DeLeys R J, Jackson D A
Nucleic Acids Res. 1976 Mar;3(3):641-52. doi: 10.1093/nar/3.3.641.
Covalently closed relaxed SV40 DNA [SV40(I')] generated by polynucleotide ligase closure of nicked circular SV40 DNA was analyzed by agarose gel electrophoresis. The DNA can be resolved into a series of bands differing in superhelical density whose intensities are approximately symmetrical about a central most intense band. Densitometric analysis of the gel pattern has revealed that the distribution of DNA species conforms to a Boltzmann distribution and has enabled us to derive an equation for the free energy of superhelix formation for SV40 DNA. We believe the observed bands reflect the time-averaged distribution of thermally induced fluctuations in DNA chain conformation in solution at the time of ligase catalyzed phosphodiester bond formation. Densitometric analysis of native supercoiled SV40 DNA, partially unwound in the presence of ethidium bromide, demonstrates that the separation between adjacent bands is approximately half that seen with SV40(I'). Agarose gel electrophoresis was also used to measure the change in average base rotation angle as a function of temperature by a procedure independent of ethidium dye binding.
通过对切口环状SV40 DNA进行多核苷酸连接酶封闭所产生的共价闭合松弛SV40 DNA [SV40(I')],采用琼脂糖凝胶电泳进行分析。该DNA可分离成一系列超螺旋密度不同的条带,其强度围绕最中间的最强条带大致对称。对凝胶图谱的光密度分析表明,DNA种类的分布符合玻尔兹曼分布,并使我们能够推导出SV40 DNA超螺旋形成自由能的方程。我们认为观察到的条带反映了在连接酶催化磷酸二酯键形成时,溶液中DNA链构象热诱导波动的时间平均分布。对在溴化乙锭存在下部分解旋的天然超螺旋SV40 DNA进行光密度分析表明,相邻条带之间的间距约为SV40(I')的一半。琼脂糖凝胶电泳还用于通过一种与溴化乙锭染料结合无关的方法来测量平均碱基旋转角度随温度的变化。