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

• US 7,294,487 B2
• Filed: 04/23/2001
• Issued: 11/13/2007
• 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): LU, FRANK WEI MIN
• Application Number:
  US09/840,722
• Publication Number:
  US 20030027141A1
• Time in Patent Office: 2,395 Days
• Field of Search: 435/6, 435/91.1, 435/91.2, 536/23.1, 536/24.3
• US Class Current: 435/91.2
• 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 of subjecting a DNA molecule to a DNA synthesis reaction, comprising the steps of:

a) preparing a DNA molecule by attaching a first linker sequence at one end of the DNA molecule and attaching a second linker sequence, different from said first linker sequence, at the other end of the DNA molecule; and

b) subjecting said DNA to a DNA synthesis reaction with a primer set comprising;

i) a first primer set population, wherein the 5′

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

sequence of primers of said first primer set population comprises a specificity region; and

ii) a second primer set population, wherein the 5′

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

sequence of primers of said second primer set population comprises a specificity region;

wherein said specificity regions of said first and second primer set populations comprise random combinations of A, T, G and C.

2. The method of claim 1, wherein the specificity region of the primers of the first primer set is 3, 4, 5, 6, 7 or 8 base pairs long.

3. The method of claim 1, wherein the specificity region of the primers of the second primer set is 3, 4, 5, 6, 7 or 8 base pairs long.

4. The method of claim 1, wherein the products of said DNA synthesis reaction are analyzed.

5. The method of claim 4, wherein said analysis of products is by polyacrylamide gel electrophoresis.

6. The method of claim 4, wherein said analysis of products is by capillary gel electrophoresis.

7. The method of claim 4, wherein said analysis of products is by mass spectrophotometry.

8. The method of claim 4, wherein said analysis of products is by a filtration and extraction device.

9. The method of claim 4, wherein said analysis of products comprises quantifying amplification products.

10. The method of claim 9, wherein said quantifying is by measuring the ratio of each product to a co-amplified reference-gene.

11. The method of claim 9, wherein said quantifying is by measuring the ratio of each product to a panel of reference-genes.

12. The method of claim 9, wherein said analysis of products is by Real-Time PCR.

13. The method of claim 4, wherein said analysis of products is performed in a multi-well plate.

14. The method of claim 4, wherein said analysis of products is performed on a membrane.

15. The method of claim 4, wherein said analysis of products is performed on a solid matrice.

16. The method of claim 15, wherein said solid matrice is a DNA chip.

17. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a different cell or tissue.

18. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cancerous cell or tissue.

19. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a pharmaceutical compound.

20. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a teratogenic compound.

21. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a carcinogenic compound.

22. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a toxic compound.

23. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a biological response modifier.

24. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a hormone, a hormone agonist or a hormone antagonist.

25. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a cytokine.

26. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or DNA derived from a cell or tissue treated with a growth factor.

27. The method of claim 1, the DNA molecule is derived from a normal cell or tissue or the DNA derived from a cell or tissue treated with the ligand of a known biological receptor.

28. The method of claim 1, more than one sample of DNA are used, wherein the DNA samples are derived from a cell or tissue type obtained from different species.

29. The method of claim 1, more than one sample of DNA are used, wherein the DNA samples are derived from a cell or tissue type obtained from different organisms.

30. The method of claim 1, more than one sample of DNA are used, wherein the DNA samples are derived from a cell or tissue at different stages of development.

31. The method of claim 1, more than one sample of DNA are used, wherein the DNA samples are derived from a normal cell or tissue and derived from a cell or tissue that is diseased.

32. The method of claim 1, more than one sample of DNA are used, wherein the DNA samples are derived from a cell or tissue cultured in vitro under different conditions.

33. The method of claim 1, the DNA molecule is derived from a cell or tissue from two organisms of the same species with a known genetic difference.

34. The method of claim 1, wherein the first and second primers are employed to amplify the DNA molecule.

35. The method of claim 34, wherein said amplification is performed with an array of combinations of alternate amplification primers.

36. The method of claim 34, further comprising, identifying the amplified DNA.

37. The method of claim 36, wherein said identification is based upon length of the amplified DNA.

38. The method of claim 36, wherein said identification is performed by a computer program.

39. The method of claim 35, wherein said array of amplifications is performed in a multi-well plate.

40. The method of claim 34, wherein said amplification comprises polymerase chain reaction, nucleic acid sequence based amplification, transcription mediated amplification or strand displacement amplification.

41. The method of claim 34, wherein a label is incorporated into said amplified DNA.

42. The method of claim 41, wherein said label is incorporated by means of a labeled primer.

43. The method of claim 41, further comprising determining a nucleotide sequence of the amplified products.

44. The method of claim 41, wherein said label is a chromophore.

45. The method of claim 41, wherein said label is a fluorophore.

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

47. The method of claim 41, wherein said label is a dye.

48. The method of claim 45, 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, and Texas Red.

49. The method of claim 1, wherein the first and second primers are employed to sequence the DNA molecule.

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

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