Method for analyzing gene expression patterns

• US 7,378,236 B1
• Filed: 08/14/1995
• Issued: 05/27/2008
• Est. Priority Date: 06/17/1994
• Status: Expired due to Fees

• Current Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Original Assignee: Board of Trustees of the Leland Stanford Junior University (Stanford University)
• Inventors: Brown, Patrick O., Shalon, Tidhar Dari
• Primary Examiner(s): FORMAN, BETTY J
• Application Number:
  US08/514,875
• Time in Patent Office: 4,670 Days
• Field of Search: 435/6, 435/172.3, 435/78, 435/3, 435/19, 435/80, 536/23.1, 536/24.31, 536/813
• US Class Current: 435/6.16
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2545/101 with an internal standard/control
• CPC Class Codes: C12Q 2565/513 characterised by the pattern of the arrayed oligonucleotides

Abstract

A method and device for detecting or monitoring the treatment status of a selected physiological state or disease condition. The device has a subarray of genes which show a statistically significant change in gene expression level when compared with the control expression levels for that gene. The method involves applying a reporter-labeled messenger nucleic acid fraction to the array in the device, and comparing the pattern of gene expression on the array with that produced by labeled messenger nucleic acid from control cells. Also disclosed is a method of constructing the array.

Claims

1. A method of constructing a subarray of distinct gene sequences whose expression levels are specifically related to differences between test cells relative to control cells, comprising:

(a) obtaining and preparing reporter-labeled copies of messenger nucleic acid from said control cells in a population of control individuals, and from said test cells in a population of test individuals having a shared phenotype that is not present in control individuals, wherein the reporter is compatible with a fluorescence detection system,

(b) applying the reporter-labeled nucleic acids from test and control cells to a microarray of distinct gene sequences, under conditions effective to hybridize the reporter-labeled nucleic acids to complementary genes sequences on the microarray, wherein the microarray comprises at least 1000 distinct gene sequences per cm² and the distinct gene sequences at each position in the microarray correspond to a single nucleic acid molecule of interest and are at least 50 subunits in length, wherein the microarray has a hydrophobic surface formed by the support material or by a coating applied to the support, wherein each said position in the microarray is formed by applying a volume of aqueous reagent solution comprising a distinct gene sequence and wherein said hydrophobic surface prevents spreading of aqueous reagents applied to said surface via reagent bead formation,

(c) comparing the pattern of reporter levels for nucleic acids from the test cells with that of nucleic acids from the control cells,

(d) identifying those test-cell distinct gene sequences on the microarray which show a significant elevation or reduction in reporter levels, when compared with control reporter levels, and

(e) forming a subarray of said test-cell distinct gene sequences.

2. The method of claim 1, wherein each distinct gene sequence is disposed at a separate, defined position in said microarray and is present in a defined amount between about 0.1 femtomole and about 100 nanomoles.

3. The method of claim 1, wherein said distinct gene sequences are obtained from multiple tissue sources.

4. The method of claim 1, wherein the reporter-labeled test-cell nucleic acids from each test individual are applied to a separate microarray, wherein the separate microarrays are composed of the same distinct gene sequences, and said identifying step includes identifying those distinct gene sequences on the separate microarrays which show a statistically significant elevation or reduction in reporter levels, when compared with control levels.

5. The method of claim 1, wherein the reporter-labeled test-cell nucleic acids from the test individuals are pooled and applied to the microarray.

6. The method of claim 1, wherein (i) the test- and control-cell nucleic acids each are labeled with an independently detectable reporter, (ii) the reporter-labeled nucleic acids from the test and control cells are applied to the same microarray, and (iii) said identifying step includes detecting the levels of the two reporters at each gene sequence position on the microarray.

7. The method of claim 1, wherein the distinct gene sequences are expressed sequence tag (EST) genes.

8. The method of claim 7, wherein the microarray includes at least 1000 such EST genes.

9. The method of claim 1, for the construction of a subarray of genes whose gene expression levels in peripheral blood cells are affected by a selected condition, wherein the test cells are peripheral blood cells so affected, and the control cells are peripheral blood cells from normal individuals.

10. The method of claim 1, for the construction of a subarray of genes whose gene expression levels are specifically related to a tumor state, wherein the test cells are neoplastic cells from a selected tissue, and the control cells are normal cells from the same tissue type.

11. The method of claim 1, for the construction of a subarray of genes whose gene expression levels are specifically related to a genetic disease, wherein the test cells are cells from a tissue whose functioning is affected by the disease, and the control cells are cells from the same tissue in a normal individual.

12. The method of claim 1, for the construction of a subarray of genes whose gene expression levels are specifically related to a virus-infected cell, wherein the test cells are virus-infected cells, and the control cells are uninfected cells of the same cell type from a non-infected individual.

13. The method of claim 1, for the construction of a subarray of genes whose gene expression levels are specifically related to immune cells under immunological challenge, wherein the test cells are immunologically challenged immune cells, and the control cells are non-challenged immune cells of the same cell type.

14. The method of claim 1, for the construction of a subarray of genes whose gene expression levels are specifically related to drug response in a given test cell type, wherein the test cells are cells exposed to the drug, and the control cells are cells of the same type not exposed to the drug.

15. The method of claim 1, wherein the number of positions in the microarray of distinct gene sequences is about 1000 or more.

16. The method of claim 15, wherein the number of positions in the microarray of distinct gene sequences is about 10,000 or more.

17. The method of claim 1, wherein the test-cell distinct gene sequences show an average of at least 2 fold elevation or reduction in reporter levels, when compared with control reporter levels.

18. A method of comparing gene expression patterns between test cells and control cells comprising:

(a) constructing a subarray using the method of claim 1,

(b) preparing reporter-labeled copies of messenger nucleic acids obtained from test cells associated with a physiological state or disease condition, wherein the reporter is compatible with a fluorescence detection system,

(c) contacting said reporter-labeled nucleic acids with said subarray containing only distinct gene sequences which are characterized by a statistically significant increase or decrease in gene expression level, when compared with the level of gene expression of the same genes in control cells, said contacting being carried out under conditions effective to hybridize said nucleic acid to complementary-sequence genes in said subarray, and wherein the distinct gene sequences at each position in the subarray correspond to a single nucleic acid molecule of interest,

(d) detecting levels of reporter-labeled nucleic acids associated with the distinct gene sequences in said subarray,

(e) using the detected levels to identify a pattern of gene expression, and

(f) comparing said pattern of gene expression with a known pattern of gene expression associated with control cells.

19. The method of claim 18, wherein the distinct gene sequences in the microarray are expressed sequence tag (EST) genes.

20. The method of claim 18, wherein the reporter-labeled nucleic acids are fluorescent-labeled nucleic acids.

21. The method of claim 18, for detecting or monitoring the treatment status of a selected disease state that affects a given tissue or organ type, and said test cells are from the same tissue or organ.

22. The method of claim 18, for detecting or monitoring the status of a selected disease state affecting a given tissue or organ type and a selected type of peripheral blood cells, wherein said test and control cells are the selected peripheral blood cells.

23. The method of claim 18, wherein the distinct gene sequences are characterized by an average of at least 2 fold increase or decrease in gene expression level, when compared with the level of gene expression of the same genes in control cells.

24. A method of determining the relative amounts of a polynucleotide in first and second mixtures of polynucleotides, comprising:

(a) labeling the polynucleotides from the first and second mixtures with first and second reporters, respectively, where the first and second reporters are independently detectable and are compatible with a fluorescence detection system,

(b) concurrently contacting both mixtures of labeled polynucleotides, under hybridization conditions, with a microarray of distinct polynucleotides located at discrete positions on the surface of a substrate comprising a density of about 1,000 or more positions per cm²,

wherein the distinct polynucleotides at each position in the microarray correspond to a single nucleic acid molecule of interest and are at least 50 subunits in length,

wherein the microarray has a hydrophobic surface formed by the support material or by a coating applied to the support, wherein each said position in the microarray is formed by applying a volume of aqueous reagent solution comprising a distinct gene sequence and

wherein said hydrophobic surface prevents spreading of aqueous reagents applied to said surface via reagent bead formation, and

(c) detecting fluorescence associated with the first and second reporters at each position in the microarray as a measure of the relative amounts of the corresponding polynucleotides in the first and second mixtures.

25. The method of claim 24, wherein the substrate is glass.

26. The method of claim 24, wherein the first and second mixtures are obtained from first and second cell types.

27. The method of claim 24, for use in determining the relative levels of expression of a gene in a first and second cell type, wherein the labeled polynucleotides are obtained from mRNAs of the first and second cell types.

28. The method of claim 24, for use in determining the relative levels of expression of a gene in a first and second cell type, wherein (i) the labeled polynucleotides are obtained from mRNAs of the first and second cell types, and (ii) the distinct polynucleotides on the microarray are cDNAs.

29. The method of claim 24, wherein the substrate is non-porous.

30. The method of claim 24, wherein the substrate is relatively hydrophobic.

31. The method of claim 24, wherein the number of positions in the microarray of distinct polynucleotides is about 1000 or more.

32. The method of claim 31, wherein the number of positions in the microarray of distinct polynucleotides is about 10,000 or more.