Methods for analysis of gene expression

• US 20030204322A1
• Filed: 06/18/2003
• Published: 10/30/2003
• Est. Priority Date: 01/28/2000
• Status: Abandoned Application

• Current Assignee: Altheadx, Inc.
• Original Assignee: Althea Technologies Inc. (Ajinomoto Co., Inc.)
• Inventors: Monforte, Joseph, Wong, Linda, Shaler, Thomas, Stephens, Kathy, Tan, Yuping, Loehrlein, Christine, Pollart, Dan
• Application Number:
  US10/465,071
• Publication Number:
  US 20030204322A1
• Time in Patent Office: Days
• 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, 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 obtaining the target sequences comprises performing reverse transcription of mRNA.

3. The method of claim 2, wherein the mRNA comprises mRNA derived from cultured cells.

4. The method of claim 2, wherein said mRNA comprises mRNA derived from cultured cells subjected to a specific treatment.

5. The method of claim 4, wherein said specific treatment comprises a chemical exposure, an environmental stress, or an exposure to one or more viable organisms or cells.

6. The method of claim 1, 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.

7. The method of claim 1, 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.

8. The method of claim 7, wherein one or more of the reference sequences comprises sequences endogenously present in the cDNA.

9. The method of claim 7, wherein one or more of the reference sequences comprises sequences exogenously added to the cDNA.

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

11. The method of claim 10, 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

12. The method of claim 10, wherein the modified nucleotide comprises one or more non-nucleotide linkers, alkyl chains, or abasic nucleotides.

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

14. The method of claim 1, wherein at least one universal primer further comprises a label.

15. The method of claim 14, 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.

16. The method of claim 1, 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.

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

18. The method of claim 17, 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.

19. The method of claim 18, 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.

20. The method of claim 17, wherein each of the one or more semi-universal primers comprises a unique label.

21. The method of claim 1, wherein multiplex amplifying comprises providing the universal primer in an excess concentration relative to the target-specific primer.

22. The method of claim 21, wherein a universal primer:

target-specific primer concentration ratio ranges from about 5;

1 to about 100;

1.

23. The method of claim 21, wherein a universal primer:

target-specific primer concentration ratio is about 10;

1.

24. The method of claim 1, wherein an annealing temperature of the universal primer is higher than an annealing temperature of the target-specific primer.

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

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

27. The method of claim 1, wherein multiplex amplifying the cDNA comprises attenuating an amplification of abundant target genes.

28. The method of claim 27, wherein attenuating the amplification of abundant target genes comprises using one or more modified target-specific primers.

29. The method of claim 28, wherein the one or more modified target-specific primer comprises a blocking group attached at a 3′

end of the modified target-specific primer.

30. The method of claim 28, wherein the one or more modified target-specific primer comprises one or more abasic nucleotides or mismatch nucleotides.

31. The method of claim 28, 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.

32. The method of claim 28, wherein the one or more modified target-specific primer comprises a blocking group attached at a 3′

end of a reverse target-specific primer.

33. The method of claim 28, 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.

34. The method of claim 28, 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.

35. The method of claim 1, 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.

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

37. The method of claim 35, wherein altering the length comprises inserting nucleotides within a universal primer or a target-specific primer.

38. The method of claim 37, 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.

39. The method of claim 35, wherein altering the length comprises incorporating a non-nucleotide linker into a universal primer or a target-specific primer.

40. The method of claim 35, wherein altering the length comprises cleaving the one or more universal primers or the one or more target-specific primers.

41. The method of claim 35, wherein one or more of the universal primers or one or more of the plurality of target-specific primers comprise semi-universal primers.

42. The method of claim 1, wherein the plurality of amplification products comprises a plurality of labels at predetermined molar ratios.

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

44. The method of claim 42, wherein the plurality of labels is incorporated on a plurality of oligonucleotide primers.

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

46. The method of claim 45, wherein separating the one or more members of the plurality of amplification products comprises performing mass spectrometry.

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

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

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

87. A pool of amplification products prepared by the method of claim 1.

59. 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;

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.

60. The method of claim 59, wherein performing the plurality of multiplexed amplifications comprises combining the plurality of target sequences, one or more target-specific primers, and one or more universal primers

61. The method of claim 60, wherein at least one of the one or more universal primers or one or more target-specific primers comprises a label.

62. The method of claim 61, 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.

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

64. The method of claim 61, wherein the first or second label comprises a high-affinity intercalating dye.

65. The method of claim 60, 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.

66. The method of claim 60, 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.

67. The method of claim 66, 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.

68. The method of claim 60, wherein separating the plurality of multiplexed amplification products comprises shifting the mobility of member amplification products relative to one another.

69. The method of claim 68, 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.

70. The method of claim 59, wherein separating comprises applying each set of multiplex amplification products to a separation platform at different times.

71. The method of claim 59, 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.

72. The method of claim 71, wherein the solid phase assay comprises a bridge amplification or rolling circle amplification.

73. The method of claim 59, wherein one or more of the performing, separating and detecting is performed in a high throughput format.

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

88. A pool of amplification products prepared by the method of claim 59.

75. 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;

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.

76. The method of claim 75, wherein amplifying the plurality of target sequences comprises using one or more universal primers.

77. The method of claim 75, wherein amplifying the plurality of target sequences comprises using one or more target-specific primers.

78. The method of claim 77, wherein the one or more universal primers or the one or more target-specific primers comprise one or more non-nucleotide linkers.

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

80. The method of claim 75, wherein separating and detecting the amplification products is performed using microfluidic devices.

81. The method of claim 75, wherein the high throughput platform comprises an HPLC for separating the amplification products and a mass spectrometer for detecting the amplification products.

82. The method of claim 75, wherein the high throughput platform comprises one or more miniaturized scale platforms.

83. The method of claim 75, 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.

84. The method of claim 75, wherein one or more of the amplifying, separating and detecting steps is performed at a rate of about 1000 samples per hour.

85. The method of claim 75, 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.

86. The method of claim 85, wherein the solid phase amplification assay comprises a bridge amplification or rolling circle amplification.

89. A pool of amplification products prepared by the method of claim 75.

105. The kit of claim 83, further comprising:

e) software for storing and analyzing data obtained from the amplification reactions.

106. The kit of claim 83, wherein the universal-primers comprises labeled primers.

107. The kit of claim 83, wherein the simultaneous amplification of the reference-genes allows quantitation of the amplification products.

90. A system for analyzing gene expression, the system comprising:

a) an amplification module for producing a plurality of amplification products from a pool of target sequences, the amplification module comprising at least one pair of universal primers and at least one pair of target-specific primers;

b) a detection module for detecting one or more members of the plurality of amplification products, wherein the detection module detects a presence, absence, or quantity of the one or more members, and generates a set of gene expression data comprising a plurality of data points; and

c) an analyzing module in operational communication with the detection module, the analyzing module comprising a computer or computer-readable medium comprising one or more logical instructions which organize the plurality of data points into a database and one or more logical instructions which analyze the plurality of data points.

91. The system of claim 90, wherein one or more of the amplification module, the detection module, and the analyzing module a comprise high throughput system.

92. The system of claim 90, wherein the at least one pair of universal primers or the at least one pair of target-specific primers comprise one or more abasic nucleotides.

93. The system of claim 90, wherein the amplification module comprises a unique pair of universal primers for each target sequence.

94. The system of claim 90, wherein the amplification module comprises components to perform 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 amplification reaction, a solution phase amplification reaction, or a combination thereof.

95. The system of claim 90, wherein the detection module comprises a mass spectrometer.

96. The system of claim 90, wherein the detection module comprises an electrophoretic device.

97. The system of claim 90, wherein the one or more logical instructions for analyzing the plurality of data points comprises software for generating a graphical representation of the plurality of data points.

98. The system of claim 90, wherein the one or more logical instructions which analyze the plurality of data points are embodied in system software which performs combinatorial analysis on the plurality of data points.

99. The system of claim 90, wherein the one or more logical instructions for analyzing the plurality of data points comprises software for performing difference analysis upon the plurality of data points.

100. The system of claim 90, the analyzing module further comprising an output file.

101. A composition for preparing a plurality of amplification products from a plurality of mRNA target sequences, the composition comprising:

one or more pairs of universal primers; and

one or more pairs of target-specific primers, wherein the target-specific primers comprise one or more regions complementary to the one or more pairs of universal primers and one or more regions complementary to one or more target mRNA sequences.

102. The composition of claim 101, wherein one or more members of the one or more pairs of universal primers or one or more pairs of target-specific primers comprises a non-nucleotide linkage.

103. The composition of claim 101, wherein one or more members of the one or more pairs of universal primers or one or more pairs of target-specific primers comprise one or more cleavable nucleotides.

104. A kit for obtaining a multiplex set of amplification products of target genes and references-genes, the kit comprising:

a) at least one pair of universal primers;

b) at least one pair of target-specific primers;

c) at least one pair of reference gene-specific primers; and

d) one or more amplification reaction enzymes, reagents, or buffers.