Visual quantification of DNA double-strand breaks in bacteria.
In this paper, we describe a method for the visualization of double-strand breaks in a single electrostretched Escherichia coli DNA molecule. We also provide evidence that electrostretched or migrated DNA under neutral microgel electrophoresis conditions is made up of individual chromosomes. Using the neutral microgel electrophoresis technique, DNA migration (stretching) was measured and the number of DNA double-strand breaks were counted following exposure of E. coli cells to 0, 12.5, 25, 50, or 100 rad of X-rays. The use of an intense fluorescent dye, YOYO and custom-made slides have helped us in visualizing individual bacterial DNA molecules. Bacterial DNA appears similar in structure compared to electrostretched DNA from human lymphocytes. We were able to detect changes in DNA migration (stretching) induced by an X-ray dose as low as 12.5 rad and an increase in the number of DNA breaks induced by a dose as low as 25 rad. The extent of DNA migration and number of breaks were directly correlated to X-ray dosage.
Singh
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Department of Bioengineering, University of Washington, Bioelectromagnetics Research Laboratory, Box 357962, Seattle, WA, USA. narendra@u.washington.edu
Stephens
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Singh
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Lai
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D001583:Benzoxazoles; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000032:analysis* / Q000528:radiation effects; D004587:Electrophoresis, Agar Gel / Q000379:methods; D004926:Escherichia coli; D005456:Fluorescent Dyes; D006801:Humans; D008214:Lymphocytes; D008856:Microscopy, Fluorescence; D009690:Nucleic Acid Conformation; D011806:Quinolinium Compounds; D011829:Radiation Dosage
D001583:Benzoxazoles; D004269:DNA, Bacterial; D005456:Fluorescent Dyes; D011806:Quinolinium Compounds; C075296:1,1'-((4,4,7,7-tetramethyl)-4,7-diazaundecamethylene)bis-4-(3-methyl-2,3-dihydro(benzo-1,3-oxazole)-2-methylidene)quinolinium