Stellwagen Nancy C
Department of Biochemistry, University of Iowa, Iowa City, IA 52242, USA.
Electrophoresis. 2006 Mar;27(5-6):1163-8. doi: 10.1002/elps.200500612.
The electrophoretic mobilities of curved and normal DNA molecules of the same size have been measured in polyacrylamide gels containing various acrylamide concentrations and cross-linker ratios. Ferguson plots were constructed to extrapolate the observed mobilities to zero gel concentration. The DNA samples were two 147-bp restriction fragments, called 12A and 12B, obtained from the MspI digestion of plasmid pBR322, and head-to-tail multimers of each fragment. Fragment 12A is stably curved and migrates anomalously slowly in polyacrylamide gels; fragment 12B has the conformation of normal DNA and migrates with normal electrophoretic mobilities. The extrapolated mobilities of the curved fragment 12A and its multimers at zero gel concentration are lower than the extrapolated mobilities of the normal fragment 12B and its multimers. The free solution mobility of the curved fragment 12A, measured by CE, is also lower than that of the normal fragment 12B. The combined results indicate that the extrapolated mobilities observed for curved DNA molecules at zero polyacrylamide gel concentration reflect the intrinsic differences in their free solution mobilities.
在含有不同丙烯酰胺浓度和交联剂比例的聚丙烯酰胺凝胶中,测量了相同大小的弯曲和正常DNA分子的电泳迁移率。构建了弗格森图,将观察到的迁移率外推到零凝胶浓度。DNA样品是从质粒pBR322的MspI消化中获得的两个147bp的限制性片段,分别称为12A和12B,以及每个片段的头对尾多聚体。片段12A是稳定弯曲的,在聚丙烯酰胺凝胶中迁移异常缓慢;片段12B具有正常DNA的构象,以正常的电泳迁移率迁移。弯曲片段12A及其多聚体在零凝胶浓度下的外推迁移率低于正常片段12B及其多聚体的外推迁移率。通过毛细管电泳测量的弯曲片段12A的自由溶液迁移率也低于正常片段12B的迁移率。综合结果表明,在零聚丙烯酰胺凝胶浓度下观察到的弯曲DNA分子的外推迁移率反映了它们在自由溶液迁移率方面的内在差异。