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Technics
Technics employed during the project
Increasing the capacity and versatility of the COMET assay
The COMET Assay with enzyme modification
Comet assay modified for detection of specific lesions with the use of repair endonucleases. The image is of a typical comet (stained with DAPI) from a moderately damaged cell.
Developing assays for DNA repair
DNA repair chips: These ‘chips’ are aimed at measuring the functionality of repair enzymes involved in BER and NER. Two different parallelized and miniaturized in vitro assays on glass support have been set up based on fluorescence detection to assess quantitatively repair activity (Sauvaigo et al. 2004; Guerniou et al. 2005; patents FR0101605 and FR0216435).
OLI Repair Chip
The OLI Repair Chip uses oligonucleotides containing base lesions, attached to a support. The repair activity of a cell extract toward the base lesions is determined upon incubation of the substrate (support + oligos) with the cell extract. At each base lesion site, the decrease in fluorescent signal is linked to the presence of a specific functional enzyme in the extract . Repair of up to twelve lesion types might be followed simultaneously. The OLI Repair Chip is specifically devoted to measurement of glycosylase activities.
PLASMI Repair Chip
The PLASMI Repair Chip is dedicated to measurement of excision/synthesis activities of cell extracts after recognition and removal of specific lesions created in different preparations of plasmid DNA spotted onto a glass surface. This assay provides insights into repair activities of both BER and NER pathways. Lesions are created on the plasmids by physical and chemical treatments. Upon incubation with extract, lesions are eliminated and the synthesis of a new DNA strand allows incorporation of a fluorescent label. Photoproducts, inter- and intra-strand adducts, oxidized and alkylated bases are examples of lesions created on the plasmid DNA.
