 1, 17 or 31, wherein the homologous and heterologous probes are bound to recombinase in the presence of a co-factor.

37. The method of claim 36, wherein the RecA-like recombinase is derived from Escherichia coli.

38. The method of claim 36, wherein the cofactor is selected from a group consisting of GTP S, a mixture of ATP S and ADP, a mixture of ADP and AlF₄⁻

(aluminum nitrate and sodium fluoride), a mixture of ATP and AlF₄⁻

(aluminum nitrate and sodium fluoride), a mixture of dADP and AlF₄⁻

(aluminum nitrate and sodium fluoride), or a mixture of dATP and AlF₄⁻

(aluminum nitrate and sodium fluoride).

39. The method of claim 38, wherein the cofactor is GTP S.

42. A kit for improving specificity of homologous pairing or strand exchange or a combination of homologous pairing and strand exchange between a double-stranded target nucleic acid sequence and a homologous nucleic acid probe according to the method of claim 1, comprising a RecA-like recombinase, a cofactor, at least one heterologous probe, a solid phase designed to trap a complex of a double-stranded nucleic acid target sequence and at least one homologous probe having a label or ligand, and washing buffer, wherein the combination of recombinase-bound heterologous probe and recombinase-bound homologous probe improves the specificity of the homologous pairing or strand exchange or combination of homologous pairing and strand exchange relative to a method wherein recombinase-bound homologous probe is used alone.

43. The kit of claim 42, wherein the weight ratio of the homologous probe to the heterologous probe is about 1:

1 to about 1;

500.

44. The method of claim 1 further comprising the step of enriching or isolating the target sequence.

45. The method of claim 1 further comprising the step of detecting the target sequence.

46. The kit of claim 42, wherein the double-stranded nucleic acid target sequence is double-stranded target DNA.

47. The kit of claim 42, wherein the homologous probes are at least two homologous probes.

48. The kit of claim 47, wherein the homologous probes are two homologous probes which are sufficiently complementary to each other.

49. The kit of claim 42, wherein the homologous probe is at least 15 nucleic acid residues in length.

50. The kit of claim 49, wherein the homologous probe is 15 to 2000 nucleic acid residues in length.

51. The kit of claim 42, wherein the homologous probe is a double-stranded DNA probe.

52. The kit of claim 42, wherein the homologous probe is a single-stranded DNA probe.

53. The kit of claim 42, wherein the heterologous probe is at least 15 bases.

54. The kit of claim 42, wherein the heterologous probe is a single-stranded DNA probe.

55. The kit of claim 42, wherein the heterologous probe is a RNA probe.

56. The kit of claim 42, wherein the RecA-like recombinase is derived from a prokaryote.

57. The kit of claim 56, wherein the RecA-like recombinase is derived from Escherichia coli.

58. The kit of claim 56, wherein the RecA-like recombinase is RecA.

17. A method for enriching or isolating or a combination of enriching and isolating a double-stranded nucleic acid target sequence in a sample by using a RecA-like recombinase, comprising the steps of:

(a) providing a homologous probe which has a label or a ligand and which is bound to at least one recombinase, and a heterologous probe bound to said at least one recombinase;

(b) reacting the recombinase-bound homologous probe and the recombinase-bound heterologous probe with the double-stranded nucleic acid target sequence under conditions which promote the formation of a complex of the double stranded nucleic acid target sequence and the homologous probe in the reaction mixture, wherein the weight ratio of the homologous probe to the heterologous probe is about 1;

5 to about 1;

500;

(c) contacting the reaction mixture with a solid support designed to selectively bind the complex of the double-stranded nucleic acid target sequence and the homologous probe;

(d) removing non-bound molecules from the solid support; and

(e) treating the solid support to release the double-stranded nucleic acid target sequence which is selectively bound to the solid support through the complex.

18. The method of claim 17, wherein the double-stranded nucleic acid target sequence is double-stranded target DNA.

19. The method of claim 18, wherein the sample is a cDNA or a genomic DNA library, and the double-stranded target DNA is inserted to a vector capable of transforming or transfecting a suitable bacterial host.

20. The method of claim 19 further comprising the steps of:

(a) providing suitable host cells which are capable of being transformed or transfected by the double-stranded target DNA inserted into a vector;

(b) transforming or transfecting the host cells with the target DNA fraction released from the solid phase; and

(c) selecting transformed or transfected cells containing said target DNA.

21. The method of claim 18, wherein the sample is a clinical specimen containing a mixture of cDNA or genomic DNA.

22. The method of claim 17, wherein the label or ligand is biotin or digoxigenin.

23. The method of claim 17, wherein the solid phase comprises magnetic or paramagnetic beads surfacely bound to avidin, streptavidin, or anti-digoxigenin antibody.

24. The method of claim 17, wherein at least two types of homologous probes are provided.

25. The method of claim 24, wherein two types of homologous probes which are sufficiently complementary to each other are provided.

26. The method of claim 17, wherein the RecA-like recombinase is derived from prokaryote.

27. The method of claim 26, wherein the RecA-like recombinase is derived from Escherichia coli.

28. The method of claim 17, wherein the heterologous probe is at least 15 nucleic acid residues in length.

29. The method of claim 17, wherein the homologous probe is at least 15 nucleic acid residues in length.

30. The method of claim 17, wherein the weight ratio of the homologous probe to the heterologous probe is about 1:

1 to about 1;

500.

31. A method for detecting a double-stranded nucleic acid target sequence in a sample of cells by in situ hybridization using a RecA-like recombinase, comprising the steps of:

(a) providing a homologous probe and a heterologous probe, wherein the probes are bound to the recombinase;

(b) adding the recombinase-bound homologous probe and the recombinase-bound heterologous probe to the sample of cells under a condition to promote the reaction of the homologous probe with the double-stranded nucleic acid target sequence; and

(c) removing the recombinase-bound homologous probes which have not reacted with the double-stranded nucleic acid target sequence;

wherein said double-stranded nucleic acid target sequence is detected in a sample of cells by in situ hybridization using a RecA-like recombinase.

32. The method of claim 31, wherein the weight ratio of the homologous probe to the heterologous probe is about 1:

5 to about 1;

250.

33. The method of claim 31, wherein at least two types of homologous probes are provided.

34. The method of claim 33, wherein two types of homologous probes which are sufficiency complementary to each other are employed.

35. The method of claim 31, wherein the heterologous probe is of 15 bases or longer.

40. A method for targeting a double-stranded nucleic acid target sequence in a sample of living cells by an in vivo gene targeting method using a RecA-like recombinase, comprising the steps of:

(a) providing a recombinase-bound homologous probe and a recombinase-bound heterologous probe;

(b) introducing the recombinase-bound homologous probe and the recombinase-bound heterologous probe into the living cells;

(c) incubating the living cells containing the recombinase-bound homologous probe and the recombinase-bound heterologous probe for a sufficient period of time to allow the recombinase-bound homologous probe to be transformed into the genome of the cells;

wherein said double-stranded nucleic acid target sequence is targeted in a sample of living cells by an in vivo gene targeting method using a RecA-like recombinase.

41. The method of claim 40, wherein the weight ratio of the homologous probe to the heterologous probe is about 1:

5 to about 1;

250.