title: Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.
abstract: DNA damage induced with ionizing radiation is considered one of the main causes of cell inactivation. Several methods including gel electrophoresis, pulsed-field gel electrophoresis, neutral filter elution method, neutral sedimentation and electron microscopy have been applied to analyze this type of DNA damage. A new method employing an atomic force microscope (AFM) for nanometer-level-structure analysis of DNA damage induced with gamma-irradiation is introduced in this report. Structural changes of plasmid DNA on a molecular size scale of about 3 kbp were visually analyzed by AFM after irradiation with 60Co gamma-rays at doses of 1.9, 5.6, and 8.3 kGy. Three forms of plasmid DNA, closed circular (intact DNA), open circular (DNA with a single strand break) and linear form (DNA with a double strand break) were visualized by dynamic force mode AFM after gamma-irradiation. The torsional feature of the plasmid DNA was visualized better with AFM than with a transmission electron microscope (TEM). All three forms of plasmid DNA were observed in the sample irradiated with gamma-rays at the dose of 1.9 kGy. Open circular and linear forms were observed in the samples irradiated with gamma-rays at doses of 5.6 and 8.3 kGy, though no closed circular form was observed. A shortening of the length of a linear form of DNA irradiated with 5.6 and 8.3 kGy gamma-rays was observed by AFM. Structural changes of DNA after gamma-irradiation were visualized by AFM at nanometer level resolution. In addition, shortening of the length of the linear form of DNA after radiation exposure was observed by AFM.
authors.affiliation: Division of Radiobiology and Biodosimetry, National Institute of Radiological Sciences, 9-1, Anagawa-4-chome, Inage-ku, 263-8555, Chiba, Japan. drhmmura@nirs.go.jp
authors.forename: M
authors.initials: M
authors.lastname: Murakami
authors.forename: H
authors.initials: H
authors.lastname: Hirokawa
authors.forename: I
authors.initials: I
authors.lastname: Hayata
mesh_terms: D003037:Cobalt Radioisotopes / Q000378:metabolism; D004247:DNA / Q000528:radiation effects* / Q000648:ultrastructure; D004249:DNA Damage; D004270:DNA, Circular / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004587:Electrophoresis, Agar Gel / Q000379:methods*; D005720:Gamma Rays; D018625:Microscopy, Atomic Force / Q000379:methods*; D008854:Microscopy, Electron; D010957:Plasmids / Q000235:genetics
chemical_list: D003037:Cobalt Radioisotopes; D004270:DNA, Circular; D004247:DNA