Short unligated sticky ends enable the observation of circularised DNA by atomic force and electron microscopies

Abstract

A comparative study of the stabilisation of DNA sticky ends by divalent cations was carried out by atomic force microscopy (AFM), electron microscopy and agarose gel electrophoresis. At room temperature, molecules bearing such extremities are immediately oligomerised or circularised by addition of Mg2+or Ca2+. This phenomenon, more clearly detected by AFM, requires the presence of uranyl salt, which stabilises the structures induced by Mg2+or Ca2+. DNA fragments were obtained by restriction enzymes producing sticky ends of 2 or 4 nucleotides (nt) in length with different guanine plus cytosine (GC) contents. The stability of the pairing is high when ends of 4 nt display a 100% GC-content. In that case, 95% of DNA fragments are maintained circular by the divalent cations, although 2 nt GC-sticky ends are sufficient for a stable pairing. DNA fragments with one blunt end and the other sticky appear as dimers in the presence of Mg2+. Dimerisation was analysed by varying the lengths and concentrations of DNA fragments, the base composition of the sticky ends, and also the temperature. Our observation provides a new powerful tool for construction of inverted dimers, and circularisation, ligation analysis or short bases sequence interaction studies.

Atomic Force Microscopy Study of the Effects of Mg2+ and Other Divalent Cations on the End-to-End DNA Interactions

“…In the presence of 200 mM Na + , the end stability seen with 10 mM Mg 2+ is lost ( Figure 1G). These data confirm that Mg 2+ is needed for complementary end-strand stabilization, as proposed by Révet and Fourcade (8). Our data show that the effect depends on the Mg 2+ concentration so that the yield of circles approaches 18% at 50 mM MgCl 2 solution.…”

The relations between neurite development and the subcellular structures of hippocampal neuron somata

“…Although AFM has been widely used to study the subcellular morphological characteristics of many biological samples and cells (Dufrene, 2001;Haga et al, 2000;McKiernan et al, 2000;Oberleithner et al, 2000;Ohta et al, 2002;OÕReilly et al, 2001;Radler et al, 2000;Ret and Fourcad, 1998;Yamane et al, 2000;Yamashina and Katsumata, 2000) including neurons (Melling et al, 2001;Nagayama et al, 1996;Scholl et al, 2000;Tojima et al, 1998), neurites (Ohshiro et al, 2000), synaptic vesicles and membranes (Sritharan et al, 1998), the interior structures of the exocytic apertures of nerve cells (Tojima et al, 2000), and so on, there are still several difficulties in imaging fine subcellular parts of the cells with AFM, including the immobilization of neurons, the softness of the samples, and the salt crystals formed from the fixative solution that would cover the fine structures if the samples were faxed for stiffness. In our present study, three methods were taken to solve these problems.…”

A nucleoprotein-hairpin in transcription regulation

“…was treated with 0.02% uranyl acetate solution and then rinsed with Millipore water and dried with filter paper (Revet and Fourcade, 1998). …”