Identification and Characterization Solutions for Oxidation Resistance Genes
Oxidative damage to DNA is a constant threat to genomic integrity and results when DNA is attacked by reactive oxygen species (ROS), a byproduct of normal aerobic metabolism. To mitigate oxidative DNA damage, cells have two lines of defense. The first line of defense is provided by enzymes that detoxify ROS. The second line of defense is provided by DNA repair enzymes that repair oxidative DNA damage.
Creative Bioarray provides our customers with solutions to identify and characterize gene products that prevent or repair oxidative DNA damage. In addition, we perform subcellular localization of the identified gene products. This solution is expected to provide a scientific basis for the study and treatment of oxidative stress-related diseases in humans.
Identification of Oxidation Resistance Genes
Our identification scheme is based on papillation assay performed on E. coli and human cDNA libraries.
We use an E. coli strain that is inherently unable to repair oxidation-induced DNA damage, and use the lacZ allele as a reporter gene for oxidative mutagenesis in the bacterial strain. Colonies in the oxidized DNA damage state will appear dark blue, but colonies repaired by the oxidation resistance genes product will be white but with a small amount of blue papillation.
Library transformation. Transform E. coli cell stock with cDNA library first by any standard method of E. coli transformation or electroporation.
Colony selection. The surface of most colonies will be covered with hundreds of dark blue papillae. Colonies that lack papillae or contain reduced numbers of papillae are obvious. These colonies contain potential candidate cDNAs that could provide antimutagenic function by preventing ROS from acting on DNA or repairing the damage they produce.
Confirmation that the antimutant phenotype is reproducible and photographic documentation of the colonies is performed.
Characterization of Oxidation Resistance Genes
Qualitative determination of mutant resistance strength
The mutation resistance intensity of each candidate was ranked according to the number of papillae present on individual colonies. This roughly reflects the mutation frequency and is a consistent feature of each clone.
Candidate clones can be classified into three groups based on the strength of their mutator-resistant phenotype.
- Strongly mutant resistant (≤5 papillation)
- Moderate antimutants (5-20 papillation)
- Weakly mutant resistant (≥20 papillation)
Quantification of mutation frequency
For specific candidate cDNAs, purified plasmid DNA is used to re-transform the E. coli strain. The lac reversion frequency is also determined.
Subcellular Localization of Candidate cDNAs
Since the above assays can detect genes whose products act in any subcellular compartment of eukaryotic cells, an important step in characterizing new proteins is to determine in which subcellular compartment the protein acts normally.
The localization region of the candidate cDNA is initially delineated by bioinformatics and biological data.
In vitro cells (e.g. HeLa cells) are treated with an oxidizing agent (e.g. H2O2), a suitable probe is selected based on the candidate cDNA biological data, and then immunofluorescence microscopy (or other suitable means) is used to determine the localization of the candidate cDNA in human cells.
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All services and products are for scientific use only, not for medical use！
