Protocol for the quantitative assessment of DNA concentration and damage (fragmentation and nicks).
An ultrasensitive protocol is presented for the quantitative assessment of fragmented and nicked dsDNA using PicoGreen and consists of four methods. The first quantifies the concentration of DNA, whereas the second (quantitative complement of the Comet assay) quantifies the degree of DNA fragmentation seen in a typical DNA agarose electrophoresis gel. Both methods have sensitivity of 5 pg of DNA. The third method (quantitative counterpart of the electrophoresis-based qualitative apoptotic and necrotic DNA assays) quantifies the polyethylene glycol-fractionated small-size (0-1 kb) fragmented DNA. This method also detects up to 5 pg of damaged DNA and requires a minimum sample of quantity 0.2 ml of 2.5 microg ml(-1). The fourth method measures the percentage of DNA nicks by alkaline DNA unwinding and requires up to 15 pg of DNA sample. The time required for processing 10 DNA samples is 1/2, 1, 13 and 1 h for the first, second, third and fourth method, respectively.
Georgiou
Christos D
CD
Department of Biology, Section of Genetics, Cell Biology and Development, University of Patras, 26100-Patras, Greece. c.georgiou@upatras.gr
Papapostolou
Ioannis
I
Grintzalis
Konstantinos
K
D000818:Animals; D002417:Cattle; D004249:DNA Damage; D004260:DNA Repair; D003850:Deoxyribonuclease I; D013950:Thymus Gland
D003850:Deoxyribonuclease I