Riesner D, Steger G, Zimmat R, Owens R A, Wagenhöfer M, Hillen W, Vollbach S, Henco K
Institut für Physikalische Biologie, Heinrich-Heine-Universität, Düsseldorf, Federal Republic of Germany.
Electrophoresis. 1989 May-Jun;10(5-6):377-89. doi: 10.1002/elps.1150100516.
Temperature-gradient gel electrophoresis (TGGE) is applied to analyze conformational transitions and sequence variations of nucleic acids and protein-nucleic acid interactions. A linear and highly reproducible temperature-gradient is established perpendicular or parallel to the direction of the electrophoresis. The instrument consists of an electrically insulated metal plate, which is heated at one edge and cooled at the other edge by two thermostating baths and is used as an ancillary device for commercial horizontal gel electrophoresis instruments. Biopolymers are separated in TGGE according to size, shape and thermal stability of their conformational transitions. If the temperature-gradient is established perpendicular to the electrophoresis, monomolecular conformational transitions of nucleic acids show up as continuous transition curves; strand-separation leads to discontinuous transitions. In the studies on viroid RNA it was shown that natural circular viroid RNA undergoes one highly cooperative transition detected by TGGE as a drastic retardation in mobility. Oligomeric replication intermediates of viroids exhibit coexisting structures which could not be detected by any other technique. Double-stranded satellite RNA from cucumber mosaic virus is a mixture of sequence variants, all of which have the identical length of 335 nucleotides. In TGGE six different strains were resolved. Sequence variants of viroids were analyzed by hybridizing viroid RNA to (-)strand viroid RNA transcripts from viroid cDNA clones. Sequence variations lead to mismatches in the double strands and thereby to a shift of the transition curve to lower temperature. Mutations in plasmids, particularly in cloned inserts, were detected by mixing plasmids of two different clones, linearizing, denaturing, renaturing, and searching for shifts in the transition curves, which are generated by mismatch-formation during the renaturation of (+)- and (-)strands from different clones. Examples are given for different viroid clones and HIV-clones from one and the same patient. In another example, clones with point mutations from site-directed mutagenesis are analyzed and selected by TGGE. TGGE is also applied to study the effect of amino acid exchanges in the Tet repressor from E. coli on the thermal stability of the repressor and on the mode of binding of the repressor to the operator DNA. The results are discussed under the aspect that TGGE may be applied as routine analytical laboratory procedure.
温度梯度凝胶电泳（TGGE）用于分析核酸的构象转变、序列变异以及蛋白质 - 核酸相互作用。在垂直或平行于电泳方向上建立线性且高度可重复的温度梯度。该仪器由一块电绝缘金属板组成，其一端由两个恒温浴加热，另一端冷却，用作商业水平凝胶电泳仪器的辅助装置。生物聚合物在TGGE中根据其构象转变的大小、形状和热稳定性进行分离。如果温度梯度垂直于电泳方向建立，核酸的单分子构象转变表现为连续的转变曲线；链分离导致不连续转变。在类病毒RNA的研究中表明，天然环状类病毒RNA经历一次高度协同的转变，通过TGGE检测为迁移率的急剧减慢。类病毒的寡聚复制中间体呈现共存结构，这是任何其他技术都无法检测到的。来自黄瓜花叶病毒的双链卫星RNA是序列变体的混合物，所有变体的长度均为335个核苷酸。在TGGE中分辨出六种不同的菌株。通过将类病毒RNA与来自类病毒cDNA克隆的（ - ）链类病毒RNA转录本杂交来分析类病毒的序列变体。序列变异导致双链中的错配，从而使转变曲线向较低温度偏移。通过混合两个不同克隆的质粒、线性化、变性、复性，并寻找转变曲线的偏移来检测质粒中的突变，特别是克隆插入片段中的突变，这种偏移是由来自不同克隆的（ + ）链和（ - ）链复性过程中形成错配产生的。给出了来自同一患者的不同类病毒克隆和HIV克隆的示例。在另一个示例中，通过TGGE分析和选择来自定点诱变的点突变克隆。TGGE还用于研究大肠杆菌中Tet阻遏物的氨基酸交换对阻遏物热稳定性以及阻遏物与操纵子DNA结合模式的影响。在TGGE可作为常规分析实验室程序这一方面对结果进行了讨论。
