Ultraviolet cross-linking of DNA to microporous membranes
S. M. Kalachikov, V. A. Adarichev, G. M. Dymshits
Institute of Cytology and Genetics, Siberian Division, Russian Academy of Sciences, Novosibirsk
Abstract: Various techniques of DNA immobilization onto nitrocellulose and nylon micropo-rous membranes have been compared. Despite a strong primary adsorption of DNA onto these membranes during blotting procedures, poor retention of the target DNA and low hybridization signals are obtained after hybridization and washings. Covalent cross-linking of DNA upon UV irradiation leads to a quantitative immobilization of target DNA. Quantum yield of DNA photoimmobilization estimated for a single base in DNA is about 10-4. UV irradiation dose sufficient for immobilization of DNA fragment of a known length can be calculated by the formula Ilc=(22.3±4.8)c/l, where l is the DNA fragment length (in base pairs), c is the DNA part (%) to be immobilized. The UV irradiation dose about 0.6-0.8 kJ/m2 is optimal for most hybridization experiments. Doses higher than 0.8—1 kJ/m2 may cause a loss in the hybridization efficiency. Under optimal immobilization conditions, hybridization signals increasing five-fold for nitrocellulose membranes and fifty-fold for uncharged nylon membranes as compared with baking these membranes in vacuum.
Russian Journal of Bioorganic Chemistry 1992, 18 (1):52-62