Method for high-density microarray medicated gene expression profiling
First Claim
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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.
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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.
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Citations
14 Claims
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1. A method for identifying gene expression changes within a bacterial species comprising:
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(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. - View Dependent Claims (3, 4, 5, 6, 7, 8, 11, 12, 13, 14)
(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.
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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:
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(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.
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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:
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(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.
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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:
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(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.
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2. A method for identifying gene expression changes within a bacterial species comprising:
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(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.
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9. A method for quantitating the amount of protein specifying RNA contained within a genome comprising:
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(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.
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10. A method for quantitating the amount of protein specifying RNA contained within a genome comprising:
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(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.
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Specification