LENGTH-INDEPENDENT SEPARATION OF DNA RESTRICTION FRAGMENTS IN 2-DIMENSIONAL GEL-ELECTROPHORESIS
LENGTH-INDEPENDENT SEPARATION OF DNA RESTRICTION FRAGMENTS IN 2-DIMENSIONAL GEL-ELECTROPHORESIS
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DOI：
10.1016/0092-8674(79)90200-9
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发表时间：
1979-01-01
期刊：
影响因子：
64.5
通讯作者：
LERMAN, LS
中科院分区：
文献类型：
--
作者：
FISCHER, SG;LERMAN, LS
当双螺旋 DNA 暴露在有利于聚丙烯酰胺凝胶部分熔化的条件下时，其电泳迁移率会发生急剧的协同转变，导致迁移率大幅降低。在本实验中，转变是在 60°C 的均匀温度下实现的。 C在脲-甲酰胺混合物的浓度梯度中，噬菌体的每个EcoRI片段。或大肠杆菌 DNA 在变性剂的特征浓度下表现出迁移率转变。梯度中向更高变性剂浓度移动的片段的突然延迟导致尖锐区域的图案，其顺序取决于核苷酸序列，而不是大小，并且仅非常轻微地依赖于最后一个片段被延迟后的时间。当与垂直方向的琼脂糖长度依赖性电泳相结合时，该系统提供片段的二维分离。大肠杆菌 DNA 的 EcoRI 消化物的 250 多个片段的清晰分辨率证明了该系统的分辨能力。来自同基因λ的相应片段。在相同的位置发现了大肠杆菌的溶原菌，并且该溶原菌特有的其他片段是明显的。
When double helical DNA is exposed to conditions favoring partial melting in polyacrylamide gels, its electrophoretic mobility undergoes a sharp cooperative transition, resulting in a large reduction in mobility. In the present experiments, where the transition is effected at a uniform temperature of 60.degree. C in a concentration gradient of a urea-formamide mixture, each EcoRI fragment of [phage] .lambda. or Escherichia coli DNA exhibits the mobility transition at a characteristic concentration of the denaturant. The sudden retardation of fragments moving toward higher denaturant concentration in the gradient results in a pattern of sharpened zones in an order depending upon nucleotide sequence, rather than size, and only very slightly dependent upon the time after the last fragment was retarded. When combined with length-dependent electrophoresis in agarose in the perpendicular direction, this system provides a 2-dimensional separation of fragments. The resolving power of the system is demonstrated by the clear resolution of over 250 fragments of the EcoRI digest of E. coli DNA. Corresponding fragments from an isogenic .lambda. lysogen of E. coli are found in the same positions, and additional fragments unique to the lysogen are evident.