Chen H S, Chang H T
Department of Chemistry, National Taiwan University, Taipei, ROC.
Anal Chem. 1999 May 15;71(10):2033-6. doi: 10.1021/ac981356k.
A new and simple method was demonstrated for separating phi X-174/Hae III DNA restriction fragments and DNA markers V and VI, respectively, without filling capillaries with polymer solutions prior to analysis. Using this novel method, poly(ethylene oxide) (PEO) solutions containing ethidium bromide migrated into capillaries by electroosmotic flow (EOF) during the separation. Two DNA fragments (123 and 124 bp) in markers V and VI were well-resolved. RSD values for the separation of phi X-174/Hae III DNA restriction fragments were less than 0.52% for 3 runs using a single 75-micron capillary and less than 3.96% using three different 75-micron capillaries. A highly viscous polymer solution prepared from 3% PEO was also used for separation of DNA markers V and VI. Theoretical plates up to 11.91 million/m and separation times of less than 7 min were achieved in the separation of phi X-174/Hae III DNA restriction fragments using a 10-micron capillary and a 2% PEO solution. Advantages of this method include simplicity, short separation times, the ability to use highly viscous polymer solutions for separating small DNA fragments, and the possibility of introducing several different polymer solutions into capillaries to extend the DNA separation range.
展示了一种新的简单方法，可分别分离φX-174/Hae III DNA限制片段以及DNA标记物V和VI，在分析前无需用聚合物溶液填充毛细管。使用这种新方法，在分离过程中，含溴化乙锭的聚环氧乙烷（PEO）溶液通过电渗流（EOF）迁移到毛细管中。标记物V和VI中的两个DNA片段（123和124 bp）得到了很好的分离。使用单个75微米毛细管进行3次运行时，φX-174/Hae III DNA限制片段分离的相对标准偏差（RSD）值小于0.52%，使用三个不同的75微米毛细管时小于3.96%。由3% PEO制备的高粘性聚合物溶液也用于分离DNA标记物V和VI。使用10微米毛细管和2% PEO溶液分离φX-174/Hae III DNA限制片段时，理论塔板数高达1191万/m，分离时间小于7分钟。该方法的优点包括操作简单、分离时间短、能够使用高粘性聚合物溶液分离小DNA片段以及有可能将几种不同的聚合物溶液引入毛细管以扩展DNA分离范围。
