Révet B, Fourcade A
Laboratoire de Microscopie Cellulaire et Moléculaire, CNRS URA 147, Institut Gustave-Roussy, F-94805 Villejuif Cedex, France.
Nucleic Acids Res. 1998 May 1;26(9):2092-7. doi: 10.1093/nar/26.9.2092.
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.
通过原子力显微镜（AFM）、电子显微镜和琼脂糖凝胶电泳对二价阳离子稳定DNA粘性末端进行了比较研究。在室温下，带有此类末端的分子通过添加Mg2+或Ca2+会立即发生寡聚或环化。这种现象通过AFM能更清晰地检测到，需要存在铀酰盐，它能稳定由Mg2+或Ca2+诱导的结构。通过限制性内切酶获得了长度为2或4个核苷酸（nt）、鸟嘌呤加胞嘧啶（GC）含量不同的粘性末端的DNA片段。当4 nt的末端GC含量为100%时，配对稳定性很高。在这种情况下，95%的DNA片段被二价阳离子维持为环状，尽管2 nt的GC粘性末端足以实现稳定配对。在Mg2+存在下，一端为平端而另一端为粘性末端的DNA片段会呈现为二聚体。通过改变DNA片段的长度和浓度、粘性末端的碱基组成以及温度来分析二聚化。我们的观察为反向二聚体的构建、环化、连接分析或短碱基序列相互作用研究提供了一种新的有力工具。
