ATOMIC-FORCE MICROSCOPY OF LONG AND SHORT DOUBLE-STRANDED, SINGLE-STRANDED AND TRIPLE-STRANDED NUCLEIC-ACIDS

Atomic force microscopy (AFM, also called scanning force microscopy) i s proving to be a useful technique for imaging DNA. Thus it is importa nt to push the limits of AFM imaging in order to explore both what typ es of DNA can be reliably imaged and identified and also what substrat es and methods of sample preparation are suitable. The following advan ces in AFM of DNA are presented here. (i) DNA molecules as short as 25 bases can be seen by AFM. The short single-stranded DNAs imaged here (25 and 50 bases long) appeared globular in the AFM, perhaps because t hey are all capable of intramolecular base pairing and because the DNA s were in a Mg(II) buffer, which facilitates intramolecular cross-brid ging. (ii) AFM images in air of short double-stranded DNA molecules, 1 00-200 bp, gave lengths consistent with A-DNA. (iii) AFM images of pol y(A) show both short bent lumpy molecules with an apparent persistence length of 40 nm and long straight molecules with an apparent persiste nce length of 600 nm. For comparison, the apparent persistence length for double-stranded DNA from (phi)X-174 under the same conditions was 80 nm. (iv) Structures believed to be triple-stranded DNA were seen in samples of poly(dA) poly(dT) and poly(dG). poly(dC). These structures were twice as high as double-stranded DNA and the same width. (v) Ent ire molecules of lambda DNA, similar to 16 mu m long, were imaged clea rly in overlapping scans. (vi) Plasmid DNA was imaged on oxidized sili con, although less clearly than on mica.