Measurement of DNA synthesis and strand breaks using alkaline sucrose density gradient centrifugation.
Alkaline sucrose density gradient (ASDG) centrifugation is probably an only method to detect elongation of "pulse-labeled" replication products in cells. If the cells are pulse-labeled after being exposed to some DNA-damaging agents, their "post-replication repair" can be measured by ASDG technique. With non-damaged cells, normal replication in replicon size can be observed, too. In addition, the method is also applicable to measure single strand breaks. We have modified this classical method to reproducibly detect very long single-stranded DNA at the megabase level. Here, the protocols are optimized to DT40 cells.
Yamada
Kouichi
K
The National Institute of Health and Nutrition, Toyama, Shinjuku-ku, 162-8636 Tokyo, Japan.
Takezawa
Jun
J
D000818:Animals; D002460:Cell Line; D002498:Centrifugation / Q000379:methods*; D002645:Chickens; D004249:DNA Damage; D004261:DNA Replication; D013395:Sucrose
D013395:Sucrose