Imaging DNA with atomic force microscopy

Why use AFM to image DNA?

Imaging DNA is a key application for atomic force microscopy in life sciences because the AFM outperforms other imaging methods on multiple aspects. With our AFMs and our experience in sample preparation it is the most efficient and cost-effective method to visualize DNA constructs for research and quality control applications (from DNA in buffer to the image below in <30 minutes). In contrast to other microscopes, AFMs directly image DNA dried on a mica substrate, no stains or contrast agents required!

M13 double-stranded DNA with various degrees of supercoiling and some fragmentation

Double-stranded plasmid DNA showing various degrees of supercoiling and a small number of DNA fragments.

What can you do with an AFM?

DNA of the bacteriophage M13, a popular tool in molecular biology, illustrates the different structures. M13 DNA is is commercially available in both single-strand and double-stranded form. Its imaging by AFM shows dramatic differences in secondary structure of ssDNA and dsDNA of the same length, and how storage or buffer conditions can affect their integrity. Imaging DNA constructs can provide critical information in quality control of DNA synthesis, for example in gene therapy.

ssDNA of the M13 bacteriophage with color-indexed topography

ssDNA of the M13 bacteriophage with color-indexed topography, 2x2 um scan

Topography images of single-stranded M13mp18 DNA illustrates how ssDNA folds in tight secondary structures (left: 4×4 um scan, right: 2×2 um).

AFM image of M13 bacteriophage double-stranded DNA, 5x5 um scan, topography image with color indexed height

AFM image of M13 bacteriophage double-stranded DNA, 2x2 um scan, topography image with color indexed height

The double-stranded M13mp18 DNA has the typical appearance of dsDNA plasmids with varying degrees of supercoiling (left: 5×5 um scan, right: 2×2 um).

Correctly stored aliquot of M13 dsDNA with various degrees of supercoiling

Fragmented M13 dsDNA after improper storage at room temperature

AFM can also detect the effects of storage conditions on DNA quality. The left image is the M13 dsDNA made fresh from a frozen aliquot. The right image is the same DNA stored at room temperature for several days, showing an increase in DNA fragments.

AFM of a DNA ladder with DNA lengths from 50 to 1,350 base pairs.

Segmented length measurements of a DNA ladder from 50 to 1,350 base pairs.

Length measurements on a DNA ladder with fragment lengths between 50 and 1,350 base pairs in 50 bp intervals.

Measuring and visualizing M13 ssDNA molecules

AFM data is quantitative in 3D, allowing measurements on single M13 ssDNA molecules.

fork in a DNA fragment with protein caps and line profiles for height measurements

line profiles showing height of DNA (red) and protein caps (black)

Topography image showing an apparent fork in a linear DNA fragment decorated with protein caps. The line profiles show the measured height of the double-stranded DNA (red) and the protein caps (black).

ssDNA of the M13 bacteriophage visualized in 3D

dsDNA of the M13 bacteriophage visualized in 3D

M13 ssDNA and dsDNA visualizations, 3D rendered and shaded.

Literature and protocols

Hansma, H. G.; Revenko, I.; Kim, K.; Laney, D. E. Atomic Force Microscopy of Long and Short Double-Stranded, Single-Stranded and Triple-Stranded Nucleic Acids. Nucleic Acids Res. 1996, 24 (4), 713–720. https://doi.org/10.1093/nar/24.4.713.
Main, K. H. S.; Provan, J. I.; Haynes, P. J.; Wells, G.; Hartley, J. A.; Pyne, A. L. B. Atomic Force Microscopy—A Tool for Structural and Translational DNA Research. APL Bioeng. 2021, 5 (3), 031504. https://doi.org/10.1063/5.0054294.
Lyubchenko, Y. L. Preparation of DNA and Nucleoprotein Samples for AFM Imaging. Micron Oxf. Engl. 1993 2011, 42 (2), 196–206. https://doi.org/10.1016/j.micron.2010.08.011.
Lyubchenko, Y. L.; Shlyakhtenko, L. S. Imaging of DNA and Protein–DNA Complexes with Atomic Force Microscopy. Crit. Rev. Eukaryot. Gene Expr. 2016, 26 (1), 63–96. https://doi.org/10.1615/CritRevEukaryotGeneExpr.v26.i1.70.
Fotiadis, D.; Scheuring, S.; Müller, S. A.; Engel, A.; Müller, D. J. Imaging and Manipulation of Biological Structures with the AFM. Micron 2002, 33 (4), 385–397. https://doi.org/10.1016/S0968-4328(01)00026-9.
Hamon, L., Pastré, D., Dupaigne, P., Le Breton, C., Le Cam, E., & Piétrement, O. (2007). High-resolution AFM imaging of single-stranded DNA-binding (SSB) protein–DNA complexes. Nucleic acids research, 35(8), e58. https://doi.org/10.1093/nar/gkm147
Fuentes-Perez, M. E., Dillingham, M. S., & Moreno-Herrero, F. (2013). AFM volumetric methods for the characterization of proteins and nucleic acids. Methods (San Diego, Calif.), 60(2), 113–121. https://doi.org/10.1016/j.ymeth.2013.02.005