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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 and González, E LU orcid (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
; ; ; and
publishing date
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
  • pmid:8783903
  • scopus:0029810921
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'--&gt;3', 3'--&gt;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'--&gt;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}},
}