Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes
(1996) In Revista latinoamericana de microbiologia 38(1). p.7-31- Abstract
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... (More)
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.
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- author
- Trujillo, L E ; Pupo, E ; Miranda, F ; Pérez, E and González, E LU
- publishing date
- 1996-01-01
- type
- Contribution to journal
- publication status
- published
- keywords
- Bacteriological Techniques, Bacteriophage lambda/genetics, Cloning, Molecular, DNA Modification Methylases/analysis, DNA Restriction Enzymes/analysis, DNA, Viral/metabolism, Deoxyribonuclease HpaII/metabolism, Drug Contamination, Escherichia coli/genetics, Exonucleases/analysis, Phosphoric Monoester Hydrolases/analysis, Radioactive Tracers, Sensitivity and Specificity
- in
- Revista latinoamericana de microbiologia
- volume
- 38
- issue
- 1
- pages
- 7 - 31
- publisher
- Mexico Asociacion Mexicana De Microbiologia
- external identifiers
-
- scopus:0029810921
- pmid:8783903
- ISSN
- 0187-4640
- language
- English
- LU publication?
- no
- id
- 35dc2e86-b0ac-4066-98c3-e1b93daa426a
- alternative location
- https://europepmc.org/abstract/med/8783903
- date added to LUP
- 2019-02-08 13:57:23
- date last changed
- 2024-01-15 14:13:28
@article{35dc2e86-b0ac-4066-98c3-e1b93daa426a, abstract = {{<p>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.</p>}}, author = {{Trujillo, L E and Pupo, E and Miranda, F and Pérez, E and González, E}}, issn = {{0187-4640}}, keywords = {{Bacteriological Techniques; Bacteriophage lambda/genetics; Cloning, Molecular; DNA Modification Methylases/analysis; DNA Restriction Enzymes/analysis; DNA, Viral/metabolism; Deoxyribonuclease HpaII/metabolism; Drug Contamination; Escherichia coli/genetics; Exonucleases/analysis; Phosphoric Monoester Hydrolases/analysis; Radioactive Tracers; Sensitivity and Specificity}}, language = {{eng}}, month = {{01}}, number = {{1}}, pages = {{7--31}}, publisher = {{Mexico Asociacion Mexicana De Microbiologia}}, series = {{Revista latinoamericana de microbiologia}}, title = {{Correspondence between radioactive and functional methods in the quality control of DNA restriction and modifying enzymes}}, url = {{https://europepmc.org/abstract/med/8783903}}, volume = {{38}}, year = {{1996}}, }