Method of preparing normalized and/or subtracted cDNA

• US 7,049,065 B2
• Filed: 08/24/2001
• Issued: 05/23/2006
• Est. Priority Date: 08/25/2000
• Status: Expired due to Fees

• Current Assignee: Kabushiki Kaisha Dnaform, Riken
• Original Assignee: Riken, Yoshihide Hayashizaki
• Inventors: Hayashizaki, Yoshihide
• Primary Examiner(s): FREDMAN, JEFFREY NORMAN
• Application Number:
  US09/935,592
• Publication Number:
  US 20020106666A1
• Time in Patent Office: 1,733 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR

Abstract

A method of preparing normalized and/or subtracted cDNA; a method in which the cDNA that is normalized and/or subtracted is in the form of uncloned cDNA (cDNA tester); a method of preparing normalized and/or subtracted cDNA comprising the steps of: (a) preparing cDNA tester; (b) preparing normalization and/or subtraction RNA driver; (c) conducting normalization and/or subtraction in two steps in any order, or conducting normalization/subtraction as a single step and mixing the normalization/subtraction RNA driver with said cDNA tester; (d) adding an enzyme capable of cleaving single strand sites on RNA drivers nonspecifically bound to cDNA tester; (e) removing said single strand RNA driver cleaved in step d) from the tester and removing tester/driver hybrids; and (f) recovering the normalized and/or subtracted cDNA; and a method of efficiently preparing normalized and/or subtracted long-chain, full-coding, and full-length cDNA libraries are provided.

Claims

1. A method of preparing normalized and/or subtracted cDNAs comprising the steps of:

I) preparing uncloned full-length or full-coding length cDNA tester;

II) preparing polynucleotide drivers for normalization and/or subtraction;

III) conducting normalization and/or subtraction and removing tester/driver hybrids, non-specifically hybridized polynucleotide drivers and non-hybridized polynucleotide drivers; and

IV) recovering the normalized and/or subtracted full-length or full-coding length cDNA.

2. The method of claim 1, wherein the cDNA tester of step I) is a reverse transcript of mRNA in the form of uncloned cDNA.

3. The method of claim 1 wherein said cDNA tester is single-stranded.

4. The method of claim 1, wherein in step III), normalization is conducted first, followed by subtraction.

5. The method of claim 1, wherein in step III), subtraction is conducted first, followed by normalization.

6. The method of claim 1, wherein in step III), said tester and normalization and subtraction drivers are mixed together and normalization and subtraction are conducted as a single step.

7. The method of claim 1, wherein step III) comprises the addition of an enzyme capable of cleaving single-stranded RNA driver nonspecifically bound to single-stranded cDNA and the cleaved single-stranded RNA driver is removed.

8. The method of claim 7 wherein said enzyme is single-strand-specific RNA endonuclease.

9. The method of claim 7 wherein said enzyme is either selected from the group consisting of RNase I, RNaseA, RNase4, RNaseT1, RNaseT2, RNase2, and RNase3, or comprises a mixture thereof.

10. The method of claim 7 wherein said enzyme is RNase I.

11. The method of claim 1, wherein said cDNA tester is prepared by CAP-trapping the 5′

end of RNA.

12. The method of claim 1, wherein the preparation of said full-length or full-coding length cDNA tester comprises the following steps:

(1) synthesizing first strand cDNA by means of reverse transcriptase forming mRNA/cDNA hybrids;

(2) chemically binding a tag molecule to the diol structure of the 5′

CAP(^7MeG_pppN) site of mRNA forming hybrids;

(3) trapping full-length or full-coding length cDNA hybrids; and

(4) removing single-stranded mRNA by digestion with an enzyme capable of cleaving single-stranded mRNA.

13. The method of claim 12 wherein said tag molecule is digoxigenin, biotin, avidin, or streptavidin.

14. The method of claim 1, wherein said polynucleotide driver for normalization and/or subtraction is RNA and/or DNA.

15. The method of claim 14, wherein said DNA driver is cDNA.

16. The method of claim 1, wherein said normalization driver comprises cellular mRNA from the same library, from the same tissue, or the same cDNA population as the cDNA to be normalized.

17. The method of claim 1, wherein said normalization driver comprises single-stranded cDNA obtained from the same library, the same tissue, or the same cDNA population as the cDNA to be normalized.

18. The method of claim 1, wherein said subtraction driver comprises cellular mRNA from a library, tissue, or cDNA population differing from the cDNA to be subtracted.

19. The method of claim 1, wherein said subtraction driver comprises single-stranded cDNA from a library, tissue, or cDNA population differing from the cDNA to be normalized.

20. The method of claim 1, further comprising a step V) of preparing a complementary strand of the recovered cDNA and cloning the resulting double-stranded cDNA.

65. The method of claim 1, wherein said tester/driver hybrids are bound to tag molecules.

66. The method of claim 65, wherein said tag molecule is avidin, streptavidin, biotin, digoxigenin, an antibody, or an antigen.

67. The method of claim 1, wherein said tester/driver hybrids are removed through the use of a matrix.

68. The method of claim 67, wherein said matrix is comprised of magnetic beads or agarose beads.

69. The method of claim 68, wherein said magnetic beads or agarose beads are covered by or bound to a tag molecule that binds to a tag molecule bound to a tester/driver hybrid.

70. The method of claim 68, wherein said magnetic beads or agarose beads are covered by or bound to a tag molecule that binds to avidin, streptavidin, biotin, digoxigenin, an antibody, or an antigen bound to a tester/driver hybrid.

71. The method of claim 69, wherein said tag molecule covering said beads or bound to said beads is an antiantigen antibody, antibiotin antibody, antiavidin antibody, antistreptavidin antibody, or antidigoxigenin antibody.

72. The method of claim 1, wherein said tester/driver hybrid is removed using streptavidin/phenol.

73. The method of claim 1, wherein hydroxyapatite and nonlabeled RNA are employed to remove said tester/driver hybrid.

74. The method of claim 1 employed to prepare one, two, or more libraries.

75. The method of claim 1, in which subtraction is performed and normalization is performed to a R₀T value of from 5 to 10.

76. The method of claim 12, wherein the chemical tagging is performed on ice.

21. A method of preparing normalized and/or subtracted full-length or full-coding length cDNAs comprising the steps of:

I) preparing cDNA tester not cloned in a plasmid;

II) preparing polynucleotide drivers for normalization and/or subtraction;

III) conducting normalization and/or subtraction and removing tester/driver hybrids, non-specifically hybridized polynucleotide drivers and non-hybridized polynucleotide drivers; and

IV) recovering the normalized and/or subtracted full-length or full-coding length cDNA.

22. The method of claim 21, wherein in step III), normalization is conducted first, followed by subtraction.

23. The method of claim 21, wherein in step III), subtraction is conducted first, followed by normalization.

24. The method of claim 21, wherein in step III), said tester and normalization and subtraction drivers are mixed together and normalization and subtraction are conducted as a single step.

25. The method of claim 21, wherein step III) comprises the addition of an enzyme that cleaves single-stranded RNA driver nonspecifically bound to single-stranded cDNA and the cleaved single-stranded RNA driver is removed.

26. The method of claim 25, wherein said enzyme is single-strand-specific RNA endonuclease.

27. The method of claim 25, wherein said enzyme is either selected from the group consisting of RNase I, RNaseA, RNase4, RNaseT1, RNaseT2, RNase2, and RNase3, or comprises a mixture thereof.

28. The method of claim 25, wherein said enzyme is RNase I.

29. The method of claim 21, wherein said cDNA tester is prepared by CAP-trapping the 5′

end of RNA.

30. The method of claim 21, wherein said normalization driver comprises cellular mRNA from the same library, the same tissue, or the same cDNA population the cDNA is to be normalized.

31. The method of claim 21, wherein said normalization driver comprises single-stranded cDNA obtained from the same library, the same tissue, or the same cDNA population as the cDNA to be normalized.

32. The method of claim 21, wherein said subtraction driver comprises cellular mRNA from a library, tissue, or cDNA population differing from the cDNA to be subtracted.

33. The method of claim 21, wherein said subtraction driver comprises single-stranded cDNA from a library, tissue, or cDNA population differing from the cDNA to be normalized.

34. The method of claim 21, further comprising a step V) of preparing a complementary strand of the recovered full-length or full-coding length cDNA and cloning the resulting double-stranded full-length or full-coding length cDNA.

35. A method of preparing normalized and subtracted full-length or full-coding length cDNA comprising the steps of:

I) preparing cDNA tester;

II) preparing polynucleotide drivers for normalization and subtraction;

III) conducting the normalization and subtraction as a single step by mixing together the tester and the drivers;

IV) removing non-specifically hybridized polynucleotide drivers; and

V) recovering the normalized and subtracted full-length or full-coding length cDNA.

36. The method of claim 35, wherein the cDNA tester is cloned or uncloned cDNA.

37. The method of claim 35, wherein the cDNA tester is the reverse transcript of mRNA in the form of uncloned cDNA.

38. The method of claim 35, wherein the cDNA tester is single-stranded.

39. The method of claim 35, wherein step IV) comprises the addition of an enzyme capable of cleaving single-strand RNA driver nonspecifically bound to single-stranded cDNA and the cleaved single-stranded RNA driver is removed.

40. The method of claim 39, wherein said enzyme is single-strand-specific RNA endonuclease.

41. The method of claim 39, wherein said enzyme is either selected from the group consisting of RNase I, RNaseA, RNase4, RNaseT1, RNaseT2, RNase2, and RNase3, or comprises a mixture thereof.

42. The method of claim 39, wherein said enzyme is RNase I.

43. The method of claim 35, wherein said cDNA tester is prepared by CAP-trapping 5′

end of RNA.

44. The method of claim 35, wherein the preparation of said full-length or full-coding length cDNA tester comprises the following steps:

(1) synthesizing first strand cDNA by means of reverse transcriptase forming mRNA/cDNA hybrids;

(2) chemically binding a tag molecule to the diol structure of the 5′

CAP(^7MeG_pppN) site of mRNA forming hybrids;

(3) trapping full-length or full-coding length cDNA hybrids; and

(4) removing single-stranded mRNA by digestion with an enzyme that cleaves single-stranded mRNA.

45. The method of claim 44, wherein said tag molecule is digoxigenin, biotin, avidin, or streptavidin.

46. The method of claim 35, wherein said polynucleotide driver for normalization and/or subtraction is RNA and/or DNA.

47. The method of claim 46, wherein said DNA driver is cDNA.

48. The method of claim 35, wherein said normalization driver comprises cellular mRNA from the same library, the same tissue, or the same cDNA population as the cDNA to be normalized.

49. The method of claim 35, wherein said normalization driver comprises single-stranded cDNA obtained from the same library, the same tissue, or the same cDNA population as the cDNA to be normalized.

50. The method of claim 35, wherein said subtraction driver comprises cellular mRNA from a library, tissue, or cDNA population differing from the cDNA to be subtracted.

51. The method of claim 35, wherein said subtraction driver comprises single-stranded cDNA from a library, tissue, or cDNA population differing from the cDNA to be normalized.

52. The method of claim 35, further comprising a step VI) of preparing a complementary strand of the recovered full-length or full-coding length cDNA and cloning the resulting double-stranded full-length or full-coding length cDNA.

53. A method of preparing normalized and/or subtracted full-length or full-coding length cDNA comprising the steps of:

(a) preparing cDNA tester;

(b) preparing normalization and/or subtraction RNA driver;

(c) conducting normalization and/or subtraction in two steps in any order, or conducting normalization/subtraction as a single step and mixing the normalization/subtraction RNA driver with said cDNA tester;

(d) adding an enzyme that cleaves single-stranded sites on RNA drivers non-specifically bound to cDNA tester;

(e) removing said single-stranded RNA driver cleaved in step d) from the tester and removing tester/driver hybrids; and

(f) recovering the normalized and/or subtracted full-length or full-coding length cDNA.

54. The method of claim 53, wherein the cDNA tester is cloned or uncloned cDNA.

55. The method of claim 53, wherein the cDNA tester is a reverse transcript of mRNA in the form of uncloned cDNA.

56. The method of claim 53, wherein said cDNA tester is single-stranded.

57. The method of claim 53, wherein in step c), normalization is conducted first, followed by subtraction.

58. The method of claim 53, wherein in step c), subtraction is conducted first, followed by normalization.

59. The method of claim 53, wherein in step c), said tester and normalization and subtraction drivers are mixed together and normalization and subtraction are conducted as a single step.

60. The method of claim 53, wherein said normalized and/or subtracted cDNA is full-length or full-coding length cDNA.

61. The method of claim 53, wherein the enzyme of said step d) is either selected from the group consisting of RNase I, RNaseA, RNase4, RNaseT1, RNaseT2, RNase2, and RNase3, or comprises a mixture thereof.

62. The method of claim 53, wherein the enzyme of said step d) is RNase I.

63. The method of claim 53, wherein said cDNA tester is prepared by CAP-trapping the 5′

end of RNA.

64. The method of claim 53, further comprising the step g) of preparing a complementary strand of the recovered cDNA and cloning the resulting double-stranded cDNA.