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US 2003/0113710 A1Published application
Methods for identifying genes essential to the growth of an organism
OwnerChristine Marie Debouck, Jeffrey L. Mooneyinventors
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Abstract
The present invention provides methods for identifying genes essential to the growth of an organism using a grid prepared from a genomic library of a selected organism. Genes identified as essential by this method and proteins encoded thereby are also provided. In addition, methods of using these genes and proteins encoded thereby are disclosed.
Claims22
A method of identifying genes essential to growth of a selected organism comprising:
- (a) preparing a genomic library of a selected organism;
- (b) providing a plurality of identical grids, each grid comprising a surface on which is immobilized at predefined regions on said surface a plurality of defined materials derived from the genomic library;
- (c) mutagenizing the selected organism;
- (d) growing a test culture comprising mutagenized selected organism and a control culture comprising non-mutagenized selected organism under a set of defined conditions;
- (e) harvesting surviving cells from the cultures;
- (f) extracting and isolating DNA from harvested cells of the test culture;
- (g) extracting and isolating RNA or DNA from harvested cells of the control culture;
- (h) generating labeled polynucleotide probes from the isolated DNA of the test culture;
- (i) generating labeled polynucleotide probes from the isolated RNA or DNA of the control culture;
- (j) hybridizing the labeled probes generated from the isolated DNA of the test culture to a first identical grid to produce a test hybridization pattern;
- (k) hybridizing the labeled probes generated from the isolated RNA or DNA of the control culture to a second identical grid to produce a control hybridization pattern;
- (l) comparing the hybridization patterns to identify genes essential for growth of the selected organism;
- and
- (m) confirming that said identified gene is essential for growth of the selected organism.
11 more elements
A method of identifying genes essential to growth of a selected organism by identifying conditionally lethal mutant genes, which comprises:
- (a) preparing a genomic library of a selected organism:
- (i) in an integration vector;
- or (ii) in an expression vector;
- (b) providing a grid comprising a surface on which is immobilized at predefined regions on said surface a plurality of defined materials derived from the genomic library;
- (c) mutagenizing the selected organism;
- (d) growing the mutagenized organism under permissive and non-permissive conditions to identify mutagenized organisms containing conditionally lethal mutant genes;
- (e) transforming such organisms containing said conditionally lethal mutant genes with the genomic library of step (a);
- (f) growing the transformed cells under the same non-permissive conditions as step (d) to identify transformed cells in which conditionally lethal mutant genes have been complemented;
- (g) harvesting surviving cells;
- (h) extracting and isolating DNA from the harvested cells;
- (i) generating labeled polynucleotide probes from the isolated DNA;
- (j) hybridizing the labeled probes generated from the isolated DNA to a grid, whereby such probes that hybridize to the grid identify genes essential for growth of the selected organism.
9 more elements
Dependencies are inferred from the claim text.