A fast comet assay variant for solid tissue cells. The assessment of DNA damage in higher plants.
The single-cell gel electrophoresis or comet assay, under high alkaline conditions, detects low levels of DNA damage. In it, broken DNA migrates from the nucleus to the anode providing images similar to comets. To adapt this assay to solid tissue cells, nuclei were directly obtained from Allium cepa L. roots. The surface of each single fresh sharply cut meristem was exposed to a small drop of 50 mM Sörensen buffer at pH 6.8, placed on a regular agarose-coated slide. By immediately adding low melting point agarose at 30 degrees C, nuclei resulted embedded in agarose. A final layer of this agarose ended the preparative steps. Conventionaly prepared leukocytes were used as a control. The treatment with detergent (lysis step of the conventional assay) proved to be unnecessary for the nude nuclei. A 20 min-long electrophoresis (at 0.65 V. cm-1, 230 mA and 10 degrees C) was more sensitive than a 10 min-long one for detecting the differential response of plant nuclei to 2 and 4 Gy of gamma-irradiation. A short fixation in methanol transformed the preparations into semi-permanent ones, without altering their later DNA staining by ethidium bromide. The use of instantaneously isolated nuclei simplifies and expands the use of this technique to any eukaryotic cell from solid tissues.
Navarrete
M H
MH
Departamento de Biología, Universidad Autónoma de Madrid, Canto Blanco, Spain. navarret@ccuam3.sdi.uam.es
Carrera
P
P
de Miguel
M
M
de la Torre
C
C
D000490:Allium / Q000528:radiation effects*; D000704:Analysis of Variance; D002467:Cell Nucleus / Q000528:radiation effects / Q000648:ultrastructure; D004247:DNA / Q000097:blood / Q000528:radiation effects; D004249:DNA Damage; D018744:DNA, Plant / Q000032:analysis / Q000528:radiation effects; D004586:Electrophoresis / Q000379:methods; D005720:Gamma Rays; D006801:Humans; D007962:Leukocytes / Q000528:radiation effects*; D018519:Meristem; D008856:Microscopy, Fluorescence; D009152:Mutagenicity Tests / Q000379:methods
D018744:DNA, Plant; D004247:DNA