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

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 - Gonzalez de Valdivia, Ernesto I

PY - 1996

Y1 - 1996

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

KW - Cloning, Molecular

KW - DNA Modification Methylases

KW - DNA Restriction Enzymes

KW - DNA, Viral

KW - Deoxyribonuclease HpaII

KW - Drug Contamination

KW - Escherichia coli

KW - Exonucleases

KW - Phosphoric Monoester Hydrolases

KW - Radioactive Tracers

KW - Sensitivity and Specificity

M3 - Journal article

C2 - 8783903

SN - 0034-9771

VL - 38

SP - 31

EP - 37

JO - Revista Latinoamericana de Microbiologia

JF - Revista Latinoamericana de Microbiologia

IS - 1

ER -