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首页> 外文期刊>Journal of chromatography, A: Including electrophoresis and other separation methods >Size exclusion chromatography of plasmid DNA isoforms
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Size exclusion chromatography of plasmid DNA isoforms

机译：质粒DNA亚型的大小排阻色谱

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There is considerable interest in using size exclusion chromatography (SEC) to analyze and purify specific plasmid isoforms, but there is currently no fundamental understanding of the effects of plasmid size and morphology on plasmid behavior in SEC. Experiments were performed for plasmids from 3.0 to 17.0 kbp in size. The linear and open-circular isoforms were generated from the supercoiled plasmid by appropriate enzymatic digestion. SEC retention data were obtained using a Sephacryl S-1000 SF resin packed column and an Agilent HPLC system over a range of flow rates using buffers of different ionic strength and composition. The plasmid partition coefficients, K-P, were evaluated from the first statistical moment of the chromatographic peak. The partition coefficient decreased with increasing plasmid size as expected; K-P varied from 0.299 to 0.045 for supercoiled plasmids of 3.0 to 17.0kbp. The partition coefficient also increased with increasing ionic strength due to the compaction of the DNA associated with the shielding of the intramolecular electrostatic interactions. For any plasmid size, the supercoiled isoform had the highest K-P followed by the open-circular and then the linear isoform, consistent with independent estimates of the plasmid radius of gyration as determined by static light scattering. The experimental data were analyzed using available theoretical models for the partitioning of linear and cyclic polymer chains in well-defined pore geometries. These results provide important insights into the behavior of different plasmid isoforms in size exclusion chromatography.
机译：使用尺寸排阻色谱法（SEC）分析和纯化特定质粒同工型引起了相当大的兴趣，但目前尚无基本了解质粒大小和形态对SEC中质粒行为的影响。对大小为3.0至17.0 kbp的质粒进行了实验。通过适当的酶消化，从超螺旋质粒产生线性和开放环状同工型。使用Sephacryl S-1000 SF树脂填充柱和Agilent HPLC系统，使用不同离子强度和组成的缓冲液，在一定流速范围内获得SEC保留数据。从色谱峰的第一个统计时刻开始评估质粒分配系数K-P。分配系数随着质粒大小的增加而降低;对于3.0至17.0kbp的超螺旋质粒，K-P在0.299至0.045之间变化。由于与分子内静电相互作用的屏蔽相关的DNA的压缩，分配系数也随着离子强度的增加而增加。对于任何质粒大小，超螺旋同工型都具有最高的K-P，其次是开环，然后是线性同工型，这与通过静态光散射确定的质粒回转半径的独立估计值一致。使用可用的理论模型对实验数据进行了分析，以在明确定义的孔几何结构中分配线性和环状聚合物链。这些结果为大小排阻色谱法中不同质粒同工型的行为提供了重要的见识。

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