An overview of the visualisation and quantitation of low and high MW DNA adducts using the trapped in agarose DNA immunostaining (TARDIS) assay.
The ability to detect and quantify specific DNA adducts benefits genome stability research, drug development and the evaluation of environmental mutagens. The trapped in agarose DNA immunostaining (TARDIS) assay was developed as a means of detecting and quantifying melphalan and cisplatin DNA adducts at the single-cell level and has since been adapted to quantify topoisomerase-DNA complexes. The method relies on salt-detergent extraction of agarose-embedded cells. Genomic DNA and any covalently attached molecules remain in place in the agarose, while other cellular constituents are removed. Drug-DNA or topoisomerase-DNA complexes are then detected and quantified by sensitive immunofluorescence using adduct-specific antibodies. Here, we give a perspective of the TARDIS assay including a comparison with other methods for quantifying topoisomerase-DNA covalent complexes and provide technical details required to set up and perform the assay.
Institute for Cell and Molecular Biosciences, Medical School, Catherine Cookson Building, Framlington Place, Newcastle University, Newcastle upon Tyne, NE2 4HH, UK.
Ian G
IG
Cowell
Michael J
MJ
Tilby
Caroline A
CA
Austin
D004247:DNA / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis* / Q000378:metabolism; D026942:DNA Topoisomerases / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012685:Sepharose / Q000737:chemistry*
D018736:DNA Adducts; D004247:DNA; D012685:Sepharose; D026942:DNA Topoisomerases