Method for high-density microarray mediated gene expression profiling

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.

What is claimed is:  
     
         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.    
     
     
         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.    
     
     
         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.  
     
     
         9 . A method for identifying gene expression changes within a genome comprising: 
 (a) providing a comprehensive micro-array synthesized from DNA comprised in a prokaryotic or eukaryotic speices;    (b) generating a control set of fluorescent cDNA from total or polyadenylated RNA, the RNA isolated from the species of step (a), the fluorescent cDNA comprising at least one first fluorescent label and at least one different second fluorescent label;    (c) mixing the control set of fluorescent cDNA labeled with the at least one first label with the control set of fluorescent cDNA labeled with the at least second first label to for a dual labeled control cDNA;    (d) hybridizing the dual labeled control set of fluorescent cDNA of step (c) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;    (e) measuring the signal generated by the hybridization of the dual labeled control set of fluorescent cDNA to the comprehensive micro-array of step (c);    (f) subjecting the prokaryote or eukaryote of step (a) to a gene expression altering condition whereby the gene expression profile of the prokaryote or eukaryote is altered to produce a modified prokaryote or eukaryote;    (g) generating an experimental set of fluorescent cDNA from total or polyadenylated RNA, the RNA isolated from the modified prokaryote or eukaryote of step (e), the fluorescent cDNA comprising the first fluorescent label and the different second fluorescent label to step (b);    (h) mixing the experimental set of fluorescent cDNA labeled with the at least one first label with the experimental set of fluorescent cDNA labeled with the at least second first label to form a dual labeled experimental cDNA;    (i) hybridizing the experimental set of fluorescent cDNA of step (h) to the comprehensive micro-array of step (a), wherein hybridization results in a detectable signal generated from the fluorescent cDNA;    (j) measuring the signal generated by the hybridization of the second set of fluorescent cDNA to the comprehensive micro-array of step (g); and    (k) comparing signal generated from the dual labeled control hybridization with the dual labeled experimental hybridization to identify gene expression changes within a prokaryotic or eukaryotic species.    
     
     
         10 . A method according to  claim 9  wherein the first fluorescent label and the second fluorescent label is independently selected from the group consisting of cy3 and cy5.  
     
     
         11 . A method according to  claim 9  wherein the prokaryotic or eukaryotic genome is comprised within an organism selected from the group consisting of enteric bacteria, Bacillus, Acinetobacter, Streptomyces, Methylobacter, Pseudomona, cyanobacteria, yeasts, filamentous fungi, plant cells and animal cells.  
     
     
         12 . A method according to  claim 11  wherein yeast are selected from the group consisting of Saccharomyces, Zygosaccharomyces, Kluyveromyces, Candida, Hansenula, Debaryomyces, Mucor, Pichia and Torulopsis.  
     
     
         13 . A method according to  claim 11  wherein cyantobacteria are selected from the group consisting of Rhodobacter and Synechocystis.  
     
     
         14 . A method according to  claim 11  wherein filamentous fungi are selected from the group consisting of Aspergillus and Arthrobotrys.  
     
     
         15 . 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 from, 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.    
     
     
         16 . 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 from, the first fluorescent signal into the second fluorescent signal to provide a quantitated measure of the amount of protein specifying RNA for each gene.    
     
     
         17 . 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 either  claim 15  or  16 .  
     
     
         18 . 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  claim 15  or  16 .