Testing the quality of electron microscope mapping data for DNA molecules with sequence-specific ligands.
A procedure for the testing of Electron Microscope (EM) mapping data for DNA molecules with site-specific bound ligands is suggested. The difficulty of distinguishing DNA molecule ends on electron micrographs indicates that their true orientations are not known. This in turn presents problems in obtaining correct maps relating to their alignment, and complicates checking the maps' validity. For these reasons a computer simulation of the EM study of double-stranded DNA molecules with site-specific bound ligands was carried out. The knowledge of the true orientations of the simulated DNA molecules allowed us to examine their final orientations after alignment. We used the number of improper-oriented molecules as the quantitative measure of the map quality. Detailed investigation based on this parameter permitted us to invent the criterion for the map validity, and to suggest the procedure for the testing of alignment of real DNA molecules. This procedure implies multiple randomization of initial orientations of the DNA molecules and minute analysis of the final maps. Most of the molecular, statistical and experimental parameters inherent to EM investigation of site-specific binding, such as the number of specific binding sites (N), the mean number of bound ligands (A), the length of the DNA molecules (L), the specific/non-specific ratio of binding (K), together with the standard deviation of DNA molecule lengths (HL) were tested for their influence upon the quality of EM mapping data. An empirical equation for the ultimate values of these parameters has been found, allowing us to predict the success of EM mapping.
Podtelezhnikov
A A
AA
Institute of Molecular Genetics Russian Academy of Sciences, Moscow.
Kurakin
A V
AV
Vologodskii
A V
AV
Cherny
D I
DI
D000465:Algorithms; D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008024:Ligands; D008854:Microscopy, Electron / Q000379:methods*
D008024:Ligands; D004247:DNA