The enzyme-modified comet assay: Enzyme incubation step in 2 vs 12-gels/slide systems.
8-Hydroxy-2'-Deoxyguanosine
/ analysis
Alkylating Agents
/ toxicity
Cell Line
Comet Assay
/ instrumentation
DNA Damage
DNA-Formamidopyrimidine Glycosylase
/ pharmacology
Dose-Response Relationship, Drug
Electrophoresis, Agar Gel
Gels
Humans
Lymphocytes
/ drug effects
Reproducibility of Results
Time Factors
Titrimetry
/ methods
12 minigels
2 gels
Comet assay
Enzyme incubation
Formamidopyrimidine DNA glycosylase
Titration
Journal
Mutation research. Genetic toxicology and environmental mutagenesis
ISSN: 1879-3592
Titre abrégé: Mutat Res Genet Toxicol Environ Mutagen
Pays: Netherlands
ID NLM: 101632149
Informations de publication
Date de publication:
Sep 2019
Sep 2019
Historique:
received:
09
08
2018
revised:
09
10
2018
accepted:
18
11
2018
entrez:
29
9
2019
pubmed:
29
9
2019
medline:
24
3
2020
Statut:
ppublish
Résumé
The enzyme-modified comet assay is a commonly used method to detect specific DNA lesions. However, still a lot of errors are made by many users, leading to dubious results and even misinterpretations. This technical note describes some critical points in the use of the enzyme-modified comet assay, such as the enzyme concentration, the time of incubation, the format used and the equipment. To illustrate the importance of these conditions/parameters, titration experiments of formamidopyrimidine DNA glycosylase (Fpg) were performed using the 2 gels/slide and the 12 minigels/slide formats (plus the 12-Gel Comet Assay Unit™). Incubation times of 15 and 30 min, and 1 h were used. Results showed that the 12 minigels/slide system requires a lower volume and concentration of Fpg. A longer time of incubation has a bigger impact when using such format. Moreover, the paper describes how to perform and interpret a titration experiment when using the enzyme-modified comet assay.
Identifiants
pubmed: 31561901
pii: S1383-5718(18)30309-7
doi: 10.1016/j.mrgentox.2018.11.005
pii:
doi:
Substances chimiques
Alkylating Agents
0
Gels
0
8-Hydroxy-2'-Deoxyguanosine
88847-89-6
DNA-Formamidopyrimidine Glycosylase
EC 3.2.2.23
Types de publication
Comparative Study
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
402981Informations de copyright
Copyright © 2018 Elsevier B.V. All rights reserved.