An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity
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An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity. / Vodenkova, Sona; Azqueta, Amaya; Collins, Andrew; Dusinska, Maria; Gaivao, Isabel; Møller, Peter; Opattova, Alena; Vodicka, Pavel; Godschalk, Roger W. L.; Langie, Sabine A. S.
In: Nature Protocols, Vol. 15, 2020, p. 3844–3878.Research output: Contribution to journal › Journal article › Research › peer-review
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TY - JOUR
T1 - An optimized comet-based in vitro DNA repair assay to assess base and nucleotide excision repair activity
AU - Vodenkova, Sona
AU - Azqueta, Amaya
AU - Collins, Andrew
AU - Dusinska, Maria
AU - Gaivao, Isabel
AU - Møller, Peter
AU - Opattova, Alena
AU - Vodicka, Pavel
AU - Godschalk, Roger W. L.
AU - Langie, Sabine A. S.
PY - 2020
Y1 - 2020
N2 - This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.This protocol describes a comet-based in vitro assay for detecting base and nucleotide excision repair activity for use in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues.
AB - This optimized protocol (including links to instruction videos) describes a comet-based in vitro DNA repair assay that is relatively simple, versatile, and inexpensive, enabling the detection of base and nucleotide excision repair activity. Protein extracts from samples are incubated with agarose-embedded substrate nucleoids ('naked' supercoiled DNA) containing specifically induced DNA lesions (e.g., resulting from oxidation, UVC radiation or benzo[a]pyrene-diol epoxide treatment). DNA incisions produced during the incubation reaction are quantified as strand breaks after electrophoresis, reflecting the extract's incision activity. The method has been applied in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues. Once extracts and substrates are prepared, the assay can be completed within 2 d.This protocol describes a comet-based in vitro assay for detecting base and nucleotide excision repair activity for use in cell culture model systems, human biomonitoring and clinical investigations, and animal studies, using isolated blood cells and various solid tissues.
KW - OXIDATIVELY DAMAGED DNA
KW - CANCER PATIENTS
KW - POLYPHENOLIC COMPOUNDS
KW - OCCUPATIONAL-EXPOSURE
KW - INCISION ACTIVITY
KW - EXPRESSION LEVELS
KW - TELOMERE LENGTH
KW - MINERAL FIBERS
KW - CROSS-LINKS
KW - CELLS
U2 - 10.1038/s41596-020-0401-x
DO - 10.1038/s41596-020-0401-x
M3 - Journal article
C2 - 33199871
VL - 15
SP - 3844
EP - 3878
JO - Nature Protocols
JF - Nature Protocols
SN - 1754-2189
ER -
ID: 252467085