TY - JOUR
T1 - Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes
AU - Trujillo, L E
AU - Pupo, E
AU - Miranda, F
AU - Pérez, E
AU - González, E
PY - 1996/1/1
Y1 - 1996/1/1
N2 - 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.
AB - 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.
KW - Bacteriological Techniques
KW - Bacteriophage lambda/genetics
KW - Cloning, Molecular
KW - DNA Modification Methylases/analysis
KW - DNA Restriction Enzymes/analysis
KW - DNA, Viral/metabolism
KW - Deoxyribonuclease HpaII/metabolism
KW - Drug Contamination
KW - Escherichia coli/genetics
KW - Exonucleases/analysis
KW - Phosphoric Monoester Hydrolases/analysis
KW - Radioactive Tracers
KW - Sensitivity and Specificity
M3 - Article
C2 - 8783903
SN - 0187-4640
VL - 38
SP - 31
EP - 37
JO - Revista latinoamericana de microbiologia
JF - Revista latinoamericana de microbiologia
IS - 1
ER -