Combinatorial oligonucleotide PCR: a method for rapid, global expression analysis

• US 6,221,600 B1
• Filed: 10/08/1999
• Issued: 04/24/2001
• Est. Priority Date: 10/08/1999
• Status: Expired due to Fees

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Aldaz, C. Marcelo, Gaddis, Sara S., MacLeod, Michael C.
• Primary Examiner(s): Brusca, John S.
• Assistant Examiner(s): SIU, STEPHEN C
• Application Number:
  US09/414,847
• Time in Patent Office: 564 Days
• Field of Search: 435/6, 435/91.2, 531/23.1, 531/24.3
• US Class Current: 435/6.1
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection

Abstract

The present invention relates to a method for the detection of gene expression and analysis of both known and unknown genes. The invention is a highly sensitive, rapid and cost-effective means of monitoring gene expression, as well as for the analysis and quantitation of changes in gene expression for a defined set of genes and in response to a wide variety of events. It is an important feature of the present invention that no single molecular species of cDNA gives rise to more than one fragment in the collection of products which are subsequently amplified and representative of each expressed gene. This achievement is facilitated by immobilizing the cDNA prior to digesting and then digesting with sequentially with two frequently cutting enzymes. Linker oligomers are ligated to each cut site following the respective digestion. Primers, complementary to the oligomer sequence with an additional 3′ variable sequence are used to amplify the fragments. Using an array of fragments theoretically facilitates the amplification of all of the possible messages in a given sample.

Claims

1. A method comprising:

a) obtaining a DNA comprising an anchorable moiety;

b) cleaving said DNA with a first restriction endonuclease;

c) ligating a linker molecule to cleaved DNA produced in step b;

d) immobilizing linker ligated DNA through said anchorable moiety;

e) cleaving DNA immobilized in step d with a second restriction endonuclease;

f) ligating a second linker molecule to DNA cleaved in step e;

g) amplfying DNA ligated in step f.

2. The method of claim 1, wherein said DNA is immobilized prior to cleaving with said first endonuclease.

3. The method of claim 1, wherein said DNA is non-genomic DNA.

4. The method of claim 1, wherein said DNA is cDNA.

5. The method of claim 1, wherein said anchorable moiety comprises a means of adhering.

6. The method of claim 5, wherein said means of adhering comprises a means of establishing a non-covalent interaction.

7. The method of claim 5, wherein said means of adhering comprises a means of establishing a covalent interaction.

8. The method of claim 5, wherein said means of adhering comprises a ligand.

9. The method of claim 5, wherein said means of adhering is biotin.

10. The method of claim 5, wherein said means of adhering comprises an antibody.

11. The method of claim 1, wherein said anchorable moiety is located at the 3′

end.

12. The method of claim 4, wherein said cDNA is reverse transcribed from messenger RNA.

13. The method of claim 12, wherein said reverse transcription is initiated at an oligo dT.

14. The method of claim 12, wherein said reverse transcription is initiated at a random hexamer.

15. The method of claim 13, wherein said oligo dT is biotinylated.

16. The method of claim 15 wherein said cDNA is immobilized on a substrate by means of said biotinylated oligo dT.

17. The method of claim 16, wherein said substrate is streptavidin.

18. The method of claim 1, wherein the order of said first and said second restriction endonucleases is reversed.

19. The method of claim 1, wherein said amplification is initiated at primers comprising a sequence complementary to said first and said second linkers respectively.

20. The method of claim 1, wherein amplification is carried out with a primer set comprising:

a) a first amplification primer, wherein the 5′

sequence of said primer is complementary to said first linker sequence and the 3′

sequence comprises a specificity region;

b) a second amplification primer, wherein the 5′

sequence of said primer is complementary to said second linker sequence and the 3′

sequence comprises a specificity region.

21. The method of claim 20, wherein said amplification is performed with an array of combinations of alternate amplification primers.

22. The method of claim 20 wherein said DNA fragment is preamplified.

23. The method of claim 1, further comprising identifying the amplified DNA.

24. The method of claim 23, wherein said identification is based upon length.

25. The method of claim 23, wherein said identification is performed by a computer program.

26. The method of claim 21, wherein said amplification is performed in a multi-well plate.

27. The method of claim 20, wherein the specificity region of the first amplification primer is 3,4,5,6,7 or 8 base pairs long.

28. The method of claim 20, wherein the specificity region of the second amplification primer is 3,4,5,6,7 or 8 base pairs long.

29. The method of claim 1, wherein said amplification comprises polymerase chain reaction, nucleic acid sequence based amplification, transcription mediated amplification, strand displacement amplification or ligase chain reaction.

30. The method of claim 1, wherein said first restriction endonuclease has a four base pair recognition site.

31. The method of claim 1, wherein said first restriction endonuclease has a recognition site of five, six, seven or eight base pairs.

32. The method of claim 30, wherein said first restriction endonuclease is NlaIII, DpnII, Sau3AI Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.

33. The method of claim 1, wherein said second restriction endonuclease has a four base pair recognition site.

34. The method of claim 1, wherein said second restriction endonuclease has a recognition site of five, six, seven or eight base pairs.

35. The method of claim 33, wherein the restriction endonuclease is NlaIII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.

36. The method of claim 1, wherein a label is incorporated into said amplified DNA.

37. The method of claim 36, wherein said label is incorporated by means of a labeled primer.

38. The method of claim 36, further comprising partial nucleotide sequence identification of the amplified products by the identity of the label.

39. The method of claim 36, wherein said label is a chromophore.

40. The method of claim 36, wherein said label is a fluorophore.

41. The method of claim 36, wherein said label is an affinity label.

42. The method of claim 36, wherein said label is a dye.

43. The method of claim 37, wherein the 5′

end of said primer comprises an amino moiety and a flurophore is covalently attached by the reaction of a succinimido ester of the flurophore to the 5′

amino-modified primer.

44. The method of claim 40, wherein said fluorophore is Alexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy2, Cy3, Cy5,6-FAM, Fluorescein, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, ROX, TAMRA, TET, Tetramethylrhodamine, or Texas Red.

45. The method of claim 1, wherein the products of said amplification are analyzed.

46. The method of claim 45, wherein said analysis of amplification products is by polyacrylamide gel electrophoresis.

47. The method of claim 45, wherein said analysis of amplification products is by capillary gel electrophoresis.

48. The method of claim 45, wherein said analysis of amplification products is by mass spectrophotometry.

49. The method of claim 45, wherein said analysis of amplification products is by energy transfer.

50. The method of claim, 45, wherein said analysis of amplification products is by OIA technology.

51. The method of claim 45, wherein said analysis of amplification products utilizes fluorescently-labeled latex beads.

52. The method of claim 45, wherein said analysis of amplification products comprises quantifying amplification products.

53. The method of claim 52, wherein said quantifying is by measuring the ratio of each amplified product to a co-amplified reference-gene.

54. The method of claim 52, wherein said quantifying is by measuring the ratio of each amplified product to a panel of co-amplified reference-genes.

55. The method of claim 52, wherein said analysis of amplification products is by Real-Time PCR.

56. The method of claim 45, wherein said analysis of amplification products is performed in a multi-well plate.

57. The method of claim 45, wherein said analysis of amplification products is performed on a membrane.

58. The method of claim 45, wherein said analysis of amplification products is performed on a solid matrice.

59. The method of claim 58, wherein said solid matrice is a DNA chip.

60. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a different cell or tissue.

61. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cancerous cell or tissue.

62. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived a cell or tissue treated with a pharmaceutical compound.

63. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a teratogenic compound.

64. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a carcinogenic compound.

65. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a toxic compound.

66. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a biological response modifier.

67. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a hormone, a hormone agonist or a hormone antagonist.

68. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a cytokine.

69. The method of claim 1, performed on DNA derived from a normal cell or tissue and on DNA derived from a cell or tissue treated with a growth factor.

70. The method of claim 1, performed on DNA derived from a normal cell or tissue and on the DNA derived from a cell or tissue treated with the ligand of a known biological receptor.

71. The method of claim 1, performed on DNA derived from a cell or tissue type obtained from a different species.

72. The method of claim 1, performed on DNA derived from a cell or tissue type obtained from a different organism.

73. The method of claim 1, performed on DNA derived from a cell or tissue at different stages of development.

74. The method of claim 1, performed on DNA derived from a normal cell or tissue and on the DNA derived from a cell or tissue that is diseased.

75. The method of claim 1, performed on DNA derived from a cell or tissue cultured in vitro under different conditions.

76. The method of claim 1, performed on the DNA derived from a cell or tissue from two organisms of the same species with a known genetic difference.

84. The method of claim 1, wherein said anchorable moiety is located at the 5′

end.

77. A kit for detection of gene expression comprising:

a) a first restriction enzyme;

b) a second restriction enzyme;

c) a first, ligatable, oligonucleotide tag;

d) a second, ligatable, oligonucleotide tag;

e) a first amplification primer, wherein the 5′

sequence of said primer is complementary to said first oligonucleotide tag and the 3′

sequence comprises a specificity region;

f) a second amplification primer, wherein the 5′

sequence of said primer is complementary to said second oligonucleotide tar and the 3′

sequence comprises a specificity region;

g) software capable of analyzing data generated from said kit.

78. The kit of claim 77, wherein said first restriction enzyme is a four base pair cutter.

79. The kit of claim 78, wherein said first restriction endonuclease is NlaIII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.

80. The kit of claim 77, wherein said second restriction enzyme is a four base pair cutter.

81. The kit of claim 80, wherein said second restriction endonuclease is NlaII, DpnII, Sau3AI, Hsp92II, MboI, NdeII, Bsp1431, Tsp509 I, HhaI, HinP1I, HpaII, MspI, TaqalphaI, MaeII or K2091.

82. The kit of claim 77, wherein said first amplification primer comprises the sequence GCTGTCTAGACG (SEQ ID NO:

1).

83. The kit of claim 77, wherein said second amplification primer comprises the sequence CGGTGATGCATC (SEQ ID NO:

2).