Methods and systems for dynamic gene expression profiling

• US 20030077611A1
• Filed: 04/01/2002
• Published: 04/24/2003
• Est. Priority Date: 10/24/2001
• Status: Abandoned Application

• Current Assignee: Primera Biosystems, Inc. (Qiagen NV)
• Original Assignee: Sention
• Inventors: Slepnev, Vladimir I.
• Application Number:
  US10/113,034
• Publication Number:
  US 20030077611A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 2525/149 incorporating a coding sequence
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs

Abstract

The invention provides compositions, methods and systems for dynamic transcription profiling of two or more samples. The method of the invention comprises the uses of sample-specific primers for cDNA synthesis and for subsequent amplification of the synthesized cDNAs. The levels of abundance of genes are compared between samples for the identification of differentially expressed genes.

Claims

1. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5′

terminal and At rich at its 3′

terminal;

(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;

(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

2. The method of claim 1, wherein said step (a) comprises reverse transcribing RNA from two or more sample sources into first strand cDNA, and wherein said cDNA is differentially tagged according to their sources.

3. The method of claim 1, wherein said plurality of first strand cDNAs is synthesized by reverse transcription using total RNAs or mRNAs derived from said first sample.

4. The method of claim 1, wherein a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer is used for said amplifying so as to generate one or more sample-specific amplified products.

5. The method of claim 1, wherein at least one of said two or more samples is derived form the group consisting of:

a normal sample, a disease sample, a sample at a given development stage or condition, a sample prior to a given treatment stage or condition, a sample after a given treatment stage or condition, and a sample at a given culturing stage or condition.

6. The method of claim 1, wherein at least one of said two or more samples is derived from the group consisting of:

an animal, an organ, a tissue type, and a cell type.

7. The method of claim 1, wherein said sample-specific sequence in said first oligonucleotide primer is 15-30 nucleotides in length.

8. The method of claim 1, wherein said sample-specific sequence is 20-24 nucleotide in length.

9. The method of claim 1, wherein said first oligonucleotide primer further comprises a sequence of 5′

oligo(dT)_nVN 3′

, where n is at least 5;

V is dATP, dGTP, or dCTP; and

N is dTTP (or dUTP), dATP, dGTP, or dCTP.

10. The method of claim 1, wherein said first oligonucleotide primer is provided as a mixture of primers comprising [5′

-(specific sequence tag)_20-24T_12-16AN-3′

, 5′

-(specific sequence tag)_20-24T_12-16CN-3′

, and 5′

-(specific sequence tag)_20-24T_12-16GN-3′

], wherein said specific sequence tags are identical or different for each primer in said mixture.

11. The method of claim 10, wherein n is 12-16.

12. The method of claim 10, wherein in said first oligonucleotide primer, said sample-specific sequence tag is located at the 5′

of oligo(dT)_nVN.

13. The method of claim 1, further comprising synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag, wherein step (b) amplifies at least a subset of said second strand cDNAs so as to generate one or more sample-specific amplified products.

14. The method of claim 13, wherein said second oligonucleotide primer further comprises a second sequence which is complementary to a subset of said first strand cDNAs so as to permit the synthesis of one or more second strand cDNAs.

15. The method of claim 14, wherein in said second oligonucleotide primer, said second sequence is located 3′

of said first arbitrary sequence.

16. The method of claim 14, wherein said second oligonucleotide further comprises a sequence of (Z)_m between said first and second sequences, where Z is a nucleotide which can form base pair with any of A, T, G, or C, and m is at least 2.

17. The method of claim 16, wherein m is 4.

18. The method of claim 14, wherein said second sequence is 5-10 nucleotides in length.

19. The method of claim 18, wherein said second sequence is 6-7 nucleotides in length.

20. The method of claim 13, wherein said first arbitrary sequence within said second oligonucleotide primer is 15-30 nucleotides in length.

21. The method of claim 13, wherein said first arbitrary sequence within said second oligonucleotide primer comprises a A-T rich region and a G-C rich region.

22. The method of claim 21, wherein said G-C rich region is located at 5′

of said A-T rich region.

23. The method of claim 13, wherein said second oligonucleotide primer used is the same for said two or more samples to be compared.

26. The method of claim 14, wherein said second sequence within said second oligonucleotide primer is gene-family-specific.

27. The method of claim 14, wherein said second sequence within said second oligonucleotide primer is a sequence encoding a peptide specific for a protein family.

28. The method of claim 27, wherein said second sequence comprises a sequence encoding a signature sequence motif for a specific protein family.

29. The method of claim 28, wherein said protein family is selected from the group consisting of:

receptor tyrosine kinases, G protein coupled receptors, seven transmembrane receptors, ion channels, cytokine receptors, tumor markers, MAPK cascade kinases, transcriptional factors, GTPases, ATPases, and development protein markers.

30. The method of claim 1, wherein said first strand cDNA is synthesized in a solution without attaching to a solid support.

31. The method of claim 1, wherein said first strand cDNA is synthesized attaching to a solid support.

32. The method of claim 31, wherein said solid support is a microparticle or an inner wall of a reaction tube.

33. The method of claim 13, wherein said method further comprises separating said one or more second strand cDNA from said plurality of first strand cDNA before amplifying said one or more second strand cDNAs.

34. The method of claim 4, wherein said third oligonucleotide primer is linked to a detectable label.

35. The method of claim 34, wherein said detectable label is selected from a group consisting of:

fluorescent labels, radioactive labels, colorimetrical labels, magnetic labels, and enzymatic labels.

36. The method of claim 35, wherein said detectable label is a fluorescent label.

37. The method of claim 34, wherein said third oligonucleotide primer used for each of said two or more samples is labeled with a sample-specific label.

38. The method of claim 1, wherein said one or more amplified products are sampled at a predetermined time or cycle interval during the amplification.

39. The method of claim 38, wherein the abundance is detected for each sampled amplified product.

47. The method of claim 1, wherein said difference in the expression profile of said one or more genes is measured by a ratio of sample-specific detectable labels on amplified products from said genes between two or more samples.

48. The method of claim 1, wherein said method further comprises generating an amplification plot, calculating a Ct of amplification for each of said one or more genes, and measuring the difference in the expression profile by a ratio of said Cts.

49. The method of claim 1, wherein said method further comprises collecting one or more genes which are differentially expressed and identifying the sequence identities of said one or more genes by DNA sequencing.

50. The method of claim 1, wherein said amplifying is performed by PCR.

24. The method of 4, wherein said amplifying further comprises using a fourth oligonucleotide primer which comprises said first arbitrary sequence tag of said second oligonucleotide primer.

25. The method of claim 24, wherein said fourth oligonucleotide primer used is the same for said two or more samples to be compared.

40. The method of 1, wherein said method further comprises separating said one or more amplified products before detecting the abundance of said one or more amplified products.

41. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by chromatography.

42. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by measurement of fluorescence.

43. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by measurement of optical density.

44. The method of claim 40, wherein said one or more amplified products are separated and their abundance detected by mass spectrometry.

45. The method of claim 40, wherein said one or more amplified products are separated by electrophoresis.

46. The method of claim 45, wherein said one or more amplified products are separated by capillary electrophoresis.

51. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;

(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;

(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

52. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;

(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;

(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(d) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

53. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5′

terminal and AT rich at its 3′

terminal;

(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;

(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;

(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

54. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;

(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;

(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;

(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

55. A method for comparing gene expression profiles of two or more samples, said method comprising:

(a) synthesizing a plurality of first strand cDNAs from a first sample using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag comprises at least one artificial nucleotide;

(b) selectively synthesizing one or more second strand cDNAs complementary to said first strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;

(c) amplifying said one or more second strand cDNA so as to generate one or more sample-specific amplified products;

(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said first sample; and

(e) comparing the expression profile of said one or more genes in said first sample with an expression profile of said one or more genes in a second sample, wherein a difference in the expression profile indicates differential expression of said one or more genes in the two samples.

56. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:

(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5′

terminal and At rich at its 3′

terminal;

(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;

(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and

(d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.

57. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:

(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;

(b) selectively amplifying at least a subset of said cDNA so as to generate one or more sample-specific amplified products;

(c) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and

(d) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.

58. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:

(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said sample-specific sequence tag is GC rich at its 5′

terminal and At rich at its 3′

terminal;

(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;

(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;

(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and

(e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.

59. A method of identifying a modulator which regulates one or more gene expression in a sample, said method comprising:

(a) synthesizing a plurality of first strand cDNAs, before contacting said sample with said modulator, using a first oligonucleotide primer comprising a sample-specific sequence tag, wherein said first oligonucleotide primer comprises at least one degenerate nucleotide;

(b) synthesizing one or more second strand cDNAs using a second oligonucleotide primer comprising a first arbitrary sequence tag;

(c) amplifying said second strand cDNAs so as to generate one or more sample-specific amplified products;

(d) detecting the abundance of one or more said sample-specific amplified products, wherein said abundance determines an expression profile of one or more genes in said sample; and

(e) comparing the expression profile of said one or more genes in said sample before contacting with said modulator with an expression profile of said one or more genes in said sample after contacting said modulator, wherein a difference in the expression profile indicates said modulator regulating one or more gene expression in said sample.

60. A composition for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said sample-specific sequence tag is GC rich at its 5′

terminal and AT rich at its 3′

terminal.

61. The composition of claim 60, further comprising a second oligonucleotide primer which comprises a first arbitrary sequence tag.

62. The composition of claim 61, further comprising a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer.

63. The composition of claim 62, further comprising a fourth oligonucleotide primer which comprises said first arbitrary sequence tag.

64. The composition of claim 60, wherein said second primer further comprises a second sequence which is complementary to a sequence of said first strand cDNA.

65. The composition of claim 60, further comprising one or more components selected from the group of:

a reverse transcriptase, a DNA polymerase, a reaction buffer for said reverse transcriptase, a reaction buffer for said DNA polymerase, and dNTPs.

66. A composition for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said first oligonucleotide primer comprises at least one degenerate nucleotide.

67. A kit for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said sample-specific sequence tag is GC rich at its 5′

terminal and AT rich at its 3′

terminal, and packaging material thereof.

68. The kit of claim 67, further comprising a second oligonucleotide primer which comprises a first arbitrary sequence tag.

69. The kit of claim 67, further comprising a third oligonucleotide primer comprising said sequence-specific sequence tag of said first oligonucleotide primer.

70. The kit of claim 68, further comprising a fourth oligonucleotide primer which comprises said first arbitrary sequence tag.

71. The composition of claim 68, wherein said second primer further comprises a second sequence which is complementary to a sequence of said first strand cDNA.

72. The composition of claim 67, further comprising one or more components selected from the group of:

a reverse transcriptase, a DNA polymerase, a reaction buffer for said reverse transcriptase, a reaction buffer for said DNA polymerase, and dNTPs.

73. A kit for detecting the level of gene expression, comprising a first oligonucleotide primer, wherein said first oligonucleotide primer comprises a sample-specific sequence tag and wherein said first oligonucleotide primer comprises at least one degenerate nucleotide, and packaging material thereof.