DNA on Mica—A Model Procedure for Nucleic Acid Binding

DNA Binding Theory

DNA and mica are both negatively charged, and so it is necessary to modify the mica surface or the DNA counter ion to allow binding. The counterion method is done by adsorbing the DNA onto the mica in the presence of a divalent (+2 charged) ion, like Ni2+. The divalent ion will serve as a counterion on the negatively charged DNA backbone and will also provide additional charge to bind the mica. This is shown schematically below:

Figure 1: Negatively charged DNA may be bound to negatively charged mica in the presence of divalent counterions, such as Ni⁺².

DNA Binding Procedure

The following procedure is adapted from these sources:

• Dunlap, D.D., A. Maggi, M.R. Soria & L. Monaco (1997) “Nanoscopic Structure of DNA Condensed for Gene Delivery.” Nucl. Acids Res. 25, 3095.
• Kasas, S., N.H. Thomson, B.L. Smith, H.G. Hasma, X. Zhu, M. Guthold, C. Bustamante, E.T. Kool, M. Kashlev & P.K. Hasma (1997) “Escherichia coli RNA polymerase activity observed using atomic force microscopy.” Biochemistry 36, 461.
• Lyubchenko, Y.L. & L.S. Shlyakhtenko (1997) “Direct Visualization of Supercoiled DNA in situ with Atomic Force Microscopy.” Proc. Natl. Acad. Sci. USA 94, 496.

Many other references regarding DNA imaging are listed in the Biological Applications Bibliography; contact Bruker for a copy.

1. Obtain the required materials:
   • Mica substrates
   • DNA: BlueScript II SK9(+) double stranded plasmid DNA, 2961 base pairs, 1 mg/ml in 10 mM Tris, 1 mM ethylenediaminetetraacetic acid (EDTA) from Stratagene, La Jolla, CA.
   • Buffer solution: 10 mM HEPES and 5 mM NiCl2 pH 7.6 (for loose binding and air imaging), or NiCl₂ (for tight binding and fluid imaging)
2. Dilute DNA in buffer solution to a final concentration of 2.5 ng/µl.
3. Glue a piece of mica to a metal support as described in Sample Preparation.
4. Cleave the mica substrate with a piece of adhesive tape.
5. Place 30 µl of the DNA solution in the center of the mica disk. The DNA will bind to the mica within 1 minute.
6. Load the prepared sample onto the AFM scanner and assemble the fluid cell. It may be helpful to wait for the temperature of the buffer to stabilize (20 minutes or more) before imaging.

The sample is now ready for TappingMode imaging.

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