Solid-State DNA Sizing by Atomic Force Microscopy

Abstract

Atomic force microscopy (AFM) allows rapid, accurate, and reproducible visualization of DNA adsorbed onto solid supports. The images reflect the lengths of the DNA molecules in the sample. Here we propose a solid-state DNA sizing (SSDS) method based on AFM as an analytical method for high-throughput applications such as finger-printing, restriction mapping, +/- screening, and genotyping. For this process, the sample is first deposited onto a solid support by adsorption from solution. It is then dried and imaged under ambient conditions by AFM. The resulting images are subjected to automated determination of the lengths of the DNA molecules on the surface. The result is a histogram of sizes that is similar to densitometric scans of DNA samples separated on gels. A direct comparison of SSDS with agarose gel electrophoresis for +/- screening shows that it produces equivalent results. Advantages of SSDS include reduced sample size (i.e., lower reagent costs), rapid analysis of single samples, and potential for full automation using available technology. The high sensitivity of the method also allows the number of polymerase chain reaction cycles to be reduced to 15 or less. Because the high signal-to-noise ratio of the AFM allows for direct visualization of DNA-binding proteins, different DNA conformations, restriction enzymes, and other DNA modifications, there is potential for dramatically improving the information content in this type of analysis.

Single molecule transcription profiling with AFM

“…The mean length in bp for pools of six different linear DNA molecules are plotted as dark squares (n = 10-40 molecules). Backbone contour length in nanometres is converted to bp using the nominal pitch of duplex beta DNA (0.33 nm/bp) [189]. Fragments range in size from 230 to 4731 bp (90-1561 nm).…”

“…One key difference between the study by Feng et al [189] and the current one is that in their work individual molecules were deposited from solution rather than being generated as in situ fragments from larger DNA molecules. In terms of surface chemistry, the current method differs in that it includes increased surface fixation avidity, which enables the use of higher ionic strength washing solutions.…”

“…In spite of its simplicity, backbone contour length appears remarkably accurate for the molecule sizes tested (∼300-20 000 bp) [167,168,[186][187][188][189]. While the conditions varied among studies, single measurement sizing accuracy (defined here as population CV) better than ±2-5% was reported for most cases for distances larger than 1000 bp.…”

“…While the conditions varied among studies, single measurement sizing accuracy (defined here as population CV) better than ±2-5% was reported for most cases for distances larger than 1000 bp. Only three studies presented data for shorter distances and only Fang et al [189] have reported analysis of fragments shorter than 500 bp. For these smaller fragments, the sizing accuracy appears to be between ±7% and ±10%.…”

“…In terms of surface chemistry, the current method differs in that it includes increased surface fixation avidity, which enables the use of higher ionic strength washing solutions. These differences in technique have been reported to alter the observed chain length of DNA, which is an anionic polymer [189]. To better characterize measurement accuracy in our system, a series of six DNA fragments, 230 to 4731 bp, were measured using the AFM to determine backbone contour length.…”