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Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes

Abstract:

We evaluated the use of two radiolabeled λ DNA/Hpa II substrates to detect 5'→3, 3'→5' single and double stranded DNA dependent exonuclease phosphatase activities found as contaminants in restriction and modifying enzyme preparations. Looking for the meaning of the radioactive assays results in a real cloning experience, we performed a cloning simulation assay using the same conditions established for the radioactive assay (enzyme units and pmols of DNA ends). As a result, we found that for degradation percentages of the radioactive DNA substrate per enzyme unit below 0.5, the false positives in the cloning simulation assay were less than 5 %. This conditions could ensure a good performance of the enzyme preparations for cloning experiments. Finally, we described the use of the radiolabeled [γ32P] ATP λ Hpa II DNA substrate to detect 5'→3' single stranded DNA dependent exonuclease and phosphatase contaminating activities in some critical steps of the purification process of the restriction enzyme Kpn I.