Method for high-density microarray medicated gene expression profiling

• US 6,607,885 B1
• Filed: 10/11/2000
• Issued: 08/19/2003
• Est. Priority Date: 10/15/1999
• Status: Expired due to Fees

• Current Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Original Assignee: E. I. du Pont de Nemours and Company (Corteva, Inc.)
• Inventors: Larossa, Robert A., Wei, Yan
• Primary Examiner(s): Horlick, Kenneth R.
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US09/686,383
• Time in Patent Office: 1,042 Days
• Field of Search: 435/5, 435/6, 435/91.1, 435/91.2, 435/252.31, 435/252.33, 435/252.35, 435/252.34, 435/252.5, 536/23.1, 536/24.33, 536/24.3, 536/24.32, 422/50, 422/68.1
• US Class Current: 506/4
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 1/689 for bacteria
• CPC Class Codes: C12Q 2600/158 Expression markers

Abstract

The global effect on genes under different environmental conditions can be determined by a comprehensive gene expression profile. The present invention provides a method to monitor the changes in comprehensive cellular gene expression levels at single length resolution by using a high-density microarray prepared with a comprehensive collection of ORFs of a genome. Under different environmental conditions, directly and indirectly affected genes can be detected as the gene expression levels are induced or repressed in comparison to the control.

Claims

1. A method for identifying gene expression changes within a bacterial species comprising:

(a) providing a comprehensive micro-array synthesized from DNA comprised in a bacterial species;

(b) generating a first set of labeled probes from bacterial RNA, the RNA isolated from the bacterial species of step (a);

(c) hybridizing the first set of labeled probes of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the labeled probe;

(d) measuring the signal generated by the hybridization of the first set of labeled probe to the comprehensive micro-array of step (c);

(e) subjecting the bacterial species of step (a) to a gene expression altering condition whereby the gene expression profile of the bacterial species is altered to produce a modified bacterial species;

(f) generating a second set of labeled probes from bacterial RNA, the RNA isolated from the modified bacterial species of step (e);

(g) hybridizing the second set of labeled probes of step (f) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the labeled probe;

(h) measuring the signal generated by the hybridization of the second set of labeled probes to the comprehensive micro-array of step (g); and

(i) comparing signal generated from the first hybridization to the signal generated from the second hybridization to identify gene expression changes within a bacterial species.

3. A method according to either claim 1 or 2 wherein the bacterial species is selected from the group consisting of enteric bacteria, Bacillus, Acinetobacter, Streptomyces, Methylobacter, Pseudomonas, Rhodobacter and Synechocystis.

4. A method according to either claim 1 or 2 wherein the signal generating label is selected from the group consisting of fluorescent moieties, chemiluminescent moieties, particles, enzymes, radioactive tags.

5. A method according to claim 4 wherein the signal generating label is a fluorescent moiety and is selected from the group consisting of cy3 and cy5.

6. A method according to either claim 1 or 2 wherein the comprehensive micro-array contains at least 75% of all open reading frames in the bacterial species.

7. A method according to claim 6 wherein the comprehensive micro-array contains from about 2000 to about 6000 open reading frames.

8. A method according to either claim 1 or 2 wherein the gene expression altering condition is selected from the group consisting of a condition altering the genotype of the bacterial species, a condition altering the growth of the bacterial species , exposure to mutagens , antibiotics, UV light, gamma-rays, x-rays, phage, macrophages, organic chemicals, inorganic chemicals, environmental pollutants, heavy metals, changes in temperature, changes in pH, conditions producing oxidative damage, DNA damage, anaerobiosis, depletion or addition of nutrients, addition of a growth inhibitor, and desiccation.

11. A method for identifying gene expression changes within a bacterial species according to either claim 1 or 2 providing for quantitating the amount of protein specifying RNA contained within a genome according to a method comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of open reading frames synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each open reading frame;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each open reading frame; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each open reading frame.

12. A method for identifying gene expression changes within a bacterial species according to either claim 1 or 2 providing for quantitating the amount of protein specifying RNA contained within a genome according to a method comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of genes synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each gene;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each gene; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each gene.

13. A method for identifying gene expression changes within a genome according to claim 8 providing for quantitating the amount of protein specifying RNA contained within a genome according to a method comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of open reading frames synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each open reading frame;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each open reading frame; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each open reading frame.

14. A method for identifying gene expression changes within a genome according to claim 8 providing for quantitating the amount of protein specifying RNA contained within a genome according to a method comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of genes synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each gene;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each gene; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each gene.

2. A method for identifying gene expression changes within a bacterial species comprising:

(a) providing a comprehensive micro-array synthesized from DNA comprised in a bacterial species;

(b) generating a first set of fluorescent cDNA from bacterial RNA, the RNA isolated from the bacterial species of step (a);

(c) hybridizing the first set of fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;

(d) measuring the signal generated by the hybridization of the first set of fluorescent cDNA to the comprehensive micro-array of step (c);

(e) subjecting the bacterial species of step (a) to a gene expression altering condition whereby the gene expression profile of the bacterial species is altered to produce a modified bacterial species;

(f) generating a second set of fluorescent cDNA from bacterial RNA, the RNA isolated from the modified bacterial species of step (e);

(g) hybridizing the second set of fluorescent cDNA of step (f) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;

(h) measuring the signal generated by the hybridization of the second set of fluorescent cDNA to the comprehensive micro-array of step (g); and

(i) comparing signal generated from the first hybridization to the signal generated from the second hybridization to identify gene expression changes within a bacterial species.

9. A method for quantitating the amount of protein specifying RNA contained within a genome comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of open reading frames synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each open reading frame;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each open reading frame; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each open reading frame.

10. A method for quantitating the amount of protein specifying RNA contained within a genome comprising:

(a) providing a comprehensive micro-array comprising a multiplicity of genes synthesized from genomic DNA comprised in a prokaryotic or eukaryotic organism;

(b) generating a set of fluorescent cDNA from total or poly-adenylated RNA isolated from the prokaryotic or eukaryotic organism of step (a);

(c) generating a set of fluorescent DNA from genomic DNA isolated from the prokaryotic or eukaryotic organism of step (a);

(d) hybridizing the fluorescent cDNA of step (b) to the comprehensive micro-array of step (a), wherein hybridization results in a first fluorescent signal generated from the fluorescent cDNA for each gene;

(e) hybridizing the fluorescent DNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a second fluorescent signal generated from the fluorescent DNA for each gene; and

(f) dividing, for each open reading frame, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each gene.