Scanning force microscopy of DNA deposited onto mica: equilibration versus kinetic trapping studied by statistical polymer chain analysis.
This paper reports a study of the deposition process of DNA molecules onto a mica surface for imaging under the scanning force microscope (SFM). Kinetic experiments indicate that the transport of DNA molecules from the solution drop onto the surface is governed solely by diffusion, and that the molecules are irreversibly adsorbed onto the substrate. A statistical polymer chain analysis has been applied to DNA molecules to determine the deposition conditions that lead to equilibrium and those that result in trapped configurations. Using the appropriate conditions, DNA molecules deposited onto freshly cleaved mica, are able to equilibrate on the surface as in an ideal two-dimensional solution. A persistence length of 53 nm was determined from those molecules. DNA fragments that were labeled on both ends with a horseradish peroxidase streptavidin fusion protein were still able to equilibrate on the surface, despite the additional protein-surface interaction. In contrast, DNA molecules deposited onto glow-discharged mica or H+-exchanged mica do not equilibrate on the surface. These molecules adopt conformations similar to those expected for a simple projection onto the surface plane, suggesting a process of kinetic trapping. These results validate recent SFM application to quantitatively analyze the conformation of complex macromolecular assemblies deposited on mica. Under equilibration conditions, the present study indicates that the SFM can be used to determine the persistence length of DNA molecules to a high degree of precision.
Rivetti
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C
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Guthold
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M
Bustamante
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C
D000327:Adsorption; D000538:Aluminum Silicates; D004247:DNA / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D018625:Microscopy, Atomic Force / Q000379:methods*; D008956:Models, Chemical; D009010:Monte Carlo Method; D009690:Nucleic Acid Conformation; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000737:chemistry
D000538:Aluminum Silicates; D011506:Proteins; D004247:DNA; C011934:mica