Amplification of DNA to produce single-stranded product of defined sequence and length

• US 20030207279A1
• Filed: 05/01/2002
• Published: 11/06/2003
• Est. Priority Date: 04/25/2002
• Status: Active Grant

• Current Assignee: Geneohm Sciences Incorporated (Becton Dickinson & Co)
• Original Assignee: Geneohm Sciences Incorporated (Becton Dickinson & Co)
• Inventors: Crothers, Donald M., Koenigsberger, Carol
• Granted Patent: US 6,815,167 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2521/301 Endonuclease
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/204 specific length of the oligonucleotides
• CPC Class Codes: C12Q 2531/113 PCR
• CPC Class Codes: C12Q 2565/519 characterised by the capture moiety being a single stranded oligonucleotide

Abstract

The present disclosure relates to methods for generating single-stranded DNA molecules of defined sequence and length. Specifically, a region of template containing target sequence is amplified by PCR or RCA, exogenous sequence is introduced by primers or probes used in amplification, double-stranded amplification products are converted to single-stranded amplification products, and single-stranded amplification products are trimmed to produce short single-stranded DNA molecules of defined sequence and length.

Claims

1. A method for generating a single-stranded DNA molecule of defined sequence and length comprising the following steps:

amplification of a template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of double-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

conversion of each said double-stranded amplification product to a single-stranded amplification product; and

trimming each said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length.

2. The method of claim 1, wherein polymerase chain reaction (PCR) is used for said amplification step.

3. The method of claim 1, wherein rolling circle amplification (RCA) is used for said amplification step.

4. The method of claim 1, wherein said amplification step is carried out in linear mode.

5. The method of claim 1, wherein said amplification step is carried out in non-linear mode.

6. The method of claim 1, wherein said template is genomic DNA or cDNA.

7. The method of claim 1, wherein said template is RNA.

8. The method of claim 1, wherein said at least one primer comprises at least one sequence having an addressable ligand attached thereto.

9. The method of claim 8, wherein said addressable ligand is biotin.

10. The method of claim 1, wherein said at least one exogenous nucleotide sequence introduced by said at least one primer comprises self-complementary sequences that form hairpin structures.

11. The method of claim 10, wherein said self-complementary sequences that form hairpin structures comprise at least one restriction enzyme recognition site for a restriction enzyme involved in said trimming step.

12. The method of claim 11, wherein said restriction enzyme involved in said trimming step is a Type II or Type IIS restriction enzyme.

13. The method of claim 12, wherein said Type II restriction enzyme is EcoRI.

14. The method of claim 12, wherein said Type IIS restriction enzyme is FokI.

15. The method of claim 1, wherein said at least one exogenous nucleotide sequence introduced by said at least one primer comprises sequences that form at least one restriction enzyme recognition site for a restriction enzyme involved in said trimming step upon addition of at least one auxiliary oligonucleotide.

16. The method of claim 15, wherein said restriction enzyme involved in said trimming step is a Type II or Type IIS restriction enzyme.

17. The method of claim 16, wherein said Type II restriction enzyme is EcoRI.

18. The method of claim 16, wherein said Type IIS restriction enzyme is FokI.

19. The method of claim 15, wherein said at least one auxiliary oligonucleotide comprises at least one sequence having an addressable ligand attached thereto.

20. The method of claim 19, wherein said addressable ligand is biotin.

21. The method of claim 1, wherein said conversion step comprises digestion of one strand of said double-stranded amplification product comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer, using 5′

→

3′

exonuclease.

22. The method of claim 21, wherein said 5′

→

3′

exonuclease is T7 or lambda exonuclease.

23. The method of claim 21, wherein said at least one exogenous nucleotide sequence introduced by said at least one primer comprises modified nucleotides that confer resistance to digestion using 5′

→

3′

exonuclease or sensitivity to digestion using 5′

→

′

3′

exonuclease.

24. The method of claim 23, wherein said modified nucleotides confer resistance to digestion using 5′

→

3′

exonuclease.

25. The method of claim 24, wherein said modified nucleotides are phosphorothioate derivates.

26. The method of claim 23, wherein said modified nucleotides confer sensitivity to digestion using 5′

→

3′

exonuclease.

27. The method of claim 26, wherein said modified nucleotides are phosphorylated.

28. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is between 10 and 100 nucleotides in length.

29. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is between 10 and 50 nucleotides in length.

30. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is 15 nucleotides in length.

31. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is 17 nucleotides in length.

32. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is 21 nucleotides in length.

33. The method of claim 1, wherein said single-stranded DNA molecule of defined sequence and length is 30 nucleotides in length.

34. A method for generating a single-stranded DNA molecule of defined sequence and length comprising the following steps:

amplification of a template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of double-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

nicking each said double stranded amplification product at one end of a defined sequence and cleaving said double stranded amplification product at the other end of said defined sequence to generate a DNA molecule of defined sequence and length; and

separating a single stranded DNA molecule of defined sequence and length from the remainder of the amplification product comprising its complement and the primer duplexes of the amplification product.

35. The method of claim 34, wherein said single stranded DNA molecule of defined sequence and length is separated from the remainder of the amplification product by heating under conditions allowing said single stranded DNA molecule of defined sequence and length to separate from its complement while leaving the the primer duplexes of the amplification product intact.

36. The method of claim 35 wherein said at least one primer has an addressable ligand attached thereto.

37. The method of claim 36, wherein said addressable ligand is biotin.

38. The method of claim 37, wherein said remainder of the amplification product comprising its complement and the primer duplexes of the amplification product are removed by attachment to magnetic beads carrying streptavidin that binds to said biotin attached to the 5′

end of at least one primer.

39. A method for generating a single-stranded DNA molecule of defined sequence and length comprising:

amplification of a template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer containing at least one exogenous nucleotide sequence not present in the template, wherein said amplification generates a plurality of single-stranded amplification products comprising at least one target nucleotide sequence and at least one exogenous sequence introduced by said at least one primer; and

trimming each said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length.

40. The method of claim 39 wherein said amplification is rolling circle amplification in the linear mode.

41. The method of claim 39 wherein said template is genomic DNA or cDNA.

42. The method of claim 39, wherein said template is RNA.

43. The method of claim 39, wherein said at least one primer comprises at least one sequence having an addressable ligand attached thereto.

44. The method of claim 43, wherein said addressable ligand is biotin.

45. The method of claim 39, wherein said at least one exogenous nucleotide sequence introduced by said at least one primer comprises self-complementary sequences that form hairpin structures.

46. The method of claim 45, wherein said self-complementary sequences that form hairpin structures comprise at least one restriction enzyme recognition site for a restriction enzyme involved in said trimming step.

47. The method of claim 46, wherein said restriction enzyme involved in said trimming step is a Type II or Type IIS restriction enzyme.

48. The method of claim 47, wherein said Type II restriction enzyme is EcoRI.

49. The method of claim 47, wherein said Type IIS restriction enzyme is FokI.

50. The method of claim 46, wherein said at least one exogenous nucleotide sequence introduced by said at least one primer comprises sequences that form at least one restriction enzyme recognition site for a restriction enzyme involved in said trimming step upon addition of at least one auxiliary oligonucleotide.

51. The method of claim 50, wherein said restriction enzyme involved in said trimming step is a Type II or Type IIS restriction enzyme.

52. The method of claim 51, wherein said Type II restriction enzyme is EcoRI.

53. The method of claim 51, wherein said Type IIS restriction enzyme is FokI.

54. The method of claim 51, wherein said at least one auxiliary oligonucleotide comprises at least one sequence having an addressable ligand attached thereto.

55. The method of claim 54, wherein said addressable ligand is biotin.

56. A method for generating a single-stranded DNA molecule of defined sequence and length comprising:

amplification of a template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer containing at least one exogenous nucleotide sequence not present in the template, wherein said amplification generates a plurality of amplification products comprising at least one target nucleotide sequence and at least one exogenous sequence introduced by said at least one primer, wherein said amplification products comprise single-stranded or double-stranded amplification products and further wherein any said double-stranded amplification products may be converted to single-stranded amplification products; and

trimming said single-stranded amplification products to generate at least one single-stranded DNA molecule of defined sequence and length.

57. A method for identifying at least one organism or individual, comprising:

obtaining template comprising at least one target nucleotide sequence from said organism or individual, wherein said template comprises genomic DNA, cDNA, or RNA;

amplifying said template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of double-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

conversion of said double-stranded amplification products to single-stranded amplification products;

trimming said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length;

determining the mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length; and

using at least one said mass or nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

58. The method of claim 57, wherein mass spectroscopy is used to determine said mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length.

59. The method of claim 57, comprising determining said mass of each said single-stranded DNA molecule of defined sequence and length and using at least one said mass of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

60. The method of claim 57, comprising determining said nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length and using at least one said nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

61. The method of claim 57, wherein a multiplicity of organisms or individuals is identified.

62. A method for identifying at least one organism or individual, comprising:

obtaining template comprising at least one target nucleotide sequence from said at least one organism or individual, wherein said template comprises genomic DNA, cDNA, or RNA;

amplifying said template comprising at least one target nucleotide sequence, said amplification being directed by at least one primer comprising at least one exogenous nucleotide sequence not present in the target nucleotide sequence, wherein said amplification generates a plurality of single-stranded amplification products comprising said at least one target nucleotide sequence and said at least one exogenous nucleotide sequence introduced by said at least one primer;

trimming each said single-stranded amplification product to generate a single-stranded DNA molecule of defined sequence and length;

determining the mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length; and

using at least one said mass or nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

63. The method of claim 62, wherein mass spectroscopy is used to determine said mass or nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length.

64. The method of claim 62, comprising determining said mass of each said single-stranded DNA molecule of defined sequence and length and using at least one said mass of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

65. The method of claim 62, comprising determining said nucleotide sequence of each said single-stranded DNA molecule of defined sequence and length and using at least one said nucleotide sequence of at least one single-stranded DNA molecule of defined sequence and length to identify at least one organism or individual.

66. The method of claim 62, wherein a multiplicity of organisms or individuals is identified.