The effect of ethanol on the structure of DNA confined to mica in the presence of Mg2+was examined by varying the ethanol concentration and imaging the DNA by atomic force microscopy. Contour length measurements of the DNA show a transition from all-B-form at 0% ethanol to all-A-form at >25% ethanol. At intermediate ethanol concentrations, contour lengths suggest that individual molecules of air-dried DNA are trapped with mixed compositions of A-form and B-form. The relative composition depends on the ethanol concentration. Fitting the length distributions at intermediate ethanol concentrations to a simple binomial model results in an upper bound estimate for the A-form and B-form domains of approximately 54 bp in the individual molecules. In addition to length changes, the apparent persistence length of DNA decreases with increasing ethanol concentration. At high concentrations of ethanol (>20%), DNA formed several higher order structures, including flower shaped condensates and toroids.
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.
Current techniques used to measure lengths of DNA fragments in atomic force microscope (AFM) images require a user to operate interactive software and execute tedious error-prone cursor selections. An algorithm is proposed which provides an automated method for determining DNA fragment lengths from AFM images without interaction from the computer operator (e.g. cursor selections or mouse clicks). The approach utilises image processing techniques tailored to characteristics of AFM images of DNA fragments. The automated measurements have a mean absolute deviation of less than 1 pixel when compared to manual image-based measurements. The DNA length determined from the histogram of calculated lengths is accurate to within 3% of the actual DNA length in solution. For fragments that are 250 base-pairs long, the precision is estimated to be within 17 nm, which is about 20% of the total length. This precision was confirmed when the algorithm easily resolved fragments in one image that differed by only 17 nm. Fragment sizes up to 2000 base-pairs have been tested and successfully sized. This algorithm is being developed as part of a new solid-state DNA sizing technique for applications such as genotyping and construction of physical genome maps.
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