CHARACTERIZATION OF THE UNUSUAL MOBILITY OF LARGE CIRCULAR DNAS IN PULSED FIELD-GRADIENT ELECTROPHORESIS
CHARACTERIZATION OF THE UNUSUAL MOBILITY OF LARGE CIRCULAR DNAS IN PULSED FIELD-GRADIENT ELECTROPHORESIS
复制标题
DOI：
10.1093/nar/16.3.925
复制
发表时间：
1988-02-11
影响因子：
14.9
通讯作者：
BEVERLEY, SM
中科院分区：
文献类型：
--
作者：
BEVERLEY, SM
大的环状扩增DNA（30和85 kb）存在于甲氨蝶呤耐药的主要利什曼原虫出现迁移的脉冲场梯度电泳（PFGE），表现出脉冲时间依赖性的流动性和迁移沿着不同的明显路径相对于大型线性染色体DNA。定量研究表明，相对脉冲时间依赖性实际上是由大的线性DNA的迁移率特性赋予的。迁移路径差异的一个影响因素是超螺旋和线性DNA迁移率的固有电压依赖性的可变性，以及不对称/不均匀的电压梯度。某些线性染色体在迁移率的电压依赖性中表现出先前未描述的脉冲-时间依赖性。当酶促松弛或物理切口时，大的环状DNA不能使用任何脉冲时间离开孔，这在常规电泳中也观察到。这些发现与PFGE理论及其在利什曼原虫和其他物种环状DNA扩增研究中的应用有关。
Large circular amplified DNAs (30 and 85 kb) present in methotrexate-resistant Leishmania major appear to migrate anomalously in pulsed field-gradient electrophoresis (PFGE), exhibiting pulse time-dependent mobility and migrating along a different apparent path relative to the large linear chromosomal DNAs. Quantitative studies indicate that the relative pulse-time dependence is actually conferred by the mobility properties of the large linear DNAs. One contributing factor to the difference in migration path is variability in the intrinsic voltage-dependence of mobility of supercoiled and linear DNAs, in combination with the asymmetrical/inhomogeneous voltage gradients. Certain linear chromosomes exhibit a previously undescribed pulse-time dependence in the voltage-dependence of mobility. When enzymatically relaxed or physically nicked the large circular DNAs fail to leave the well using any pulse time, a property also observed in conventional electrophoresis. These findings are relevant to PFGE theory, and its application to the study of circular DNA amplification in Leishmania and other species.