Methods for analysis of gene expression

• US 6,618,679 B2
• Filed: 01/27/2001
• Issued: 09/09/2003
• Est. Priority Date: 01/28/2000
• Status: Expired due to Term

• Current Assignee: Altheadx, Inc.
• Original Assignee: Althea Technologies Inc. (Ajinomoto Co., Inc.)
• Inventors: Monforte, Joseph, Wong, Linda, Shaler, Thomas, Stephens, Kathy, Loehrlein, Christine, Pollart, Dan, Tan, Yuping
• Primary Examiner(s): MORAN, MARJORIE A
• Application Number:
  US09/771,372
• Publication Number:
  US 20020160361A1
• Time in Patent Office: 955 Days
• Field of Search: 435/6, 435/91.2, 702/20, 356/320, 356/328, 356/344, 536/24.33, 536/25.3, 706/13
• US Class Current: 702/20
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 2525/15 incorporating a consensus or conserved sequence
• CPC Class Codes: C12Q 2525/155 incorporating/generating a new priming site
• CPC Class Codes: C12Q 2525/161 incorporating target specific and non-target specific sites
• CPC Class Codes: C12Q 2537/143 Multiplexing, i.e. use of multiple primers or probes in a single reaction, usually for simultaneously analyse of multiple analysis
• CPC Class Codes: G16B 25/00 ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
• CPC Class Codes: G16B 25/10 Gene or protein expression profiling; Expression-ratio estimation or normalisation

Abstract

This invention provides methods, compositions and kits for gene expression analysis and gene expression profiling. The methods of the invention are highly sensitive; have a wide dynamic range; are rapid and inexpensive; have a high throughput; and allow the simultaneous differential analysis of a defined set of genes. The methods, compositions and kits of the invention also provide tools for gene expression data collection and relational data analysis.

Claims

1. A method for analyzing gene expression comprising:

a) obtaining a plurality of target sequences, wherein the plurality of target sequences comprises cDNA;

b) multiplex amplifying said plurality of target sequences, wherein multiplex amplifying comprises combining the plurality of target sequences, a plurality of target-specific primers, and one or more universal primers, and wherein the universal primer is provided in an excess concentration relative to the target-specific primer, thereby producing a plurality of amplification products;

c) separating one or more members of the plurality of amplification products;

d) detecting one or more members of the plurality of amplification products, thereby generating a set of gene expression data;

e) storing the set of gene expression data in a database; and

f) performing a comparative analysis on the set of gene expression data, thereby analyzing the gene expression.

2. The method of claim 1, wherein a universal primer:

target-specific primer concentration ratio ranges from about 5;

1 to about 100;

1.

3. The method of claim 1, wherein a universal primer:

target-specific primer concentration ratio is about 10;

1.

13. The method of claim 1, 4 or 7, wherein obtaining the target sequences comprises performing reverse transcription of mRNA.

14. The method of claim 13, wherein the mRNA comprises mRNA derived from cultured cells.

15. The method of claim 13, wherein said mRNA comprises mRNA derived from cultured cells subjected to a specific treatment.

16. The method of claim 15, wherein said specific treatment comprises a chemical exposure, an environmental stress, or an exposure to one or more viable organisms or cells.

17. The method of claim 1, 4 or 5, wherein multiplex amplifying comprises simultaneously amplifying a plurality of cDNA in the same reaction mixture;

wherein said plurality of target-specific primers comprises one or more target-specific primer pairs, each pair comprising a forward target-specific primer and a reverse target-specific primer; and

wherein the one or more universal primers comprises one or more universal primer pairs, each pair comprising a forward universal primer and a reverse universal primer.

18. The method of claim 1, 4 or 5, wherein said plurality of target sequences further comprises one or more reference sequences, wherein a portion of the one or more reference sequences is homologous to at least one member of the plurality of target-specific primers.

19. The method of claim 18, wherein one or more of the reference sequences comprises sequences endogenously present in the cDNA.

20. The method of claim 18, wherein one or more of the reference sequences comprises sequences exogenously added to the cDNA.

21. The method of claim 1, 4 or 5, wherein at least one member of the plurality of target-specific primers or universal primers further comprises a modified nucleotide.

22. The method of claim 21, wherein the modified nucleotide prevents amplification of one or more portions of the at least one member of the plurality of target-specific primers or universal primers.

23. The method of claim 21, wherein the modified nucleotide comprises one or more non-nucleotide linkers, alkyl chains, or abasic nucleotides.

24. The method of claim 1, 4 or 5, wherein at least one member of the plurality of target-specific primers or universal primers further comprises a cleavable linker.

25. The method of claim 1, 4 or 5, wherein at least one universal primer further comprises a label.

26. The method of claim 25, wherein the label comprises one or more of a chromaphore, a fluorophore, a dye, a releasable label, a mass label, an affinity label, a friction moiety, a hydrophobic group, or an isotopic label.

27. The method of claim 1, 4 or 5, wherein each member of the plurality of target-specific primers comprises a first sequence that is derived from a target gene of interest and positioned within a 3′

region of the member, and a second sequence that is complementary to the universal primer and positioned within a 5′

region of the member.

28. The method of claim 1, 4 or 5, wherein the one or more universal primers comprise one or more semi-universal primers.

29. The method of claim 28, wherein each of the one or more semi-universal primers comprises a unique label.

30. The method of claim 28, wherein the one or more semi-universal primers comprise primers which are complementary to one or more forward target-specific primers, one or more reverse target-specific primers, or a combination thereof.

31. The method of claim 30, wherein the one or more semi-universal primers comprise a first semi-universal primer that is complementary to all of the one or more forward target-specific primers, and a second semi-universal primer that is complementary to all of the one or more reverse target-specific primers.

32. The method of claim 1, 4 or 5, wherein obtaining a plurality of target sequences comprises providing two or more target sequences having two or more target-specific primer annealing temperatures.

33. The method of claim 1, 4 or 5, wherein multiplex amplifying the cDNA comprises amplifying target genes that have comparable expression levels.

34. The method of claim 1, 4 or 5, wherein multiplex amplifying further comprises altering the length of one or more of the universal primers or one or more of the plurality of target-specific primers prior to combining.

35. The method of claim 34, wherein altering the length comprises adding nucleotides to an end of a universal primer or a target-specific primer.

36. The method of claim 34, wherein altering the length comprises incorporating a non-nucleotide linker into a universal primer or a target-specific primer.

37. The method of claim 34, wherein altering the length comprises cleaving the one or more universal primers or the one or more target-specific primers.

38. The method of claim 34, wherein one or more of the universal primers or one or more of the plurality of target-specific primers comprise semi-universal primers.

39. The method of claim 34, wherein altering the length comprises inserting nucleotides within a universal primer or a target-specific primer.

40. The method of claim 39, wherein altering the length of a target-specific primer comprises inserting nucleotides between a universal sequence and a target-specific sequence of the target-specific primer.

41. The method of claim 1, 4 or 5, wherein the plurality of amplification products comprises a plurality of labels at predetermined molar ratios.

42. The method of claim 41, wherein the plurality of labels is incorporated on a single oligonucleotide primer.

43. The method of claim 41, wherein the plurality of labels is incorporated on a plurality of oligonucleotide primers.

44. The method of claim 1, 4 or 5, wherein separating the one or more members of the plurality of amplification products comprises performing one or more size separation techniques.

45. The method of claim 44, wherein separating the one or more members of the plurality of amplification products comprises performing HPLC or FPLC.

46. The method of claim 44, wherein separating the one or more members of the plurality of amplification products comprises performing mass spectrometry.

47. The method of claim 44, wherein separating the one or more members of the plurality of amplification products comprises employing an electrophoresis platform.

48. The method of claim 47, wherein the electrophoresis platform comprises one or more of a capillary platform, a microcapillary platform, a microfluidics platform, an agarose gel, an acrylamide gel, an agarose/acrylamide gel or a chromatographic platform.

49. The method of claim 1, 4 or 5, wherein performing the comparative analysis comprises measuring a ratio of each target gene to each reference gene.

50. The method of claim 1, 4 or 5, wherein separating the one or more members of the plurality of amplification products comprises performing HPLC followed by mass spectroscopy.

51. The method of claim 1, 4 or 5, wherein detecting the one or more members of the plurality of amplification products comprises measuring one or more inherent properties of the amplification products.

52. The method of claim 51, wherein the one or more inherent properties comprise mass, light absorption, or an electrochemical property.

53. The method of claim 1, 4 or 5, wherein detecting the one or more members of the plurality of amplification products comprises measuring the presence, absence, or quantity of a labeled amplification product.

54. The method of claim 53, wherein the labeled amplification product comprises a singly labeled amplification product, a multiply-labeled amplification product, or a combination thereof.

55. The method of claim 53, wherein detecting comprises resolving a first signal from a singly labeled amplification product and a second signal from a multiply labeled amplification product by deconvolution of the data.

56. The method of claim 53, wherein detecting comprises resolving a first signal from a singly labeled amplification product and a second signal from a multiply labeled amplification product by reciprocal subtraction of the first or second signal from an overlapping signal.

57. The method of claim 1, 4 or 5, wherein one or more of the multiplex amplifying, separating and detecting is performed in a high throughput format.

4. A method for analyzing gene expression comprising:

a) obtaining a plurality of target sequences, wherein the plurality of target sequences comprises cDNA;

b) multiplex amplifying said plurality of target sequences, wherein multiplex amplifying comprises combining the plurality of target sequences, a plurality of target-specific primers, and one or more universal primers, wherein an annealing temperature of the universal primer is higher than an annealing temperature of the target-specific primer;

thereby producing a plurality of amplification products;

c) separating one or more members of the plurality of amplification products;

d) detecting one or more members of the plurality of amplification products, thereby generating a set of gene expression data;

e) storing the set of gene expression data in a database; and

f) performing a comparative analysis on the set of gene expression data, thereby analyzing the gene expression.

5. A method for analyzing gene expression comprising:

a) obtaining a plurality of target sequences, wherein the plurality of target sequences comprises cDNA;

b) multiplex amplifying said plurality of target sequences, wherein multiplex amplifying comprises combining the plurality of target sequences, a plurality of target-specific primers, and one or more universal primers, and wherein multiplex amplifying the cDNA further comprises attenuating an amplification of abundant target genes, thereby producing a plurality of amplification products;

c) separating one or more members of the plurality of amplification products;

d) detecting one or more members of the plurality of amplification products, thereby generating a set of gene expression data;

e) storing the set of gene expression data in a database; and

f) performing a comparative analysis on the set of gene expression data, thereby analyzing the gene expression.

6. The method of claim 5, wherein attenuating the amplification of abundant target genes comprises using one or more modified target-specific primers.

7. The method of claim 6, wherein the one or more modified target-specific primer comprises a blocking group attached at a 3′

end of the modified target-specific primer.

8. The method of claim 6, wherein the one or more modified target-specific primer comprises one or more abasic nucleotides or mismatch nucleotides.

9. The method of claim 6, wherein using one or more modified target-specific primers comprises providing a mixture of the one or more modified target-specific primers with one or more unmodified target-specific primers, at a ratio optimized for a desired amount of attenuation.

10. The method of claim 6, wherein the one or more modified target-specific primer comprises a blocking group attached at a 3′

end of a reverse target-specific primer.

11. The method of claim 6, wherein the one or more modified target-specific primers comprise primers having a phosphate group on the terminal 3′

-hydroxyl of the target-specific primer.

12. The method of claim 6, wherein the one or more modified target-specific primers comprise primers having a nucleotide penultimate to the terminal 3′

-nucleotide and attached via a 3′

-3′

phosphodiester linkage.

58. A method for analyzing gene expression comprising:

a) obtaining cDNA from a plurality of samples for a plurality of target sequences;

b) performing a plurality of multiplexed amplifications of the target sequences, thereby producing a plurality of multiplexed amplification products, wherein performing a multiplexed amplification comprises combining a plurality of target sequences, one or more target-specific primers, and one or more universal primers to form a reaction mixture, and wherein the reaction mixture comprises one or more of i) an excess concentration of the one or more universal primers relative to a target-specific primer, ii) universal primers which have a higher annealing temperature relative to the annealing temperature of a target-specific primer, or iii) modified target-specific primer for attenuation of abundant target genes;

c) pooling the plurality of multiplexed amplification products;

d) separating the plurality of multiplexed amplification products;

e) detecting the plurality of multiplexed amplification products, thereby generating a set of gene expression data;

f) storing the set of gene expression data in a database; and

g) performing a comparative analysis of the set of gene expression data.

59. The method of claim 58, wherein at least one of the one or more universal primers or one or more target-specific primers comprises a label.

60. The method of claim 59, wherein the first or second label comprises a high-affinity intercalating dye.

61. The method of claim 59, wherein a first multiplexed amplification is performed with a primer comprising a first label that produces a first signal, and a second multiplexed amplification is performed with a primer comprising a second label that produces a second signal, wherein the first and second signals are distinguishable from one another.

62. The method of claim 61, wherein the first and second signals are distinguishable by deconvolution of signals obtained from the plurality of multiplexed amplification products.

63. The method of claim 58, wherein performing the plurality of amplifications of the target sequences comprises using universal primers having two or more lengths, and wherein detecting the plurality of multiplexed amplification products comprises measuring one or more size shifts among the plurality of multiplexed amplification products.

64. The method of claim 58, wherein performing the plurality of amplifications of the target sequences comprises using target-specific primers having two or more lengths, and wherein detecting the plurality of multiplexed amplification products comprises measuring one or more size shifts among the plurality of multiplexed amplification products.

65. The method of claim 64, performing the plurality of amplifications of the target sequences comprises using universal primers comprising one or more cleavage sites, and wherein detecting the plurality of multiplexed amplification products comprises measuring one or more size shifts among the plurality of multiplexed amplification products.

66. The method of claim 58, wherein separating the plurality of multiplexed amplification products comprises shifting the mobility of member amplification products relative to one another.

67. The method of claim 66, wherein shifting the mobility comprises incorporating a friction moiety into one or more of the universal primers, thereby creating a reduction in mobility of the amplification products.

68. The method of claim 58, wherein separating comprises applying each set of multiplex amplification products to a separation platform at different times.

69. The method of claim 58, wherein performing the plurality of amplifications comprises performing a polymerase chain reaction, a transcription-based amplification, a self-sustained sequence replication, a nucleic acid sequence based amplification, a ligase chain reaction, a ligase detection reaction, a strand displacement amplification, a repair chain reaction, a cyclic probe reaction, a rapid amplification of cDNA ends, an invader assay, a solid phase assay, a solution phase assay, or a combination thereof.

70. The method of claim 69, wherein the solid phase assay comprises a bridge amplification or rolling circle amplification.

71. The method of claim 58, wherein one or more of the performing, separating and detecting is performed in a high throughput format.

72. The method of claim 71, wherein one or more of the performing, separating and detecting steps is performed at a rate of about 1000 samples per hour.

73. A method for analyzing gene expression comprising:

a) obtaining cDNA from multiple samples;

b) amplifying a plurality of target sequences from the cDNA, thereby producing a multiplex set of amplification products, wherein amplifying the plurality of target sequences comprises combining the plurality of target sequences, one or more target-specific primers, and one or more universal primers to form a reaction mixture, and wherein the reaction mixture comprises one or more of i) an excess concentration of the one or more universal primers relative to a target-specific primer, ii) universal primers which have a higher annealing temperature relative to the annealing temperature of a target-specific primer, or iii) modified target-specific primer for attenuation of abundant target genes;

c) separating and detecting the amplification products using a high throughput platform, wherein detecting generates a set of gene expression data; and

d) storing the set of gene expression data in a database; and

e) performing a comparative analysis of the set of gene expression data.

74. The method of claim 73, wherein the one or more universal primers or the one or more target-specific primers comprise one or more non-nucleotide linkers.

75. The method of claim 73, wherein separating and detecting the amplification products comprises performing mass spectrometry, polyacrylamide gel electrophoresis, HPLC, capillary electrophoresis, microcapillary electrophoresis, or a combination thereof.

76. The method of claim 73, wherein separating and detecting the amplification products is performed using microfluidic devices.

77. The method of claim 73, wherein the high throughput platform comprises an HPLC for separating the amplification products and a mass spectrometer for detecting the amplification products.

78. The method of claim 73, wherein the high throughput platform comprises one or more miniaturized scale platforms.

79. The method of claim 73, wherein one or more of the amplifying, separating and detecting steps is performed at a rate of about 100 samples per hour to about 5,000 samples per hour.

80. The method of claim 73, wherein one or more of the amplifying, separating and detecting steps is performed at a rate of about 1000 samples per hour.

81. The method of claim 73, wherein amplifying the plurality of target sequences comprises performing on or more of a polymerase chain reaction, a transcription-based amplification, a self-sustained sequence replication, a nucleic acid sequence based amplification, a ligase chain reaction, a ligase detection reaction, a strand displacement amplification, a repair chain reaction, a cyclic probe reaction, a rapid amplification of cDNA ends, an invader assay, a solution phase amplification assay, or a solid phase amplification assay.

82. The method of claim 81, wherein the solid phase amplification assay comprises a bridge amplification or rolling circle amplification.