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US 6,607,885 B1Granted
Method for high-density microarray medicated gene expression profiling
AssigneeE. I. Du Pont de Nemours and Company
InventorsRobert A. Larossa, Yan Wei
Life of this patent
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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.
Claims14
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.
7 more elements
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.
7 more elements
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.
4 more elements
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.
4 more elements
Dependencies are inferred from the claim text.