Methods for constructing subarrays and uses thereof

• US 20010051344A1
• Filed: 07/17/2001
• Published: 12/13/2001
• Est. Priority Date: 06/17/1994
• Status: Active Grant

• Current Assignee: Patrick O. Brown, Tidhar Dari Shalon
• Original Assignee: Patrick O. Brown, Tidhar Dari Shalon
• Inventors: Brown, Patrick O., Shalon, Tidhar Dari
• Granted Patent: US 7,625,697 B2
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00274 Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
• CPC Class Codes: B01J 2219/00308 interchangeably mounted in racks or blocks
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00576 fluorophore
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00619 using hydrophilic or hydrophobic regions
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00677 Ex-situ synthesis followed by deposition on the substrate
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01L 2300/0838 Capillaries
• CPC Class Codes: B01L 2400/025 tapping tip on substrate
• CPC Class Codes: B01L 3/0244 using pins
• CPC Class Codes: B01L 3/0248 : Prongs, quill pen type dispenser
• CPC Class Codes: B82Y 30/00 : Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: C12Q 1/6883 : for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/158 : Expression markers
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 2035/1037 : Using surface tension, e.g. pins or wires
• CPC Class Codes: G01N 35/109 : with two horizontal degrees of freedom

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 level expression 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 genes whose gene expression levels are specifically related to the differences between test cells relative to control cells, comprising

obtaining and preparing reporter-labeled copies of messenger nucleic acid from such control cells in a population of control individuals, and from such test cells in a population of test individuals having a shared phenotype that is not present in control individuals,

applying reporter-labeled nucleic acid from test and control cells to an array of at least 10² distinct gene sequences, under conditions effective to hybridize the nucleic acid to complementary-sequence genes on the array,

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

identifying those test-cell genes on the array which show a significant elevation or reduction in reporter levels, when compared with control reporter levels, and

forming a subarray of such identified genes.

2. The method of

claim 1

, wherein said array contains at least 10³ distinct gene sequences.

3. The method of

claim 1

, wherein said array is a microarray formed on a single, contiguous substrate, at a density of at least 10³ distinct gene sequences per cm² surface area.

4. The method of

claim 3

, wherein each distinct gene sequence is disposed at a separate, defined position in said array and is present in a defined amount between about 0.1 femtomole and 100 nanomoles.

5. The method of

claim 1

, wherein said gene sequences are obtained from multiple tissue sources.

6. The method of

claim 1

, wherein the test-cell nucleic acids from each test individual are applied to a separate array, and said identifying includes identifying those genes on the separate arrays which show a statistically significant elevation or reduction in reporter levels, when compared with control levels.

7. The method of

claim 1

, wherein the test-cell nucleic acids from the test individuals are pooled and applied to an array.

8. The method of

claim 1

, wherein the test- and control-cell nucleic acids have different fluorescent reporter, the nucleic acids from the test- and control cells are applied to the same array, and said identifying includes detecting the difference levels of the two reporters at each gene sequence on the array.

9. The method of

claim 1

, wherein the genes are expressed sequence tag (EST) genes.

10. The method of

claim 9

, wherein the array includes at least 10³ such EST genes.

11. The method of

claim 1

, for use in constructing 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 are peripheral blood cells from normal individuals.

12. The method of

claim 1

, for use in constructing 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.

13. The method of

claim 1

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

14. The method of

claim 1

, for use in constructing 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.

15. The method of

claim 1

, for use in constructing 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.

16. The method of

claim 1

, for use in constructing a subarray of genes whose gene expression levels are specifically related to drug response in a given test cell type, and the control cells the control cells are cells of the same type not exposed to the drug.

17. A method of detecting or monitoring the treatment status of a selected physiological state or disease condition, comprising

preparing reporter-labeled messenger nucleic acid obtained from test cells associated with the physiological state or disease condition,

contacting said nucleic acid with a subarray of genes which are characterized by a statistically significant increase or decrease in gene level expression, when compared with the level of gene expression of the same gene in control cells, said contacting being carried out under conditions effective to hybridize said nucleic acid species to complementary-sequence genes in said array,

detecting the levels of reporter associated with the genes in said subarray,

using said detected levels to construct a pattern of gene expression, and

comparing said gene-expression pattern with a known pattern of gene expression associated with control cells.

18. The method of

claim 17

, wherein the genes in the array are expressed sequence tag (EST) genes.

19. The method of

claim 17

, wherein the reporter-labeled nucleic acids are fluorescent-labeled nucleic acids.

20. The method of

claim 17

, 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.

21. The method of

claim 17

, for detecting or monitoring the treatment status of a selected disease state effecting a given tissue or organ type and a selected type of peripheral blood cells, and said test and control cells are the selected peripheral blood cells.

22. A gene-array device for use in detecting or monitoring the treatment status of a selected disease condition, said device comprising

a substrate, and

a subarray of genes which show a statistically significant increase or a statistically significant decrease in gene level expression when compared with the level of gene expression in a control cell type.

23. The device of

claim 22

, wherein the genes in the array are expressed sequence tag (EST) genes.

24. The device of

claim 22

, wherein each gene in the array is present in a defined amount between about 0.1 femtomole and 100 nanomoles.