[Immobilization of DNA on microporous membranes using UV-irradiation].

  • 1 January 1992
    • journal article
    • abstracts
    • Vol. 18  (1) , 52-62
Abstract
Various techniques of DNA immobilization onto nitrocellulose and nylon microporous 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.

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