A simple and sensitive pulsed field gel electrophoresis protocol to study 50 kb apoptotic DNA fragmentation in human lymphocytes.
DNA fragmentation of 50 kb is observed in apoptotic human lymphocytes as measured with pulsed field gel electrophoresis (PFGE). Standard PFGE assay involves embedding of cells into agarose blocks followed by lysis in the presence of proteinase K. In this study, we modified the PFGE protocol by omitting the proteinase K. In this study, we modified the PFGE assay by omitting the proteinase K and changing lysis solution according to the method of anomalous viscosity time dependence (AVTD). The conditions of PFGE were adjusted aiming to compress apoptotic fragments, increasing sensitivity and the number of samples that could be loaded on the same gel. Lymphocytes were irradiated with gamma-rays and apoptotic fragmentation of DNA was determined by PFGE using standard lysis with proteinase K and lysis protocol from AVTD method. Both protocols of lysis resulted in the same pattern of DNA fragments. The yield of radiation-induced apoptotic fragmentation was higher with the AVTD protocol of lysis. The novel PFGE protocol is simple and relatively non-expensive, requires only 7 h running time and gives a possibility to analyse simultaneously up to 69 samples in the same gel. The sensitivity of our protocol provides reproducible detection of 50 kb fragmentation after irradiation of human lymphocytes with 5 cGy of gamma-rays. In 2 of 6 donors tested, this DNA fragmentation was detected at dose on 2 cGy. The novel protocol can be used for quantification of 50 kb apoptotic fragments induced by different agents including low dose ionising radiations, chemicals and electromagnetic fields.
Belyaev
I Ya
IY
Department of Genetic and Cellular Toxicology, Stockholm University, S-106 91, Stockholm, Sweden. Igor.Belyaev@genetics.su.se
Harms-Ringdahl
M
M
D053938:DNA Fragmentation; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D005720:Gamma Rays; D006801:Humans; D008214:Lymphocytes / Q000528:radiation effects / Q000648:ultrastructure*; D012680:Sensitivity and Specificity