Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes
Trujillo, L.E.; Pupo, E.; Miranda, F.; Pérez, E.; González, E.
Revista latinoamericana de microbiologia 38(1): 31-37
1996
ISSN/ISBN: 0187-4640 PMID: 8783903 Document Number: 458504
We evaluated the use of two radiolabeled lambda DNA/Hpa II substrates to detect 5'-->3', 3'-->5' single and double stranded DNA dependent exonuclease and 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 stimulation 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 [gamma 33P] ATP lambda 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.