Accuracy of AFM measurements of the contour length of DNA fragments adsorbed on mica in air and in aqueous buffer

• Published in: Ultramicroscopy Vol. 92; no. 3; pp. 151 - 158
• Main Authors: Sanchez-Sevilla, Albert, Thimonier, Jean, Marilley, Monique, Rocca-Serra, José, Barbet, Jacques
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.08.2002; Elsevier Science
• Subjects: Air; Aluminum Silicates; Atomic force microscope (AFM); Atomic force microscopes; Buffers; Deoxyribonuclease EcoRI - metabolism; Deoxyribonucleases, Type II Site-Specific - metabolism; DNA - chemistry; DNA contour length; Exact sciences and technology; Image processing; Image Processing, Computer-Assisted; Instruments, apparatus, components and techniques common to several branches of physics and astronomy; Microscopy, Atomic Force - methods; Nucleic Acid Conformation; Physics; Plasmids - genetics; Plasmids - metabolism; Scanning probe microscopes, components and techniques; Image processing; 07.05.Pj; DNA contour length; 87.64Dz Subject index terms: 018; Atomic force microscope (AFM); 87.15By; 07.79.Lh; Molecular fragments; Atomic force microscopy; Sample preparation; DNA; Length measurement; Measuring methods; Calibration; Measurement accuracy; Experimental study; Edge detection; Mica
• ISSN: 0304-3991; 1879-2723
• DOI: 10.1016/S0304-3991(02)00128-6

The measurement by atomic force microscope of the contour length of DNA fragments adsorbed on mica has been made as accurate as possible by revisiting the different steps of image acquisition and processing. In air, the DNA helical rise was estimated at 2.97±0.15 Å per base pair (bp) (mean±standard deviation) by imaging a 648-bp DNA fragment and 2.95±0.14 Å per bp for a 1115-bp fragment. This confirms earlier observations suggesting that drying DNA fragments on mica in the presence of nickel induces limited conformational changes. At this point the exact nature of these conformational changes remains unknown. Simple hypotheses are the transconformation of stretches of the DNA molecules to the A-form of the double helix or alteration of the helix structure at the points of contact between DNA and mica. By contrast, in aqueous buffer, the measured helical rise was 3.14±0.15 Å per bp for the 648-bp fragment and 3.17±0.13 Å per bp for the 1115-bp fragment. Thus, measured helical rises do not depend on the fragment length and are significantly shorter than the 3.38 Å per bp measured by crystallography, but close to the 3.18 Å per bp found in NMR studies. These findings are discussed with respect to discrepancies in earlier results published in the literature.