Abrams E S, Murdaugh S E, Lerman L S
Department of Biology, MIT, Cambridge 02139, USA.
Nucleic Acids Res. 1995 Jul 25;23(14):2775-83. doi: 10.1093/nar/23.14.2775.
Melting of DNA in a segment bounded at both ends by regions of greater stability during electrophoresis in denaturing gradient gels show complex properties, not accommodated with standard melting theory. Compact bands of some DNA molecules become anomalously broadened at the retardation level in a denaturing gradient, or double bands may appear in a uniform denaturant concentration. These properties are associated only with molecules for which the distribution of stability calculated by the Poland-Fixman-Freire algorithms indicates that the region of lowest stability does not extend to an end of the molecule. Retention of helicity at the ends is shown by the difference in the effect of base substitution in the end domains and in the least stable domain. Both the appearance of double bands and band broadening can be explained by invoking a hypothetical metastable intermediate in melting, which is converted into the equilibrium melted form at a relatively slow rate, depending on both denaturant concentration and field strength. A kinetic model permits plausible rate constants to be inferred from the patterns. Despite the increased band width, sequence variants with base changes in the least stable domain result in readily detectable band shifts in the gradient.
在变性梯度凝胶电泳过程中，DNA在两端由稳定性更高区域界定的片段中的解链表现出复杂的特性，不符合标准的解链理论。一些DNA分子的紧密条带在变性梯度中的阻滞水平处异常变宽，或者在均匀的变性剂浓度下可能出现双条带。这些特性仅与那些通过波兰 - 菲克斯曼 - 弗雷雷算法计算稳定性分布表明最低稳定性区域未延伸至分子末端的分子相关。末端螺旋性的保留通过末端结构域和最不稳定结构域中碱基取代效应的差异得以体现。双条带的出现和条带变宽都可以通过引入解链过程中的一种假设的亚稳态中间体来解释，该中间体以相对较慢的速率转化为平衡解链形式，这取决于变性剂浓度和场强。一个动力学模型允许从这些模式中推断出合理的速率常数。尽管条带宽度增加，但在最不稳定结构域中发生碱基变化的序列变体在梯度中会导致易于检测到的条带迁移。
