Dougherty G, Koller T
Nucleic Acids Res. 1982 Jan 22;10(2):525-38. doi: 10.1093/nar/10.2.525.
A novel method of determining the number of superhelical turns of a covalently-closed plasmid DNA is described. It relies on the determination of the hyperchromicity, and hence the proportion of unstacked basepairs, of a partially heat-denatured sample which co-migrates during electrophoresis with nicked circular duplex DNA. The values obtained for plasmid pBR beta G DNA at 4 degrees C (-29.8 and -33.5 in the two buffers used) agree closely with the values obtained in parallel by topoisomer band-counting. Our method is less precise than band-counting but is readily applicable to determining the superhelicity of very large DNA molecules. Our results confirm earlier findings that magnesium-containing buffers cause an increase in the duplex winding angle, and hence an increase in the number of negative superhelical turns.
描述了一种确定共价闭合质粒DNA超螺旋圈数的新方法。它依赖于对部分热变性样品的增色效应的测定，从而确定未堆积碱基对的比例，该样品在电泳过程中与切口环状双链DNA共迁移。在4℃下获得的质粒pBRβG DNA的值（在两种使用的缓冲液中分别为-29.8和-33.5）与通过拓扑异构体条带计数并行获得的值非常一致。我们的方法不如条带计数精确，但很容易用于确定非常大的DNA分子的超螺旋度。我们的结果证实了早期的发现，即含镁缓冲液会导致双链缠绕角增加，从而导致负超螺旋圈数增加。
