Time-lapse imaging of conformational changes in supercoiled DNA by scanning force microscopy.
Most of the scanning force microscopy (SFM) images of supercoiled DNA on untreated mica thus far reported have not shown tight plectonemic structure seen by electron microscopy, but instead less coiled molecules and sometimes a partly "condensed" state with intimate chain-chain interactions. By observing time-lapse images of conformational changes of DNA induced by decreasing ionic strength of imaging buffer in solution SFM, we could show that the process of water rinsing, an indispensable step for preparation of dried samples, may be responsible for some of the conformational anomalies in the images previously reported. We have studied several protocols to observe supercoiled DNA molecules by SFM and discuss the merits and the demerits. Images obtained following uranyl acetate treatment may be ideal for the detection of DNA damage, as the supercoiled and nicked forms are easily distinguishable.
Nagami
Fuji
F
Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, 3-8-1 Komaba, Meguro-ku, Tokyo 153-8902, Japan.
Zuccheri
Giampaolo
G
Samorì
Bruno
B
Kuroda
Reiko
R
D004278:DNA, Superhelical / Q000737:chemistry* / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D018625:Microscopy, Atomic Force / Q000379:methods*; D009690:Nucleic Acid Conformation / Q000187:drug effects; D009942:Organometallic Compounds / Q000494:pharmacology; D009994:Osmolar Concentration; D012996:Solutions
D004278:DNA, Superhelical; D009942:Organometallic Compounds; D012996:Solutions; C005460:uranyl acetate