Chen H S, Chang H T
Department of Chemistry, National Taiwan University, Taipei, Taiwan.
J Chromatogr A. 1999 Aug 20;853(1-2):337-47. doi: 10.1016/s0021-9673(99)00380-5.
Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.
在等度条件下，利用含有溴化乙锭（EtB）的聚环氧乙烷（PEO）溶液，在电渗流（EOF）存在的情况下实现了DNA标记物V和VI的单碱基分辨率分离。此外，还开发了一种称为逐步毛细管电泳（SCE）的新方法用于DNA分析，包括PEO浓度、EtB浓度以及PEO和EtB浓度的逐步变化，其中在分离过程中利用EOF将不同的PEO溶液引入毛细管。小于80 bp的DNA片段在使用20微克/毫升EtB的等度条件下以及使用1和20微克/毫升EtB的SCE中均能被检测到，但在使用1微克/毫升EtB的等度条件下则不能。SCE中DNA分离的分辨率和速度与等度方法不同，这表明DNA在SCE中经历了不同浓度的PEO和EtB。例如，具有458和504个碱基对（bp）的DNA片段在SCE中部分分离，但在等度条件下未分离。结果进一步表明，开发梯度技术以拓宽DNA分析的分离范围并提高分辨率是值得的。
