Improvement of separation method of fragmented DNA from an apoptotic cell DNA sample for the quantitation using agarose gel electrophoresis.
In order to quantify fragmented DNA extracted from apoptotic cells, we devised a separation method which condenses fragmented DNA into a small band, separating it from larger-size DNA with agarose gel electrophoresis. Calf thymus DNA and standard fragmented DNA were loaded onto 1.0% gel for 0.5, 1.0, 1.5 and 2.0 cm length, and onto 0.7, 1.0, 1.5 and 2.0% of gels for 1 cm length. DNA was then extracted from gel slices with the UltraClean 15 DNA Purification Kit, and estimated by measuring fluorescence intensity using Hoechst No.33258 dye. DNA recovery from the gel showed constant values regardless of the amount of loaded DNA up to 1 microg/assay, and a plot of loaded DNA amounts vs. the DNA amount yielded resulted in a strait line in any gel concentration used. Our results show the best conditions to estimate DNA fragmentation rates in apoptotic cells in which fragmented DNA was separated from thymus DNA by loading on 1.0% gel for 1.0 cm length. We used our method to estimate fragmentation rates in DNA fractions extracted from apoptotic human cervical fibroblast, amnion epithelial and chorion laeve trophoblast cells by stimulation with actinomycin D. The results show that DNA fragmentation rates in these cells were consistent with the electrophoretic patterns of the DNA samples shown by their photographs.
Ohyama
K
K
Department of' Biochemistry, Tokyo University of Pharmacy & Life Science, Hachioji, Japan. ohyamak@ps.toyaku.ac.jp
Enn
P
P
Uchide
N
N
Bessho
T
T
Yamakawa
T
T
D017209:Apoptosis; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis* / Q000302:isolation & purification*; D053938:DNA Fragmentation; D003609:Dactinomycin / Q000494:pharmacology; D004587:Electrophoresis, Agar Gel; D004847:Epithelial Cells / Q000737:chemistry / Q000187:drug effects; D005321:Extraembryonic Membranes / Q000166:cytology / Q000187:drug effects; D005260:Female; D005347:Fibroblasts; D006801:Humans; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D011247:Pregnancy; D014327:Trophoblasts / Q000737:chemistry / Q000187:drug effects
D019384:Nucleic Acid Synthesis Inhibitors; D003609:Dactinomycin; D004247:DNA