TY - JOUR
T1 - Time-lapse imaging of conformational changes in supercoiled DNA by scanning force microscopy
AU - Nagami, Fuji
AU - Zuccheri, Giampaolo
AU - Samorì, Bruno
AU - Kuroda, Reiko
N1 - Funding Information:
This work was partly supported by Programmi Biotecnologie Legge 95/95 (MURST 5%) and MURST PRIN (ex quota 40%) to G.Z. and B.S. F.N. acknowledges financial support from the Marubun Research Promotion Foundation (Tokyo, Japan) and from the Italian government. The authors are also grateful to Dr. M. Ishikawa at JRCAT for the use of equipment.
PY - 2002/1/15
Y1 - 2002/1/15
N2 - 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.
AB - 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.
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U2 - 10.1006/abio.2001.5435
DO - 10.1006/abio.2001.5435
M3 - Article
C2 - 11779108
AN - SCOPUS:0037081220
SN - 0003-2697
VL - 300
SP - 170
EP - 176
JO - Analytical Biochemistry
JF - Analytical Biochemistry
IS - 2
ER -