Short unligated sticky ends enable the observation of circularised DNA by atomic force and electron microscopies.
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.
Révet
B
B
Laboratoire de Microscopie Cellulaire et Moléculaire, CNRS URA 147, Institut Gustave-Roussy, F-94805 Villejuif Cedex, France. bmrevet@igr.fr
Fourcade
A
A
D002413:Cations, Divalent / Q000494:pharmacology; D004247:DNA / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry / Q000648:ultrastructure*; D019281:Dimerization; D004587:Electrophoresis, Agar Gel; D008274:Magnesium / Q000494:pharmacology; D018625:Microscopy, Atomic Force; D008854:Microscopy, Electron; D009942:Organometallic Compounds / Q000494:pharmacology
D002413:Cations, Divalent; D004270:DNA, Circular; D009942:Organometallic Compounds; C005460:uranyl acetate; D004247:DNA; D004262:DNA Restriction Enzymes; D008274:Magnesium