The use of α-DNA as an internal standard so the detection and quantitation of DNA damage in specific genes using Southern blotting

摘要

Modified Southern blotting assays are powerful techniques for analysing DNA damage and repair in specific genes caused by. for example, ultra-violet (UV) light (I). or the anti-cancer agents mechlorethamine (HN2) (2) and cisplutin (3). The assay relies upon the availability of enzymatic or chemical reactions which can convert damaged bases to single strand breaks. In the case of UV-induced pyrimidine dinners or cisplatin lesions, strand break formation is catalysed by the enzymes T4 endonuclease V and ABC excinuclease respectively (3.4). N-alkylpurines formed by HN2 are converted to strand breaks by neutral depurination followed by alkaline hydrolysis (2). In all these methodologies. DNA from treated ceils is digested with a restriction enzyme, cleaved at damage sites, denatured, electrophoresed. Southern blotted and hybridised to a specific probe. DNA damage is determined as a function of the signal, from treated DNA relative to an untreated control.