DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication
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DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication. / Nikolova, Teodora; Göder, Anja; Parplys, Ann; Borgmann, Kerstin.
in: Methods Mol Biol, Jahrgang 1510, 2017, S. 103-113.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - DNA Fiber Spreading Assay to Test HDACi Effects on DNA and Its Replication
AU - Nikolova, Teodora
AU - Göder, Anja
AU - Parplys, Ann
AU - Borgmann, Kerstin
PY - 2017
Y1 - 2017
N2 - DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental genotoxic agents or cancer drugs. The method relies on the detection of incorporated thymidine analogues during DNA synthesis in the S phase of the cell cycle by indirect immunofluorescence. Here, we describe the procedure established in our laboratories for sequential pulse labeling of human cells with 5-chloro-2'-deoxyuridine (CldU) and 5-iodo-2'-deoxyuridine (IdU), cell lysis, and DNA fiber spreading on slides and sequential immunodetection of the incorporated thymidine analogues by primary antibodies recognizing specifically CldU or IdU alone. We describe also the laser scanning imaging, classification, and measurement of the detected DNA fiber tracks. The obtained quantitative data can be evaluated statistically to reveal the immediate or long-term effects of DNA-damaging agents, DNA repair inhibitors, and epigenetic modulators like HDAC inhibitors on DNA replication in normal and tumor cells.
AB - DNA fiber spreading assay is an invaluable technique to visualize and follow the spatial and temporal progress of individual DNA replication forks. It provides information on the DNA replication progress and its regulation under normal conditions as well as on replication stress induced by environmental genotoxic agents or cancer drugs. The method relies on the detection of incorporated thymidine analogues during DNA synthesis in the S phase of the cell cycle by indirect immunofluorescence. Here, we describe the procedure established in our laboratories for sequential pulse labeling of human cells with 5-chloro-2'-deoxyuridine (CldU) and 5-iodo-2'-deoxyuridine (IdU), cell lysis, and DNA fiber spreading on slides and sequential immunodetection of the incorporated thymidine analogues by primary antibodies recognizing specifically CldU or IdU alone. We describe also the laser scanning imaging, classification, and measurement of the detected DNA fiber tracks. The obtained quantitative data can be evaluated statistically to reveal the immediate or long-term effects of DNA-damaging agents, DNA repair inhibitors, and epigenetic modulators like HDAC inhibitors on DNA replication in normal and tumor cells.
U2 - 10.1007/978-1-4939-6527-4_8
DO - 10.1007/978-1-4939-6527-4_8
M3 - SCORING: Journal article
C2 - 27761816
VL - 1510
SP - 103
EP - 113
JO - Methods Mol Biol
JF - Methods Mol Biol
SN - 1064-3745
ER -