Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes.
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.
Trujillo
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LE
Molecular Biology Department, Center for Genetic Engineering and Biotechnology, (CIGB), Havana City, Cuba.
Pupo
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E
Miranda
F
F
Pérez
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González
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E
D001431:Bacteriological Techniques; D010582:Bacteriophage lambda / Q000235:genetics; D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000032:analysis / Q000378:metabolism / Q000592:standards*; D004262:DNA Restriction Enzymes / Q000032:analysis / Q000378:metabolism / Q000592:standards*; D004279:DNA, Viral / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D004340:Drug Contamination; D004926:Escherichia coli / Q000235:genetics; D005092:Exonucleases / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis; D011849:Radioactive Tracers; D012680:Sensitivity and Specificity
D004279:DNA, Viral; D011849:Radioactive Tracers; D015254:DNA Modification Methylases; D005092:Exonucleases; D004262:DNA Restriction Enzymes; D019003:Deoxyribonuclease HpaII; D010744:Phosphoric Monoester Hydrolases