Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis.

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2010-09-23

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Abstract

Atomic force microscopy, which is normally used for DNA imaging to gain qualitative results, can also be used for quantitative DNA research, at a single-molecular level. Here, we evaluate the performance of AFM imaging specifically for quantifying supercoiled and relaxed plasmid DNA fractions within a mixture, and compare the results with the bulk material analysis method, gel electrophoresis. The advantages and shortcomings of both methods are discussed in detail. Gel electrophoresis is a quick and well-established quantification method. However, it requires a large amount of DNA, and needs to be carefully calibrated for even slightly different experimental conditions for accurate quantification. AFM imaging is accurate, in that single DNA molecules in different conformations can be seen and counted. When used carefully with necessary correction, both methods provide consistent results. Thus, AFM imaging can be used for DNA quantification, as an alternative to gel electrophoresis.

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10.1021/jp105603k

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Jiang, Yong, Mahir Rabbi, Piotr A Mieczkowski and Piotr E Marszalek (2010). Separating DNA with different topologies by atomic force microscopy in comparison with gel electrophoresis. J Phys Chem B, 114(37). pp. 12162–12165. 10.1021/jp105603k Retrieved from https://hdl.handle.net/10161/4074.

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Scholars@Duke

Marszalek

Piotr E. Marszalek

Professor in the Thomas Lord Department of Mechanical Engineering and Materials Science

My research focuses on investigating relationships between structural and mechanical properties of biopolymers (polysaccharides, DNA, proteins), which I study at a single molecule level. My main approaches are experimental scanning probe microscopy techniques and computational methods involving Molecular Dynamics simulations and ab initio quantum mechanical calculations. The ultimate goal of this research is to understand the above-mentioned relationships at an atomic level and to apply the knowledge gained towards elucidating basic phenomena such as: molecular recognition that mediates interactions between proteins and sugars, mechanotransduction that underlies mechanical sensing and hearing in all organisms, and protein folding that is fundamental to all biology. Our DNA research is aimed at exploiting atomic force microscopy techniques to develop new ultra-sensitive assays for detecting and examining DNA damage, the process underlying carcinogenesis, and to increase our mechanistic understanding of DNA damage and repair processes. This research, in addition to its basic science aspects will lay a foundation for the future use of AFM technologies in the nanoscale DNA diagnostics with a potential to directly benefit human health.


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