Nucleic acid amplification using nicking agents

• US 20030104431A1
• Filed: 07/15/2002
• Published: 06/05/2003
• Est. Priority Date: 07/15/2001
• Status: Active Grant

• Current Assignee: Keck Graduate Institute
• Original Assignee: Keck Graduate Institute
• Inventors: Van Ness, Jeffrey, Galas, David J., Van Ness, Lori K.
• Granted Patent: US 7,112,423 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C12Q 1/6844 Nucleic acid amplification reactions
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2521/307 Single strand endonuclease
• CPC Class Codes: C12Q 2521/313 Type II endonucleases, i.e. cutting outside recognition site
• CPC Class Codes: C12Q 2521/513 Winding/unwinding enzyme, e.g. helicase
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm
• CPC Class Codes: C12Q 2531/119 Strand displacement amplification [SDA]
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof

Abstract

The present invention provides methods and kits for amplifying target nucleic acids (including whole genomes) using nicking agents. In certain aspects, the amplification does not require the use of any external oligonucleotide primers that are capable of annealing to a portion of the target nucleic acid. This invention is useful in many areas such as genetic disease diagnoses, forensic analyses and palcoarcheological studies.

Claims

1. A method for nucleic acid amplification comprising forming a mixture of

(a) a double-stranded target nucleic acid of which at least one strand has two or more nicking sites;

(b) a nicking agent capable of nicking at the two or more nicking sites; and

(c) a DNA polymerase;

under a condition allowing for amplification of at least a portion of the target nucleic acid, wherein the amplification does not require the presence of an external oligonucleotide primer (ODNP) that are capable of specific annealing to a portion of the target nucleic acid.

3. The method of claim 1 or claim 2 wherein the target nucleic acid is derived, or prepared, from a single-stranded nucleic acid.

4. The method of claim 1 or claim 2 wherein the target nucleic acid is isolated from a single cell.

5. The method of claim 4 wherein the single cell is a sperm cell.

6. The method of claim 4 wherein the single cell is an oocyte.

7. The method of claim 1 or claim 2 wherein the target nucleic acid is isolated from a tumor tissue.

8. The method of claim 1 or claim 2 wherein the target nucleic acid is isolated from bone marrow.

9. The method of claim 1 or claim 2 wherein the target nucleic acid is isolated from blood.

10. The method of claim 1 or claim 2 wherein the target nucleic acid is immobilized.

11. The method of claim 1 or claim 2 wherein the target nucleic acid is isolated from a subject with, or suspected to have, a genetic disease.

12. The method of claim 1 or claim 2 wherein the target nucleic acid comprises a whole genome of an organism.

13. The method of claim 12 wherein the organism is prokaryotic.

14. The method of claim 12 wherein the organism is eukaryotic.

15. The method of claim 14 wherein the organism is a mammal.

16. The method of claim 14 wherein the organism is a human.

17. The method of claim 1 or claim 2 wherein the target nucleic acid is a genomic DNA.

18. The method of claim 1 or claim 2 wherein the nicking agent is a nicking endonuclease.

19. The method of claim 18 wherein the nicking endonuclease is any one selected from the group consisting of N.BstNB I, N.Alv I, N.BbvC I-a and N.BbvC I-b.

20. The method of claim 1 or claim 2 wherein the DNA polymerase is 5′

→

3′

exonuclease deficient.

21. The method of claim 20 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is selected from the group consisting of exo⁻

Vent, exo⁻

Deep Vent, exo⁻

Bst, exo⁻

Pfu, exo⁻

Bca, the Klenow fragment of DNA polymerase I, T5 DNA polymerase, Phi29 DNA polymerase, phage M2 DNA polymerase, phage PhiPRD1 DNA polymerase, Sequenase, PRD1 DNA polymerase, 9°

Nm™

DNA polymerase, and T4 DNA polymerase homoenzyme.

22. The method of claim 21 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is exo⁻

Bst polymerase, exo⁻

Bca polymerase, exo⁻

Vent polymerase, 9°

Nm™

DNA polymerase, or exo⁻

Deep Vent polymerase.

23. The method of claim 1 or claim 2 wherein the DNA polymerase has a strand displacement activity.

24. The method of claim 1 wherein the amplification is performed under an isothermal condition.

25. The method of claim 1 wherein the amplification is performed at 50°

C.-70°

C.

26. The method of claim 25 wherein the amplification is performed at 55°

C.

27. The method of claim 1 wherein the amplification is performed in the presence of a strand displacement facilitator.

28. The method of claim 27 wherein the strand displacement facilitator is selected from the group consisting of BMRF1 polymerase accessory subunit, adenovirus DNA-binding protein, herpes simplex viral protein ICP8, single-stranded DNA binding proteins, phage T4 gene 32 protein, calf thymus helicase, and trehalose.

29. The method of claim 28 wherein the strand displacement facilitator is trehalose.

30. The method of claim 28 wherein the strand displacement facilitator is phage T4 gene 32 protein.

31. The method of claim 1 wherein the amplification is performed in the absence of any external ODNP.

2. A method for nucleic acid amplification comprising

(a) multiply nicking at least one strand of a double-stranded target nucleic acid with a nicking agent to provide at least two new 3′

termini in that strand;

(b) extending one or more of the at least two new 3′

termini with a DNA polymerase;

(c) nicking the extension product of step (b); and

(d) extending the nicking product of step (c) to thereby amplify at least a portion of one strand of the target nucleic acid.

32. The method of claim 2 wherein steps (a)-(d) are performed under an isothermal condition.

33. The method of claim 2 wherein steps (a)-(d) are performed at 50°

C.-70°

C.

34. The method of claim 33 wherein steps (a)-(d) are performed at 55°

C.

35. The method of claim 2 wherein steps (b) and (d) are performed in the presence of a strand displacement facilitator.

36. The method of claim 35 wherein the strand displacement facilitator is selected from the group consisting of BMRF1 polymerase accessory subunit, adenovirus DNA-binding protein, herpes simplex viral protein ICP8, single-stranded DNA binding proteins, phage T4 gene 32 protein, calf thymus helicase, and trehalose.

37. The method of claim 36 wherein the strand displacement facilitator is trehalose.

38. The method of claim 36 wherein the strand displacement facilitator is phage T4 gene 32 protein.

39. A kit for nucleic acid amplification that does not require the use of an external oligonucleotide primer, comprising a nicking agent, a 5′

→

3′

exonuclease deficient DNA polymerase, and instructions for using the kit.

40. The kit of claim 39 wherein the nicking agent is a nicking endonuclease.

41. The kit of claim 40 wherein the nicking endonuclease is N.BstNB I.

42. The kit of claim 40 further comprising a buffer for the nicking endonuclease.

43. The kit of claim 39 further comprising a buffer for the 5′

→

3′

exonuclease deficient DNA polymerase.

44. The kit of claim 39 further comprising a buffer suitable for both the nicking agent and the 5′

→

3′

exonuclease deficient DNA polymerase.

45. The kit of claim 39 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is selected from the group consisting of exo⁻

Vent, exo⁻

Deep Vent, exo⁻

Bst, exo⁻

Pfu, exo⁻

Bca, the Klenow fragment of DNA polymerase I, T5 DNA polymerase, Phi29 DNA polymerase, phage M2 DNA polymerase, phage PhiPRD1 DNA polymerase, Sequenase, PRD1 DNA polymerase, 9°

Nm™

DNA polymerase, and T4 DNA polymerase homoenzyme.

46. The kit of claim 39 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is exo⁻

Bst polymerase, exo⁻

Bca polymerase, exo⁻

Vent polymerase, 9°

Nm™

DNA polymerase, or exo⁻

Deep Vent polymerase.

47. The kit of claim 39 wherein the 5′

→

3′

exonuclease deficient DNA polymerase has a strand displacement activity.

48. The kit of claim 39 further comprising a strand displacement facilitator.

49. The kit of claim 48 wherein the strand displacement facilitator is selected from the group consisting of BMRF1 polymerase accessory subunit, adenovirus DNA-binding protein, herpes simplex viral protein ICP8, single-stranded DNA binding proteins, phage T4 gene 32 protein, calf thymus helicase, and trehalose.

50. The kit of claim 48 wherein the strand displacement facilitator is trehalose or phage T4 gene 32 protein.

51. The kit of claim 39 wherein the instructions for using the kit comprise instructions for whole genome amplification.

52. A method for whole genome amplification comprising

(a) multiply nicking at least one strand of a whole genome with a nicking agent;

and

(b) amplifying at least a portion of the whole genome using a nicking product of step (a) as a template.

53. The method of claim 52 wherein step (b) is performed by a polymerase chain reaction (PCR).

54. The method of claim 53 wherein the PCR is carried out in the presence of a random or a partially random primer.

55. The method of claim 52 wherein the whole genome is isolated from a single cell.

56. The method of claim 55 wherein the single cell is a sperm cell.

57. The method of claim 55 wherein the single cell is an oocyte.

58. The method of claim 55 wherein the whole genome is isolated from a tumor tissue.

59. The method of claim 55 wherein the whole genome is isolated from a subject with, or suspected to have, a genetic disease.

60. The method of claim 59 wherein the whole genome is a human genome.

61. The method of claim 59 wherein the whole genome is a mammalian genome.

62. The method of claim 52 wherein the nicking agent is a nicking endonuclease.

63. The method of claim 62 wherein the nicking endonuclease is any one selected from the group consisting of N.BstNB I, Alw I, N.BbvC I-a and N.BbvC I-b.

64. The method of claim 52 wherein step (b) is performed in the presence of a 5′

→

3′

exonuclease deficient DNA polymerase.

65. The method of claim 64 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is selected from the group consisting of exo⁻

Vent, exo⁻

Deep Vent, exo⁻

Bst, exo⁻

Pfu, exo⁻

Bca, the Klenow fragment of DNA polymerase I, T5 DNA polymerase, Phi29 DNA polymerase, phage M2 DNA polymerase, phage PhiPRD1 DNA polymerase, Sequenase, PRD1 DNA polymerase, 9°

Nm™

DNA polymerase, and T4 DNA polymerase homoenzyme.

66. The method of claim 65 wherein the 5′

→

3′

exonuclease deficient DNA polymerase is exo⁻

Bst polymerase, exo⁻

Bea polymerase, exo⁻

Vent polymerase, 9°

Nm™

DNA polymerase, or exo⁻

Deep Vent polymerase.

67. The method of claim 52 wherein step (b) is performed in the presence of a DNA polymerase with a strand displacement activity.

68. The method of claim 52 wherein step (b) is performed in the presence of a DNA polymerase without a strand displacement activity.

69. The method of claim 52 wherein steps (a) and (b) are performed under an isothermal condition.

70. The method of claim 52 wherein step (b) is performed in the presence of a strand displacement facilitator.

71. The method of claim 70 wherein the strand displacement facilitator is trehalose.

72. The method of claim 70 wherein step (b) is performed in the absence of any external ODNP.