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Abstract
It has been shown that thermal-treatment at 100°C can denature deoxyribonucleic acid (DNA), yet this does not cause it to break down completely. To clarify the risk of gene pollution from thermal-treated recombinant DNA, the renaturation characteristics of thermal-denatured plasmid pET-28b and its persistence in aquatic environments were investigated. The results revealed that the double-stranded structure and transforming activity of the thermal-treated plasmid DNA could be recovered even if the thermal-treatment was conducted at 120°C. The presence of sodiumchloride (NaCl) and ethylenediamine tetraacetic acid (EDTA) led to the increase of renaturation efficiency of the denatured DNA. When thermal-treated plasmid DNA was discharged into simulated aquatic environments with pH values from 5 to 9, it showed a longer persistence at pH 7 and 8 than that at 5, 6 and 9; however, the denatured plasmid DNA could persist for more than 33 min at any pH. Moreover, a higher ionic strength further protected the thermal-denatured plasmids from degradation in the simulated aquatic environment. These results indicated that when the thermal-treated DNA was discharged into an aquatic environment, it might not break down completely in a short period. Therefore, there is the potential for the discarded DNA to renature and transform, which might result in gene pollution.
Published Version
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