Analysis of radiation damage of DNA by atomic force microscopy in comparison with agarose gel electrophoresis studies.
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.
Murakami
M
M
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
Hirokawa
H
H
Hayata
I
I
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
D003037:Cobalt Radioisotopes; D004270:DNA, Circular; D004247:DNA