ZINC-IMIDAZOLE POSITIVE - A NEW METHOD FOR DNA DETECTION AFTER ELECTROPHORESIS ON AGAROSE GELS NOT INTERFERING WITH DNA BIOLOGICAL INTEGRITY

摘要

Nucleic acids separated by gel electrophoresis are commonly detected within the gel matrix with ethidium bromide staining, followed by gel irradiation with ultraviolet (UV) light. When the separated nucleic acids are to be recovered for further characterization or use, this methodology is unsuitable (i) because a significant number of chemical lesions to the nucleic acid molecules are caused, heavily compromising their biological activity, and (ii) because of health hazards due to accumulative direct contact with ethidium bromide and exposure to UV-light. As an alternative, for preparative purposes, a new nontoxic detection method employing zinc and imidazole salts is described. After electrophoresis, the gel is first washed in distilled water to substantially remove remaining electrophoresis reagents, then incubated in 40 mM zinc sulfate for 10 min to allow binding of Zn2+ to the DNA. and subsequently washed with distilled water to remove unbound Zn2+ from gel regions devoid of DNA. On soaking in 0.2 M imidazole for a Few minutes, zinc-DNA complexes are visualized as deep-white (positive) stained bands against a slightly opaque background. The sensitivity is similar to that of ethidium bromide. Gels can be kept in distilled water for months without loss of staining. After zinc chelation, e.g. with EDTA, it is Feasible to quantitatively recover chemically intact and biologically active DNA from the gels, as shown by reelectrophoresis and transformation experiments. [References: 5]